Abstract:Objective To explore the distribution and characteristic of class 1 integrons among Shiga toxin-producing E.coli (STEC) in China，and to elucidatethe type of cassettes.Methods Antibiotics susceptibility was tested by the disk diffusion method，class 1 integron was detected by PCR assay and PCR products were sequenced and analyzed in eight isolates. Results Four isolates were multiple-drug resistant whose antibiogram were ampicillin，tetracycline，erythromycin，sulfamethoxazole/trimethoprim and ciprofloxacin，but two isolates conferred resistance of streptomycin and spectinomycin simultaneously.Only the two isolates carried class 1 integrons ranging in 1 000 bp which located on the plasmid of 7 800 bp.The sequenced PCR product demonstrated that the 1 000 bp integron harbored aad A1 gene cassettes conferred the resistance of streptomycin and spectinomycin; and linked with sul 1 and qacEΔ1 gene conferred the resistance of sulfamethoxazole and quaternary disinfectants. Conclusion The class 1 integrons exists among STEC isolates in China and determines the resistant antibiotics.

To explore the therapeutic effects of metallothionein (MT) on the H-22 liver neoplasm of BALB/c mice after administration with different concentrations of egg-milk with MT. Methods Seventy BALB/c mice were randomly divided into four groups:normal control (n=10)，tumor control (n=20)，low dose MT egg-milk group (n=20) and high dose MT egg-milk group (n=20).The mice excluding the normal control were injected with H-22 cells subcutaneously to build liver neoplasm models before they were administrated with egg-milk with MT for continuous two weeks.Lymphocyte proliferation ability and IL-2 activity in their spleen were measured with MTT method，the percentages of CD4⁺，CD8⁺ and CD25⁺ T cells were measured with flow cytometry，the killing activities of CTL and NK cells were detected with ³H-TdR method，the activities of IFN-γ and TNF-α were measured with cytopathic effect inhibition (CPEI) and L929 killed in vitro methods，respectively.The tumor volume of mice was detected at the 21th to 31th day. Results As compared with those in the tumor group，the tumor volume of the mice with H-22 cell hepatoma after administration with egg-milk with low dose and high dose MT decreased significantly at the 27th，29th and 31th day (P<0.05).The lymphocyte stimulating indexes in the low dose and high dose MT groups were significantly higher than that in the tumor control (P<0.01).Compared with the tumor control，the positive percentage of CD4⁺ T cells and the ratio of CD4⁺/CD8⁺ T cells increased significantly (P<0.01)，and the positive percentage of CD25⁺ T cells decreased significantly (P<0.05).The percentage of G₀/G₁ cells and the percentage of apoptotic cells in the high dose MT group were obviously lower than those in the tumor control (P<0.05)， and the percentage of G₂/M cells in the high dose MT group was obviously higher than that in the tumor control (P<0.05).The killing percentages of NK cells and CTL cells and the IL-2 and TNF-α activities in the high dose MT group were significantly higher than those in the tumor control (P<0.01 or P<0.05).While MT egg-milk had no effect on the IFN-γ activity. Conclusion Egg-milk with MT could build up the immune function of the mice with liver neoplasm and play a therapeutic effect on the tumor to some extent.

To investigate the change regularity of cell cycle progression in Jurkat T cells damaged by ionizing radiation.Methods The time- and dose-effects of cell cycle progression in Jurkat T cells after X-irradiation with different doses(0.075，0.5，1.0，2.0，4.0 and 6.0 Gy) were detected with flow cytometry. Results For the time-effect，the percentage of Jurkat T cells in S phase immediately increased after irradiation with 2.0 Gy X-rays，and that of G₂ + M phase cells increased significantly 8 h after irradiation as compared with that in the sham-irradiation (P<0.001).For the dose-effect，the percentage of G₀/G₁ phase cells 18 h after irradiation with 0.075 Gy and 0.5-6.0 Gy was significantly lower than that in the sham-irradiation in a dose-dependent pattern (P<0.001)，while the percentages of S and G₂ + M phase cells increased significantly (P<0.001). Conclusion Ionizing radiation with 0.5-6.0 Gy X-rays could induce S delay and G₂ arrest in Jurkat T cells in a dose-dependent pattern.

To study the changes of apoptosis and cell cycle progression of Hela cells after the poly (ADP-ribose) polymerase (PARP) was inhibited by its inhibitor 3-aminobenzamid (3-AB) and the mechanisms of PARP interaction with Hela cells damaged by irradiation. Methods Hela cell line was used.Flow cytometry (FCM) was used to examine the PARP expression of control and 3-AB groups at 0,2,4,8,12 h after administration with 5 mmol•L^-1 3-AB.The percentage of apoptotic cells and cell cycle progression of control, irradiation,3-AB plus irradiation groups were measured with FCM at 2,8,12,24 h after exposure to 2 Gy irradiation following administration with 5 mmol•L^-1 3-AB. Results The percentage of Hela cells with positive expression of PARP protein decreased after administration with 3-AB and there was significant difference between 3-AB plus irradiation group and control group (P<0.01)，and the positive expression rate of PARP was (7.60±1.36)% at 2 h after treatment with 3-AB; the percentage of apoptotic cells in the 3-AB plus irradiation group at 2,8,12,24 h after 2 Gy irradiation were higher than those in the irradiation group (P<0.01 or P<0.05), and the percentages of G₂ cells in the 3-AB plus irradiation group were lower than those in the irradiation group (P<0.01 or P<0.05) at different time points.Conclusion 3-AB could rapidly inhibit the PARP expression of Hela cells，enhance the apoptosis and block G₂ arrest induced by irradiation.

To study the anti-HBV activity and toxicity of a novel MCC-478 devivative 030705 with specific structure in vitro. Methods Anti-HBV activities in HepG2.2.15 cells were analyzed by Southern blotting in experimental groups with different doses of 030705 （0.01, 0.03, 0.10, 0.30 and 1.0 μmol•L^-1）and Adefovir Dipioxil positive control groups(0.10, 0.30, 1.0, 3.0 and 10.0 μmol•L^-1). Concentration of 50% inhibitation (IC₅₀)and concentration of 90% inhibitation (IC₉₀) were obtained. The inhibitory effect of HBeAg was analyzed in HepG2.2.15 cells by ELISA method. The cytotoxicities were tested by MTT method in the experimental groups（10, 30, 100, 300 and 1 000 μmol•L^-1） in HepG2 cells. Concentration of 50% extinction (CC₅₀) was obtained. The inhibitory effects of mitochondrial DNA contents in experimental groups（0.10, 1.0 and 10 μmol•L^-1）were tested by Dot blotting in HepG2 cells. While didenoxycytidine (ddC) groups were as positive control groups and untreated groups were as negative groups. Results Compared with Adefovir Dipioxil positive control groups, the inhibitory effects of the test compound 030705 on HBV DNA had no obvious difference（P>0.05）. Anti-HBV activity of the test compound 030705 was similar to that of Adefovir Dipioxil in HepG2. 2.15 cells. The inhibitory rates of HBeAg in experimental groups （0.01, 0.03, 0.10, 0.30 and 1.0 μmol•L^-1 030705）were 5.94%, 6.08%, 6.32%, 10.31% and 12.49%, respectively in HepG2.2.15 cells; compared with negative control group, there was no obvious difference（P>0.05）. CC₅₀ value of the test compound was 2 014 μmol•L^-1 (>1 000 μmol•L^-1) in HepG2 cells. The inhibitory rates of mitochondrial DNA content from positive drug ddC （0.10, 1.0 and 10 μmol•L^-1）were 38.43%,46.51%,56.51%, respectively in HepG2 cells; compared with negative control group, there was obvious difference（P＜0.05）. The inhibitory rates of mitochondrial DNA content from the test compound 030705 with the same concentrations were 7.00%,5.81%,5.78%, respectively in HepG2 cells; compared with negative control group, there was no obvious difference（P＞0.05）. Conclusion Anti-HBV activity of the test compound 030705 was similar to that of Adefovir Dipioxil in HepG2.2.15 cells. CC₅₀ value of the test compound is 2014 μmol•L^-1 (>1 000 μmol•L^-1) in HepG2 cells. No apparent reductions of mitochondrial DNA content are observed in HepG2 cells. The test compound 030705 with specific structure may be a new promising anti-HBV agent.

To explore the effect of mesenchymal stem cells (MSCs) on nervous function in rats with focal cerebral ischemia.Methods The MSCs were cultivated，purified，and proliferated in vitro by density gradient and adherence to plastic dishes method.The models of Wistar rats were prepared after middle cerebral artery occlusion (MCAO) of right lasted 90 min and reperfusion 1 h.Wistar rats were randomly divided into normal control group (A,n=10)，sham operation group (B,n=10)，no-handle group after cerebral ischemia/reperfusion (C,n=10)，free-serm DMEM transplantation group after cerebral ischemia/reperfusion (D,n=10)，MSCs transplantation group after cerebral ischemia/reperfusion (E,n=10).After identified by flow cytometry，5 μL 5-bromo-2-deoxyuridine(BrdU) labeled MSCs (4×10⁵• μL^-1) and 5 μL serum-free DMEM were respectively injected intracerebraly into ischemic boundary zone of right in D and E groups.Immunohistochemical method was used to detect the expression and survival of BrdU-labeled MSCs in vivo.Nervous function behavioral tests were performed on 1st，3th，7th and 28th day after transplantation by forelimb use asymmetry test and postural reflex test.Results MSCs were successfully purified and proliferated in vitro. The MSCs expressed CD29，CD44, but didn’t expressed CD34，CD45，CD31 identified by flow cytometry.transplanted MSCs survived and were localized to the ischemic boundary zone.Behavioral tests of every group were improved with time prolonged.However，MSCs transplantation group was significantly better than any other groups (P<0.05) .And on 7th day after MSCs transplantation，nervous function was significantly improv ed than other time points in MSCs transplantation group (P<0.01).Conclusion The intracerebral administration of MSCs into ischemic boundary zone can promote the recovery of nervous function of rats with focal cerebral ischemia.Forelimb use asymmetry test can objectively evaluate motor function for a long time.

To isolate and cultivate hepatic stem cells(HSCs) from rat fetal liver in vitro and identify their biological features.Methods Collagenase perfusion method and mechanical cutting method were used to isolate HSCs from rat fetal liver which were then cultivated by H-DMEM containing 10% fetal bovine serum.The cell surface antigen expression of HSCs was observed with immunocytochemical method under confocal laser scanning microscope.Results The isolated HSCs from rat fetal liver grew to monolayer 5 d after cultivation in vitro.They presented pykno-round cells and distinct borderline under the light microscope.After 8 d the cells grew like epithelium.These cells expressed AFP antigen，CK 18 and CK19. Conclusion The cultivated cells are proved to be HSCs and can proliferate quickly in vitro.

To clone the human bone morphogenetic protein-2 (hBMP-2) gene cDNA and construct its replication-deficient type 5 adenoviral vector.Methods Using osteoblastic sarcoma tissue to extract cell total RNA，hBMP-2 cDNA was amplified by RT-PCR.The hBMP-2 cDNA was then inserted into cloning vector pGEM-T.The recombinant plasmid was screened and the recombinant plasmid which was confirmed by restriction enzyme，PCR and sequence analysis was gotten.To construct the recombinant adenoviral vector，the BMP-2 cDNA was subcloned into a shuttle vector and the recombinant pShuttle-BMP2 vector was obtained.It was subcloned into Adeno-X adenoviral genomic DNA and the pAd-BMP2 DNA was obtained. Results A length of 1 200 bp BMP-2 cDNA was cloned，and it was subcloned into adenoviral vector，the recombinant adenovirus was constructed successfully.After measuring the titre of virus（1×10¹¹ nfu•L^-1），the target gene was ev aluated by PCR.Conclusion The hBMP-2 adenoviral is obtained.The successful construction of pAd-BMP2 may provide us useful target for the further research.

To evaluate the protective ability of fusion protein of Hsp65-MOMP-T-epitopes(H-ctm1) against C.trachomatis(Ct) genital tract infection .Methods Female C57BL/6 mice aged 6—8 weeks were divided into H-ctm1，heat-killed EBs(HK-EBs) and PBS group（28 mice in each group，9 for detection of the amount of Ct IFU in vaginal tract of mice; 10 for histopathology; 9 for analysis of hydrosalpinx situation）.They were immunized by H-ctm1，HK-EBs and PBS, respectively.7 d before inoculation with Ct through vagina，progesterone was administered to all the experimental mice by subcutaneous injection.The number of Ct shedding from genital tract，inflammation score of genital tract，and situation of hydrosalpinx were evaluated between the three groups. Results On the 3rd and 6th day after infection，there was no significant difference of the amount of Ct in vaginal tract of mice between H-ctm1 and HK-EBs immunized groups; however，they were lower than that in PBS-immunized group（P＜0.01）.On the 9th and 18th day afterinfection，the amount of Ct in vaginal tract in H-ctm1 immunized group was lower than that in HK-EBS immunized group（P＜0.01 or P＜0.05）.6 d after infection，the inflammation scores in H-ctm1 immunized mice and HK-EBs immunized mice were lower than that in PBS immunized mice（P＜0.01 or P＜0.05）.12 d after infection，there were significant differences of vaginal inflammation scores between H-ctm1 immunized mice and HK-EBS immunized mice or PBS immunized mice（P＜0.05 or P＜0.01）.Hydrosalpinx was not found in H-ctm1 or HK-EBS immunized mice 40 d after infection.In contrast，unilateral or bilateral hydrosalpinx was present in all nine PBS immunized mice. Conclusion Protective immune response can be induced in the mice immunized with H-ctm1 following the Ct infection of genital tract.H-ctm1 as a candidate vaccine can shorten the duration of Ct infection effectively，which is better than HK-EBs.

To explore the possibility of human vascular endothelial growth factor 165 (hVEGF₁₆₅) to transfect rabbit mesenchymal stem cells (MS Cs)，and establish the experimental foundation of angiogenesis tissue engineering organization and the treatment of ischemic disorders. Methods pcDNA3.0-VEGF₁₆₅ eukaryotic expression vector was constructed，the vector was used directedly to transfect MSCs.The supernatant then was collected and the soluble protein of hVEGF gene expression was analysed with ELISA method.The proliferation capability of human umbilical vein endothelial cells (HUVEC) stimulated by the supernatant was measured with MTT methods，untreated MSCs and transfected MSCs were used as control groups. Results hVEGF eukaryotic expression vector was successfully constructed and transfected into the MSCs.Compared with the control group，concentration of hVEGF protein in the supernatant of the cultured cells increased significantly after the MSCs were tranfected with pcDNA3.0-VEGF₁₆₅ for 24，48，72 h (P<0.05)，hVEGF protein was still found till the 12th progeny.The proliferation rate of HUVEC cells in the culture containing 2% supernatant of transfected cells was obviously higher than that of the control group (P<0.05). Conclusion hVEGF gene could be successfully transfected into MSCs and could express effectively.

To explore the feasibility and safety of repairing common bile duct with laser-welding assited with glue after placing a degradable stent in it. Methods A total of 45 local healthy hybrid dogs were chosen and treated with choledochotomy.According to the primary repairing methods，the dogs were divided into three groups averagely and randomly：single suturing group(SSG),laser-welding group(LWG) and laser-welding assisted with chemical glue group(LWACGG).Before repairing，a degradable sent PLA was inserted into the common bile duct.The anastomotic time of common bile duct was recorded，measure the intention of the bursting pressure and the diameter of the anastomotic stoma were measured，the pathologic changes of the anastomotic stoma and appearance of stents were observed under electron microscope.Results Compared with the single suturing group，the anastomotic time in laser-welding group and laser-welding assisted with chemical glue group was more shorter (P<0.05); at the 1st，4 th and 12 th week after operation，there were not any significant differences of the intention of the bursting pressure between the single suturing group and laser-welding assisted with chemical glue group(P>0.05)，but it in laser-welding group was more lower than those in both groups mentioned above(P<0.05).At the 4 th，12 th week after operation， the diameters of the stoma didn’t dilate obviously in the laser-welding group and laser-welding assisted with chemical glue group compared with before anastomosis，but it dilated markedly in the single suturing group.Histological observation result showed less fibrosis and less inflammation reaction in the laser-welding and laser-welding assisted with chemical glue groups， but more serious inflammation could be seen in the single suturing group. The SEM photos showed the surface of the stents turned from initially smooth into rough and the cross-sections turned from compact into porous during degradation. Conclusion It is possible and safe of repairing common bile duct with laser welding assisted with chemical glue after placing a degradable stent in it.

To construct an eukaryotic expression plasmid containing the truncated gene encoding the part of herpes simplex virus type 1 glycoprotein B(HSV-1,gB) and evaluate its effects on cell-mediated immunity.Methods The part encoding sequence of the glycoprotein B 14-507 amino acid was amplified from HSV-1 SM44 DNA genome by polymerase chain reaction (PCR)，and then was directionally cloned into eukaryotic expression vector pcDNA₃，the recombinant vector pcDNA₃-gBt was confirmed by the restrictived endonuclease analysis，PCR and sequence analysis.It was employed to evaluate immune response of the mice inoculated triply with the DNA vaccine.The transformation of CD4⁺/CD8⁺T lymp hocyte was detected by means of antibody and the influence of cell-mediated immunity represented by CTL was assayed through CFSE/PI flow cytometry.Results The construction of the HSV-1 gBt DNA vaccine was identified; cytotoxic activity was strengthened; the amount of CD4⁺ T cell from BALB/c mice immuned with pcDNA₃-gBt was higher than mice immuned with plasmid pcDNA₃and saline，the tendacy was the same for CTL，but the amount of CD4⁺ T cell and CD4⁺/CD8⁺ from BALB/c mice immuned with pcDNA₃-gBt had no obvious difference with mice immuned with plasmid pcDNA₃ and saline.Conclusion The HSV-1 gBt DNA vaccine is powerful to induce cell-mediated immunity .

To investigate the immunohistochemical exprssions of IL-1 and VEGF in wound healing process for possible application in determination of incised wound age.Methods Fifty one male SD rats were divided into 17 groups at random， the rat back skin in thirteen groups was incised，then the rats of these groups were sacraficed for different interval (5,10, 30 min and 1,3,5,7,9, 12 h and 1,3,5 , 7 d);another three groups were used as postmortem control，the incisition wound was performed at 5,10,30 after the rat were sacraficed，rat skin from the group without any additional treatment was used as normal control.Immunohistochemical study on the expressions of IL-1 and VEGF were performed on rats skin incised wounds and postmortem incised wounds，and the normal rat skin . Results In antermortem skin incisions，the positive stain of IL-1 was found as early as 0.5 h after incised and lasted till 5 d in epithelial and macrophages of granullation tissue，the rate of IL-1 positive cells was low in the wound specimens in 3 h，and maximized in the wound specimens at 5 h（49.87%±3.42%）and 5 d（47.56%±7.52%）,then the rate decreased to norml.The positive expression of VEGF was found at 3 h after incision and lasted till 10 d.There were no changes of VEGF and IL-1 in the postmortem incised group compared with normal control group. Conclusion The immunohistochemical expressions of VEGF and IL-1 change regularly according to different wound time，VEGF and IL-1 are considered as potentially useful markers for wound time determination in forensic practice.

To explore the effects of donepezil hydrochloride on learning and memory function in normal adult rats. Methods According to weight,40 Wistar rats were randomly divided into 2 groups: normal control group and donepezil hydrochloride group(n=20).The learning and memory function in rats were determined with Morris water maze and Step-down tests.Results Compared with control group，latency，distance，average speed and starting angle of rats treated by donepezil hydrochloride did not change significantly from the first day to the sixth day （P>0.05）;the time in platform， the time of platform quadrant in 2 minutes，times passing platform， the percentage of distance in platform / total distance and average speed also did not change significantly on the 7th day in Morris water maze test（P>0.05） ; Error times of rats treated by donepezil hydrochloride did not decrease on the 1st and 2nd day（P>0.05）,latency did not prolong in the 2nd day in Step-down test（P>0.05） Conclusion Donepezil hydrochloride do not improve learning and memory function in normal adult rats.

To study the effects of hypoxic tolerance on learning and memory in mice.Methods Forty mice were divided into 3 groups by weight randomly：control group，run 1 hypoxic group and run 4 hypoxic group. Morris water maze test，step-down test and step through test were used to observe the effect of hypoxic tolerance　on learning and memory in mice .Results In the Morris water maze test，compared with control group，the total swimming time of run 1 hypoxic mice increased significantly on the 1st,2nd,4th,5th day and the total swimming distance of run 1 hypoxic mice also increased evidently on the 2nd,3th,5th day (P<0.05); compared with run 1 hypoxic mice，the total swimming time of run 4 hypoxic mice decreased significantly in the first five days(P<0.01　or P<0.05)，and the total swimming distance of run 4 hypoxic mice decreased evidently on the 1st,4th,5th day (P<0.01 or P<0.05)，and the starting angle of run 4 hypoxic mice diminished evidently on the first day(P<0.05); compared with control group，the total swimming distance of run 4 hypoxic mice decreased evidently on the first day (P<0.05).In the step-down test，compared with control group，wrong times on the 2nd day of run 1 hypoxic mice increased evidently(P<0.05);compared with run 1 hypoxic mice，latency and wrong times on the 2nd day of run 4 hypoxic mice also decreased significantly(P<0.05).Conclusion Learning and memory of the mice injuried by run 1 hypoxic can be improved by run 4 hypoxic tolerance.

To investigate the therapeutic effects of glucagon-like peptide-1 （GLP-1） analog on rats with experimental diabetes mellitus.Methods Eighty-four Wistar rats were divided randomly into normal control group，high fat forage group，low-dose GLP-1 analog group，middle-dose GLP-1 analog group，high-dose GLP-1 analog group,diabetes mellitus model group and positive control group.Diabetes mellitus animal models were induced with high fat forage and injection of 40 mg•kg^-1 STZ.After 7 and 14 d of GLP-1 analog therapy，the level of fasting plasma glucose(FPG) was detected with enzyme-chemical method.After 14 d of GLP-1 analog therapy，the levels of serum total cholesterols (TC)，triglyceride (TG)，low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were detected with enzyme-chemical method. The levels of serum insulin and C-peptide were measured with immunoradiometric assay method .Results After 14 d of GLP-1 analog injection，food intake in high-dose GLP-1 analog（100 μg•kg^-1）group was less than that in DM group（P<0.01）; Water intake in middle-dose GLP-1 analog（10 μg•kg^-1） group and high dose （100 μg•kg^-1）group were less than that in DM group（P<0.01）. There were no statistical differences of body weight between all therapeutic groups and DM group(P<0.05).The levels of FPG in middle-dose GLP-1 analog（10 μg•kg^-1 ） group and high dose （100 μg•kg^-1）group were lower than that in DM group（P<0.01）.The level of TG in high-dose GLP-1 analog（100 μg•kg^-1）group was lower than that in DM group（P<0.01）.The levels of LDL in middle-dose GLP-1 analog（10 μg•kg^-1） group and high dose （100 μg•kg^-1）group were lo wer than that in DM group（P<0.01）.The levels of serum insulin and C-peptide in middl e-dose GLP-1 analog（10 μg•kg^-1） group and high dose （100 μg•kg^-1）group were higher than that in DM group（P<0.05）. Conclusion GLP-1 analog can paly an important role in treatment of experimental diabetes mellitus through multiple ways.

To study the effects of atorvastatin on the expressions of peroxisome proliferators-activated receptor gamma（PPARγ）and matrix metalloproteinase-9（MMP-9）in atherosclerosis（AS） tissue. Methods Atherosclerotic rabbit models on aortas were established with the high-cholesterol diet，and animals were divided into control，model and atorvastatin groups(n=8).The expressions of PPARγ and MMP-9 and the effects of atorvastatin on them were observed by means of immunohistochemistry. Results The expression of PPARγ in model group(14.38%±2.58%)was higher than that in control group（7.82%±0.96%）（P<0.01），and the expression of MMP-9 in model group（17.13%±1.58%）was also higher than that in control group（2.72%±0.36%）（P<0.01）.The expression of PPARγ in atorvastatin group（16.11%±2.35%）was higher than in model group（P<0.01），and the expression of MMP-9 in atorvastatin group（12.11%±2.05%）was lower than that in model group （P<0.01）. Conclusion Atorvastatin can increase the expression of PPARγ and inhibit the expression of MMP-9.These results suggest that atorvastatin has anti-inflammatory effects which are probably related to its activating PPARγ and inhibiting MMP-9.

To study the relationship between activity and virulence of secretory acid proteinase and extracellular phosphlipase of different Candida albicans isolated from various specimens .Methods 190 strains of Candida albicans were isolated from various specimens (sputum,blood,thrush,wound,secretion of vaginitis) of patients with Candida albicans infection in hospital. Milk-plate medium and egg-yolk medium were used respectively to test the activities of secretory acid proteinase and extracellular phosphlipase of Candida albicans.The suspension of two forms of Candida albicans （5×10⁶CFU•mL^-1） were injected to vein of tail in mice respectively in virulence test.The mortality and mean survival time of mice were observed in 1 month.The virulence was appreciated with the mean survival time of mice. Results All of 190 strains，the positive detectable rates of secretory acid proteinase and extracellular phosphlipase were 83.68% and 85.26%，respectively.Animal experimental results showed the activities of secretory acid proteinase and extracellular phospolipase in hyphal forms were significantly higher than that in spore form(P<0.01,P<0.05).The mortality of mice injected with hyphal forms was higher than that of mice injected with spore forms (P<0.05)and the mean survival time of mice injected with hyphal forms was shorter than that of mice injected with spore forms(P<0.01). Conclusion Secretory acid proteinase and extracellular phosphlipase are important virulence factors of Candida albicans.The activities of secretory acid proteinase and extracellular phosphlipase can be used as direct parameters for virulence of Candida albicans.

To study the toxic effect of exotoxin A of Pseudomonas aeruginosa on keratocytes.Methods Three-dimensional gels of type I collagen containing rabbit keratocytes were incubated in the presence of different concentrations of exotoxin A (0.1,1.0,10 mg•L^-1)，cultivated for 24 h at 37℃， the change of keratocytes in morphology was observed under the light microscope，and the amount of lactate dehydrogenase(LDH) was measured by enzyme linked immunosorbent assay (EL ISA).Results The LDH contents in different concentrations(0.1,1.0,10 mg•L^-1)of exotoxin A groups were higher than that in the group without exotoxin A (P<0.01）.The normal keratocytes were long atractoid.When keratocytes were incubated in the presence of different concentrations of exotoxin A,they changed into cycloid .Conclusion Exotoxin A of Pseudomonas aeruginosa can injury keratocytes and its effect is in a dose-dependent manner.

To investigate the expression of connexin CX43 using hybridization techniques after GABA restraining receptor gene was transferred into the supramammillary of hypothalamus in Kainic acid (KA) induced epileptic rats.Methods Twenty-four male Wistar rats were divided into four groups: control group (n=2),surgery group (n=2),KA group (n=10) and GABA restraining receptor gene transferred group (GABA group) (n=10).No treatment was given to the control group.Physiological saline (1 μL) was injected into right amygdala of rat in surgery group.In KA group,KA (1 μL,1 μg) was injected into the amygdala of rat to build epileptic model.In GABA group,the GABA restraining receptor gene (400 nL,40 ng) was transferred into the supramammillary of hypothalamus by HVJ-liposome 48 h before KA was injected into amygdala.The expressions of CX43 mRNA and morphological changes different time (3 h,6 h,24 h,3d,7d) were observed by in situ hybridization in each group.Results In control group and surgery group,the morphological manifestations were normal,and the hippocampus structures were complete.In KA group,swell and degenerative hippocampus neurons were showed and deteriorated with time.In GABA group,the degeneration and necroses of hippocampus neurons were relatively alleviated.The positive expression of CX43 mRNA was few in control group and surgery group.And in KA group it increased with time.In GABA group,the positive expression of CX43 mRNA was fewer than that of KA group at every period. Conclusion The expression of CX43 mRNA in hippocampus can be decreased by transferring GABA receptor gene into hypothalamus.

To determine the role of epidermal growth factor receptor (EGFR) signaling in endochondral ossification.Methods Long bones, from EGFR knockout mice (EGFR-/-),wild type (EGFR+/+) and (or) heterozygous mice (EGFR+/-) were collected and Trichrome Masson staining,in situ hybridization and tartrate-resistant acid phosphatase (TRAP) staining were used to observe endochondral ossification and recruitment of osteoclasts; Osteoclast formation was observed by addition of EGFR tyrosine kinase inhibitor AG1478 to the cultured osteoclasts from bone marrow cells. Results Due to the same phenotype of EGFR+/+ and EGFR+/-,they were regarded as EGFR+/+ in our study.EGFR deficiency caused delayed primary endochondral ossification of cartilage anlage and delayed osteoclast recruitment.The hypertrophic cartilage area in EGFR-/-mice was about 5 times longer than that in EGFR+/+ mice.There was different distribution of MMP-9 between EGFR+/+ and EGFR-/- in E16.5,but there was no difference about the quantity of MMP-9 in osteoclasts between EGFR-/- and EGFR+/+.However,inhibition of EGFR signaling with AG1478 significantly decreased osteoclast formation compared with control group with DMSO (P<0.01). Conclusion Inhibition of EGFR signaling can delay endochondral ossification,osteoclast recruitment and decrease osteoclast formation.

To explore the effect of glycine on the intestinal barrier of rats with obstructive jaundice. Methods The male Wistar rats weighing 220—260g were randomly assigned to three groups(n=20): sham operation group (A), common bile ligation gruop(B), common bile duct ligation plus glycine group(C). Except sham operation group (A), rat models with obstructive jaundice in the other groups were set up by double ligation of the common bile duct. The rats in group C was fed with 5% glycine solution for 5 d. From the second day after the operation , the rats in group C were fed according to the preoperative standard. On the 21th day after operation, the rats were killed and serum bilirubin (BIL) and the amount of nitric oxide (NO) in intestinal tissue were determined, the morphological changes of the intestine were observed, the villus height and mucosal thickness were measured. Results Compared with group A, the serum BIL and the amount of NO in intestinal tissue were increased and the villus height and mucosal thickness were reduced in group B (P<0.01). As compared with group B, the amount of NO were reduced in group C (P<0.01), the architecture distortion in the intestinal tissue was alleviated, an increase was noted in the villus height and mucosal thickness (P<0.01). In group C, the BIL increased (P<0.01), however there were no significant differences of the amount of NO, villus height, mucosal thickness (P>0.05) compared with group A. Conclusion Glycine can decrease the level of NO in the intestinal tissue and improve the damaged intestinal barrier obviously.

To discuss the effect of growth hormone (GH) on acute gastric mucosal lesion (AGML) in rats.Methods 120 Wistar rats were divided into four groups randomly: blank control group(n=8),simple model group(n=32),model+rhGH group(n=40) and model+cimetidine group(n=40).Animal models of AGML were set up through soak and tie of rats.GH was injected to the rats in experimental groups,while cimetidine was used in the positive control group.Morphologic changes of rat gastric mucosa in each group were observed and compared on different days(0,4,8,12 d) after stress through gross looking,light microscope,and electron microscope.Ulcer index(UI),secretion amount of gastric acid,pH value and gastric mucosal were compared.Results Compared with blank control and model+cimetidine groups,the histomorphologic changes of gastric mucosa were obviously alleviated in model+rhGH group: only part of epithelial cells swelled,red blood cells were seldom seen,neutrophils infiltration reduced obviously,no remarkable changes were observed in the submucosal tissue.Mucosal cells were in good state and degeneration were seen in only a few cells under the electronic microscope.In addition,UI and the degree of gastric mucosal atrophy in model+rhGH group were lower than that in control group significantly(P<0.05),and the difference became more and more obvious with the prolongation of duration of administration of GH. Conclusion GH can strengthen the mucosal barrier by promoting the growth and reparation of gastric mucosa,and promote restoration of gastric mucosa in both structure and function,thereby play curative effect on AGML.

To explore the relationship between dipeptidyl peptidaseⅡ(DPPⅡ) and the pathogenesis of congenital cataract.Methods Eighty SCR rats with congenital cataract were randomly divided into four groups:8,10,12 and 14 weeks groups(n=20).Normal Wistar rats at each age were used as control groups.Immunohistochemical experiments using the streptavidin-biotin immunoper-oxidase method were used to observe the immunoreactivity changes of DPPⅡ in lens in SCR rats at different time(8,10,12,14 weeks). Results The immunoreactivities of DPPⅡ in the lens epithelial cells and fibres of cataractous lenses of all ages were higher than those in control groups.At the 12th and 14th week,immunoreactive staining of DPPⅡwas found to extend into the perinuclear region.Conclusion The immunoreactivity of DPPⅡ is enhanced in cataractous rat lenses.DPPⅡ may be participated in the proteolytic modification of lens proteins during cataractogenesis.

Abstract:Objective To explore the relationship between dipeptidyl peptidaseⅡ(DPPⅡ) and the pathogenesis of congenital cataract.Methods Eighty SCR rats with congenital cataract were randomly divided into four groups:8,10,12 and 14 weeks groups(n=20).Normal Wistar rats at each age were used as control groups.Immunohistochemical experiments using the streptavidin-biotin immunoper-oxidase method were used to observe the immunoreactivity changes of DPPⅡ in lens in SCR rats at different time(8,10,12,14 weeks).Results The immunoreactivities of DPPⅡ in the lens epithelial cells and fibres of cataractous lenses of all ages were higher than those in control groups.At the 12th and 14th week,immunoreactive staining of DPPⅡwas found to extend into the perinuclear region.Conclusion The immunoreactivity of DPPⅡ is enhanced in cataractous rat lenses.DPPⅡ may be participated in the proteolytic modification of lens proteins during cataractogenesis.

Abstract:Objective To evaluate the effect of basic fibroblast growth factor (bFGF) on sheep bone marrow mesenchymal stem cells (BMSCs) proliferation and differentiation in vitro. Methods The sheep BMSCs were isolated and expanded in vitro　and divided into experimental group and control group. bFGF(2 μg•L^-1) was added into cell culture medium of experimental group. In control group, no bFGF was added. The morphological differences of BMSCs between two group were observed. Growth curves and alkaline phosphatase(ALP) activity were assessed. In order to observe the influence of bFGF on differentiation potential of BMSCs, osteogenic and adipogenic inductions were conducted on BMSCs in two groups. Results Fibroblast colonies appeared 1 week after seeding of the mononuclear cells. 2 weeks later, more colonies formed and each colony became larger . The cell number was 2.5 times of the control when the first passage cells grew to confluence and the ALP activity in experimental group was lower than that in control group(P<0.05). Osteogenic and adipogenic inductions were confirmed by Von Kossa and oil red-“O” staining respectively.Conclusion bFGF can increase sheep BMSCs colony and proliferation efficiency and inhibit ALP activity while retain the potential of osteogenic and adipogenic differentiation in vitro.

Abstract:Objective To explore the cytocompatibility of biodegradable PEG-PLA-PGL/RGD ［pol（ethyleneglycol)-b-poly(l-lactide)-b-poly(l-glutamic acid）］ with human umbilical vein endothelial cells (HUVECs) and provide a basis for the development of biodegradable material for vessel scaffold. Methods The HUVESCs cultivated in vitro were inoculated onto PEG-PLA-PGL/RGD membrane and their growth and cell count were observed by phase contrast microscopy. Immunohistochemistry was used to examine the existence of factor Ⅷ expressed by HUVESs.The proliferation of cells was observed by MTT method.Results The observation result of phase contrast microscopy showed that endothelial cells grew well in PEG-PLA-PGL/RGD membrane and showed no significant difference with control. Cells attached to the PEG-PLA-PGL/ RGD and stretched after grafting 4—6 h.After 3 d,cells began to grow in colony rapidly.After 5 d,colony began to fuse and seemed like stone,the cells were shuttle and polygon in shape. Counting of HUVECs in 1 week: growth rate of cells in experimental group didn’〖KG-3〗t decrease significantly as compared with control（P>0.05）. There were no significant differences of the proliferation index of cells by MTT method 1, 3,5 and 7 d after inoculation between experimental and control groups (P>0.05). Conclusion Tissue engineering blood vessel with normal cytocompatibility to a certain degree can be constructed through seeding cultured HUVECs on PEG-PLA-PGL/RGD scaffold and PEG-PLA-PGL/RGD is a perfect biodegradable carrier material.

Abstract:Objective To study efficacy enhancing and side-effects reducing of Phellinus linteus (PL) on radiotherapy or chemotherapy in mice bearing tumor. Methods MFC were implanted subcutaneously to the right anterior limb of mice and the mice were randomly divided into thirteen groups: control, CTX groups （20, 10 and 5 mg•kg^-1）and  groups of PL（2.0,1.0 and 0.5 g•kg^-1）with CTX（20, 10 and 5 mg•kg^-1）,respectively.Mice in control group were administered intragastricly at a dose 0.01 mL•g^-1 for 10 d；PL were administered intragastricly for 10 d；CTX were administered parenterally every other day for 10 d. The body weight and the tumor weight of mice were measured.HepA were implanted subcutaneously to the lateral side of tibia of the right posterior limb of mice and the mice were randomly divided into five groups: control, radiotherapy group and groups of PL（2.0,1.0 and 0.5 g•kg^-1）with radiotherapy, respectively. Mice in control group were administered intragastricly with distilled water at a dose of 0.01 mL•g^-1 for 14 d；PL was administered intragastricly for 14 d；after PL was administered for 5 d, X-rays were utilized at a dose of 0.5 Gy one time everday for 5 d. The body weight and the tumor weight of mice were measured. Results Compared with control group, the tumor weights decreased significantly in CTX groups and groups of PL with CTX (P<0.01). Compared with the groups of the equal-dose CTX, the tumor weights in groups of PL with CTX respectively (P<0.05 or P<0.01). Compared with control group, the body weights decreased in CTX groups (P<0.05 or P<0.01) ,but the body weights in groups of PL with CTX didn’t change remarkably. Compared with control group, the tumor wei ghts decreased in radiotherapy group and groups of PL with radiotherapy (P<0.01). Compared with radiotherapy group ,the tumor weights decreased in groups of PL with radiotherapy (P<0.05 or P<0.01).Compared with control group, the body weight decreased in radiotherapy group (P<0.05),but the body weights in groups of PL with radiotherapy didn’t change remarkably.Conclusion PL is in coordination with radiotherapy or chemotherapy on anti-tumor,and PL can enhance efficacy and reduce side-effects of radiotherapy or chemotherapy.

Abstract:Objective To investigate the gene expressions of IL-1 and TNF-α in bronchoalveolar lavage fluid(BALF) and lung tissues in mice with pulmonary fibrosis(PF) at various periods and to study the effects of cytokines on pulmonary fibrosis. Methods Forty male mice were randomly divided into two groups:experimental group(n=30) and control group(n=10), and poured respectively by bleomycin solutions and saline and killed on the 3rd, 7th,14th,28th and 56th day. RT-PCR method was used to analyse the mRNA expressions of IL-1 and TNF-α.Results ① The expressions of IL-1 mRNA and TNF-α mRNA in BALF in experimental group were higher than those in control group(P<0.05),and peaked on 14th day. ②The mRNA expressions of IL-1 and TNF-α in lung tissues in experimental group were higher than those in control group(P<0.01), and peaked on the 7th and 14th day. Conclusion The mRNA expression time of IL-1 and TNF-α are different, by which the progression and activities of PF can be judged. They play an important role in the stage of pulmonary injury in PF.

Abstract:Objective To reconfirm the association between KPNB3 gene polymorphism with schizophrenia in Chinese population. Methods Two single nucleotide polymorphisms (SNPs), rs2588014 and rs626716 at the KPNB3 locus, were detected in 304 Chinese Han family trios consisting of fathers, mothers and affected offsprings with schizophrenia with PCR-based restriction fragment length polymorphism (RFLP) analysis. The Hardy-Weinberg equilibrium for genotypic distribution was estimated by the goodness-of-fit test. The UNPHASED program was applied to perform the transmission disequilibrium test (TDT), the haplotype analysis and the pair-wise measure of linkage disequilibrium (LD) between these 2 SNPs. Results The genotypic distribution of both rs2588014 and rs626716 was not deviated from the Hardy-Weinberg equilibrium (P>0.05). The TDT showed significantly biased transmission from parents to affected offsprings for rs626716（χ²=9.31，P=0.002 3）.The allelic frequency transmitted from the heterozygote parents didn’〖KG-3〗t deviate for 50% at rs2588014 (χ²=3.44, P=0.064). The haplotype analysis also showed rs2588104(c)-rs626716(c) was transmitted by parents（χ²=9.8,P=0.006 8）. Conclusion There may be genetic association between KPNB3 gene polymorphism and schizophrenia in the Han descent population in the north of China.

Abstract:Objective To investigate the association between schizophrenia and the proline dehydrogenase (PRODH) gene rs2058672in chromosome 22q11. Methods The PCR-based restriction fragment length polymorphism (RFLP) technique was used to detect the G/A single nucleotide polymorphism (SNP), rs2058672 locus, at the PRODH gene in 163 Chinese Han family trios consisting of fathers, mothers and affected offsprings with schizophrenia. Results The distribution of genotypic frequency of rs2058672 did not deviate from the Hardy-Weinberg equilibrium; The haplotype-based haplotype relative risk test ( HHRR) did not show a significant difference between transmitted and non-transmitted alleles in family trios of schizophrenia patient (χ²=0.197, P>0.05 ); transmission disequilibrium test (TDT) did not show significantly biased transmission from parents to affected offspring (χ²=0.163, P>0.05); there were not correlations between allelic and genotypic frequencies and the clinical symptoms of schizophrenia (P>0.05 ). Conclusion The rs2058672 locus of PRODH gene may not be the susceptible locus of schizophrenia in Chinese Han population.

Abstract:Objective To identify the relationship between the CACNA1H gene and childhood absence epilepsy(CAE).Methods Exons 6 to 12 of CACNA1H gene were sequenced in 100 CAE trios of Han population in China.Variants in these regions were then analyzed.Results Fourteen variants were identified only in 32 CAE patients that were not present in 191 normal gender-matched controls.Twelve of them had not been previously reported.These 14 variants included 2 nonsynonymous variants,3 synonymous variants,9 intronic variants,including a single nucleotide polymorphism (SNP) 52037C>T in intron11 found in 17 CAE patients.The result of transmission disequilibrium test (TDT) indicated that 52037C>T in intron11 was in significant transmission disequilibrium in CAE trios（χ²＝9.783,P=0.001 76）.Conclusion CACNA1H is a susceptibility gene for CAE in the Chinese Han population.

Abstract:Objective To investigate the association between KPNB3 gene at 13q32 and schizophrenia.Methods The method of PCR-RFLP was conducted to examine the genotypes of KPNB3 gene in 382 patients with schizophrenia and 389 healthy Han people in the north of China.And then the frequencies of genotypes and alleles of KPNB3 gene were statistically computed.Results The χ² goodness-fit test showed the genotypic distribution of three SNPs (rs626716,rs624066 and rs2761072) did not deviate from Hardy-Weinberg equilibrium either in the patient group or in the control group(P>0.05).There were no significantly differences of the allelic and genotypic frequency distributions of KPNB3 between the patients and the controls(P>0.05).Conclusion The KPNB3 gene may not be the susceptible locus of schizophrenia.

Abstract:Objective To investigate the genetic association between APOD gene and schizophrenia in the North Chinese Han population.Methods The PCR-based restriction fragment length polymorphism (RFLP) technique was conducted to examine the genotypes of rs2280250 locus of APOD gene in 291 family trios consisting of fathers,mothers,and affected offsprings with schizophrenia,and then the frequencies of genotype and allele of rs2280520 locus of APOD gene were statistically computed.Results ① The distribution of genotypic frequency of rs2280250 did not deviate from the Hardy-Weinberg equilibrium either in the patient group (χ²=0.178,df=1,P=0.673) or in the parent group (χ²=0.597,df=1,P=0.440); ②The transmission disquilibrium test (TDT) showed the allele transmitted from heterozygotic parents to affected offsprings did not bias from 50%(χ²=0.030,P=0.862); ③The haplotype-based haplotype relative risk test (HHRR) showed there was no significant difference of frequency distribution of allele of rs2280250 between patient group and parent group (χ²=0.055,P=0.815).Conclusion The polymorphism of rs2280250 at the APOD locus may not be associated with schizophrenia in North Chinese Han population.

Abstract:Objective To investigate the association of susceptibility of bronchial asthma with GPRA gene polymorphism in North Chinese Han children.Methods The techniques of PCR and restriction fragment polymorphism (RFLP) were used to examine single nucleotide polymorphisms ( SNPs) on rs324374 of GPRA gene in 117 children with bronchial asthma ( case group ) and 121 healthy individuals ( control group).Results The genotype distributions on rs324374 of GPRA gene in case group and control group did not deviate from Hardy-Weinberg equilibrium.The frequency distribution of rs324374 in case group as follows: 22 were CC (18.80%),66 were CT (56.40%),29 were TT (24.80%); the frequency distributions on rs324374 in control group as follows: 30 were CC(24.79%),61 was CT(50.42%),30 were TT(24.79%).And there was no significant difference of frequency distribution of three genotypes between case group and control group（P>0.05）and there was no significant difference of frequency distribution of allele C and T between two groups either（P>0.05）.Conclusion The mutant allele of rs324374 of GPRA gene may not be associated with bronchial asthma in children.

Abstract:Objective To explore the expressions of DNA repair enzyme 6-methyiguanine-DNA methyltransferase（MGMT） and cell multiplication nuclear antigen ( Ki-67) in human astrocytomas and clinical significances．Methods Sixty  nine specimens of human astrocytomas (grade Ⅰ-Ⅱ: 34 cases, grade Ⅲ:21 cases,grade Ⅳ:14 cases) were selected.The expressions of MGMT and Ki-67 were observed with immunohistochemical technique,and the relation between MGMT and Ki-67 was evaluated.Results The positive rate of MGMT in astrocytoma was 60.9%, the positive rates of grade Ⅰ-Ⅱ,grade Ⅲ and grade Ⅳ were 88.2%，23.5% and 50.0%,respectively.There were significant differences between various grades of tumor （P=0.003 1），and the expression of MGMT had negative correlation with tumor grades （r=－0.351 1）.The positive rate of Ki-67 was 62.3%,and obviously increased with the grades of tumor （P=0.000 0)，and the expression of ki-67 had positive correlation with tumor grades（r=0.690 4, P=0.000 0）; the expression of MGMT decreased with the increase of expression of Ki-67（P=0.051）,the expression of MGMT had negative correlation with Ki-67 expression （r＝－0.291 5,P＝0.015 1）. Conclusion The expressions of MGMT and Ki-67 have close correlation with the degrees of malignancy in human astrocytomas.MGMT and Ki-67 may be used as biological parameters to estimate the prognosis of astrocytoma.

Abstract:Objective To study the gene expression profiles of ovarian cancer by cDNA microarray,and preliminarily analyze the function of part of differential expression genes. Methods BiostarH-40s gene microarray containing 4 097 genes was used to analyze gene expression patterns in tissue samples from 5 cases of human ovarian serous cystadenocarinoma(cy5-dUTP present ) and 5 cases of normal ovarian tissues (cy3-dUTP present).Results 163 genes of differential expression were found in more than 4 cases of ovarian cancer,the expression of 66 genes increased (up-regulated),the expression of 97 genes decreased(down-regulated). 37 differential genes with difinite gene function classification were found including three protocogene and tumor suppressor genes，one cyclin protein gene,three cytoskeletal and movement protein genes，one DNA binding,transcription and transcription factors gene,two cell receptor genes,five immune-related protein genes,seven metabolism genes,two protein translation and synthesis genes,three growth-related genes and seven other types of genes.Conclusion Gene expression profiles of ovarian cancer can be detected by cDNA microarray,and differential expression genes and their gene function classification of ovarian cancer are found.

Abstract:Objective To explore the association between the C46T gene polymorphism in the exon-1 region of the coagulation factorⅫ(FⅫ) and venous thrombosis. Methods PCR-RFLP method was used to investigate the distribution frequency of the C46T gene polymorphism in the 87 venous thrombosis patients and 129 healthy controls.The relation of the C46T gene polymorphism with venous thrombosis was analyzed by Logistic un-conditional regression.Results There were three kinds of genotypes including 46C/C,46C/T,46T/T in the study population.The distribution frequency of 46C/C genotype in the venous thrombosis patients (6.9%) was higher than that in the healthy controls(4.65%).There was no relativity between 46C/C genotype and venous thrombosis.（χ²=1.036，P=0.309，OR=1.86，95%CI：0.57—6.12）.Conclusion It is not deemed that the C46T gene polymorphism of FⅫ is an independent risk factor in the venous thrombosis and the relativity between the deep venous thrombosis and C46T gene polymorphism is going on research.

Abstract:Objective To study the inhibitory effects of Probucol on the expressions of serum promatrix metalloproteinase-2 (pro-MMP-2) and matrix metalloproteinase-2 (MMP-2) in patients with coronary heart disease(CHD). Methods The levels of serum Pro-MMP-2 and MMP-2 were detected by zymograph and total cholesterol(TC),triglyceride(TG),high density lipoprotein cholesterol (HDL-C),low density lipoprotein cholesterol(LDL-C )were measured by enzyme method in 30 CHD patients before and after treatment with Probucol.Results After treatment of Probucol,the expression levels of pro-MMP-2,MMP-2,TC and LDL-C in serum were decreased than those before treatment，respectively;there were obvious differences of various indexes mentioned above between before and after treatment（P<0.05）.The expression levels of HDL-C and TG in serum had no significant difference between before and after treatment（P>0.05）.Conclusion Probucol can inhibit the expressions of pro-MMP-2 and MMP-2 in serum as same as TC and LDL-C.Probucol,as one of TIMPs,may be applied to stabilize plaque.

Abstract:Objective To explore the clinical application significance of quantitative detection of minimal residual leukemia cells in peripheral blood in acute leukemia patients.Methods Using PCR amplification of quantitative method of limited dilution,the FLT3 gene was detected in blast cells of peripheral blood from 25 newly diagnosed cases of acute leukemia,and the number of minimal residual leukemia cells in newly diagnosed cases and the cases after one course of chemotherapy was calculated respectively.10 healthy subjects were used as control group.Results ①The positive rate of FLT3 gene in 25 newly diagnosed cases of acute leukemia sample was 80%(20/25),the positive rate in acute myeloblastic leukemia（AML） patients was 88.2%（15/17）and in acute lymphocytic leukemia（ALL）was 62.5%(5/ 8).②The mean DNA content in peripheral blood in 20 cases with FLT3 positive expression in newly diagnosed group was （2.36±1.25)×10⁸ μg•L^-1,being equal to (18.66±8.79)×10⁶ leukemia cells in every microliter peripheral blood.After one course of chemotherapy ,there was 1 case  without remission,the DNA content in peripheral blood was 1.69×10⁷μg•L^-1,being equal to 1.01×10⁶ leukemia cells in every microliter peripheral blood;there were 3 cases with partial remission,the mean DNA content in peripheral blood was (0.57±0.24)×10⁶ μg•L^-1,being equal to (1.82±0.19)×10³ leukemia cells in every microliter peripheral blood.There were 9 cases with complete remission with FLT3 negtive expression,and 7 cases with complete remission with FLT3 positive expression,the mean DNA content in peripheral blood was (0.16±0.06)×10⁶ μg•L^-1,being equal to (1.86±1.31)×10² leukemia cells in every microliter peripheral blood.Conclusion The quantitative and periodic detection of minimal residual leukemia cells would help to evaluate leukemia chemotherapy efficiency and to adjust treatment scheme in time.

Abstract:Objective To study the expressions of insulin-like growth factor Ⅰ (IGF-Ⅰ ) and its receptor (IGF-1R) in endometriosis. Methods Immunohistochemical assay was adopted to detect the levels of IGF-Ⅰ and IGF-1R in specimen of endometria from the ectopic and eutopic endometrium from 30 cases of clinical endometriosis 25 normal endomentrium were used as control. Results IGF-Ⅰ and its receptor expressed strongly in glandular epithelial cells of endometrium in 3 groups;IGF-Ⅰ expressed poorly in stromal cells of endometrium in 3 groups;while IGF-Ⅰ didn’〖KG-3〗t express. The expression levels of IGF-Ⅰ in endometrium in patients with endometriosis were higher than that in controls(P<0.01). The expression level of IGF-1R in eutopic endometrium was higher than those in normal and ectopic endometrium(P<0.01).Conclusion IGF-Ⅰ play an important role in pathogenesis of endometriosis.

Abstract:Objective To investigate the correlation between the expressions of tumour suppressor gene PTEN and oncogene C-myc in colorectal carcinoma. Methods The expressions of PTEN and C-myc protein in normal colorectal mucosa(n＝8), adjacent non-cancerous tissues(n＝10) and primary colorectal carcinoma tissues(n＝60) were observed by S-P immunohistochemical assay. Results Of 60 colorectal carcinoma tissues, C-myc protein was detected in 46 cases. The expression rate of C-myc in the primary colorectal carcinoma tissues(76.70%) was significantly higher than that in the normal(0) and adjacent non-cancerous tissues(10%) (P<0.01). Overexpression of C-myc was significantly correlated with hepatic metastasis(r＝5.540, P<0.05) and was not related with TNM staging(r＝0.013, P>0.05). The positive expression rate of PTEN protein in the primary colorectal carcinoma tissues (25.00%,15/60) was significantly lower than that in the normal(100%) and adjacent non-cancerous tissues(90%) (P<0.01). PTEN protein expression was positively correlated with pathological differentiational degree(r＝0.869, P<0.05), negatively with TNM staging, lymph nodes metastasis and hepatic metastasis(r＝－0.791,r＝－0.741, r＝－0.634, P<0.05). The level of C-myc protein expression was negatively correlated with PTEN expression (r＝－0.322, P<0.001). Conclusion C-myc protein overexpression can not be used as a clinicopathological parameter to dignose colorectal cancer. The expression of PTEN is closely related to clinicopathological features in colorectal carcinoma, and could be used as a marker of tumour burden of colorectal carcinoma.

Abstract:Objective To study the expressions of Hsp70,P27，Bcl-2 in mucoepidermoid carcinoma of salivary glands and clinical significances.Methods Forty cases of mucoepidermoid carcinoma of salivary glands were chosen and the expressions of Hsp70,P27, Bcl-2 were detected by the method of immunohistochemistry,10 cases of polymorphic adenoma were chosen as control group.Results Both Hsp70 and P27 were stained with aliomycin in nucleus and cytoplasm,the positive rates of them were 75% and 27.5%, respectively,there were significant differences compared with control group (P<0.01).Bcl-2 was stained with aliomycin or brown,the positive expression rate was 62.5% and there was no significant difference compared with control group (P>0.05).The expressions of Hsp70 and P27 had significant differences in different clinical stages,different histological grades and lymph node metastasis（P<0.05 or P<0.01）,the expression of Bcl-2 had significant difference in different clinical stages (P<0.05),while no significant difference in different histological grades and lymph node metastasis of mucoepidermoid carcinoma of salivary glands (P>0.05). Conclusion The expressions of Hsp70 and P27 can be used to estimate prognosis of patients with mucoepidermoid carcinoma.Hsp70 and Bcl-2 can inhibit apoptosis by different ways.

Abstract:Objective To explore the relation of the expressions of MDM2 and VEGF in osteosarcoma with the pathological parameters and prognosis of the tumor.Methods The expressions of MDM2 and VEGF were detected with immunohistochemical(SP) method in specimens from 56 cases of osteosarcoma.The correlation between the expressions of MDM2,VEGF and pathological grade,metastasis and prognosis was analyzed statistically.while 8 cases of fibrous dysplasia of bone were used as negative control group.Results The positive rates of MDM2 and VEGF in osteosarcoma were 64.3%(36/56) and 67.9%(38/56)， respectively.MDM2 and VEGF didn’[KG-*3]t express in negative control group.The expression of MDM2 and VEGF were not significantly correlated to the pathological grades of the osteosarcoma,but which were significantly correlated with tumor metastasis and prognosis (P<0.05).The five-year survial rate in patients with positive expression of MDM2 and (or) positive expression of VEGF was significantly lower than that in negative control group(P<0.05).The patients with positive reaction for both MDM2 and VEGF had the lowest survival rate, but the patients with negative reaction for both MDM2 and VEGF had the highest survival rate,the difference was significant (P<0.01).Conclusion There is an intimate correlation between the overexpression of MDM2 and VEGF in osteosarcoma and its unfavorable prognosis.MDM2 cooperating with VEGF probably plays a role in angiogenesis of osteosarcoma and is an useful parameter to evaluate prognosis of osteosarcoma.

Abstract:Objective To explore the relationship between therapeutic effects of ⁸⁹SrCl₂ on osteodynia induced by multiple bone metastasis and ET,CGRP，TXB₂ and 6-K-PGF_1a.Methods ⁸⁹SrCl₂ treatment was used in 39 cases of multiple bone metastasis,the serum content of ET,CGRP，TXB₂ and 6-K-PGF_1a were detected with radioimmunological method pre-treatment and 1, 3,6 months post-treatment,respectively;the ET/CGRP value was calculated.Results The content of ET showed no obvious changes 1 month post-treatment compared with pre-treatment.However,the ET contents increased significantly 3 and 6 months post-treatment compared with 1 month post-treatment and before treatment (P<0.01).CGRP content increased significantly 1 month post-treatment compared with pre-treatment （P<0.05）.However,ET/CGRP value had no significant difference 1 month post-treatment compared with pre-treatment. ET/CGRP increased remarkably 3 and 6 months post-treatment compared with pre-treatment and 1 month post-treatment (P<0.01).Also,TXB₂ content had no significant changes between pre- and post-treatment.While 6-K-PGF_1a content demonstrated no obvious changes 1 month post-treatment compared with pre-treatment.6-K-PGF_1a content increased markedly 3 and 6 months post-treatment compared with pre-treatment and 1 month post-treatment (P<0.01). Conclusion The mechanism of releasing pain through ⁸⁹SrCl₂ strategy is related to ET,CGRP,6-K-PGF_1a and ET/CGRP.

Abstract:Objective To evaluate the application values of time-resolves fluroimmunoassay (TRFIA) technique for CA15-3 and CEA detection in mammary gland diseases.Methods TRFIA was used to detect the serum concentrations of CA15-3 and CEA in normal control group (n=57),hyperplasia group (n=206),benign mammary neoplasm group (n=136),mammary cancer with out recurrence or metastasis group (n=89) and mammary cancer with recurrence or metastasis group (n=39).Results There were no significant differences of CA15-3 and CEA serum concentrations between control group,mammary hyperplasia group and benign mammary neoplasm group (P>0.05).However,the CA15-3 and CEA concentrations in mammary cancer without recurrence or metastasis group were significantly higher than those in the former three groups (P<0.05).Also,the CA15-3 and CEA concentrations in mammary cancer with recurrence or metastasis group were remarkably higher than those in the former three groups(Ｐ<0.01) and mammary cancer without recurrence or metastasis group (Ｐ<0.05).While CA15-3 and CEA concentrations in the 3-month post-operation group showed no significant difference with those in the former three groups(Ｐ>0.05),though,were distinctly lower than those in the mammary cancer without or with recurrence or metastasis groups (P<0.05,Ｐ<0.01).Conclusion The atraumatic detection of CA15-3 and CEA may be used as indexes in diagnosis,therapy，and curative effect estimation post-operation and following-up observation for mammary diseases.

Abstract:Objective To observe the effects of glycemic control in patients with newly diagnosed type 2 diabetes mellitus(T2DM) by continuous subcutaneous insulin infusion (CSII). Methods A total of 179 patients with newly diagnosed T2DM received 3 weeks’ treatment of CSII. Before-after study in the same patient was applied. Intravenous glucose tolerance test (IVGTT) was performed, and blod glucose, HbA_1c, insulin, C-peptide (C-P) were measured before and after CSII. The same index was observed 3 and 8 weeks after drug withdrawal. Results Of  all 179 patients,159 cases were chosen. Before CSII, the average fasting plasma glucose (FPG) was (15.27±2.32)mmol•L^-1, postprandial plasma glucose (PPG)was (22.18±3.43)mmol•L^-1, and HbA_1c was 14.95%±2.42%. 3 weeks after CSII, FPG, PPG and HbA1c were significantly decreased［(5.52±1.30)mmol•L^-1，(7.18±1.21)mmol•L^-1 and 10.86%±1.33%, respectively］(P<0.001 or P<0.05). The patients had different degree restoration of the first-phase insulin secretion. 3 and 8 weeks after drug withdrawal, FPG, PPG and HbA_1c were significantly reduced (P<0.001 or P<0.05). Conclusion The excellent glycemic control can be induced by 3 weeks’ treatment of CSII in newly diagnosed T2DM.