Objective To investigate the effect of ionizing radiation on caspase-3 protein expression of EL-4 cells. Methods Mouse lymphoma EL-4 cell line was used. Flow cytometry (FCM) was used to examine caspase-3 protein expression and apoptosis. For dose-effect experiment, caspase-3 protein expression and apoptosis were measured 24 h after X-irradiation with doses of 0.5, 1.0, 2.0, 4.0 and 6.0 Gy. For time course experiment, caspase-3 protein expression was measured at 2, 4, 8, 12, 24 and 72 h after 4.0 Gy X-irradiation. Results In dose-effect experiment, it was demonstrated that the caspase-3 protein expression of EL-4 cells was increased significantly 24 h after X-irradiation with the doses of 0.5, 2.0, 4.0 and 6.0 Gy compared with sham-irrad (P<0.05 or P<0.01). It was also showed that apoptosis of EL-4 cells was induced significantly 24 h after X-irradiation with the doses of 0.5, 1.0 and 4.0 Gy compared with sham-irrad (P<0.05 or P<0.01). In time course experiment, the result showed that the caspase-3 protein expression of EL-4 cells was increased significantly 8, 12 and 24 h after 4.0 Gy X-irradiation compared with sham-irrad (P<0.01 or P<0.001). Conclusion Caspase-3 protein expression and apoptosis in EL-4 cells could be induced by X-rays.

Objective To study the role of α_Vβ₃ integrin in extracellular matrix (ECM) regulation of the expression and activation of matrix metalloproteinase-2 (MMP-2). Methods The difference between human melanoma cells M21 and M21-L cells was that M21-L didn′t express α_Vβ₃ integrin. Human melanoma cells M21 and M21-L cells were cultured on three dimensional (3D) type I collagen gel, fibronectin (FN) and ECM gel. To prepare serum-free conditioned medium (SFCM), cells were harvested by trypsinization and counted using a hemacytometer and 2×10⁵ cells/well were seeded in 24-well tissue culture plates coated with these gels,respectively. After 12　h, cells were washed three times with phosphate-buffered saline, followed by incubation in serum-free medium. SFCM was collected 48 h later and stored at -80℃ until being used. Then,the expression of MMP-2 was examined by zymography, and the expression of MT1-MMP mRNA was examined by reverse transcription polymerase chain reaction (RT-PCR). Results Induction of MMP-2 activation was specific to human melanoma cells M21 cultured on type I collagen gel， FN and ECM gel. Pro-MMP-2 protein was expressed in human melanoma cells M21-L, but no active from of MMP-2 was detectable. MT1-MMP mRNA was expressed in human melanoma cells M21, and enhanced expression of MT1-MMP mRNA was seen in cells cultured on type I collagen gel，FN and ECM gel. MT1-MMP mRNA was expressed in human melanoma cells M21-L,but cells cultured on these gels did not influence the expression of MT1-MMP mRNA. Conclusion These ECM components can regulate expression and activiation of MMP-2 by integrin α_Vβ₃.

Objective To study the effects of Rh₂(a ginsenoside extracted from ginseng) on PC-3M (androgen independent cell lines) apopotosis and new gene GRIM-19 expression. Methods Rh₂ agents were divided into three groups:high dose group (Rh₂40 mg·L^-1),middle dose group (Rh₂30 mg·L^-1)and low dose group(Rh₂15 mg·L^-1),which incubated with PC-3M for 24 h， respectively， in the following experiments. Morphological assessment of apoptosis was performed by acridine orange (AO) stained fluorescence microscope to detect the apoptosis of PC-3M cells after treated with Rh₂ agents; RT-PCR was used to measure the level of GRIM-19 mRNA in PC-3M cells treated with Rh₂ agents;GRIM-19 protein expression of PC-3M cells treated with Rh₂ agent was measured by Western blotting. Results After various doses of Rh₂ interacted with PC-3M cells for 24 h, AO assay showed that cells changed from green color into orange one and typical exocytosis phenomena were observed in Rh₂ 15 mg·L^-1 group. The consequences of RT-PCR and Western blotting demonstrated that GRIM-19 expression was intensified both in gene and protein levels with the increasing Rh₂ doses. The expressions of GRIM-19 in Rh₂ 15 mg·L^-1 group and 30 mg·L^-1 group were higher than that in control group（P<0.05）, and in Rh₂ 40 mg·L^-1 group the expression of GRIM-19 had a remarkable difference compared with control group （P<0.01）. Conclusion Rh₂ can lead to apoptosis of PC-3M cells that correlated with GRIM-19 gene and protein expression upregulation, the expression level increases with the Rh₂ in a dose-dependent manner.

Objective To construct an eukaryotic expression vector of human ERβ (hERβ) full length gene which named pEGFP-C1-hERβ and transfect it into hormone-independent prostate cancer cell line PC-3M. Methods The complete gene of hERβ（1 593 bp） was obtained with the technique of RT-PCR by using human ovary tissue mRNA as template which obstained from the ovarian cyst patients. The PCR product was connected with pMD18-T vector and sequenced automatically，and then the connected product and the expression vector pEGFP-C1 were digested by restrictive enzyme Hind Ⅲ and BamH Ⅰ， respectively，and the pEGFP-C1-hERβ vector was constructed by using gene recombinant technique. The plasmid was detected by endonuclease digestion and PCR，and then was transfected to PC-3M cells by lipid reagent. Results The clone of the complete hERβ gene and the construction of expression vector were finished successfully by endonuclease digestion and sequenced automatically. The product of the endomuclease digestion was as long as the human complete hERβ gene (1 612 bp)，and sequence analysis suggested that the hERβ sequence detected by PCR was identical to that published in GenBank (NM_001437). The expression of green fluorescence protein (GFP) was observed under the fluorescence microscope. Conclusion The eukaryotic expression vector pEGFP-C1-hERβ is constructed and it expresses in PC-3M cells successfully.

Objective To establish stable insect cell line expressing mIL-4 on the basis of construction of inset expression vector pIZT/V5-His. Methods Amplified mIL-4 and pIZT/V5-His were treated with EcoRI and XbaI, and mIL-4 were ligated into pIZT/V5-His vector using T4 DNA ligase. Sf9 cells were transfected with recombinant DNA and ELISA was employed to detect soluble mIL-4 production by transfected Sf9 cells. Results Transfected Sf9 cells could significantly produce mIL-4 (1 mg·L^-1) compared with control group(0.003 mg·L^-1) . Conclusion Inset expression vector pIZT/V5-His is an ideal expression vector for mIL-4 production in transfected cells.

Objective To isolate and identify the structures of the phenolic compounds from Geum aleppicum Jacq..Methods The structures of six compouds were determined by the physical and chemical data and spectroscopic analysis after the purification by thin-layer chromotography and silical gel column chromatography. Results Six phenolic compounds were isolated from Geum aleppicum Jacq.,and their structures were established to be benzoic acid (Ⅰ),gallate acid (Ⅱ),salicylic acid (Ⅲ),vanilin (Ⅳ),3,4,5-trihydoxybenzoic dehyde(Ⅴ) and 3,4,5-trihydoxybenzoic acid,ethyl ester (Ⅵ). Conclusion The compounds Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ,Ⅵ are isolated from this plant for the first time.

Objective To study the rehabilitative effect of velvet antler polypeptides-PLGA compound membrane on peripheral nerve injury. Methods Two doses (3 and 15 mg·g^-1) of velvet antler polypeptides-PLGA compound membrane (thickness 50 μm) were used to wrap the sutures of severed sciatic nerves, which were compared with nerve sutures only. 2, 4 and 6 weeks post-operation, general morphological, electrophysiological, histological and electron microscopic observations and examinations were investigated. Results There were no ulcers in the nars and toes in the treatment group, light conglutination appeared in nerve anastomosis and around tissues. At 6th week post-operation,conglutination was alleviated obviously than 2 weeks post-operation; findings from latency inducing potentials of calf triceps dominated by sciatic nerves showed that recover ratios (%) in treatment group (2 weeks:9.07±1.44，8.02±1.41;6 weeks: 49.87±9.69,50.11±6.11) were better than those in control group(2 weeks:2.52±1.83;6 weeks:30.31±6.32) obviously in each time (P<0.01); high dose group was better than low dose group (P<0.05), and 6 weeks post-operation recover ratios (%) were better than 2 weeks ones (P<0.05); regenerative myelination fiber counts, diameter and section area in treatment group were better than those in control group in each time obviously (P<0.01), high dose group was better than low dose group (P<0.05), and recovery ratios at 6th week post-operation were better than 2 weeks ones (P<0.05); maturation of myelination fiber in high dose group was more evident than control and low dose groups respectively, 6 weeks post-operation maturation of myelination fiber was more obvious. Conclusion Velvet antler polypeptides-PLGA compound membrane can elevate the curative effect of rehabilitation of injured peripheral nerves.

Objective To clone and express the genes of hβ₂GPⅠ-DI and hβ₂GPⅠ-DI₂ in E.coli M15. Methods Target gene β₂GPⅠ-DI was cloned by PCR,the PCR products were connected into dimmer,the monomer and dimmer were inserted into PGEM-T easy vector and sequenced. Then they were inserted into an expression vector PQE30 and expressed in E.coli M15. The recombinant proteins were identified by Western blotting. Results The expected protein of hβ₂GPⅠ-DI and hβ₂GPⅠ-DI₂ recombinant protein were synthesized, their relative molecular mass were 9 000 and 17 000. The two proteins could be recognized by hβ₂GPⅠ mAb with Western blotting. Conclusion The monomer and dimmer of hβ₂GPⅠ first domain gene are constructed successfully.

Objective To construct the double hairpin siRNA and green fluorescent protein (GFP) expressing vector pU6/double-siRNA/Neo/GFP/1A/2A to interfere 1A and 2A gene of Coxsackie virus B4. Methods 21 bp fragments of the Coxsackie virus B4 2A and 1A gene were chosen as the targets and 65 bp complimentary fragments were synthesized, then the target gene fragments were cloned into pSilencer2.1U6 Neo and pGCsi-U6/Neo/GFP/siNeGative, respectively, then the double siRNA expressing vector pU6/double-siRNA/Neo/GFP/1A /2A was constructed by restrict endonuclease digestion,elctrophoresis isolation and reclaimer, ligatied by T4 DNA ligase; then the double siRNA expressing plasmid was transfected into Hela cells, and the GFP was observed under fluorescent microscope. Results The correct results showed that the recombinant plasmid had the correct special fragments and DNA sequence detected by restrict endonuclease digestion, electrophoresis and DNA sequencing; and GFP was also observed in Hela cells tansfected with pU6/double-siRNA/Neo/GFP/1A /2A under fluorescent microscope more than 15 d after transfection. Conclusion The double siRNA expressing vector pU6/double-siRNA/Neo/GFP/1A/2A is constructed successfully; it has the correct target viral gene sequences and can express GFP gene in Hela cells more than 15 d after transfection.

Objective To study the effects of human insulin-like growth factor 1 (hIGF-1) gene transfection on the proliferation of NIH3T3 fibroblasts. Methods The plasmid of pcDNA3.1-hIGF-1 was transfected into NIH3T3 fibroblasts by using Lipofectin method. The positive cell clones were selected with G418 and cultured for 4 weeks. The stable expression of hIGF-1 in the positive cells was determined by in situ hybridization and immunocytochemical analysis. MTT assay and flow cytometer analysis were used to observe the proliferation of NIH3T3 fibroblasts. Results hIGF-1 mRNA and protein expressed in NIH3T3 fibroblasts transfected with pcDNA3.1-hIGF-1 by in situ hybridization and immunocytochemical analysis. MTT assay showed the A value of transfected NIH3T3 fibroblasts rose, compared with untransfected NIH3T3 fibroblasts group, the difference was significant (P<0.01). Cellular proportion in S period (59.3%) was increased and it in G₁ periods was decreased (27.2%) after transfection by flow cytometer measurement. Conclusion The stable expression of hIGF-1 in NIH3T3 fibroblasts transfected with pcDNA3.1-hIGF-1 is obtained. Gene transfection of hIGF-1 can stimulate the proliferation of NIH3T3 fibroblasts.

Objective To study the protective mechanism of Gross Saponin Tribulus Terrestris (GSTT) on the neonatal rat ventricular cardiocytes injured by hypoxia and the effect on protein kinase C（PKC）. Methods The hypoxia-ischemia model was performed by treating the cultured neonatal rat ventricular cardiocytes with NaCN. The effect of GSTT(100 and 30 mg·L^-1) on the contents of εPKC and δPKC were detected by flow cytometry and laser confocal microscopy system. Results GSTT up-regulated εPKC and δPKC expressions. The contents of εPKC and δPKC in GSTT 100 mg·L^-1 group（1 325.00±53.25，810.55±36.89；66.22±6.23，40.12±2.21） were increased significantly than those in model group（792.00±32.36，492.40±30.15；32.70±2.78，29.28±4.82）（P<0.001，P<0.01；P<0.001，P<0.01）, and were higher than those in GSTT 30 mg·L^-1 group(998.00±23.21, 710.00± 38.12 ; 55.35±5.15, 35.68±3.89) （P<0.01，P<0.05；P<0.01，P<0.05）. Conclusion GSTT can protect cardiocytes through activating PKC signaling pathway.

Objective To clone aquaporin 8 (AQP8) cDNA of testis in Wistar rats and construct the eucaryotic expression vector of enhance green fluorescent protein (EGFP) and AQP8 fusion protein.Methods The full sequence of AQP8 was fished by RT-PCR and sequenced, then it was recombined in the downstream of the pEGFP-C1 gene. Results The sequence of AQP8 cDNA of Wistar rat was homology with NM_019158（access number logged in GenBank），but there were 4 different bases：P135-137 of NM_019158 partly were C，A and G，but the sequencing result was absence；P311 of NM_019158 was A，but the sequencing result was G. The recombinant pEGFP-C1-AQP8 was identified by enzyme digestion. Conclusion The recombinant pEGFP-C1-AQP8 is constructed successfully.

Objective To study the dose- and time-effect relationship bet ween lipopolysaccharide (LPS) and CD14 expression in the cell line J774A.1 of mouse macrophages. Methods The cell line J774A.1 was stimulated with different doses of LPS，and the CD14 surface-positive cells from the cell line J774A.1 were detected by flow cytometry（FCM）45 min after stained with FITC-CD14 monoclonal antibody. The changes of CD14 surface expression in the cell line J774A.1 at different time（1,2,4,8 and 16 h）after stimulated with LPS were observed. After the best stimulant time was selected，the cell line J774A.1 was treated with different doses（1,10,50,100,500 and 1 000 μg·L^-1）of LPS and the changes of CD14 surface expression were observed. Results In view of time-effect，compared with control group（the positive rate was 12.50±3.71），LPS significantly enhanced the CD14 expression at 1，2 and 4 h after stimulation（the positive rates were 23.80±5.07，23.04±2.88 and 28.22±1.54,respectively） when the dose of LPS was 1 μg·L^-1 （P<0.01）. When the concentration of LPS was 10 μg·L^-1, CD14 expression strengthened at 1 and 2 h after stimulation（the positive rates were 40.85±6.05 and 26.63±6.17, respectively）（P<0.05，P<0.01）.LPS obviously stimulated CD14 expression at 1 h after stimulation（the positive rate was 47.40±2.85）when the dose of LPS was 50 μg·L^-1（P<0.01）. In the dose-effect, the stimulant effect of LPS occurred when LPS was administered only below the dose of 10 μg·L^-1 at 4 h after stimulation（P<0.01）. Conclusion LPS can significantly stimulate the expression of CD14 in the cell line J774A.1 below the dose of 50 μg·L^-1 within 4 h.

Objective To study the neuroprotective effects of citicoline (CC) on the toxicity induced by 6-OHDA towards dopaminergic mesencephalic neurons in primary culture-Parkinson′s disease (PD) model in vitro and its mechanism. Methods Mesencephalic neurons in culture were prepared from embryonic 15-day Wistar rats. Cultures were treated for 6, 8, 10 d with various concentrations of CC (2,1,0.1,0.01 and 0.001 mmol·L^-1). At 11th day, the cultures were co-treated with the toxin 6-OHDA (50 μmol·L^-1) for 0.5 h, the cells were collected. Seven groups were categorized as follows:CC (2,1, 0.1, 0.01 and 0.001 mmol·L^-1) +6-OHDA, control and 6-OHDA group. The cell viability was evaluated with MTT assay. Intracellular free Ca²⁺，Ca²⁺i， was labeled by using the fluorescent dye Fluo3-AM and detected by flow cytometer. By measuring the intracellular Rhodamine 123 fluorescence density with flow cytometer, mitochondrial membrane potential (MMP) was evaluated. Results Cultures were treated with 2, 1 and 0.1 mmol·L^-1 CC, the viability of cell was increased (P<0.05). 1, 0.1, 0.01 and 0.001 mmol·L^-1 CC significantly attenuated 6-OHDA-induced neurotoxic effects. As compared with 6-OHDA, the viability of neurons increased, the mean Ca²⁺i was significantly lower in cells treated with CC(1, 0.1, 0.01 and 0.001 mmol·L^-1 ) plus 6-OHDA (49.30±7.62)% than that in 6-OHDA group without CC treatment (P<0.01). The densities of Ca²⁺i in CC (1, 0.1, 0.01 and 0.001 mmol·L^-1) plus 6-OHDA groups were (32.23±1.87)%,(17.09±7.45)%,(21.71±8.89)%,(29.18±4.71)%, respectively，and the density of mean Ca²⁺i in 6-OHDA group was (49.30±7.62)%. MMP significantly increased（P<0.01）. Conclusion CC has an important effect on dopaminergic cell survival in vitro in a validated model of PD. The neurotoxic effect of 6-OHDA can be reduced by CC and CC can increase the viability of neurons, decrease Ca²⁺i, maintain MMP at a relatively higher level.

Objective To establish a retroviral vector of small hairpin RNA (shRNA) expression in which the sense and antisense sequences targeting wild type human p53 were linked together with a 9-nucleotide loop to detect the interfering role of p53 gene in mammalian cells. Methods The human H₁ promoter was inserted into the upstream of the cytomegalovirus (CMV) promoter by using Xhol I and EcoR I sites. The resistanting hygromycin gene was replaced with the human CD₄ gene. These oligonucleotides were annealed and ligated the downstream of the H₁ promoter. All oligonucleotides were synthesized with Qiagen. HEK293 was infected with the shRNA vector and the expression of p53 was detected 72 h after infection by flow cytometry. Results The retroviral vector was successfully constructed and the expression of P53 protein was definitely suppressed in the HEK293 72 h after infection. Conclusion The applying of shRNA expression vector is possible to provide a prompt and promising method for evaluating the gene function of mammalian cells.

Objective To investigate the effect of radix salviae milliorhize on hemodynamics of portal hypertensive rats. Methods 60 Wistar rat models with portal hypertension were induced by CCl₄. Among them 56 rats with portal hypertension were divided at random into 4 groups: radix salviae 3 d group, radix salviae 10 d group, radix salviae 15 d group, and control group. After administration, the blood flow of vena portae, superior mesenteric artery, superior mesenteric artery and pressure of vena portae, average arterial pressure were measured. Results Compared with 0.9% Natrii Chloridi control group, the blood flow of vena portae, superior mesenteric artery, superior mesenteric artery and pressure of vena portae, average artery pressure in radix salviae milliorrhize 3, 10 and 15 d groups were decreased significantly (P<0.05), but between radix salviae 3 d group and 10 d group, there were no significant differences (P>0.05). Conclusion Radix salviae milliorhize can be employed for treating portal hypertension. Radix salviae milliorrhize can decrease the blood flow and portal pressure, moreover, the effect can be enhanced with the elongation of treatment time.

Objective To study the inhibitory effect of triethyltin chloride (TETC) on the proliferation of cultured rat C6 glioma cells. Methods MTT assay and light microscope were performed to evaluate the inhibitory effect of 0.5,1.0 and 2.0 μmol·L^-1 TETC on the proliferation of rat C6 glioma cells for 48 h,and electron microscope was performed to observe the ultrastructure changes of C6 glioma cells treated by 0.5,1.0 and 2.0 μmol·L^-1TETC for 48 h. Results 0.5,1.0 and 2.0 μmol·L^-1 TETC could inhibit the proliferation of cultured C6 glioma cells in vitro,and the inhibitory rate were 15.62%,36.16% and 41.92%,respectively. The inhibitory rate of TETC on the proliferation of C6 glioma cells evaluated by MTT assay increased in a dose-dependent manner,and there was significant difference compared with control and compared with each other among every two adjacent-dose groups(P<0.05). The nuclear chromatic condensation and margination were found in C6 glioma cells treated by 0.5,1.0 and 2.0 μmol·L^-1 TETC for 48 h by electron microscope,which showed the early ultrastructure changes of apoptosis. Conclusion TETC can inhibit the proliferation of cultured rat C6 glioma cells in viro.

Objective To study the feasibility of reconstructing trachea by implanting autogenous costal perichondrium tracheal prosthesis. Methods Ten dogs were used. Three pieces of costal perichondrium of every dog were wrapped on a silicon bar used as skeleton and implanted into sternohyoid muscle adjacent to trachea to establish blood supply. Four weeks later, silicon bar was removed and perichondrium ring served as tracheal prosthesis. Trachea resection and prosthesis replacement was performed. Viability of dogs, gross and microscopic pathological changes were studied to evaluate the effect of prosthesis replacement. Results The respiratory tracts of the 8 survival dogs were not obstructed. Their diet, activity and bark were not different from those of the normal dogs. With abundant blood supply,the prosthesis fused tightly with the surrounding tissue. Anastomotic orifice healed without cicatricial tissue and granulation tissue. The internal surfaces of prosthesis were smooth and glossy with white tunica mucosa covered. Under light microscope, internal surfaces of prosthesis were found covered by pseudostratified columnar epithelium with few cilia. Under tracheal mucous membrane, there were fibrous membrane, neonate chondrocytes and striated muscle cells stratum successively. Fibrous membrane was composed of collagen and fibroblasts with a few inflammatory cells scattered. A small number of neonate chondrocytes were found under fiber membrane. Plasma of these chondrocytes looked pale after staining, which indicated that these chondrocytes were neonate. Striated muscle cells connected with the neonate chondrocytes firmly. Neonate capillaries and micro blood vessels of various diameters were abundant in striated muscle stratum. Conclusion It is feasible to reconstruct dog trachea using prosthesis constructed with autogenous costal perichondrium.

Objective To study the damage effects of different doses ofcadmium (Cd²⁺) on human normal liver cell line HL-7702 cells and its mechanism. Methods HL-7702 cell cultures were exposed to cadmium within a dose range of 5-40 μmol·L^-1. The inhibition of the cells during 8 d was determined by cell counting and the cell growth curve was made. The cytotoxicity of cells during 72 h was determined by MTT assay. Effects of cadmium on the distribution of cell cycle of HL-7702 cells were monitored by flow cytometry analysis (FCM). The apoptotic rate of cells induced by cadmium was determined by Annexin V-FITC/PI double fluorescent staining flow cytometry analysis(FCM) at the same time. Results Cadmium at different concentrations inhibited the growth and proliferation of HL-7702 cells in a dose-dependent manner and the cell growth curve changed accordingly. After treated by different concentrations of cadmium for 24,48 and 72 h, the cell inhibitory rates increased with the elevating of doses. After 48 and 72 h exposure of cadmium, the data showed that certain doses of cadmium might induce S blocking, and this effect was in time- and dose-dependent manner. After 24,48 and 72 h exposure of cadmium，the data suggested that certain doses of cadmium might induce apoptosis. Conclusion Cadmium can significantly inhibit the growth and proliferation of HL-7702 cells and result in S blocking and apoptosis may be responsible for the inhibition.

Objective To investigate the protective effect of taurine on primary cultured neonatal rat myocardial cells and the inhibitory effect on cardiac fibroblast proliferation. Methods The myocardial cells were divided into six groups:control group, H₂O₂ damage group, H₂O₂ damage group treated with ligustrazine and H₂O₂ damage group treated with taurine with different doses (80, 40 and 20 mmol·L^-1). Morpholoical changes of myocardial cells damaged for 6 h by 0.5 mmol·L ^-1 H₂O₂ under the inverted microscope were observed. Meanwhile, cell survival ability and the contents of LDH, SOD and MDA were measured. Cardiac fibroblasts were randomly divided into 7 groups and co-cultured for 72 h with taurine with doses of 0, 10, 20, 40, 80, 160 and 320 mmol·L^-1 and proliferation ability was assessed by MTT method. Results Absorption values in H₂O₂ damage groups treated with taurine (80, 40 and 20 mmol·L^-1) were 0.479±0.055，0.437±0.052 and 0.421±0.062,respectively. They were higer than that in H₂O₂ damage group(0.304±0.050)（P<0.001 or P<0.01）；LDH release and MDA content were remarkly decreased and SOD contents were increased in 80 and 40 mmol·L^-1 taurine groups, as compared with H₂O₂ damage group (P<0.01 or P<0.05). Cardiac fibroblast proliferation was inhibited significantly in 40-320 mmol·L^-1 taurine groups than that in control group(P<0.05, P<0.01 or P<0.001). Conclusion Taurine can protect cultured neonatal rat myocardial cells from free radical damage and inhibit cardiac fibroblast proliferation.

Objective To identify the status of Turki human remain and its relation to Khitan nobles by the means of molecular biology. Methods MtDNA was successfully extracted from Turki human remain. Through four overlapping primers, the nucleotide sequence of 360 bp length was gotten. Then it was contrasted with the cambridge reference sequence (CRS), and the sequence was analyzed with that of Khitan nobles basing the methods of phylogenetics. Results The result showed that Turki had closest relationship with Qidan noble individuals. And it was closer with Yeluyuzhi′s family in the two Qidan noble families. It was proved that Turki was the noble of Khitan. Besides, it showed that Turki had closest relationship with modern out Mongolia contrasted with the reported populations. And the 5 mutation sites of Turki were all hot mutation sites of Mongolian. Conclusion Turki is north Mongolian.

Objective To study the effects of quercetin (QUE) on proliferation of rat glioma C6 cell line in vitro. Methods The cells were divided into 5 treatment groups（10,25,50,75 and 100 μmol·L^-1 QUE）, blank control and menstruum control group. The rat C6 cells were cultivated to 1×10⁶·mL^-1 in the RPMI 1640 medium, then added into 96 holes board with various doses of QUE by 3 holes per group,and MTT assay was used to observe the proliferation of the cells treated for 24，48 and 72 h. The change of cell cycle was also observed by flow cytometry (FCM) after the cells were treated with 50 and 100 μmol·L^-1 QUE for 48 h. The changes of the protein P53 and Bcl-2 of C6 cells treated with 50 μmol·L^-1 QUE for 48 h were detected by immunocytochemical methods. Results With the augmentation of QUE and the extension of the treated time, the C6 cell growth was inhibited, the A values decreased and the cell number in G₀／G₁ phase was increased，the cell numbers in S and G₂/M phases were cut down, and the decreased expression of Bcl-2 protein and the increased expression of P53 protein were also observed after treatment with QUE. Conclusion Inhibitory effect of QUE on C6 cell line is proved to be dependent on the treated time of the drug and the dose of QUE, and the induced apoptosis of C6 cells is implemented by the means of up-regulation of P53 protein expression and down-regulation of Bcl-2 protein expression.

Objective To construct mIL-12 recombinant retrovirus vector and evaluate the effect of mIL-12 expression on glioma in rats. Methods mIL-12 DNA was cloned into vector pLEGFP using standard procedures to develop recombinant plasmid pLEGFP-mIL 12 ,then it was transferred into PA317 cells. Results Products of enzyme digestion and PCR of recombinant plasmid pLEGFP-mIL12 were analysed using electrophoresis and a 2 290 bp fragment of DNA as same as mIL-12 gene in size was found, transfected NIH3T3 expressed GFP-mIL12 fusion protein. ls can produce retrovirus which can infect NIH3T3 cells,and the expression of mIL-12 gene can be detected in NIH3T3 cells.

Objective To investigate the effects of polylactic acid-polyglycolic acid-polyethylene (PLGA-PEG) stent on matrix metalloproteinase (MMPs) in rabbits with atherosclerosis. Methods 30 adult healthy rabbits were divided randomly into two groups:control group (n=15)　and PLGA-PEG group (n=15). Right iliac arteries of rabbits of PLGA-PEG group were injured by balloon denuded endothelization. PLGA-PEG stents were implanted in rabbits in PLGA-PEG group. MMP-2,9 and pro MMP-2,9 levels of PLGA-PEG group were determined before implantation,7 and 30 d later by SDS-PAGE zymography. MMP-2,9 and pro MMP-2,9 levels were determined in control groups. Results The concentrations of MMP-2,9 and pro MMP-2 in PLGA-PEG group were much higher than those in control group (11 568±2 219,10 364±1 429,13 649±1 894 INT·mm² vs 6 128±1 562,8 519±2 167,8 413±2 156 INT·mm²,P<0.05). There were no significant differences of pro MMP-9 between two groups (P>0.05). In PLGA-PEG group, the levels of MMP-2,9 and pro MMP-2,9 didn′t elevate significantly 7 d later　(P>0.05). However the MMP-2 and pro MMP-2 concentrations were still higher 30 d after implantation （13 935±2 167，15 628±1 739 INT·mm² vs 11 568±2 219，13 649±1 894 INT·mm²，P<0.01）. Conclusion The serum levels of MMP-2 and pro MMP-2 increase significantly in rabbits with atherosclerosis. The MMP-2 level also increases after implantation.

Objective To observe the effect of Ixeris sonchifoila, a kind of Chinese traditional medicine on noise-induced hearing loss. Methods Fourty-two guinea pigs were divided into two groups. The animals were exposed under 110 dB (n=21) and 120 dB (n=21) noise, separately. Each group was divided into medicine group (n=7), saline group(n=7) and control group (n=7). Ixeris sonchifoila was used in the medicine group and saline in the saline group immediately after the noise exposure by abdomen injection. The threshold was recorded 7 and 14 d later by ABR. Cochlea patch was performed to find hair cell loss. Results By using Ixeris sonchifoila, the threshold shift was significantly reduced (P<0.01) and the hair cells had less injuries in both of 110 and 120 dB groups, the threshold shift of 14 d later was less than that of 7 d later in medicine groups (P<0.01). Conclusion Ixeris sonchifoila can reduce the hair cell injury induced by noise and improve hearing, the effect depends on the degree of noise and the time of Ixeris sonchifoila injection.

Objective To investigate the effects of 5-hydroxytryptamine (5-HT) on apoptosis of cultured human cytotrophoblast cells and its possible mechanism involved in pathogenesis of pregnancy-induced hypertension syndrome. Methods Human cytotrophoblast cells from three term pregnancy (37-39 weeks) women were cultured and divided into four groups: normal control group, 5-HT groups (0.1,1 and 10 μmol·L^-1). The cells were cultivated for 3,6,12 and 24 h, respectively. Apoptotic index was measured by TdT-mediated biotinyated-dUTP nick end labeling (TUNEL) and morphological features of apoptotic cells were observed under electron microscope. Results When cells were cultivated for 3,6,12 and 24 h, compared with the normal control group (1.21±0.97,2.30±1.24,3.89±2.32 and 7.34±5.59,respectively), apoptotic indexes of cytotrophoblast cells in 10 μmol·L^-1 5-HT group were significantly increased (8.40±2.48,17.82±5.43,29.53±7.41,45.56±12.10, respectively)（P<0.05）；the apoptotic indexes in 1 μmol·L^-1 5-HT group were increased to 17.96±3.48 and 23.76±8.47（P<0.05） when cells were cultivated for 12 and 24 h; but there was no significant difference between 0.1 μmol·L^-1 5-HT group and ic cytotrophoblast cells in various groups were found under electron microscope. Apoptotic cytotrophoblast cells were obviously compact and the chromatins were formed as mass and apoptotic bodies were also found under electron microscope. Conclusion 5-HT can promote the apoptosis of human cytotrophoblast cells and its mechanism may be correlated with pregnancy induced-hypertension syndrome.

Objective To explore the effects of semiconductor laser on proliferation of the multidrug-resistant model of human osteosarcoma cell line (R-OS-732). Methods Four test groups and one control group were set up. The test groups were divided into A,B,C and D groups according to the irradiation time (output power was 200 mW). The time of laser irradiation of A,B,C and D groups were 5,10, 20 and 40 min， repectively. The control group (E) wasn′t treated with laser irradiation. MTT method was used to observe the proliferation of R-OS-732， and the morphological changes of the cells were observed by inverted microscope. Results The survival rates of cells in all test groups under condition of semiconductor laser irradiation with 200 mW output power and 532 nm weavlength, compared with control group (P<0.05); and it increased with the irradiation dose of semiconductor laser. The cellular density decreased under light microscope. Conclusion The semiconductor laser can result in the inhibition of proliferation of R-OS-732 cultured in vitro.

Objective To study the role of Bid in simulated ischemia/reperfusion injury in cultured neonatal rat cardiocytes and its possible mechanism. Methods A cell culture model of neonatal rat(born within 3 days) cardiocytes was used to establish simulated ischemia/reperfusion model. Unrelated siRNA or Bid specific siRNA was transfected into rat cardiocytes by Oligofectamine. The activation of Bid and caspase-8 was detected by Western blotting. The apoptotic rate of cardiocytes was determined by flow cytometry. Results Bid and caspase-8 of cardiocytes were activatied in simulated ischemia/reperfusion injury model. The activation of caspase-8 of cardiocytes in Bid-specific siRNA group decreased significantly compared with those in simulated ischemia/reperfusion group and unrelated siRNA group; the apoptotic rate of cardrocytes (7.4%) was lower than those in simulated ischemia/reperfusion group (51.2%) and unrelated siRNA group (48.7%) (P<0.01), and approached the rate in control group (4.6%). Conclusion Bid mediates the apoptosis of rat cardiocytes in simulated ischemia/reperfusion injury.

Objective To identify the biomechanical feasibility of the extra-pedicular technique for screw insertion in thoracic. Methods Five fresh adult cadaveric thoracic spine from T1 to T8 were harvested and divided into forty single vertebrae. Pedicle screw and extra-pedicular screw were inserted in each specimen respectively. Pull-out strength of pedicle screw and extra-pedicular screw through sagital axis of vertebral were measured and compared statistically. Results Two kinds of insertion result were obserbed in extra-pedicular group,A and B. A:19 screws were inserted through transverse into vertebrae directly; B: 21 screws were inserted into vertebrae through lateral cortex of pedicle. In extrape-dicular group,along the sagital axis,the mean pull-out strength of A was (827.01±260.00) N, and the mean pull-out strength of B was (954.25±254.00) N . The mean pull-out strength of all screws in extra-pedicular group was (890.63±42.00) N, and the mean pull-out strength of screws in intrapedicular group was (1 001.23±220.00) N .Compared with pull-out strength of screws in intrapedicular group, the mean pull-out strength of all screws in extra-pedicular screw was decreased by 11.04%, the difference was no significant (P>0.05). Conclusion The extra-pedicular technique is feasible in biomechanics.

Objective To study the efficient expression of a Kunitz-type protease inhibitor(KPI) domain of the Alzheimer′s disease related to amyloid β-protein precursor, and produce rhKPI/AβPP by optimizing fermentation papameters on large scale using Pichia pastoris as host system. Methods KPI/AβPP gene was inserted into expression vector pPICZα and the resulted recombinant vector was transformed into the Pichia pastoris. Then KPI/AβPP was expressed in 80L fermentor with optimizing parameters and the broth was harvested and purified with SP Sepharose. Results KPI/AβPP gene could be induced by methanol and expressed with the maximal yield of 1.0 g·L^-1. The expressed product was purified from the fermentating culture. Analytic results revealed that resulted product with relative molecular mass of about 6 750 inhibited the serine protease activities. Conclusion rhKPI/AβPP can be expressed in Pichia pastoris on large scal. A stable fermentation and purification technology is successfully set up.

Objective To investigate the renoprotective effect of reduced glutathione on streptozotocin-induced diabetic rats and its possible mechanism. Methods Streptozotocin-induced diabetic rats received aminoguanidine 50 mg·kg^-1·d^-1 and reduced glutathione 400 mg·kg^-1·d^-1 intraperitoneally respectively or synchronously for 8 weeks. The expression of TGF-β1 mRNA and protein in renal cortex were determined by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry respectively. The mean glomerular area (MGA) and volume(MGV) were measured by image analysis system. The changes of creatinine clearance rate(Ccr),the kidney weight/body weight ratio and the urinary albumin excretion rate(UAER) were determined. Results By the end of 8 weeks, the Ccr, UAER, MGA, MGV, kidney weight/body weight ratio, the contents of TGF-β1 mRNA in renal cortex were increased significantly in DM groups compared with the blank control group (P<0.05 or P<0.01), and they were decreased significantly in aminoguanidine and reduced glutathione respectively or synchronously treatment groups compared with DM control group (P<0.05 or P<0.01). Conclusion Reduced glutathione has renoprotective effect on streptozotocin-induced diabetic rats, which may be at least partly correlated with suppression effect on increased oxidative stress as well as overexpression of TGF-β1 in renal tissue.

Objective To investigate the effects of microencapsulated NGF expressing NIH3T3 cells on the repair of peripheral nerve defect in rats. Methods Thirty SD male rats with 10 mm defect of sciatic nerve were randomly divided into three groups (n=10). Group A: The nerve defect was bridged with heterogeneous nerve, the neural regeneration room was formed and filled with microencapsulated NGF expressing NIH3T3 cells. Group B: Autogenous nerve grafting was performed. Group C: The nerve defect was bridged with heterogeneous nerve, the neural regeneration room was formed and filled with microencapsulated NIH3T3 cells. The walking track analysis, electrophysiological and morphological observation were performed 12 weeks after operation. Results There were no significant differences of sciatic nerve function index, nerve concluctive velocity, histological changes of regenerative nerve, maturation degree of regenerative nerve fiber between group A and group B 4,8,12 weeks after operation; group A and B were surperior to group C (P<0.05). Conclusion There is good biocompatibility between the microencapsules and peripheral nerve tissue of rat in vivo. The microencapsulated NGF expressing NIH3T3 cells is good bridge and can promote regeneration of peripheral nerve.

Objective To investigate the effect of resveratrol on proliferation inhibition, cell cycle arrest and apoptosis of Jurkat cell line in acute T lymphoblast leukemia. Methods MTT assay was used to determine the cell vitality. Wright-Giemsa，Hoechest 33258/PI staining and transmission electron microscope technique were used to detect the apoptosis status of Jurkat cells. The cell cycle arrest was analyzed by flow cytometry. Results Resveratrol had 64.01% inhibitory rate on the growth of Jurkat cells at 0.2 mmol·L^-1 and inhibited the growth of Jurkat cells in dose- and time-dependent manner. 24 h after treated with resveratrol,the typical features of apoptosis were observed under light and electron microscope in all treatment groups. Some nuclei showed bright blue under fluorescence microscope in the resveratrol-treated Jurkat cells, and the number of cells with bright blue fluorescence increased with time. Nuclei condensation and fragmentation were observed. Cell shrinkage, chromatin condensation, and marginalization were found by Wrigh-Giemsa staining and transmission electron microscope technique. By flow cytometry, 62.57% of the cells were arrested at the S phase after exposured to 0.05 mmol·L^-1 resveratrol for 48 h, the rate of apoptotic cells to total cells was 12.01% in 0.05 mmol·L^-1 treatment groups, and that in the control groups was 2.05%. Conclusion Resveratrol can inhibit the proliferation, cause S-phage arrest and induce the apoptosis of Jurkat cells.

Objective To observe the protective effects of Diemailing Injection (DMLI) on experimental myocardial infarction in rats and its mechanism. Methods The experimental myocardial infarction model was induced by left anterior descending coronary occulusion for 24 h in rats. The rats were randomly divided into sham group, myyocardial infaction model group, DMLI groups with different doses (2.5,5.0,10.0 mL·kg^-1) (n=20). The changes of myocardial infarction size (MIS), aspartate aminotransferase(AST),actate dehydrogenase(LDH), creatine phosphokinase (CK), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) content in serum, endothelin (ET) and angiotensinⅡ (AngⅡ) levels in plasma, and low shearing specific viscosity, middle shearing specific viscosity and high shearing specific viscosity of blood and specific viscosity of plasma were determined. At the same time, myocardial free fatty acid (FFA) contents of infarction and noninfarction area were determined. Results In rats treated by DMLI (in doses of 2.5,5.0 and 10.0 mL·kg^-1 i.v after coronary occulusion), the MIS was significantly reduced (P<0.05 or P<0.01), the AST, LDH and CK activities in serum, the ET and AngⅡ levels in plasma, and viscosity of blood and plasma were declined (P<0.05 or P<0.01), while SOD and GSH-Px activities in serum were increased significantly (P<0.05 or P<0.01). In addition, myocardial FFA contents of infarction and noninfarction area were decreased markedly (P<0.05 or P<0.01).Conclusion DMLI has protective effects on experimental myocardial infarction,which may be related to improving myocardial metabolism, increasing the activity of antioxidize, eliminating the free radicals, decreasing ET and AngⅡ levels in plasma, and decreasing blood viscosity etc.

Objective To construct phosphate-specific transport system 1 (PstS1)-BCG recombinant vaccine. Methods PstS1 gene was amplified from the genome of mycobacterium tuberculosis H37Rv and was cloned into E.coli - M.bovis BCG shuttle plasmid PMD31 to get recombinant plasmid S-PMD31. The S-PMD31 was introduced into E. coli and PstS1 expressed in high level induced by IPTG. S-PMD31 was then introduced into BCG by transformation or electroporation. Results 1 100 bp PstS1 gene was amplified from the genome of mycobacterium tuberculosis H37Rv, and then was cloned into E.coli - M. Bovis BCG shuttle plasmid PMD31. The PstS1 gene was correcctly inserted into shuttle plasmid PMD31 confirmed by restriction endonuclease digestion analyzing and DNA sequencing. PstS1 protein could be expressed in E.coli and then was introduced into M.bovis BCG by electroporation. Conclusion The recombinant PstS1-BCG vaccine is successfully constructed. It is anticipate that the PstS1-BCG vaccine can improve the immunogenicity and protective efficacy of BCG vaccine.

Objective To study the effects of low dose radiation（LDR）on expression of apoptosis-related gene mRNA of transplantation tumor tissues of human glioma (U251)-bearing nude mice.Methods Thirty -five tumor-bearing nude mice were divided into seven groups: sham-irradiated group：0 mGy；D₁ group：75 mGy；D₂ group：4 Gy；D₁-12 h+D₂ group：between D₁ and D₂ was 12 h; D₁-24 h+D₂ group：between D₁ and D₂ was 24 h；D₁-48 h+D₂ group：between D₁ and D₂ was 48 h；D₁-72 h+D₂ group：between D₁ and D₂ was 72 h. The expression of apoptosis-related gene mRNA of transplantation tumor tissues of tumor(U251)- bearing nude mice was detected by in situ hybridization. Results There were basal expressions of p53, Bcl-2, Bax mRNA in all groups. The transcription levels of p53 and Bax were slightly up-regulated and Bcl-2 was slightly down-regulated in D₁（75 mGy）group and D₂（4 Gy） group, but there was no significant difference compared with sham-irradiated group (0 mGy)（P>0.05）; While the differences of the levels of p53, Bcl-2, Bax between all D₁+D₂ groups and sham-irradiated group were significant (P＜0.05 or P＜0.01). Conclusion LDR could up-regulate the transcription level of p53, Bax and down-regulate Bcl-2 to some extent, and it maybe have synergic effect with high dose radiation.

Objective To observe the suppression of HBsAg and HBeAg expression by DNAzyme and LNAzyme located at HBV pre-area of HBV. Methods Eecoding sequence of 10-23 DNAzyme thiolmodificated 10-23DNAzyme and LNAzyme that were directed against Pre C/C region of HBV were designed and synthesized. Experimental groups and control groups were set up. The experimental groups included 10-23 DNAzyme group,S-10-23 DNAzyme group and LNAzyme group. The control groups include blank control group, simple lipofectamine group,simple 10-23DNAzyme group and random 10-23 DNAzyme group. In the dosege of 0.16,0.64,1.28,1.60,1.92 μmol·L^-1 and the time of 12,24,36,48,60,72,84 and 96 h, the suppression of HBsAg and HBeAg expression by 10-23 DNAzyme and LNAzyme in 2.2.15 cells were studied. Results The suppression of HBsAg and HBeAg expression by 10-23 DNAzyme and LNAzyme in 2.2.15 cells were significant. The inhibitory effects caused by LNAzyme was more significant than that by thiolmodified 10-23 DNAzyme whose inhibitory effects were more significant than that of 10-23 DNAzyme. The inhibitory rates of LNAzyme and 10-23 DNAzyme thiolmodification reached (91.6±8.4)%, (78.4±2.0)% on HBsAg，respectivelly and (90.1±5.2)% , (76.4±4.8)% on HBeAg. The inhibitory effects of LNAzyme and thiolmodification of 10-23 DNAzyme were found 12 h after they were added to 2.2.15 cells, and optimized at 48 h, effective inhibitory time for LNAzyme was 84 h,for thiolmodification 10-23 DNAzyme was 72 h. Addition of LNAzyme and 10-23 DNAzyme to 2.2.15 cells didn′t exert cytotoxicity. Conclusion 10-23 DNAzyme and LNAzyme have demonstrated significant inhibitory effects on the HBsAg and HBeAg expressions in 2.2.15 cells. Morever, the inhibitory effects of LNAzyme is more significant than that of DNAzyme. LNAzyme is a specific anti-HBV therapeutic agent.

Objective To evaluate dentin-resin interface on bond strengths under different thermal cycling treatments in vitro. Methods Sixty intact non-carious human molars were selected, the occlusal enamel were removed using a low-speed-saw to expose a dentin surface ,dentin surface were polished with 300 grit，600 grit silicon carbide paper successively. Sixty specimens were divided randomly into three groups according to different thermal cycling treatments, in each group ,two bonding agents were used: Single Bond，a total etch bonding system;and Adper Prompt,a self etch bonding system, with ten teeth in each agent. In group Ⅰ (n=20),there was no thermal cycling treatment ,after preserved in 37℃ water for 24 h,shear bond tests were performed. Another forty specimens were assigned as group Ⅱ (n=20) and group Ⅲ (n=20). Specimens were submitted to thermal cycling, 500 cycles in group Ⅱ and 1 000 cycles in group Ⅲ. The treatment temperature in groups Ⅱand Ⅲ was 5℃-55℃.　The dwell time was 30s in each bath with a transfer time of 20 s. After 24 h storage in distilled water at 37℃, shear bond strength tests were performed. Shear bond strength tests of all groups were conducted in a instron 1121 universal testing machine at a cross-head speed of 1 mm·min^-1 .After shear bond strength test, one of Single Bond fractured specimens and one of Adper Prompt fractured specimen were selected in each group to be examined under SEM. Results Shear bond strengths (MPa)after　0,500 and 1 000 thermal cycles were: Single Bond 18.12±3.36,17.62±3.61 and 16.93±2.66; Adper Prompt:13.22±2.76,12.90±2.93 and 11.17±2.63. There were no significant differences of shear bond strengths among different thermal cycling treatment groups （P>0.05）; Single Bond provided significantly higher shear bond strengths than Adper Prompt in all groups (P<0.05) . All fractures of interfaces showed resin tag create with Single Bond more than with Adper Prompt. Conclusion In short terms,thermal cycling dosen′t influence significantly the shear bond strength of the resin-dentin interface. Compared two dentin-bonding systems used in this study,total etch bond system provide significantly higher strength than self etch bond system.

Objective To investigate the association SLC25A12 and SCN2A2 gene single nucleotide polymorphisms(SNPs) and susceptibility to autism among 105 Japanese family trios consisting of fathers, mothers, and affected offsprings with autism. Methods Genomic DNA was isolated from the whole blood samples. The PCR-single stranded conformational polymorphism (SSCP) technique was used to test genotype of SNPs (rs3770448,rs3769955) at SLC25A12 and SCN2A2 genes. Results The distributions of genotypic and allelic frequencies of rs3770448 and rs3769955 were not deviated from the Hardy-Weinberg equilibrium. The results of transmission disequilibrium test (TDT) indicated that the allelic frequency transmitted from the heterozygote parents didn′t deviate 50%. Conclusion The polymorphism of rs3770448 in the SLC25A12 and rs3769955 in the SCN2A2 locus may not be associated with autism. But the association of the other SNPs at the SLC25A12 and SCN2A2 locus with the illness can not be ruled out.

Objective To investigate the expressions of decorin (DCN) mRNA and protein in colorectal carcinoma tissues. Methods Immunohistochemistry and in situ hybridization technique were used to detect DCN protein and mRNA expressions in 30 cases of colorectal carcinoma tissues and 20 cases of relative normal tissues. Results The positive expression rate of DCN mRNA in 30 cases of colorectal carcinoma was 66.67%(20/30), while the positive expression rate of DCN mRNA in 20 cases of relative normal tissue was 85.00%(17/20). The difference between them was not significant (P>0.05). DCN protein didn′t express in both 20 cases of relative normal tissue and 30 cases of colorectal carcinoma. Conclusion DCN mRNA can express in both colorectal carcinoma and relative normal tissue.

Objective To measure the magnitude of galvanic currents produced by different occluding metallic contact. Methods The circuits of instantly different metallic contact were simulated in artificial saliva,and electrical potential of every couple and 15 currents of instant contact were measured. To simulate night sleep, after 8 h soaking, 15 currents were obtained in addition. Results During early soaking, gold/Co-Cr alloy produced the highest current (6.17 μA),while there were significant differences in currents between gold/zinc-free amalgam，gold /zinc-containing amalgam and other couples (P<0.05) after 8 h soaking. In comparison between currents produced by couples before and after 8 h soaking, the currents produced by Co-Cr alloy/ zinc-free amalgam proved to be no significant difference (P>0.05)，and the current value was low all the time. Potential of zinc-containing amalgam was more negative than other alloys,and it was always under anodic behavior during the experiment and was corrupted acceleratedly. Conclusion Couples of gold/titatium,gold/zinc-containing amalgam,zinc-free/zinc-containing amalgam appearing in oral will do harm to the patients′health,while Co-Cr/zinc-free amalgam can coexist in oral . Corrosion behavior of amalgam is affected by different contents of zinc. dental alloys；galvanic corrosion；galvanic current

Objective To study the expressions of TNFR1,TRAF2 and TNFR2 in laryngeal squamous carcinoma and the relationship with the biological characteristics of laryngeal squamous carcinoma. Methods By using immunohistochemical method and TUNEL method，the expressions of TNFR1, TNFR2，TRAF2 and the apoptotic index were examined in 33 laryngeal squamous carcinoma tissues. Results In the laryngeal squamous carcinoma cells, the positive rate of TNFR1 was 63.6%, the positive rate of TNFR2 was 6.1%, the positive rate of TRAF2 was 69.7%. Neither TNFR1 nor TNFR2 was related to the biological characteristic of laryngeal squamous carcinoma (P>0.05), and TRAF2 was related to the tumor grade only (P<0.05). The expression of TNFR1 was strongly associated with the expression of TRAF2 (P<0.01). The apoptotic indexes of the positive expression tissues of TNFR1 and TRAF2 （3.12±1.47,2.85±1.19） were significantly lower than those of the negative expression tissues （4.28±1.34,5.12±0.81）（P<0.05 and P<0.01）. Conclusion TRAF2 could be a new diagnostic index in laryngeal squamous carcinoma. The negative expression of TNFR2 may participate in the progress of laryngeal squamous carcinoma.

Objective To investigate the relationship between promoter methylation of RASSF1A gene and laryngeal squamous cell carcinoma. Methods A methylation-specific PCR was performed to detect the promoter methylation of RASSF1A gene in 48 tumor tissues, 48 corresponding normal tissues and 48 normal blood plasma. The expression of RASSF1A protein was determined by means of Western blotting. Results The methylation in RASSF1A gene was detected in 34 (70.83%) tumor tissue samples, 11 (22.92%) corresponding normal tissue samples and 6 (12.50%) blood plasma samples, respectively. The methylation degree of tumor tissue was higher than that of corresponding normal tissues and normal blood plasma (P<0.05). 27 （56.25%）of 48 tumor tissue samples and 8（16.67%）of 48 corresponding normal tissue samples expressed less RASSF1A protein. The level of expression of RASSF1A protein in tumor tissues was lower than that in corresponding normal tissues (P<0.01). 25（73.53%）of 34 methylated tumor tissue samples expressed less RASSF1A protein, while only 1（14.29%） of 7 unmethylated tumor tissue samples expressed less RASSF1A protein (P<0.05). Conclusion The promoter methylation of RASSF1A is associated with the carcinogenesis of laryngeal squamous cell carcinoma. It may be a useful marker for diagnosis of laryngeal squamous cell carcinoma.

Objective To assess the efficacy of NaClO irrigation of root canal at different temperatures. Methods Thirty human teeth with single root-canal mandible premolar were instrumented using standard technique, then were divided into 3 groups, carrying on root-canal irrigation. group A: 5.25% NaClO+System B, group B:5.25% NaClO+15% EDTA, group C:5.25% NaClO+System B+15% EDTA. After the teeth root were split, the scanning electron microscope was used to observe the coronal third ,middle third and apical third parts. Results The amount of remaining debris on root canal wall in group C decreased significantly,compared with group A and B. The differences of coronal third and middle third between group A and B, group B and C,group A and C were significant （P<0.05, P<0.01,P<0.05). The difference of apical third between group A and B was no significant （P＞0.05）, but there were significant differences between group A, B and C（P<0.01, P<0.05).The differences of diameters of dentinal tubules of the coronal third,middle third and apical third parts were significant between various groups (P<0.05 or P<0.01).Conclusion The application of 5.25% NaClO after heating united with 15% EDTA can strengthen the effect of root canal cleaning.

Objective To explore the relationship between pregnancy-associated plasma protein-A (PAPP-A) and occurance，development of cardiovascular diseases,and lipids. Methods 75 patients with coronary disease were divided into acute myocardial infarction (n=32),unstable angina pectoris (n=22) and stable angina pectoris (n=21) groups,and 60 subjects without coronary diseases were used as controls. The serum PAPP-A，IL-6，IL-10，lipids were measured in all patients and controls by different methods of enzymatically amplified two-step sandwith-type immunoassay，double antibody radio-immunoassay，ABC-HRP, auto biochemistic analytist. Results ①The level of PAPP-A in acute coronary syndrome（ACS，including acute myocardial infarction and unstable angina pectoris） patients was significantly higher than that in stable angina pectoris patients and controls (P<0.05). ②There were significantly associations between PAPP-A and serum totle cholesterol，ApoA1/ApoB (r=0.348，0.420，P＜0.05). ③The levels of IL-6 and IL-10 in coronary heart disease patients were significantly higher than those in controls (P<0.05),and the variations among acute myocardial infarction，unstable angina pectoris，stable angina pectoris patients were significantly (P<0.05). There were significantly associations between PAPP-A,IL-6 and IL-10（Spearman r 0.446，0.523, P<0.05）. Conclusion PAPP-A is significantly associated with occurance and development of coronary heart disease , probablely as a marker of unstable plaque in coronary heart disease .