Abstract:Objective To explore the preventive effects of egg-milk with metallothionein (MT) on the H-22 hepatocellular carcinoma in BALB/c mice. Methods Seventy female BALB/c mice were divided into four groups at random: normal control (n=10), tumor control (n=20), low dose egg-milk with MT group (n=20) and high dose egg-milk with MT group (n=20). The mice were injected with H-22 cells subcutaneously to build hepatocellular carcinoma model after they were administered with egg-milk with MT for two weeks. The developing time of tumor and tumor volume were detected. The mice were killed after one month and immune indices were measured with their spleens. The ability of lymphocyte proliferation was measured with MTT method, the percentages of CD4⁺, CD8⁺ and CD25⁺ T cells and apoptotic percentage of lymphocytes were measured with flow cytometry, the killing activities of CTL and NK cells were detected with ³H-TdR method. Results As compared with tumor group, the tumor developing speed in the mice administered with egg-milk with MT decreased significantly (P<0.05), the lymphocyte proliferation rate increased significantly (P<0.01), the positive percentages of CD4⁺, CD8⁺ T cells and the radio of CD4⁺/CD8⁺ T cells increased significantly (P<0.05 or P<0.01), the positive percentage of CD25⁺ T cells and their apoptotic percentages decreased significantly (P<0.05 or P<0.01), the toxicities of NK and CTL cells increased significantly (P<0.05 or P<0.01). Conclusion Egg-milk with MT could enhance the immune function in the mice with hepatocyte tumor and inhibit the tumor developing speed obviously.

Abstract:Objective To study the effect of ionizing radiation on apoptosis and necrosis of Jurkat T cells, and investigate the model and mechanism of cell death after ionizing radiation. Methods The time- and does-effects on apoptosis and necrosis of Jurkat T cells after X-irradiation with different doses (0.075, 0.500, 1.000, 2.000, 4.000 and 6.000 Gy) were detected by using Annexin-V/PI double staining by flow cytometry. Results For the time-effect, the apoptotic percentage of Jurkat cells increased significantly 18 h after irradiated with 2.000 Gy X-rays and kept higher level to 24 h compared with sham-irradiation group (P<0.001). The percentage of necrosis increased strikingly 4 h after irradiation, reached the peak value at 12 h and kept higher level to 48 h (P<0.001). For the does-effect, the percentages of apoptosis and necrosis of Jurkat cells didn’[KG-*3]t change markedly 18 h after irradiated with 0.075 Gy X-rays compared with sham-irradiation group. The percentages of the apoptosis and necrosis increased in a dose-dependent manner 18 h after irradiated with the higher 2.000-6.000 Gy X-rays compared with sham-irradiation group (P<0.001). Conclusion Apoptosis and necrosis of Jurkat cells may be induced by X-irradiation with the dose of above 2.000 Gy. The cell death occurs earlier and persists longer than apoptosis.

Abstract:Objective To investigate the efftcets of low dose X-ray radiation on the activity of the reactive oxygen species (ROS) and mitochondrion membrane potential (△Ψm) in spermatogenic cells of mouse testes. Methods The ROS activity and △Ψm in the cells were measured indirectly by flow cytometry with 2′, 7′-dichlorofluorescin diacetate (DCFH-DA) and Rhodamine 123 probes. Results The ROS activity increased with the increase of irradiation doses 12 h after irradiation with different doses of X-rays compared with 0 Gy (P<0.05); but the △Ψm decreased with the increase of irradiation doses compared with 0 Gy (P<0.05). After irradiation with 0.075 Gy X-rays, the ROS activity increased with time prolongation compared with 0 h (P<0.05), 　reached the peak at 12 h later, and kept the higher level; but △Ψm decreased with the time prolongation, compared with 0 h (P<0.05), and reached the lowest level at 12 h later, then recovered to the normal level gradually. Conclusion Low dose radiation can induce the the increase of ROS and decrease of △Ψm in spermatogenic cells of mouse testes, and has dose- and time-effect regulations.

Abstract:Objective To construct the recombinant plasmid pIRESEgr-IFNγ and detect its expression in Lewis lung carcinoma induced by irradiation in vitro. Methods The recombinant plasmid pIRESEgr-IFNγ containing Egr-1 promoter and IFNγ gene was constructed with gene recombinant technique. The plasmid was transferred into Lewis lung carcinoma by liposome in vitro. The correlations of dose- and time-effects in the expression of IFNγ gene induced by X-ray were detected by ELISA. Results The identification with enzymes proved that Egr-1 promoter and IFNγ gene were inserted into vector pIRES1neo correctly. After X-ray irradiation with different doses, the expression of IFNγ in the supernatant of Lewis lung carcinoma transfected by pIRESEgr-IFNγ was significantly higher than that in 0 Gy group (Ｐ＜0.001). After 5 Gy X-ray irradiation, the expression of IFNγ was the highest, being 4.39 times as much as that in 0 Gy group. The expression of IFNγ in the supernatant increased after 5 Gy X-ray irradiation, being 6.27 times as much as that in 0 h group 36 h after irradiation. Conclusion The recombinant plasmid pIRESEgr-IFNγ is constructed successfully, and it has the property of enhancing the expression of IFNγ gene induced by irradiation.

Objective To observe the effect of prenatal alcohol on blood glucose in newborn rats and study its mechanism. Methods Six female Wistar rats were divided randomly into two groups, the rats in one group were given ethanol 4 g•kg^-1•d^-1 by gavage untill delivery(ETOH group), the rats in another group were given the same volume water (control group). 12 newborn rats from both groups were chosen randomly. The mRNA level of adipose leptin and resistin of newborn rats were determined by RT-PCR. Histomorphology and parameter of islet and cells were observed by morphometry. Results Compared with controls, the birth weight of rats in ETOH group was decreased significantly (P<0.001); the insulin contents of plasma and pancreas were also decreased (P<0.05), 　however blood glucose was increased in newborn ethanol rats (P<0.05). Adipose resistin mRNA level was higher (P<0.05), but leptin level was lower (P<0.05) in newborn ethanol rats. Morphology and the amount of islets as well as cell density were normal in newborn ethanol rats (P>0.05). Conclusion Heavy ethanol consumption during pregnancy increase blood glucose level of newborn rats and decrease insulin secretion and adipokines gene linked insulin resistance which may contribute to insulin resistance in adult offspring.

Objective To investigate the effects of Ca²+and Mg²+on the GTPase activities of the wild-type and mutant FtsZs in Porphyromonas gingivalis. Methods Plasmids pEZ1 (Wt PgFtsZ), pYW1(missing 73 residues from the C-terminus of PgFtsZ, ZΔC01) and pYW2 (missing 43 residues from the N-terminus of PgFtsZ, ZΔN01) were introduced into Escherichia coli BL21(DE3)pLysS to express and purify the purpose proteins. Malachite green assay was used to measure the GTPase activities of purified Wt PgFtsZ, ZΔC01 and ZΔN01. Results GTPase activities of Wt PgFtsZ, ZΔC01 and ZΔN01 were 8.25±0.22, 7.43±0.23, 8.00±0.18, respectively, without no ion. GTPase activities of Wt PgFtsZ, ZΔC01 and ZΔN01 were 8.30±0.15, 7.51±0.22, 7.85±0.17, respectively, with 10 mmol•Ｌ^-1 Mg²⁺, and not affected by 10 mmol•L^-1 Mg²+（P>0.05）. However, GTPase activities of Wt PgFtsZ, ZΔC01 and ZΔN01 were 5.84±0.20, 5.25±0.18, 5.73±0.13, respectively, with 10 mmol•L^-1 Ca²+, and reduced by 10 mmol•L^-1 Ca²+（P<0.01）. Conclusion 10 mmol•L^-1 Mg²+ has no effect on GTPase activities of Wt PgFtsZ, ZΔC01 and ZΔN01. However, 10 mmol•L^-1 Ca²+ inhibits their GTPase activities.

Objective To observe the effects of target solubility kinase insert domain receptor(sKDR) on proliferation and apoptosis of human bladder cancer T24 cells and the feasibility and effectiveness of sKDR gene as the target of bladder cancer gene therapy .Methods Human bladder cancer T24 cells were divided into sKDR transfected group,untransfected group and contol group.Eukaryotic secretory expression plasmid PCl-sKDR was constructed and T24 cells were transfected by Lipofectamine^TM2000 and further screened by ELISA . The expressions of sKDR gene in at all groups were determined by RT-PCR,the proliferation of T24 cells in these groups was measured by MTT method,the apoptotic rates were detected by TUNEL.T24 cells treated with sKDR were transplanted subcutanuously in nude mice and the tumorgenesis ability was observed.Results The expression of KDR mRNA in the KDR- expression T24 cells were 4.16 times that in vacant vector P^CI-neo cells.Compared with P^CI-neo contol,the proliferatory inhibitory rate of cells with sKDR- expression was increased by 55.1％(P<0.01).The tumorgenesis in transfected group was significantly restrained compared with untransfected group(P<0.01).Conclusion sKDR can inhibit the proliferateion and promote the apoptosis of bladder cancer T24 cells to a certain degree.

Objective To establish cell model of Parkinson’〖KG-3〗s disease (PD) and to approach the toxic effect of rotenone on dopaminergic neurons and its mechanism . Methods PC12 cells were differentiated into dopaminergic neurons and treated with various concentrations of rotenone. The morphological changes of PC12 cells were observed after treated with rotenone（0,10,25,50,75, and 100 nmol•L^-1）for 24,48 and 72 h , the cell viability was measured by MTT assay . Immunohistochemistry and immunofluorescence were used to observe the accumulation of α-synuclein in cytoplasm. AO/EB double staining was also adopted to test apoptosis. Results After differentiation PC12 cells shaped irregularly with big and long ecptomas and multiple cell conjunctions. After the treatment of rotenone cell ecptomas vanished gradually and cell bodies became smaller and smoother. The cell viability began to decline significantly at a concentration of 50 nmol•L^-1 for 24 h (P<0.05) ，and as concentration increased, the cell viability declined in a dose-dependent manner (P<0.01).As time prolonged, the cell viability behaved the same way. There was a significant difference between each time groups (P<0.01) .Immunohistochemistry demonstrated a brown round α-synuclein immunoactive mass in cytoplasm; cell immunofluorescence showed a strong greenα-synuclein immunoactive aggregation in cytoplasm. Viable apoptotic cells，non-viable apoptotic cells and necrosis cells were observed gradually as cell concentration increased and time prolonged. Conclusion Rotenone should be toxic to dopaminergic neurons，causing inclusion of α-synuclein aggregation and inducing apoptosis. The malmetabolism of α-synuclein and apoptosis induced by rotenone might play an important role in the pathogenisis of Parkinson’〖KG-3〗s disease .

Objective To investigate the effects of mitogen-activated protein kinase p38 (p38 MAPK) on CD3/CD28-stimulated T-cell proliferation and IL-2 expression which were enhanced by shockwaves. Methods Jurkat T cells or peripheral blood mononuclear cells (PBMC) were pretreated with the p38 MAPK-inhibitor (SB203580)(The Jurkat T cells or PBMC of the control groups were not pretreated with SB203580), then treated with shockwaves, and stimulated respectively with a suboptimal dose of anti-CD3 and anti-CD28 antibodies or PHA. Finally the IL-2 productions of Jurkat T cells or the proliferation of PBMC were respectively measured. The protein tyrosine phosphorylation of p38 MAPKin Jurkat T cells treated either with shockwaves or not was measured by Western blotting with anti-phosphotyrine antibodies(Thr180/Tyr182). The expressoins of p38 MAPK in Jurkat T cells treated either with shockwaves or not were determined by Western blotting with anti-p38 MAPK antibodies. Results Compared with negative control group without shockwave treatment, the ³H-TdR incorporation of the phytohemagglutinin-stimulated PBMC, which were treated with low dose shockwaves (LDSWs) of 100, 150, 200, 250, 300, 330 impulses at (0.180±0.015) mJ•mm², increased significantly (P<0.01). Compared with negative control group, the additions of SB203580 attenuated the IL-2 expression in the cellular supernatant of CD3/CD28-stimulated Jurkat T cells or the ³H-TdR incorporation of the phytohemagglutinin-stimulated PBMC, which were affected with LDSWs of 100, 150, 200, 250, 300, 330 impulses at (0.18±0.015) mJ•mm² (P<0.01). The activation of p38 MAPK under the condition without LDSWs was obviously lower than that with LDSWs of 100, 150, 200, and 250 impulses (P<0.01), and the activation of p38 MAPK increased with the shock-wave impulses.Conclusion SB203580 prevents the enhancing effect of LDSWs on CD3/CD28-stimulated IL-2 expression. In T cells, the shockwaves enhance IL-2 expression through activating p38 MAPK.

Objective To construct the expression vectors of pSilencer3.1 -hif-1α and identify the inhibition of hif-1α in Hela299 cells after transfection with the combinative plasmids. Methods The interfering sequences of hif-1α were designed according to the sequence of hif-1α of GenBank. Three recombinant plasmids of pSilencer3.1-hif-1α were constructed by DNA ligase. Trizol reagent was used to extract the whole RNA of cells and RT-PCR assay was applied to detect the expression of hif-1α mRNA . Lysis assay was used to extract the protein from cells and Western blotting was adopted to observe the expression of HIF-1α protein. Results The vectors were identified to be right after sequencing. The mRNA level was decreased 24 h after transfection with three vectors of pSilencer-hif-1α, and the ratios of hif-1α /GAPDH in control, group transfecting with pSilencer-sihif-1α-1, group transfecting with pSilencer-sihif-1α-2, group transfecting with pSilencer-sihif-1α-3 were 0.55, 0.13, 0.33, and 0.08, respectively(P<0.05, P<0.05, P<0.01). The protein expression levels of HIF-1α 48 h after transfection with three vectors of pSilencer-hif-1αwere decreased significantly. Conclusion Effective pSilencer-hif-1α plasmids are constructed successfully and they could interfere with the expression of hif-1α in Hela299 cells.

Objective To establish a co-culture system of human bone marrow mesenchymal stem cells(hMSCs) and osteoblasts to get human osteoclasts. Methods The osteoblasts were obtained with collagenase and the alkaline phosphatase staining was used to test the form of osteoblasts; hMSCs were isolated , cultivated and proliferated by Percoll gradient centrifugation. Co-culture hMSCs and osteoblasts were cultivated together, and osteoclasts were identified with tartrate-resistant acid phosphatase (TRAP) staining. Results High homogenous hMSCs were obtained and after co-culture for 5 d with osteoblasts, they fused to form multinuclear cell clusters in micrograph and were postive in TRAP staining. Conclusion The co-culture system method is successful.With this method osteoclasts can be obtained from co-culture of hMSCs and osteoblasts.

Objective To study the condition of pheochromocytoma cells (PC12 cells) differentiation into neurons induced by nerve growth factor (NGF). Methods There were five groups :10,20,50, and 80 μg•L^-1 NGF groups and control group which contained 10% fetal calf serum. PC12 cells were exposed to NGF with different concentrations for 72 h and the lengh of neurite and max diameter of PC12 cells were observed at 24,48,and 72 h, respectively. The expression of MAP2 was detected by immunocytochemistry after PC12 cells were exposed to NGF for 72 h . Results The number of MAP2 positive cells was significantly increased in 20, 50, and 80 μg•L^-1 groups compared with control group and the most significant concentration was 50 μg•L^-1（P<0.05）. The length of total primary neurite and max diameter of PC12 cells in 20,50, and 80 μg•L^-1 NGF groups were obviously longer and bigger than those in control group and the most significant concentration also was 50 μg•L^-1（P<0.01）. Conclusion PC12 cells could differentiate into neurons induced by NGF, and 50 μg•L^-1 is the optimal concentration in which NGF could induce PC12 cells to differentiate into neurons.

Objective To isolate the rat insulin gene enhancer binding protein 1(Islet-1) gene and construct plEGFP-C1-Islet-1 recombinant retroviral expression vector. Methods The cDNA encoding the rat Islet-1 gene was isolated by RT-PCR method, the cDNA was first cloned into PGET-1 TA vector to facilitate the sequence and then subcloned into the retroviral vector plEGFP-C1. plEGFP-C1-Islet-1 was transfected into PA317 packaging cells with lipofectamine 2000. Transformants were selected in medium containing G418. Results A 1 050 bp DNA fragment was obtained by RT-PCR; plEGFP-C1-Islet-1 recombinant retroviral expression vector was identified by restrictive enzymes digestion, PA317 cells transfected with recombinant vector expressed enhancer green fluorescent protein (EGFP). Conclusion The gene encoding the rat Islet-1 is obtained and plEGFP-C1-Islet-1 expression vector is constructed successfully.

Abstract:Objective To clone human cell division cycle gene 2 (CDC2) from human liver cancer tissue and express CDC2 protein in E.coli BL-21(DE3).Methods The total RNA was extracted from liver cancer tissue and amplified by reverse transcription polymerase chain reaction (RT-PCR).The PCR products were cloned into the prokaryotic expression vector pET28a(+) followed by DNA sequencing.The recombinant pET28a(+)/CDC2( rCDC2) plasmid was transformed into E.coli.The rCDC2 was induced with IPTG and characterized by SDS-PAGE.Results The cloned CDC2 gene was composed of 894 nucleotides,and is accordance with that reported in GenBank.The prokaryotic expression vector was constructed successfully.The CDC2 protein was successfully expressed in E.coli.Conclusion The CDC2 cDNA is successfully cloned from huma n liver cancer tissue,and can express in E.coli.

Objective To study the correlation between the injury in rat hippocampus after epilepsy kindled by amygdala and the expression of glial fibrillary acidic protein （GFAP）,and the role of GFAP in experimental epilepsy.Methods The rat epilepsy model was set up by electron amygdala,the pathological changes in hippocampus after kindled epilepsy were observed by using optical microscope and electron microscope.And the number of GFAP immunoreactive positive cells in hippocampus after rat kindled epilepsy,area,circumference and integral optical density were detected by immunohistochemistry.Results By using optical microscope,the neurocytes of hippocampus in experimental group were observed to denaturalize,tumefy,destroy and disappear.And under electron microscope,the karyon was irregular,nucleoles deviated,chondriosome tumefied,crista collapsed,membrane was damaged,substance cavitated.Such changes were more evident with the increased paroxysm of epilepsy,the parameters between control group and operation group had no difference（P＞0.01）.But make a monofactorial variance analysis among cells’ number,area,circumference and integral optical density in different groups,it showed that there were significant differences between optional two groups（P<0.01);at the same time,the more serious injury in hippocampus,the more powerful GFAP immunoreaction was.Conclusion The more serious injury in hippocampus,the more powerful GFAP immunoreaction is.The correlation between astrocyte and injury of brain after epilepsy can be speculated by detecting GFAP.

Objective To explore the molecular mechanism of brain tissue injury induced by endotoxin and observe the effects of Dex on TLR4,IκBα and IL-18 mRNA expressions of cortex in the endotoxic shock rats.Methods Eighteen Wistar rats were randomly divided into LPS,LPS+DEX and control group(n=6).Except control group,all the rats were administered with LPS(4 mg•kg^-1) by intravenous injection.The rats in control group were given glucose solution at same volume.The TLR4,IκBα and IL-18 mRNA expressions were assayed 4 h after intravenous injection.Results The mean arterial pressure (MAP) and the survival rate in LPS+DEX group were significantly higher than those in the LPS group(P<0.01).TLR4 and IL-18 mRNA expressions in LPS+DEX groups were obviously lower than those in LPS group (P<0.05).The IκBα mRNA expression in LPS+DEX group was significantly higher than that in LPS group (P<0.05).Conclusion Dex may down-regulate TLR4 expression and up-regulate IκBα expression in the brain tissue,and has protective effect on central nervous system.

Objective To study the effects of panax quinquefolium 20s-protopanaxdiolsaponins（PQDS）on diabetic nephropathy（DN）rats.Methods DN model rats,made by streptozotocin(STZ),were divided into model group,PQDS 50 and 100 mg•kg^-1 groups according to fasting blood glucose value and weight.The rats were given with PQDS by oral administration for 35 d.Every 10 d,the index of blood glucose was measured.On the last day,several indexes were measured including blood glucose,serum creatinine,UN,kidney weight,urine volume (within 12 h) and the structure of kidney was observed by light microscope and electron microscope.Results Compared with model group,the blood glucose and serum creatinine levels and 12 h urine volume were decreased in PQDS 100 mg•kg^-1 group （P<0.05）,but the indexes did not change in PQDS 50 mg•kg^-1 group（P>0.05）.PQDS (50 and 100 mg•kg^-1) decreased cell matrix and intercapillary cells,protected structure of kidney,improved renal pathological lesion in diabetic rats.Conclusion PQDS has therapeutic effects on DN rats through decreasing blood glucose,improving kidney function and preserving kidney structure.

Objective To observe the protective effects of Ginsenoside-Rb(G-Rb) on experimental myocardial ischemia-reperfusion injury in rats.Methods The myocardial ischemia-reperfusion model was induced by 30 min left anterior descending coronary occulusion and 24 h reperfusion in openchest anesthetized rat.The changes of aspartate aminotransferase (AST),lactate dehydrogenase (LDH),MB isoenzyme of creatine kinase(CK-MB),superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and malondialdehyde(MDA) content in serum,and prostacycline(PGI₂) and thromboxane A₂ (TXA₂) levels in plasma were determined.At the same time,the activity of platelet aggregation was also determined.Results In rats treated with G-Rb (with dose of 50 and 100 mg•kg^-1 i.g for 7 d),the MIS was significantly reduced (P<0.05 or P<0.01),the AST,LDH and CK-MB activities in serum and the TXA₂ level in plasma were declined (P<0.05 or P<0.01),while PGI2 level in plasma and PGI₂/TXA₂ ratio were increased signficatly (P<0.05 or P<0.01).In addition,the LPO content in serum was declined,SOD and GSH-Px activities in serum were increased markedly (P<0.05 or P<0.01).The platelet aggregation rate(PAR) at 1,3 and 5 min and maximum platelet aggregation rate(MOAR) were decreased markedly (P<0.05 or P<0.01).Conclusion G-Rb has protective effects on myocardial ischemia-reperfusion injury through improving free radicals metabolism,decreasing TXA₂ level in plasma,increasing PGI₂ level in plasma and PGI₂/TXA₂ ratio,and inhibitting the activity of platelet aggregation etc.

Objective To detect the effects of MT egg-milk powder on blood glucose and blood lipid level and serum zinc,copper levels in diabetic rats.Methods Sixty Wistar rats were randomly divided into normal control group(CON,n=10),diabetic group(DM,n=10),positive control group(POS,n=10)and high,middle,low doses of MT egg-milk powder groups (DM+MT,n=10)with doses of 10.0,5.0,2.5 g•kg^-1.After the rats were injected intraperitoneally with streptozosin(STZ) to establish DM models,the rats were treated with MT egg-milk powder for 40 d;the blood glucose,blood lipid level and the zinc,copper levels were observed.Results At the end of the experiment，compared with DM group, high and middle doses of MT egg-milk powder decreased triglycerides(TG),cholesterol (CHO) and low density lipoprotein(LDL) levels（P<0.05）,but high-density lipoprotein (HDL) were significantly elevated（P<0.01）.Low dose of MT decreased LDL level(P<0.01),and raised HDL level（P<0.05）.The serum zinc and copper levels were significantly elevated（P<0.05）in middle and low DM+MT groups compared with DM group.Conclusion MT egg-milk powder can morderate the lipid metabolism and enhance the serum zinc level.

Objective To establish a suitable animal model of experimental periodontitis and study the relationship between stress and inflammatory periodontal diseases.Methods Forty Wistar rats with nylon thread placed around the neck of maxillary left second molar tooth were divided randomly into two groups: stress group (the rats were treated with restraint strees for 12 h every day for 10 d) and control group.Four rats of either group were sacrificed at the day of 2,4,6,8,10, separately; the level of blood glucose and the contents of adrenocorticotropic hormone（ACTH）,orticosterone and adrenaline were measured as the stress markers,as well as the relative weight of the thymus and spleen.The furcation area of the second maxillary molar was examined histologically and histometrically.Results In stress group,all the markers for stress were significant higher than those in control group,and the thymus and spleen were atrophied（P<0.05 or P<0.01）.Marked alveolar bone resorption occurred in stress group on the 8th and 10th day（P<0.05or P<0.01）.Conclusion Wistar rat is suitable for establishing the animal model of experimental periodontitis.Restraint stress itself doesn’ t result in periodontitis whereas it modulates the progression of periodontitis.

Objective To establish the nude mouse model of human bladder carcinoma in situ and observe the formation course of human bladder transitional cell carcinoma in mouse bladders,which induced by VEGFsiRNA-transfected T24 cells or untransfected T24 cells.Methods The nude mice were transplanted with VEGFsiRNA-transfected T24 cells or untransfected T24 cells into their bladders.The imageologic alterations were observed periodicly with MRI examination and the mice were sacrificed 48 d later.The weight and the volume of the tumor were tested,and the histopathologic and cytologic test were performed at the same time.Results The total ratio of the tumor formation as 90 percent(36/40) and the tumor growth speed in transfected group was much slower than that in untransfected group.The tumor weights in transfected group and untransfected group were (1.1±0.3)g and (1.6±0.5)g,respectively, and the volumes of the tumor in transfected and untransfected groups were (805±172)mm³ and (1389±294)mm³.The differences of tumor weight and volume between two groups were both significant (P<0.05).While the cell grade and malignant degree in untransfected group were higher than those in transfected group.Conclusion The nude mouse model of human bladder carcinoma in situ can simulate the character of intra-bladder lacuna-growing of human bladder carcinoma in situ.The nude mouse model of human bladder carcinoma in situ has great importance in the research and exploration of new intro-bladder antineoplastic biological agent.It is an important way to value the effectiveness of chemical anticancer drugs preclinically.

Objective To screen efficient anti-influenza A (H₅N₁) medicine with low toxicity.Methods Normal control group,virus control group and test group were included in this study .The inhibitory effect and cytotoxicity of modified amantadine (NAM) on H₅N₁ virus and MDCK cells were detected by MTT and cytopatric effect (CPE) assay,respectively.The therapy index (TI) and 50% cell-inhibitory concentration (IC₅₀) were calculated.The mice infected with H₅N₁ virus were administered with NAM.The death rate,survival rate,death protection rate and prolong survival rate were calculated.Results The maximal no cytoxicity concentration and 50% cytoxicity concentration of NAM on MDCK cells were 235.09 mg•L^-1 and 1582.78 mg•L^-1,respectively.The IC₅₀ and TI of NAM on H₅N₁ virus were 15.32 mg•L^-1 and 103.31,respectively.The death rate (12.5%) in NAM 100 mg•kg^-1 group was significantly lower than that in virus control group (87.5%)(P<0.05),the mean survival days (13.38 d) was significantly higher than that in virus control group (9.63 d)(P<0.05),both inhibitory rate of pneumonia (99.15%) and prolongation survival rate (26.79%) were higher than those in amantadine group(69.18% and 13.39%)(P<0.05).Conclusion NAM has better antivirus activity and lower toxicity,compared with amantadine in vitro.NAM can significantly decrease mortality,increase average survival days and prolongation survival rate in vivo.

Objective To explore one simple way of preparation of chitosan extracellular matrix wire frame, and investigate its application as scaffold for tissue engineering. Methods Chitosan and acetic acid were soluted and stirred into uniform foam, and made into porous and spongiform wire frame after refrigeration and casting. The pore size and vacancy volume of the wire frame were determined under light microscope and SEM. Tissue engineered skin and cartilage were reconstructed with chitosan scaffold. Results Chitosan extracellular matrix wire frame looked porous and spongiform, its pores manifested inequality of size under light microscope, and many interlacing pores were found under SEM with pore size of 83-136 μm(average 110 μm). The three diamensions lattice had a vacancy volume of 78%. The tissue engineering skin was similar with natural skin and had bilayer structure. Keratinocytes and fibroblasts sticking on chitosan grew and proliferated, and formed into contiguous cellular layer. They increased obviously and secreted cutin and matrix. The wire frame degraded gradually. Chondrocytes sticking on wire frame grew and proliferated well, and they also secreted extracellular matrix. Generation of new cartilage tissue was found with histological observation. Conclusion The chitosan extracellular matrix wire frame has certain pore size and vacancy volume. And it is fit for cell growth and proliferation. There will be brilliant perspective for application as tissue engineering scaffold.

Objective To observe the effects of high concentration glucose on aquaporin 4(AQP4) expression in rabbit retina Müller cell cultured in vitro. Methods The rabbit retinal Müller cells were cultivated in vitro under the condition of 30, 40, 50 mmol•L^-1 high concentration glucose. The AQP4 expression was detected by immunocytochemical staining, FACS and RT-PCR after cultivated for 1,3, and 5 d. Results Green fluorescence of AQP4 expression of Müller cells in high glucose groups was weaker than that in control group(P<0.01). FACS results indicated that the position of wave crest advanced obviously and AQP4 expression deseased(P<0.01). With the increasing of glucose concentration and prolongation of time, the extent of AQP4 expression was gradually weakened. RT-PCR results revealed that on the condition of 50 mmol•L^-1 glucose for 3 d, AQP4 mRNA expression was weaker than that in control group (P<0.01). Conclusion High concentration glucose could desease the AQP4 expression of cultured retinal Müller cells. With the increasing of glucose concentration and prolongation of time, the AQP4 expression deseases gradually and it may be a protective response in diabetic retinopathy DR.

Objective To study the influence of interim overload exercise on health and it’s biochemical mechanism. Methods 45 Wistar rats were randomly divided to three groups (n=15) : no exercises group (control,C), normal exercise group (NE , the rats ran in the animal running machines at 15 m•min^-1for 5 d in one eek , exercise time was 30 min×2,and rested for 10 min after ran for 30 min) , overload exercise group(OE,the rats exercised 2 bouts,2 d in consecutive in one week,exercise time was 100 min×2,and they rested for 10 min after ran for 100 min ).After training for 8 weeks , the rats in three groups were sacrificed and 15 biochemical indexes in blood of rats were determined .Results Compared with C and NE groups , CK , LDH , and ALT increased obviously （P<0.05 or P<0.01 ）;HGB ,IgG , TB and ALB decreased （P<0.05 or P<0.01 ）in OE group;compared with C group, WBC , RBC ,HDL , TP and IgG increased obviously （P<0.05 or P<0.01 ）,and LDL decreased （ P<0.01 ）in NE group.Conclusion Interim overload exercise has some bad influence on biochemical indexes in blood of rats.

Objective To detect the effects of pancreatic kininogenase on the expression of TGF-β₁ and collagen remodeling in myocardiom of spontaneously hypertensive rats(SHR). Methods Twenty-four male SHR (aged fifteen weeks) were randomly divided into three groups: SHR group,pancreatic kininogenase group and captopril group（n=8），8 male Wistar Kyoto rats with normol blood pressure was considered as control group. Pancreatic kininogenase was given by peritoneal injection(7.2 U• kg^-1• d^-1), captopril was given by intragastric administration（10 mg• kg^-1• d^-1）, the rats in SHR group and control group were administered with 0.9% NaCl（2 mL • kg^-1• d^-1）through peritoneal injection.After four-week experiment, the pressure was measured in rats througth carotid artery ,the rats were sacrificed and left ventricular mass index, collagen volume fraction, peripheral vascular collagen area were measured.Myocardial tissue was stained with VG and pathological changes were observed. The expression of TGF-β₁ were detected by immunohistochemical technique(SP method). Results The systolic blood pressure, left ventricular mass index, collagen volume fraction, peripheral vascular collagen area and the expression level of TGF-β₁ in SHR group were obviously higher than those in control group (P<0.05). After treatment with pancreatic kininogenase for 4 weeks, all the indexes in pancreatic kininogenase group were obviously reduced compared with SHR group(P<0.05);but except systolic blood pressure,there were no significant differences of various indexes between pancreatic kininogenase and captopril groups(P>0.05).Conclusion Pancreatic kininogenase can obviously control pressure and reverse myocardial fibrosis probably by decreasing the expression of TGF-β₁ in SHR.

Objective To investigate genetic association between PCYT1A gene and schizophrenia in Chinese Han population from the Northeast area of China. Methods PCR-RFLP analysis was applied to　detect the genotype of PCYT1A gene in 115 family trios consisting of fathers, mothers and affected offspring with schizophrenia. The haplotype-based haplotype relative risk (HHRR) analysis and the transmission disequilibrium test (TDT) were used to process the genotype data. Results The genotypic frequency of the PCYT1A gene did not deviate from Hardy-Weinberg equilibrium in both the patient group （P>0.05） and the parent group （P>0.05）; The HHRR analysis did not show allelic association for the PCYT1A gene (χ²=0.011, P>0.05); the TDT analysis did not show preferential transmission of the two alleles (χ²=0.010, P>0.05). Conclusion There is no association between schizophrenia and the genetic polymorphism of rs3772108 locus of PCYT1A gene on chromosome 3.

Objective To investigate the association between single nucleotide polymorphism of DTNBP1 gene and schizophrenia in the Han people of Northern China. Methods The method of PCR-RFLP was conducted to examine the genotypes of DTNBP1 gene in 106 family trios (including patients and their parents ) , and then the genotypic and allelic frequencies of gene were statistically computed. The haplotype relative risk test (HRR) and transmission disquilibrium test (TDT) were applied to process genotype data. Results The distribution of genotypic frequency of rs9476867 was not deviated from Hardy-Weinberg equilibrium either in the parent group or in the patient sample. The rs9476867, a G to A base change, presented in the DTNBP1 locus. In the HRR test, its allelic frequency showed significant difference between case and control (χ²=0.706, P>0.05). And the TDT result did not show a genetic association between rs9476867 and schizophrenia ( χ²=0.641, P>0.05). Conclusion There is no association between rs9476867 locus on DTNBP1 gene and schizophrenia .

Objective To explore the association between sex hormone binding globulin(SHBG) in first trimester of pregnancy and subsequent development of gestational diabetes mellitus (GDM) .Methods During pregnancy, 41 patients with GDM diagnosed with glucose screening and glucose tolerance test were used as GDM group, 734 cases of pregnant women without GDM were used as control group, and the expression levels of SHBG, T, E₂ in two groups were compared. Logistic regression analysis was adopted to analyse the risk factors for GDM. Results ①The level of SHBG in GDM group (269.55 nmol•L^-1±119.35 nmol•L^-1) in first trimester of pregnancy was lower than that in control group (321.77 nmol•L^-1±124.21 nmol•L^-1) (P<0.05). ②Logistic regression analysis result showed there was independent association between SHBG level and GDM （β=0.622,P=0.025,OR=1.864,95%CI 1.083－3.207).③With the decrease of SHBG level ,the morbidity of GDM increased. Conclusion First-trimester SHBG level has independent association with subsequent GDM ,SHBG offers a potential early marker to target women who are at risk for GDM.

Objective To detect COX-2 expressions in primary tumor and axillary lymph node tissues of breast cancer, and to study the relationship between COX-2 expression in breast cancer and lymph node metastasis. Methods The expressions of COX-2 in 9 normal breast tissues, 50 primary breast cancer tissues, 19 positive axillary lymph nodes and 31 negative axillary lymph nodes were detected with immunohistochemistry S-P method. Results COX-2 staining was granular and localized to the cytoplasm of tumor cells. In normal breast tissues, COX-2 staining was granular and localized to mesenchymocyte. In negative lymph nodes, COX-2 staining was granular and localized to macrophagus cytoplasm. The positive expression rates of COX-2 in normal breast tissues, primary breast cancer tissues, positive axillary lymph nodes and negative axillary lymph nodes were 11.1%, 60.0%, 84.2%, and 32.3%, respectively. There were significant differences of COX-2 positive expression rates between normal breast tissues, primary breast cancer tissues and positive axillary lymph nodes (P<0.01). There was significantly difference of COX-2 positive expression rate between positive-lymph node primary breast cancer tissues and negative-lymph node primary breast cancer tissues (P<0.01), and also between positive axillary lymph node tissues and negative axillary lymph node tissues (P<0.001). Conclusion The patients with COX-2 positive expression possibly have axillary lymph node metastases of breast cancer.

Objective To reconstruct three-dimensional(3-D)structures of the internal capsule(including anterior limb,genu and posterior limb) with ultrathin freezing transection, and the length,width, volume, and centroid of the internal capsule were obtained and to provide an important theoretic basis for stereotactic surgery for multiple diseases of the internal capsule. Methods Three male skulls without structural diseases were selected. Each of these skulls was cut into 1-millimeter-thick layer,the photographs were taken with digital camera and the data was put into the computer. Then the border line in layers that contained the internal capsule was drawn with self-made software, the 3-D reconstruction was gotten and the data was measured. Results The midpoint of intercomissural distance was considered as original point of brain and then the centroid coordinates of the internal capsule was gotten. The centroid coordinates of the left internal capsule were －15.761 9，2.203 2 and 10.143 8 mm;the right were 17.932 0，2.656 5 and 6.384 4 mm.The volume of the left internal capsule was 4　009.173 0 mm³，the right was 4 217.234 0 mm³. After three-dimensional reconstruction, the appearance of the internal capsule looked like irregular plate-form domian. Its inner surface was convex while the external surface was concave, and the anterior limb was short while the posterior limb was high.Conclusion The reconstructed internal capsule and its parts which demonstrate the natural shape and exact position of the structures are realistic, they can be displayed in artificial colour and true colour. It provides an anatomic basis for stereotactic surgery for the treatment of the vascular disease,functional neurosurgery disease and psychiatrical surgery disease of the internal capsule.

Objective To discuss and compare the anti-tumor effects of aspirin and celecoxib on breast cancer cell MCF-7 through investigating the effects of aspirin ，celecoxib, and combined with anastrozole respectively on the growth of human breast cancer cell MCF-7.Methods The human breast cancer cell MCF-7 were treated with 2.5 ,5.0 ,and 10.0 mmol•L^-1　aspirin and 30 ,60 ,and 120 μmol•L^-1 celecoxib for 24，48，and 72 h respectively, the MCF-7 without treatment with drug was used as negative control group, the MCF-7 treated by ADM was used as positive control group, the inhibitory effect was detected by MTT assay. Besides, the MCF-7 cells were treated by anastrozole(0.5 and 1.0 μmol•L^-1), anastrozole(0.5 and 1.0 μmol•L^-1) combined with aspirin(2.5 ,5.0 ,and 10.0 mmol•L^-1)or celecoxib(30，60，and 120 μmol•L^-1) for 48 h, respectively, the inhibitory rate was detected by MTT assay. Results ① The inhibitory rate of the MCF-7 cell line treated with aspirin was reduced compared with controls, which was in time-dependent and dose-dependent manner(P<0.01).②The inhibitory rate of the MCF-7 cell line treated with celecoxib was reduced compared with controls, which was in time-dependent and dose-dependent manner(P<0.01).③The inhibitory rates of 10 mmol•L^-1 aspirin and 120 μmol•L^-1 celecoxib on MCF-7 were 68.88% and 87.00% when treated for 72 h, the inhibitory rate of 2.0 μmol•L^-1 ADM on MCF-7 was 86.30% when treated for 72 h.④The inhibitory rate of 0.5 μmol•L^-1 anastrozole combined with aspirin on MCF-7 was increased compared with that of anastrozole single (P<0.05).The inhibitory rate of 0.5 μmol•L^-1 anastrozole combined with 10.0 mmol•L^-1 aspirin on MCF-7 was increased compared with that of 0.5 μmol•L^-1 anastrozole combined with 2.5 mmol•L^-1 or 5.0 mmol•L^-1 aspirin(P<0.05).The inhibitory rates of 0.5 μmol•L^-1 anastrozole combined with 60 μmol•L^-1 or 120 μmol•L^-1 celecoxib on MCF-7 were increased compared with that of 0.5 μmol•L^-1 anastrozole single and 0.5 μmol•L^-1 anastrozole combined with 30 μmol•L^-1 celecoxib (P<0.05).The inhibitory rate of 0.5 μmol•L^-1 anastrozle combined with 120 μmol•L^-1 celecoxib on MCF-7 was increased compared with that of 0.5 μmol•L^-1 anastrozole combined with 60 μmol•L^-1 celecoxib .The inhibitory rates of 1.0 μmol•L^-1 anastrozole combined with 10.0 mmol•L^-1 aspirin, 60 μmol•L^-1 or 120 μmol•L^-1 celecoxib on MCF-7 were increased compared with that of 1.0 μmol•L^-1 anastrozole single(P<0.05).The inhibitory rate of 1.0 μmol•L^-1 anastrozole combined with 120 μmol•L^-1 celecoxib on MCF-7 was increased compared with that of 1.0 μmol•L^-1 anastrozole combined with 60 μmol•L^-1 celecoxib(P<0.05).Conclusion Aspirin and celecoxib have inhibitory effects on the growth of human breast cancer cell MCF-7 in a dose- and time-dependent manner. There is a synergistic effect on MCF-7 when anastrozole is combined with aspirin or celecoxib .

Objective To study the interspace lateral to internal carotid artery, observe and measure the boundary, area and different content in the interspaces under different visual angles.Methods The anatomy of interspace via pterional approach was studied in 15 adult cadaver brains under microscope. The observation and measurement were performed under different visual angles. The outcomes were analyzed with SPSS10.0 software. Results The difference of the interspaces shape looked like triangle. The important structure had changed under different visual angles. The length of lateral side was about 4-5 mm，lateral side’[KG-*3]s change was not obvious under different visual angles（P>0.05）. But the difference of the length between the interior side and the basal side was obvious under different visual angles（P<0.05）. Conclusion The interspace lateral to internal carotid artery is often used under angle 30° whose operating space is the biggest, and often used to help operate in other interspaces successfully. The PcoA, AchA, the branches of the ICA and oculomotor nerve are very important while operating in the interspace. The PcoA and the extremity of ICA are exposed obviously under angle 30°.

Objective To observe the branches and distribution of the tibial nerve and analyze the compressed position of it. Methods Twenty sides of adult cadavers were anatomized and the tibial nerve branches in ankle, foot, the internal and external plantar tunnel were observed. Correlated data was measured and recorded. Results ① The tendinous part of abductor hallucis muscle that was located under the muscle and consisted the surface of the internal and external plantar tunnel was（8.62±0.79）cm in length ,（3.01±0.30）cm in width,and the thickness of it was about（0.24±0.02）cm. The internal and external nerves were located in the internal and external tunnel.The length of the internal tunnel was about（4.58±0.41）cm，and the diameter was（1.11±0.10）cm. The length of the external tunnel was about（2.58±0.23）cm，the diameter was about（0.96±0.08）cm. ② Internal hell nerve was in an isolated tunnel interior the hell, which was the hell tunnel.The length of the hell tunnel was about（3.03±0.21）cm,the diameter was（1.07±0.09）cm. Conclusion The tibial nerve branches can be compressed at internal, external plantar tunnel or the heel tunnel. When making neurolysis of the tibial nerve for tarsal tunnel syndrome patients, except for the flexor retinaculum, the internal and external plantar tunnel and the hell tunnel should also be decompressed especially for the patients with the symptom of compressed single branch .

Objective To explore the expressions of proliferating cell nuclear antigen(PCNA) and cytochrome C (Cyt C) in gastric carcinoma tissue and discuss their relationship and clinical significances. Methods The expressions of PCNA and Cyt C in 59 gastric carcinoma samples and 10 normal gastric mucosa samples were detected by immunohistochemistry. Results In 10 normal gastric mucosa tissue, the positive rates of PCNA and Cyt C were 20.0% (2/10) and 10.0% (1/9), respectively. In gastric carcinoma, the positive rates of PCNA and Cyt C were 86.4% and 78.0%, respectively. The expression levels of PCNA and Cyt C in gastric carcinoma were significantly higher than those in normal gastric mucosa tissue (P<0.05 or P<0.01). The expressions of PCNA and Cyt C had no relationship with either the age of patient or the size of tumor (P>0.05), but had relationship with histological type (P<0.05). The expression levels of PCNA and Cyt-C in poorly differentiated tissue were higher than those in well differentiated tissue(P<0.05), the expression levels of PCNA and Cyt C in poorly differentiated tissue and moderately differentiated tissue were higher than those in well differentiated tissue (P<0.01). In gastric carcinoma tissue , the expressions of PCNA and Cyt C had no difference (P>0.05). Conclusion The high expression of PCNA and low expression of Cyt C are related to the occurrence and development of gastric carcinoma, and they play an important role with different methods.

Objective To investigate the value of the expressions of estrogen receptor(ER) and progesterone receptor (PR) in the cast-off cells from the fluid of latex ductal lavage in the diagnosis and treatment of breast diseases. Methods A total of 58 female out-patients were examined by fiberoptic ductoscopy (FDS). All patients were divided into 6 groups by the complaints, physical examination, ultrasound and FDS:normal group (10 persons without symptoms and abnormalities in any examination),galactostasis group (n=19), latex fill up group (n=11), intraductal papilloma group (n=2), mammary ductal ectasia group (n=7) and contralateral examination after mastectomy for breast cancer group(n=9). A total of 168 ducts were successfully examined by FDS, and the expressions of ER and PR in the cast-off cells were detected among all of the samples. Results The positive rates of ER and PR expression in the cast-off cells from the fluid of latex ductal lavage in benign breast disease groups were much higher than those in normal group (P<0.05). The positive rates of ER and PR expression in the cast-off cells from the fluid of abnormal ducts detected by FDS were also much higher than those in normal ducts (P<0.05) in the same case of breast disease. The expression rates of ER and PR in the cast-off cells in normal group showed no significant difference as compared with normal ducts detected by FDS in the abhormal breast. Conclusion The local high expressions of ER and PR are associated with the high risk of breast diseases. The detection of ER and PR expressions plays an important role in guiding the treatment and judging the risk factors of breast diseases.

Objective To study the anti-infection effects of VCM-PMMA femoral prosthesis on revision after sheep hip arthroplasty. Methods Right lateral femoral replacement on thirty Inner Mongolia sheep was taken. Gelatin sponge, having methicillin resistant staphylococcus aureus (MRSA), was implanted in the right hip. Three weeks later, the model of infected hip after arthroplasty was established in sheep. VCM-PMMA prosthesis (experimental group) and PMMA prosthesis (control group) were implanted in the right hip after the debridement. The infection control rate, the pathological changes of soft tissue and the prosthesis position were observed in the two groups after the internal of five weeks. Results The rate of infection in the right hip joints was 100% on the third week. After five weeks, the rate of infection control was 93.33% in experimental group; the rate of infection control was 33.33% in control group, the difference was significant between two groups(χ² =12.36, P<0.05). The prosthesis was on the good place by the X-ray. Pathological section was observed under microscope. Neutrophilic granulocytes were hardly found in the experimental group. Neutrophilic granulocytes were much in control group. Conclusion The anti-infection effects of VCM-PMMA prosthesis on revision after sheep hip arthroplasty are very significant.

Objective To explore the correlation between the joint line and the range of knee flexion after condylar knee joint replacement. Methods The range of knee flexion and the change of the joint line in 41 patients(50 knees) with osteoarthritis(OA) were measured and analyzed before and after total knee arthroplasty(TKA). The linear regressive analysis was used to judge the correlation between the two variables. Results The knee joint flexion was linear correlative to the joint line. It was a negative correlation if the joint line was above the anatomical position（r=－0.92,P<0.01）, and it was a positive correlation if the joint line was below the anatomical position(r=0.58，P<0.01). The knee joint could flex to more than 120°averagely if the joint line was elevated no more than 3 mm. If the joint line was elevated 3-6 mm, the joint could still flex to more than 100°.If the joint line was elevated more than 6 mm,the joint could only flex to 90°. The knees can still flex to more than 120°averagely for the patient with lowered joint line even if the lowest one was －6 mm, Conclusion For the primary TKA of OA cases by posterior stabilized implant, the joint line should be placed to the anatomical position as near as possible. The best range of flexion could be accomplished if the joint line is elevated less than 3 mm.