[Objective]To study the transfection and expression of green fluorescent protein gene in rat chondrocytes by constructing a combination lentiviral vector containing green fluorescent protein gene.[Method]293T cells were co-transfected with the vector plasmids pLentiLox 3.7,the packaging plasmids pRSV-REV,pMDL g/p RRE and the envelope plasmids VSV-G to produce lentiviral vector particles.Rat chondrocytes were isolated,cultured and infected by the lentiviral vector particles.The expression of reporter gene GFP was observed under fluorescent microscope.[Result]At 96 hours after transfection,a strong expression of GFP could be found in 293T cells.The viral titer was 3.0?103ifu/?l.GFP expression was observed in about 80% of the rat chondrocytes.[Conclusion]A combination lentiviral vector containing green fluorescent protein gene was successfully established and it could infect rat chondrocytes efficiently,which will provide a basis for gene modification of chondrocytes through RNA interference.

Objective To discuss the selection and application of internal fixators in the treatment of aged patients suffering from femoral condyle fracture. Methods 82 aged patients with femoral condyle fracture were treated from February 1995 to June 2002. According to AO/ASIF classifcation,26 cases were classified as type A,17 cases type B, and 39 cases type C. They were treated with internal fixation by 95?angle plate (19 cases), T type plate(5 cases), AO condyle supporting plate (11 cases), dynamic condylar screw (DCS) (23 cases) and Green Seligsen Henry (GSH) (24 cases). Measures of auto ilium transplant or homologous allograft bone transplant (28 cases) were also taken. Results All the cases were followed up for an average of 14.6 months. Merchan grading method was applied to evaluate their knee joint functions. The total excellent and good rate was 90.2%. Conclusions Aged patients with femoral condyle fracture should be treated specially. The fixation should be chosen according to their fracture type so as to ensure fine clinical results. The goal of the operation is to keep the integrity of articular surface of the femoral condyle and to acquire secure internal fixation.

Objective:To investigate the effects of Zinc sulfate on preventing human umbilical veins endothelial cells ( HUVECs) from oxidative stress. Methods:Hydrogen peroxide was used to stimulate HUVECs to build the oxidative stress model in vitro. HUVECs were divided into normal group, H2 O2 group, Zinc sulfate group and Zinc sulfate treated group. Method of nitrate reductase was used to detect the content of nitric oxide ( NO ) and ELISA was used to detect the content of endothelin ( ET ) in supernatant in different groups. The level of HO-1,SOD and CAT of HUVECs were measured by Western blot. Apoptosis of HUVECs was examined by TUNEL as well. Results: Zinc sulfate could enhance the content of NO and decrease the content of ET in the supernatant,which induced by hydrogen peroxide on HUVECs. Zinc sulfate could also increase the level of HO-1, SOD and CAT obviously and decrease the apoptosis cells significantly induced by H2 O2 . Conclusion: Zinc sulfate play an important role in resisting oxidative stress in HUVECs, and maybe prevent HUVECs from oxidative stress damage. Zinc sulfate is expected to be an effective medicine on improving endothelial cells anti-oxidative ability.

AIM:To investigate the effects of Compound Rhizoma Coptidis ( Rhizoma eoptidis,Radix suctellariae,Cortex phellodendri Chinensis,Fructus gardeniae and Radix et Rhizoma glycyrrhizae) on vascular calcification in rats treated with warfarin and vitamin D_3.METHODS: Thirty-two male SD rats were assigned randomly into control group,calcified group,Compound Rhizoma Coptidis prevented group and treated group.The later three groups were treated with warfarin,and subcutaneously injected with vitamin K_1 and vitamin D_3 for one week to induce extensive calcification of the aorta.Compound Rhizoma Coptidis was given before the first warfarin dose in prevented group and the drug was given after the modeling in the treated group.The control group was treated with normal saline.The calcification in the aorta was analyzed and osteoprotegerin (OPG) mRNA and protein were determined using histomorphometry,RT-PCR and immunohistoehemistry after 4 weeks of drug intervention.RESULTS : The results of 32 rats was analyzed compared to the control group,the area of darkly stained regions by Von Kossa staining and the level of calcium content in aorta wall increased significantly[(608.32±42.29) μg/g vs (1 139.47±230.03) μg/g,P ＜0.05].The OPGmRNA and protein decreased in aortic sections.No artery calcification could be detected in Compound Rhizoma Coptidis prevented group and a little artery calcification could be detected in Compound Rhizoma Coptidis treated group.The level of calcium content in aorta wall significantly lower[(854.77±12.99) μg/g,(875.78±27.23 ) μg/g].The expresion of OPGmRNA and the protein significanfly increased in Compound Rhizoma Coptidis prevented and treated groups.CONCLUSION:Compound Rhizo-ma Coptidis may prevent or regress the vegcular calcifiation,that seems dependent on the upregression of aortic OPG levels.

Objective:To study the positive expression rate of M2 subtype of macrophage cell surface molecules and the inflammatory factors of PPS in IL-4-induced M2 macrophage.Methods:The experiment was divided into 5 groups:blank control group, Model group,PPS groups(50 μg/ml,100 μg/ml and 200 μg/ml).The expression of CD206 and CD23 was used as bio-maker to confirm IL-4 induced macrophages by treating RAW264.7 with 20ng/ml of IL-4.IL-4 induced RAW264.7 cells were treated with PPS of 50μg/ml,100μg/ml and 200μg/ml for 24 h.Then the expression of CD206,CD16/32 and CD40 were analyzed by flow cytometry, and the mRNA expression of IL-1β,TNF-α,IL-10 and iNOS were detect by qRT-PCR.Results: After treated with IL-4,the positive rate of CD206 of RAW264.7 were high.After treated with PPS ,the rate of CD16/32 and CD40 in IL-4 induced RAW264.7 cells were high ,the expression of CD206 decreased,and the mRNA level of IL-1βand TNF-αincreased.Conclusion:RAW264.7 cells can be polarlized to M2 subtype macrophage by using 20 ng/ml IL-4.PPS enhances the mRNA of IL-1β,TNF-αand the expression of CD40, CD16/32 in IL-4-induced RAW264.7 cells .These results indicate that PPS can induce the M2 subtype to become M1 macrophages, can improve immune function of macrophages.

Objective To compare laparoscopic hepatectomy to open surgery in recurrent hepatolithiasis.Methods From Jan 2014 to Feb 2016,106 cases of recurrent hepatolithiasis eligible for hepatectomy were prospectively randomized into laparoscopic group (53cases) and open group (53 cases).The perioperative variables,postoperative morbidity and outcomes were analyzed and compared between the 2 groups.Results The statistical results show that the laparoscopic group and open group had similar intraoperative rate of blood loss ＞400 ml,wound infection,abdominal infection,Grade B bile leakage,score of Clavien classification system,stone clearance rate and hospitalization cost (P ＞ 0.05).The laparoscopy group had longer operation time (P ＜ 0.05),and shorter postoperative hospital stay (P ＜0.05).Binary logistic analysis showed that decreased preoperative platelet count,longer operation time,and hepatectomy in difficult places are risk factors for intraoperative blood loss ＞ 400 ml (OR ＞ 1,P ＜0.05);type of past biliary tract operation,positive bile culture during surgery and residual stones are risk factors for grade B bile leakage (OR ＞ 1,P ＜ 0.05).Conclusions Laparoscopic hepatectomy for recurrent hepatolithiasis is safe and feasible.

Objective To compare the cardiac function effect of right ventricular septum(RVS) pacing with that of right ventricular apex(RVA) pacing.Methods One hundred and six patients with indication of dual chamber pacemaker implantation were divide into two groups randomly.In each patient,influence of different pacing site to LVEF and pacing parameter were examined and left ventricular eject fractions were compared.Results All patients' operation were successful,LVEF of RVS group compared with that of RVA showed a significant difference.Conclusion The cardiac function are significantly different between right ventricular septum pacing group and right ventricular apex group.

Background Proliferative vitreoretinopathy (PVR):no blood vessels,fibrous membrane cell proliferation occurs retinal surface.The development of specific mechanisms has not been completely clarified,but the retinal pigment epithelium(RPE) and platelet-derived growth factor(PDGF) for the past few years are the research hotspot.Objective To explore the effects of hypoxia on the production of PDGF-BB and proliferation in cultured human RPE (hRPE) cells.Methods In the experimental group,10,15,20,30,40 μmol/L CoCl2 were used to mimic hypoxia environment of hRPE cells,the control group did not add CoCl2 on cultured hRPE cells.By using reverse transcriptive PCR (RT-PCR) and enzyme linked immunosorbent assay(ELISA),the expression of PDGF-BB mRNA and protein was detected,and detected proliferation of hRPE cells by MTT.The cells were divided into 6 groups,different siRNAs were transfected into PDGF-BB siRNA1 group,PDGF-BB siRNA2 group,PDGF-BB siRNA3 group (because PDGF-BB has three different siRNAs,one of which is a valid siRNA),β-actin siRNA group,unrelated sequence siRNA group,only Lip2000 was plused in the blank control group.After transfection of 4-6 hours,except for blank control group,the other five groups were added to PDGF-BB mRNA and protein and its effect on the proliferation of hRPE cells obviously CoCl2 concentrations (15 μmol/L) simulated hypoxia environment of 24 hours,the detection of PDGF-BB mRNA and protein and hRPE cell proliferation rate.Results Neither PDGF-BB mRNA or protein was found in the blank control group.The production of PDGF-BB mRNA and protein in hRPE cells cultured by different concentrations were different,with significant differences among them (F=43.737,P＜0.01;F=54.612,P＜0.05),and the expressions of PDGF-BB mRNA and protein of 15 μmol/L CoCl2 group were more than other groups,with significant differences between the two groups (all at P＜0.05).MTT showed that PDGF-BB protein and hRPE cell proliferation rate in different concentions of CoCl2 groups were different,with significant differences among them(F=95.379,P＜0.01 ; F =63.375,P＜0.05),the expression of PDGF-BB protein was more and hRPE cell proliferation rate was higher in 15 μmol/L CoCl2group than other groups,with significant differences between the two groups (all at P＜0.05),postive correlation was found between PDGF-BB protein and hRPE cell proliferation rate (r =0.994,P＜0.05).The production of PDGF-BB mRNA and protein in the different siRNA transfected groups were different,with significant differences among them (F =156.330,125.650,both at P＜0.01),and the expressions of PDGF-BB mRNA and protein of PDGF-BB siRNA2 group were more than other groups,with significant differences between them(all at P＜0.05).MTT showed that PDGF-BB protein and hRPE cell proliferation rate in different siRNA transfectedgroups were different,with significant differences among them (F =73.131,98.564,both at P＜ 0.01).The expression of PDGF-BB protein was less and hRPE cell proliferation rate was lower in PDGF-BB siRNA2 group than other groups,with significant differences between them (all at P ＜ 0.05),postive correlation was found between PDGF-BB protein and hRPE cell proliferation rate (r =0.996,P＜0.05).Conclusions Proper hypoxia can induce the expression of PDGF-BB,PDGF-BB expression can significantly promote the proliferation of hRPE cells.In the transfection targeting PDGF-BB siRNA,PDGF-BB expression is suppressed,can effectively reduce the value of hRPE cell hyperplasia.

BACKGROUND: Intra-articular injection of hormone has been applied in the treatment of arthritis because it can alleviate arthralgia rapidly, which is accompanied commonly by progressive cartilage impairments. It is not clear if supplement of growth factor like insulin effect can play a protective role in articular chondrocytes.OBJECTIVE: To observe the effects of insulin or hydrocortisone alone and the combination on the proliferation of chondrocytes.DESIGN: Grouping comparative study, the effect of one medicine was analyzed by using one-factor analysis of variance, while the combined effect was analyzed with multi-factor analysis of variance.SETTING: Guangdong Institute of Trauma Sugery.MATERIALS: Articular cartilage from the knees of New Zealand white rabbits of 4 - 6 weeks old.METHODS: This study was carried out at Guangzhou Traumatic Research Institute from Feberary 2000 to May 2001. Chondrocytes were isolated from the knee joints of New Zealand white rabbits, digested with hyaluronidase,pancreatin and type Ⅱ collagenase and exposed to insulin, hydrocortisone or the combination of insulin and hydrocortisone of different dosage. They were divided into four groups:Control group ( without adding insulin and hydrocortisone), insulin group (0. 035,0. 35,3.5,35 mg/L subgroups), hydrocortisone group(1,5,10,50,100 mg/L subgroups) and insulin(0. 35 mg/L) combined with hydrocortisone(50 mg/L) group. Their influence on chondrocytes proliferation was observed with methyl thiazolyl tetrazolium(MTT) method.sulin.at the concentration of 0. 035 mg/L( P ＜ 0.01 ), reaching the maximum at could inhibit the proliferation of chondrocytes ( P ＜ 0.05 ), which became significant with increasing concentration and no viable chondrocytes could be exposed to 0 . 35 mg/L insulin combined with 50 mg/L hydrocortisone, the promoting effect of insulin was inhibited due to negative cooperation.CONCLUSION: Insulin at low concentration could enhance the proliferation of chondrocytes, but hydrocortisone displayed inhibiting effect on the growth of chondrocytes. The function of insulin was antagonized when combined with hydrocortisone.