Objective To investigate the effect of liver function with portal vein occlusion (PVO) in various phases and the following restoration portal vein flow on intestinal mucosal cells. Methods Twenty-four healthy adults white Japanese rabbits were randomized into 1 control group and 2 experimental groups (according to portal vein clamping for 30 min and 45 min). Each experimental group's blood samples were collected from caval vein 1 h before operation, by the end of portal vein oc-cluded, 30 min, 60 rain after relief of portal vein blocking, then with restoration of portal vein flow for 2 h and rabbit guts were continuously cut to sections for HE, TUNEL staining and Bcl-2, Bax protein immunohistochemical staining to observe the injury of intestinal mucosa cell apoptosis and the relation-ship between the expressions of Bcl-2 and Bax. The levels of blood glutamate-pyruvate transaminase (ALT), glutamic oxalacetic transaminase (AST) were measured and compared. Results The levels of ALT, AST in the control group did not significantly change. Compared with control group, group B did not change significantly with PVO 30 rnin in liver enzyme and they were significantly increased after portal vein occlusion relief. The levels of ALT and AST were increased obviously at 45 min with PVO, then raised again. Down-regulation of Bcl-2 expression, up-regulation of Bax expression and the increased index of apoptotic cell were found in each experimental group. Conclusion It may be more safe with PVO for 30 min. But the following restoration portal vein flow will bring about ischemia-reperfusion injury that is mainly apoptosis in the small intestine. The index of apoptosis will be raised with time prolongation of PVO.

Objective To investigate the effect of hepatic stellate cells (HSC) on proliferation and invasion of hepatocellular carcinoma cells and the possible mechanism involved.Methods The HSC was isolated by optiprep method.Methyl thiazolyl tetrazolium(MTT) assay was used to detect the proliferation of hepatocellular carcinoma cells.The effect of invasion was measured with transwell assay.Matrix metalloproteinase-2 (MMP-2) and nuclear factor-κB (NF-κB) were detected by Western blotting.Results HSC was isolated and cultured successfully.HSC promoted the proliferation and invasion of hepatocellular carcinoma cells (proliferation:0.571 ±0.024 vs 0.803 ±0.048,1.271 ±0.044,1.973 ±0.036; invasion:25.2 ± 1.9 vs 35.8 ±3.3,44.4 ±2.7,53.9 ±3.6) (P ＜0.05).MMP-2 (1.32 ±0.22 vs 2.46 ±0.39) and NF-κB(0.85 ±0.09 vs 1.44 ±0.21) were increased obviously in hepatocellular carcinoma cells stimulated by HSC.Conclusions HSC can promote the proliferation and invasion of hepatocellular carcinoma cells.The mechanism might be related to up-regulation of the expressions of MMP-2 and NF-κB.

Objective To propose a rational clinical classification of gallstone pancreatitis for better guide and select clinical treatment scheme. Methods On the basis of severity of pancreatitis and the presence or absence of biliary obstruction, 273 cases of gallstone pancreatitis were classified into four types: non obstructive mild type (type Ⅰ) , obstructive mild type (type Ⅱ) , obstructive severe type (type Ⅲ) , non obstructive severe type (type Ⅳ). Moreover, according to the presence or absence of common bile duct stone, every type was further classified into two subtypes: subtype a and subtype b. Then, the results of clinical classification, treatment methods and prognosis were analyzed. Results Ⅰa subtype: 34 cases, Ⅰ b subtype: 112 cases; Ⅱ a subtype; 59 cases, Ⅲ subtype; 11 cases; Ⅲa subtype; 6 cases, Ⅲ b subtype: 4 cases; Ⅳa subtype: 3 cases, Ⅳb subtype: 44 cases. The overall mortality was 3.3% (9/273) , the mortality in Ⅰ type, Ⅱ type, Ⅲ type or Ⅳ type was 0, 0, 10% (1/10), 17.0% (8/47), respectively. The difference was statistically significant (P<0.05). The mortality of Ⅳ type in early operation group, traditional non-operative group, and regional intra-arterial infusion group was 30. 8% (4/13) , 25% (3/12) , 4. 5% (1/22) , respec tively. The mortality of regional intra-arterial infusion group was significantly lower than those in other two groups (P<0.05). Conclusions This 4 types and 2 subtypes classification method of gallstone pancreatitis was rational. The treatment efficacy may be improved according to the clinical classification. However, attention shall be paid to the transformation of these clinical types.

Objective To evaluate a new method for the isolation of rat pancreatic stellate cells (PSCs) and to investigate the influence of adipose derived stem cells (ADSCs) on PSCs in vitro.Methods Normal rat PSCs was isolated by collagenase and Optiprep density gradient centrifugation.The coculture system of ADSCs and PSCs was set up by transwell insert.The proliferation of PSCs was tested by CCK-8 test kit.Smoothmuscle α-actin (α-SMA) expression of PSCs were tested by Western blot.The apoptosis of HSCs was tested by flow cytometer.The cytokines in the culture solution were tested by ELISA kit.Results The quantity of PSCs was above 5 × 106 cells per rat.The purity and the viability of the cells were about 90-97 percent.After coculture for 72 h,the proliferation and activation of PSCs was inhibited by ADSCs (F =223.27,P ＜ 0.05 ; F =52.97,P ＜ 0.05) and the apoptosis of PSCs was promoted by ADSCs (F =43.62,P ＜ 0.05).more NGF and less TGF-β1 was secreted by ADSCs than by PSCs (NGF:14.68 ± 0.94 vs.8.31 ±0.86,t =4.67,P ＜0.05;TGF-β1:10.65 ±0.46 vs.70.47 ±0.99,t =21.72,P＜0.01).Conclusions ADSCs inhibit the proliferation and activation of PSCs by ADSCs secreting cytokines.

Objective To compare the difference between bone marrow stomal cell (BMSC) and adipose-derived stem cell (ADSC) of liver fibrosis in rats.Methods BMSC and ADSC of Sprague-Dawley (SD) rats were isolated and purified.The stem cell markers were detected with flow cytometry.The coculture system was set up with 0.4 μm Transwell insert semipermeable membrane.ADSC or BMSC were co-cultured with hepatic stellate cells (HSC).Normal hepatocyte cell line of rat (BRL) was co-cultured with HSC as negative control group and HSC cultured alone was blank control group.After cultured for 72 hours,the proliferation of HSC was determined by cell counting kit-8 (CCK-8) method.The expression of α-smooth muscle actin (α-SMA) of HSC was detected by Western blotting.The apoptosis of HSC was examined by flow cytometry.After BMSC,ADSC and BRL cultured alone for 72 hours,expression level vascular endothelial growth factor (VEGF),interleukin-10 (IL-10),nerve growth factor (NGF) and transforming growth factor-β1 (TGF-β1) in the culture medium were detected by enzymelinked immunosorbent assay (ELISA) method.The rats model of liver fibrosis were established.The rats were divided into BMSC treatment group,ADSC treatment group,BRL group and culture medium group,six rats in each group,which were injected with 1.5 mL BMSC,ADSC and BRL cells suspension (5 × 106) through portal vein,respectively,and same volume of culture medium was injected to the rate of culture medium group,once every two weeks for four weeks.The pathological changes of liver tissue sections were observed and liver fibrosis markers were tested.T test was performed for comparison between two samples and analysis of variance was used for comparison among multiple groups.Results BMSC and ADSC were successfully isolated and cultured.The phenotype of BMSC and ADSC was similar.Compared with blank control group and negative control group,both ADSC and BMSC could inhibit the proliferation of HSC and promote apoptosis (proliferation,2.43±0.27,2.39±0.33,1.92±0.38 and 2.18±0.31,FBMSC =25.61,FADSC =38.63,both P＜0.05 ;apoptosis rate,(5.59 ± 0.40)％,(6.82±0.57)％,(8.31± 1.03) ％ and (9.36 ± 0.54) ％,FBMSC =73.69,FADSC =97.41,both P＜ 0.05).The effects of ADSC were more significant than those of BMSC (t=5.76 and 5.18,both P＜0.05).There was difference in the cytokine levels secreted by ADSC and BMSC (NGF,(7.46 ± 0.54) pg/mL vs (3.95 ± 0.71) pg/mL,t =10.92,P＜0.05; TGF-β1,(8.79 ±0.93) pg/mL vs (6.36±0.85) pg/mL,t=7.58,P＜0.05).The cell transplantation experiment indicated that both BMSC and ADSC had significant inhibitory effect on liver fibrosis.The activity index of inflammation and degree of fibrosis in BMSC treatment group and ADSCs treatment group were 9.87±2.07,4.17 ± 0.94 and 10.13 ± 1.81,3.98 ± 0.82,which were significantly lower than those in blank control group (13.78±2.53 and 5.09±1.15)and negative control group (13.34± 1.89 and 4.95± 1.22,FBMSC=51.26 and 32.29,P＜0.05; FADSC =46.73 and 40.94,P＜0.05).The level of hyaluronic acid ((191.5±33.2) μg/L and (178.8±28.2) μg/L),type Ⅲ collagen ((19.9±5.1) μg/L and (21.7± 3.3) μg/L) and hydroxyproline ((312.6±38.8) μg/g and (325.8±28.2) μg/g) of BMSC treatment group and ADSC treatment group were significantly lower than those of negative control group and blank control group (hyaluronic acid,(282.3 ± 18.7) μg/L and (287.5 ± 26.7) μg/L),F =73.51 ; type Ⅲ collagen,(35.3± 3.3) μg/L and (32.5±4.3) μg/L,F=76.19; hydroxyproline,(458.4 ± 38.1) μg/g and (473.9 ± 63.7) μg/g,F=60.37,all P＜0.05).However,there was no difference between BMSC treatment group and ADSC treatment (all P＜0.05).Conclusions ADSC and BMSC had similar stem cell characteristics.There was difference in inhibiting the activation of HSC between ADSC and BMSC.But there was no significant difference in inhibiting liver fibrosis of rats in vivo.

Objective To develop a double-regulated replicative adenovirus carrying the Human endostatin gene(hEndo). Methods The plasmid pTPHre-hEndo was constructed by gene engineering technique, carrying human endostatin gene, in which El A gene and E1B gene were driven by human telomerase reverse transcriptase (hTERT) promoter and hypoxia response element (HRE) promoter,respectively. The pTPHre-hEndo was co-transfected with pBHGE3 in 293 cells to generate recombinant adenovirus AdTPHre-hEndo. Virus titer was measured by the TCID50 method. Virus replication assay was performed to evaluate the selective replication ability of AdTPHre-hEndo. The transgene expression of endostatin was detected by ELISA assay. Results A novel gene-viral therapeutic system AdTPHre-hEndo was constructed by gene engineering technique and its titer was 3. 25 X 1010 pfu/ml.Proliferative test revealed that AdTPHre-hEndo could proliferate selectively in telomerase positive tumors. Furthermore, in comparison with non-replicative adenovirus Ad-hEndo, the transgene expression of endostatin mediated with AdTPHre-hEndo was significantly increased (P < 0. 01).Conclusion The novel gene-viral therapeutic system AdTPHre-hEndo has the capacity to replicate in pancreatic cancer cells and expresses the endostatin efficiently, and may provide a new strategy for pancreatic cancer gene therapy.

Objecltive To intensify the coordination and nursing in the operation room in order to prevent surgical site infection after gastrointestinal operation. Methods 337 medical history of patients received gastrointestinal operation and third rate healing from 1999 to 2006 were collected. A series of intensified measures were applied to surgical site infection from 2003 gradually, including invocation of new surgical handwashing method, modified skin disinfection manner, adoption of degreasing with ethanol first before disinfection with iodophor, placement of incision protector and clean bag for incision protection after entering abdomen, changing to use new gastrointestinal anastomofic thimerosal,standardization of operation order and clean manage-ment in operation room. The incidence rate of surgical site infection after gastrointestinal operation of patients from 1999 to 2002 and from 2003 to 2006 underwent χ2 test. Results The incidence rate of surgical site in-fection after gastrointestinal operation greatly decreased after adoption of intensified nursing intervention, Signifi-cant difference existed in rate of patients with third rate healing between the year 1999 to 2002 and 2003 to 2006. Conclusions Modified nursing intervention for surgical incision after gastrointestinal operation can de-crease incision infection rate evidently.

Objective To investigate the effects of protein active composition of Scorpio on apoptosis of L1210 tumor cells for the purpose of establishing the quality evaluation method of biological effect of Scorpio. Methods L1210 cells were examined by trypan blue exclusion. The proliferation of cells was determined by improved MTT assay. Flow cytometry was performed to analyze cell apoptosis with propidium iodide (PI). Results When the concentration of protein active composition of Scorpio exceeded or equaled 37 mg/ml, the coefficient correlation of the growth inhibiting curve of L1210 cells was 0.9357, and IC50 was 175 mg/ml. The excellence time was 0 to 48 hours. When the concentration of protein active composition of Scorpio exceeded or equaled 9.25 mg/ml, the apoptosis ratio of L1210 cells was raised significantly.Conclusion The protein active composition of Scorpio might promote the apoptosis and restrain the proliferation of L1210 cells. The value of anti-tumor biological effect of the protein active composition of Scorpio was 9.25 ～ 175 mg/ml. This value may be one of the indexes for quality evaluation of biological effect of Scorpio.

Objective To investigate the effect of a dual regulation of replicating adenovirus vector carrying human endostatin gene (AdTPHre-hEndo) on pancreatic cancer. Methods Human endostatin (hEndo) gene was cloned into the genome of replicating adenovirus specific for the tumor cells by virus recombination technology. The virus titer was 3.25 × 1010pfu/ml. A Balb/c nude mouse model carring sw1990 cells pancreatic cancer was established, the expression of human endostain and inhibition of tumor cells in vivo were detected. Results We successfully constructed AdTPHre-hEndo. The inhibition on pancreatic cancer cell line SW-1990 of AdTPHre-hEndo is better than Ad-hEndo (P ＜0. 01 ), and ONYX-015 ( P ＜ 0. 05 ). The endostatin expression of AdTPHre-hEndo group was significantly higher than Ad-hEndo group and the control group (P ＜ 0. 01 ). The intratumoral MVD also decreased significantly in the treated tumors(6. 8 ±2. 5 vs. 16. 0 ±4. 6、47. 2 ± 10. 0, P ＜0. 01 ). Conclusions The recombination adenovirus can express biologically active hEndo effectively, which results in inhibiting the growth of micro-blood vessels and proliferation slowly.

Objective To construct the adenovirus vector containing mice CD40- IgG2aFc-IRS-GFP fusion gene and detect the expression of the fusion protein in transfeete 293cells.Methods The fragments of CD40, IgG2aFc were obtained and inserted into plasmid PDC316-IgG2aFc-GFP.After being verified by sequencing, Ad5-PDC316-CD40-IgG2aFc-GFP was contransfored with the bckbone vector into 293 cells.The virus titer was detected after replicating and purifing and the expression of the fusion protein was analyzed.Results A virus and plasmid were constructed successfully.The vitro infection showed that the virus can infect cells of which the fusion protein was confirmed by fluo-rescence.The expression of the fusion protein increased with the increased time and virus concentra-tion.The protein expression stopped increasing after the virus concentration came to a certain level.Conclusion CD40, IgG2aFc fragments are correctly ligated with plasmid PDC316-IgG2aFc-GFP, and the fusion protein can be expressed in 293cells.This might lay a foundation for further studies of the expresstion of virus, immune tolerance and mechanism of liver transplantation model in rats.