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 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 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 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.

Objective To investigate the effect of adipose derived stem cells (ADSCs) on hepatic stellate cells (HSCs) in vitro and on liver fibrosis in vivo.Methods ADSCs and HSCs were isolated from adipose tissue and liver respectively in SD rats.The coculture system was set up by transwell insert.The 5th passage HSCs were cultured on the 6-well plastic plate,and ADSCs or BRLs seeded on the transwell insert.The proliferation of HSCs was tested by CCK-8 test kit.Smoothmuscle α-actin (α-SMA) expression of HSCs were tested by Western blot.Rat models of liver fibrosis was established.Rats in ADSCs treatment group were infused with ADSCs and those in control group were infused with Buffalo rat liver cells (BRLs).Liver sections were studied by immunocytochemistry.Liver hydroxyproline (Hyp) content,serum laminin (LN)and hyaluronic acid (HA) were tested,the cytokines in the culture medium were assayed.Results HSCs and ADSCs were isolated successfully.After coculture for 72 h,compared with the control group,the proliferation and activation of HSCs was inhibited by ADSCs( absorbance of each group were 2.172 ±0.107,1.424 ± 0.013,1.209 ± 0.117,F =90.605,P ＜ 0.05 ; Gray-scale values of each group were 1.4 ± 0.2,152 ± 14,258 ± 18,F =283.348,P ＜ 0.05 ),ADSCs infusion inhibits liver fibrosis in model rats ( F =77.234,65.164,58.309,all P ＜ 0.05 ).More hepatocyte growth factor(HGF) and less transforming growth factor-β1 (TGF-β1) (F=1.767,P＜0.05)and nerve growth factor (NGF) (F=2.301,P＜0.05) were secreted by ADSCs than by BRLs.Conclusions ADSCs inhibit the proliferation and activation of hepatic stellate cells.Treatment with ADSCs decreases collagen deposition in the liver and inhibits liver fibrosis.

Objective To establish human pancreatic cancer xenografts in nude mice, and to investigate the antitumor efficacy of human endostatin expressed by replication-competent adenovirus AdTPHre-hE in vivo. Methods Pancreatic cancer cells AsPC-1 were injected subcutaneously in BALB/c nude mice to establish the xenografts. Tumor growth was observed and measured after AdTPHre-hE treatment. Expression of endostatin was detected by ELISA assay. The tumors were harvested for pathologic examination and immunohistochemical staining. Results Tumors grew more slowly in the AdTPHre-hE group and their sizes were markedly smaller than those of the Ad-hE group (P＜0.01)and control group(P＜0. 01). Endostatin levels were detected in the sera of nude mice in all treated groups, and endostatin expression in AdTPHre-hE group increased with time. The endostatin level in the AdTPHre-hE treated group was much higher(P＜0. 01)and increased faster than that in the Ad-hE treated group. Immunohistochemical staining for Hexon of adenovirus capsid showed more positive tumor cells in the tumor tissues treated with AdTPHre-hE. Immunohistochemical staining for FⅧ revealed a decreased microvessel density in the tumor tissues treated with AdTPHre-hE. Conclusion The replication-competent adenovirus efficiently expressed high-level endostatin and significantly inhibited tumor growth in vivo.

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 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 investigate the influence of fat-specific protein 27 (Fsp27) gene on the regulation of liver fibrogenesis in vivo.Methods Hepatic stellate cells (HSCs) were isolated from rat liver.Fsp27 gene was detected in primary HSCs and activated HSCs by real-time quantitative PCR (RTqPCR).Lentiviral vector carrying Fsp27 gene was constructed.The model of liver fibrosis was established by infusing carbon tetrachloride (CC14).The rats with liver fibrogenesis were infected by the virus.Liver sections were made to observe the structure and form of liver histocytes.The content of fibrous protein in liver and serum was detected by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay.Resukts HSCs were isolated and cultured successfully.The difference of Fsp27 gene between primary HSCs and activated HSCs was significant(P ＜ 0.01).The model of liver fibrosis was achieved.After infecting the model rats,we found the fibrosis level in treatment group was lower compared with control group.Conclusions Fsp27 treatment can decrease collagen deposition in the liver and inhibit the formation of fibrosis.