Objective To construct the inducible vector carrying green fluorescence protein(GFP). Methods We constructed an inducible vector pMD-GFP, including GFP cDNA, which allowed controlled expression of protein upon addition of 100 ?mol/L Zinc as an external inducer. The whole length of GFP cDNA were transfected into human hepatocellular cancer cells HCC-9204 by the method of lipofectin transfection. The expression of GFP was observed under fluorescence microscopy. Results The inducible vector carrying green fluorescence protein was successfully constructed. The observation under fluorescence microscopy showed that green fluorescence was spread in entire HCC-9204 cells transfected with GFP gene. Conclusion This new kind of the inducible vector could serve as a new tool and method for observing the growth and metastasis of neoplasm.

Purpose: To explore the autocrinism of vascular endothelial growth factor ( VEGF) in human hepatocellular carcinoma cell and its significance. Methods: The expression of VEGF receptors was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) in human hepatocellular carcinoma cell lines SMMC7721, HHCC and HepG2 in vitro. The expression of VEGF in human hepatocellular carcinoma cells was inhibited by synthetic antisense oligodeoxynucleotide (ODN). Phenotypic alterations in human hepatocellular carcinoma cells by antisense reduction of VEGF expression were observed. And the alterations for expression of VEGF receptors in human hepatocellular carcinoma cell membranes were analyzed by flow cytometry. Results: Flt-1 was expressed faintly in SMMC-7721 cell whereas KDR was expressed in HHCC cell and HepG2 cell in vitro. Compared to control, antisense ODN against VEGF inhibited HHCC cell proliferation in vitro in a dose-dependent manner and had no effects on SMMC-7721 cell and L929 cell. When the concentration of antisense ODN was 2.5 ?mol/L,5 ?mol/L and 10 ?mol/L, the proliferation of HHCC cell was inhibited by 26%, 41 % , and 50% respectively. Compared with control, flow cytometry analysis showed a decrease in cell number in S phase and a pre-apoptosis peak in HHCC cell treated by antisense ODN. There was no alteration of cell cycle in SMMC-7721 cell. The positive rate of KDR expression in HHCC cell membrane was 19. 2%, 29. 8% and 31. 2% in the antisense ODN, sense ODN and control groups, respectively. The expression of KDR in HHCC cell membrane was markedly inhibited by antisense ODN against VEGF compared with the control group, as measured by flow cytometry. There was no alteration for expression of Flt-1 in SMMC-7721 cell membrane. Conclusions: VEGF produced by human hepatocellular carcinoma cells acts directly upon human hepatocellular carcinoma in an autocrinal manner and may promote the proliferation and differentiation of human hepatocellular carcinoma cells via the activation of the KDR-dependent signaling pathway.

Objective To study the protective effects of ulinastatin and tumor necrosis factor-?(TNF-?) antibody on ischemia and reperfusion injury of liver in rats. Methods One hundred and twenty male SD rats were randomly divided into four groups: the normal control group, ischemia and reperfusion group, TNF-? antibody group and ulinastatin plus TNF-? antibody group. And the animals were killed after 60 minutes ischemia of liver followed by reperfusion for 1,3,6 and 12 hours. Serum alanine aminotransferase(ALT) and malondialdehyde(MDA) were detected, and liver histopathologic lesions were observed. Results After ischemia and reperfusion, the serum level of ALT and MDA remarkedly increased, and the hepatic congestion was prominent. Treatment of ulinastatin and TNF-? antibody could decrease the serum level of ALT and MDA significantly, and relieve hepatic congestion. Conclusions Ulinastatin and TNF-? antibody can suppress the inflammatory reaction induced by hepatic ischemia and reperfusion, and has protective effects on rat hepatic ischemia and reperfusion injury.

Objective[WT5”BZ] To evaluate the method and timing of operation and long term effect of congenital choledochal cysts(CCC) in adults.[WT5”HZ] Methods[WT5”BZ] The mode and timing of operation, effective rate, reoperation rate and incidence of carcinoma after operation for 70 operated patients with CCC in adults during the period from Jan, 1980 to June 1999 were analyzed retrospectively. [WT5”HZ]Results[WT5”BZ] The reoperation rate of external drainage was 86%(6/7); The effective rate of internal drainage was significantly lower than that of resection of the cyst(3/10 vs 45/49,? 2=20 94, P

ObjectiveTo investigate the feasibility of auxiliary orthotopic partial liver transplantation (APOLT) from a living donor for the treatment of Willson′s disease.MethodsThe patient was a 20-year-old girl with Willson′s disease, whose blood type was O. The donor was a man aged 21 and his blood type was A. The left lateral lobe (260?g) of patient′s liver was removed, the left lateral lobe (295?g) of donor′s liver was grafted to the patient in situ. Blood plasma exchange was carried out to the recipient before transplantation. FK506 adrenocortical hormones and cytoxan was administered after operation. ResultsThe recipient had a onset of hepatic artery thrombosis 15 days after operation, and ensuing intraabdominal hemorrhage caused by thrombolytic agent was successfully managed by laparotomy. Other postoperative complications such as hydroperitonia, pulmonary atelectasis and bile fistula were cured. Now the patient has survived 15 months with a normal copper-protein level, a mitigated extrapyramidal symptom and the grafted liver grows larger.ConclusionAPOLT is a feasible remedy for late-staged Willson′s disease.

Objective To explore the clinical manifestations, pathogenic mechanisms and treatment of Mirizzi syndrome.Method 65 patients with Mirizzi syndrome were analysed retrospectively.Results According to Csendes typing, patients with Mirizzi syndrome included type Ⅰ in 18 patients(27 6%), type Ⅱ in 25 patients (38 4%), type Ⅲ in 13 patients(20 0%), type Ⅳin 9 patients(13 8%), all patients were healed by corresponding operation and perioperative management.Conclusions Good outcome of Mirizzi syndrome patients can be achieved by accurate diagnosis,exact operation and active perioperative management.

ObjectiveTo investigate the effect of p27 KIP1 transfection on cell cycle and apoptosis of hepatocellular carcinoma cells(HCC). MethodsWe used an inducible expression system pMD neo, which allowed controlled expression of protein upon addition of zinc as an external inducer. p27 KIP1 cDNA was transfected into human HCC 9204 cell line. Expression of p27 KIP1 was analyzed and cell growth was observed. ResultsExpression of p27 KIP1 in protein and mRNA increased significantly in HCC 9204 cell line transfected with p27 KIP1 . The cell growth reduced by 35%, p27 KIP1 over expression caused cell growth arrest at G 1 by 35% ( P =0 000). Apoptotic cell index significantly increased ( P =0 000).Conclusionp27 KIP1 may cause cell cycle arrest in G 1 phase and subsequently lead to apoptosis.

To elucidate the effect of p27 KIP1 on cell cycle and proliferation of gastric carcinoma cells, we transfected the full e length cDNA of p27 KIP1 into human SGC7910 gastric cancer cells by the method of lipofectin transfection. Expression of p27 KIP1 at protein or mRNA level was analyzed by Western blotting, and RNA dot blotting, respectively. Effect of p27 KIP1 on cell growth was observed by trpan blue exclusion assay and anchorage independent growth in soft agar. Flow cytometry was applied to assess the effect of p27 KIP1 on cell cycle. The results showed that the expression of p27 KIP1 at protein or mRNA level increased evidently in SGC7901 cells transfected with p27 KIP1 . The cell growth was reduced by 42% 48h post induction with zinc as determined by cell viability assay. The rate of anchorage independent growth in soft agar decreased significantly. p27 KIP1 over expression caused cell arrest at G 1 by 36%(from 33 68% to 69 29%, P

Objective　To investigate whether or not the antisense oligodeoxynucleotide (ODN) of vascular endothelial growth factor (VEGF) can supresses the protein expression of VEGF.Methods　SMMC 7721 is a cell line that expresses VEGF.Therefore the medium conditioned by SMMC 7721 stimulates proliferation of bovine aortic endothelial cell (BAEC).Synthesized antisense ODN complementary to one region of VEGF mRNA was added into the medium of SMMC 7721.The expression of VEGF and the stimulation effect of SMMC 7721 was then determined.Result　It was found that by means of inhibition of VEGF expression,the stimulation effect of SMMC 7721 was inhibited by the VEGF antisense ODN in a dose-dependent manner.At a dosage of 1,2.5,5,10μmol/L,the inhibition rate was 63.2%,82.4%,210% and 287.5%,respectively.Conclusion　It is promising that VEGF antisense ODN works as a new gene therapeutic modaliey for antiangiogenesis of HCC.

Objective To study the role of down regulation of bcl 2 expression of human cholangiocarcinoma cells with bcl 2 mRNA cleaving ribozyme (RZ). Methods Synthetic gene of bcl 2 mRNA cleaving RZ was recombined with the expression vector of eukaryotic cells. The recombined vector was used to transfect human cholangiocarcinoma cells. The expression of bcl 2 in human cholangiocarcinoma cells was observed by immunocytochemistry and flow cytometry. Results The expression of bcl 2 in human cholangiocarcinoma cells transfected with RZ gene was lower than that of control group. Spontaneous apoptosis was observed in a few of the cells. Conclusion bcl 2 mRNA cleaving RZ can down regulate the expression of bcl 2 of cholangiocarcinoma cells notably and can also induce spontaneous apoptosis.