【Abstract】ObjectiveTo construct a recombinant eukaryotic expression vector for human endostatin in order to study the inhibitory effect of liposome-mediated endostatin gene on the growth of human liver carcinoma in nude mice. MethodsHuman endostatin cDNA including IL-2 secreting peptide was cloned into eukaryotic expression plasmid pcDNA3.0 to construct recombinant plasmid pCD-sEndo. pCD-sEndo plasmid was transferred into hepatocarcinoma cell line SMMC-7721 mediated by liposome Dosper, the expression and secretion of endostatin gene was detected by RTPCR and Western blot analysis. The suspension of SMMC-7721 cells was injected subcutaneously at the back of 32 nude mice to establish the model of human liver carcinoma. The mice were divided into 4 groups randomly, and injected with Dosper+pCD-sEndo, Dosper+pcDNA3.0, Dosper and physiological brine separately. Tumor volume was measured by stages. The mice were executed after the drug had been given for 1 week, then the microvessel density (MVD) of the tumor tissue was detected with immunohistochemical method and apoptotic index of tumor cells was measured by TUNEL-stain. ResultsThe eukaryotic expression vector pCD-sEndo was successfully constructed and was confirmed by enzyme digestion and sequence analysis. Expression of endostatin gene was detected in transfected SMMS-7721 cells by RTPCR in vitro, and endostatin protein was also detected in the supernatant of transfected SMMS-7721 cells by Western blot. In vivo study, the growth of human liver carcinoma was inhibited in the group injected with endostatin gene: the average volume of tumor in this group was significantly smaller than that in other groups （P＜0.05）; the average MVD in this group was 6.2±2.5, significantly less than that in the group injected with physiological brine (32.8±6.4), Dosper （27.8±6.4）, or Dosper+pcDNA3.0 (25.5±5.5), P＜0.05. The average apoptotic index of tumor cells in treatment group, brine group, Dosper group and Desper+pcDNA3.0 group was 24.5±7.3, 7.6±2.5, 9.5±3.0 and 11.2±3.6 respectively, it was evidently higher in the treatment group than in the latter three groups. ConclusionHuman endostatin mediated by cation liposome could decrease the microvessel number of implanted human hepatocarcinoma in nude mice. It could also accelerate apoptosis of tumor cells and inhibit growth of tumor.
Objective To explore the indications for liver transplantation among patients with hepatolithiasis. Methods Data from 1431 consecutive patients with hepatolithiasis who underwent surgical treatment from January 2000 to December 2006 were retrospectively collected for analysis. Surgical procedures included T-tube insertion combined with intraoperative cholangioscopic removal of intrahepatic stones, hepatectomy, cholangiojejunostomy, and liver transplantation. Results Nine hundred and sixty-one patients who had a stone located in the left or right intrahepatic duct underwent hepatectomy or T-tube insertion combined with intraoperative cholangioscopic removal of intrahepatic stones. The rate of residual stones was 7.5% (72/961). Four hundred and seventy patients who had a stone located in the bilateral intrahepatic ducts underwent surgical procedures other than liver transplantation; the rate of residual stones was 21.7% (102/470). Only 15 patients with hepatolithiasis underwent liver transplantation; they all survived. According to the degree of biliary cirrhosis, recipients were divided into 2 groups: a group with biliary decompensated cirrhosis (n=7), or group with biliary compensated cirrhosis or noncirrhosis group (n=8). There were significant differences in operative times, transfusion volumes and blood losses between 2 groups (P＜0.05). In the first group, 6 of 7 patients experienced surgical complications, and in the second, 8 recipients recovered smoothly with no complications. Health status, disability and psychological wellness of all recipients (n=15) were significantly improved in 1 year after transplantation as compared with pretransplantation (P＜0.05). Conclusion Liver transplantation is a possible method to address hepatolithiasis and secondary decompensated biliary cirrhosis or difficult to remove, diffusely distributed intrahepatic duct stones unavailable by hepatectomy, cholangiojejunostomy, and choledochoscopy.
Objective Ｔo summarize the advancement of ABO-incompatible liver transplantation. Methods Relevant literatures about ABO-incompatible liver transplantation, which were published recently domestic and abroad were reviewed and analyzed. Results Owing to various treatments recent years, outcomes of ABO-incompatible liver transplantation have been improved dramatically. Conclusion With effective immnosuppressive protocols and effective perioperative management, ABO-incompatible liver transplantation is feasible.
To investigate the role of platelet-activating factor (PAF), neutrophils in ischemia-reperfusion-induced liver injury and their possible mechanism, PAF and the degree of neutrophil infiltration in liver tissue and the preventive effects of PAF antagonist kadsurenone were evaluated in this study by means of a partial liver ischemia model, in which it was induced by clamping only left and median lobes of the liver without causing intestinal congestion. The present study was undertaken to find out the mechanism of liver ischemia-reperfusion injury and preventive effect of kadsurenone. The results indicate that in early stage of reperfusion liver injury possibly caused by the generation of free radicals, declined of autioxidant defence and increased Ca２＋ influx, and in the later stage of reperfusion injury was mainly mediated by accumulation of PAF in the liver, which elicits the release of polymorphonuclear leukocytes induced toxical free radical, endothelial damage, microcirculatory collapse. The authors conclude that the effectiveness of antagonist kadsurenone in protecting against ischemiareperfusioninduced liver injury is due not only to their action in preventing the direct effects of PAF, but also to their ability to inhibit both PAF priming and PAF dependent feedback processes, thus preventing escalation of auto generated inflammatory damage.
Objective To investigate the mRNA expressions of liver X receptor α (LXRα), farnesoid X receptor (FXR), steroid xenobiotic receptor (SXR) and liver receptor homolog 1 (LRH-1) gene in patients with cholesterol gallstone (CGS) disease in order to elucidate the biomolecular pathogenesis of gallstone formation. Methods Twenty-seven patients with CGS (CGS group) and 10 controls without gallstones (control group) were included in this study. Serum lipid composition (total cholesterol, triglyceride, high density lipoprotein cholesterol, apoprotein B, apoprotein A1), gallstone cholesterol concentration and biliary composition (cholesterol, bile salts, lecithin) were assayed. Biliary total lipid and cholesterol saturation index (CSI) were calculated. mRNA expressions of LRH-1, FXR, SXR and LXRα gene were determined by real-time polymorphism chain reaction. Results Serum high density lipoprotein cholesterol concentration was lower in CGS group than that in control group 〔(0.93±0.05) mmol/L vs (1.33±0.09) mmol/L, P＜0.001〕 and serum apoprotein A1 was also lower in CGS group than that in control group 〔(1.19±0.05) g/L vs (1.36±0.06) g/L, P＜0.05〕. There were no differences in serum total cholesterol, triglyceride and apoprotein B between two groups (Pgt;0.05). CSI was higher in CGS group than that in control group (1.17±0.02 vs 0.79±0.10), P＜0.001. Biliary cholesterol was also higher in CGS group than that in control group 〔(7.96±0.39) mol% vs (5.26±0.89) mol%, P＜0.01〕, while biliary total lipid was lower in CGS group than that in control group 〔(104.72±10.51) g/L vs (154.24±14.20) g/L, P＜0.05〕. There were no differences in bile salts and lecithin between two groups (Pgt;0.05). Expression of LRH-1 gene was higher in CGS group than that in control group (14.18±1.80 vs 7.22±2.22), the difference was statistically significant (P＜0.05). There were no differences in mRNA expressions of LXRα, FXR and SXR gene between two groups (Pgt;0.05). Conclusion CGS disease may be related to increased expression of LRH-1 gene.
ObjectiveTo study the development of methods assessing donor liver viability in liver transplantation.MethodsThe literature in the recent years on the methods of assessing donor liver viability was reviewed.ResultsFrom donor liver morphology to function,there have being developed many methods which assess donor liver viability,including:①donor liver appearance; ②intraoperative biopsies; ③donor liver microcirculation; ④portal pressure; ⑤enzymes levels in liver; ⑥lidocainemetabolizing activity; ⑦energy metabolism of donor liver; ⑧fat content in donor liver.ConclusionThere are many methods to assess the viability of donor liver. Each has its supericrity and defect respectively. Intraoperative biopsies, 31Pmagnetic resonance spectroscopy and portal pressure have more importance in clinical application.
Objective To observe the changes of blood biochemistry during orthotopic liver transplantation in pigs. Methods Fourteen healthy pigs were anesthetized and intubated, the right carotic artery was cannulated for blood pressure monitoring and blood biochemical sampling. The right internal jugular vein was cannulated for central venous pressure monitoring. No exogenous glucose was administrated during operation, the donor liver was perfused with Collin’s solution. During the anhepatic stage, the veno-venous bypass was established, blood was pumped from the portal and femoral veins using an extracorporeal circuit, and returned to the axillary vein. The arterial blood was sampled for monitoring arterial blood gas tensions, Na＋,K＋,Ca2＋, and blood glucose levels.Results During preanhepatic stage, blood glucose levels increased progressively, blood electrolytes and acidbase balance were stable. During anhepatic stage, pH was decreased, base deficit was increased significantly, blood glucose levels were significantly decreased, blood electrolytes were stable. During neohepatic stage, acidosis became more severe, blood potassium increased abruptly on reperfusin of the grafted liver, blood glucose was higher than that in prehepatic and anhepatic stage. Conclusion During operation of liver transplantation, metabolic acidosis developed progressively, blood glucose decreased in anhepatic stage, blood glucose and potassium increased significantly on reperfusion of the grafted liver.
Objective To investigate the influence of cationic liposomemediated endostatin gene on colorectal cancer liver metastasis. Methods Animal model for colorectal carcinoma liver metastasis were established. The plasmid expressing endostatin genelipofectAMINE were injected in vein. Results After cationic liposomemediated endostatin gene were injected in vein, the incidence of liver metastasis and mean numbers of liver tumors were decreased, survival time of animal was significantly longer. Conclusion Intravenous injection of cationic liposomemediated endostatin gene can control the development of colorectal cancer liver metastasis effectively.
Objective To study the effects of heat shock proteins (HSPs) in the course of hepatic ischemia reperfusion injury (HIRI), and analyze its mechanism. Methods The relationship between HSPs and HIRI was studied by reviewing literatures. Results HSPs was a kind of stress protein induced after cell was sitmulated by the stress. It could improve body′s tolerance to tough situation. Though hepatic ischemia reperfusion usually results in serious hepatic injury, at the same time it could induce can increase the production of HSPs that can protect liver from and lessen ischemia reperfusion injury. Conclusion HSPs can improve the tolerance to HIRI and lessen injury. In addition, HSPs is thought to be markers of HIRI, and can be used as a efficient indicator to test the level of hepatic injury and assess prognosis.
Objective To elucidate the role of the transcription factor liver activator protein (LAP, a member of the C/EBP family) in the expression of α1(I) collagen gene in activated hepatic stellate cells (HSCs). Methods Rat HSCs were prepared from SD rats by in situ perfusion and singlestep density Nycodenz gradient. Two chimeric luciferase reporter gene plasmids containing the human collagen α1(I) gene promoter fragments (-804～+1 452 or -804～+222) were constructed. Culture-activated HSCs were co-transfected with the reporter gene contructs and mammalian vector expressing LAP using the cationic-liposome mediated method, and the promoter activity was determined by measuring luciferase activity. Results The luciferase reporter gene construct containing the first intron of α1(I) collagen gene (-804～+1 452, was called as PGL3-col) had a higher level of gene expression, as compared with the construct lacking the first intron 〔was called as PGL3-col (△intron)-in activated HSCs (315±45 U/mg protein vs 220±70 U/mg protein, Ｐ＜0.05). Transient transfection of the vector expressing LAP significantly increased basal transcription from PGL3-col and PGL3-col (△intron) reporter gene vectors (587±62 U/mg protein vs 315±45 U/mg protein and 326±52 U/mg protein vs 220±70 U/mg protein respectively, both Ｐ＜0.05). Conclusion The transcription factor LAP transactivates collagen α1(I) gene in activated HSCs, and the first intron is important for α1(I) collagen gene transcription activity in activated HSCs.