Objective To investigate the effects of activities of Na+K+ATPase in plasma membranes of hepatocytes on the formation of calcium bilirubinate gallstone. MethodsThe research were studied in Xiao’s rabbit models.One hundred and three rabbits were randomly divided into the control group (Con,n=28),simple biliary obstruction group (BO,n=36),biliary obstruction and infection group (BOI,n=39).The activities of Na+K+ATPase,contents of intracellular calcium and bile acids in bile were measured in 3rd,7th,14th and 20th day. ResultsThe activities of Na+K+ATPase,contents of bile acids in bile decreased gradually and the contents of intracelluar calcium of hepatocytes increased continously in all phases of experimental groups compared with control group (P＜0.01).There was more significant changes in BOI group than in BO group (P＜0.05). Conclusion The progressive decrease of activities of Na+K+ATPase has a close relationship with the overloaded intracellular calcium and the continous decrease of bile acids contents in bile in formation of calcium bilirubinate gallstone.Infection can promote above mentioned changes and more stone formation.
To study the mechanism of cholesterol gallstone formation, rabbit models were induced by feeding with high cholesterol diet. Bile acids were tested with bi-wavelengh thin layer scan and low density lipoprotein receptor activity of hepatocytes binding to 125I-LDL were tested with radio immunoassay in different feeding phases as 1,2,3 and 4-week groups, as well as the control group. The results showed that cholesterol gallstones in 2,3 and 4-week groups were induced in respectively. The contents of glucocholic acid (GCA) in bile were decreased significantly (vs control group, Ｐ＜0.05). The Bmax values of LDL receptor of hepatocytes binding to 125I-LDL were decreased significantly (Ｐ＜0.05). Kd values of those gradually increased (Ｐ＜0.05). These suggest that the decreased activity of LDL receptor of hepatocytes would reduce the synthesis of GCA, thus resulting in the formation of cholesterol gallstones.
OBJECTIVE: To validate the hemostatic properties of collagen sponge made in China. METHODS: The experimental model of superficial cut of liver was established in 20 Sprague-Dawley adult rats, which were divided into two groups randomly. Collagen sponge or gelatin sponge was used to cover the cut respectively. Hemostatic result was observed. Afterwards, standard liver trauma model by resection left front liver lobe was made, wound was treated with collagen sponge or gelatin sponge respectively. Hemostatic result was observed. Concurrent hemostatic time and bleeding amount were noted. At 7, 14 and 20 days after operation, intra-abdominal adhension, infection and healing state of liver were observed by exploratory laparotomy. The histological changes of regenerate liver tissue were observed by microscopy. RESULTS: Collagen sponge adhered to wound well. Concurrent hemostatic time and bleeding amount in collagen sponge group were superior to those of gelatin sponge (P lt; 0.05). The histological examination showed that collagen sponge was absorbed and degraded rapidly, regenerative hepatocytes could be induced. CONCLUSION: Collagen sponge has fine hemostatic properties and can induce regeneration of hepatocytes effectively. It is worth popularizing for its convenience in clinical application and its properties of rapid degradation and absorption.
This research aims to construct a lentiviral expression vector carrying the extracelluar domain (ED) of human hepatocyte growth factor receptor (C-Met), and to express it in transfected 293T cells. The extracellular domain of C-Met was amplified by RT-PCR, ligated with lentiviral expression vector p RRL-CMV-ED, and then expressed in 293T cell line. The expressed protein was purified and identified by RT-PCR and Western blot. The enzyme digestion and sequence analysis showed that the lentiviral expression vector p RRL-CMV-ED was constructed correctly. The size of amplified genes was about 2 700 bp. The purified protein with Ni-affinity column was about 105 kD analyzed by SDS-PAGE. The Western blot and ELISA results showed that the expressed protein which could bind to HGF specifically was the extracelluar domain of human hepatocyte growth factor receptor. This research may lay a foundation for further study of anti-C-MET monoclonal antibody and neutralizing antibody.
ObjectiveTo investigate the interventive effect of xuebijing for injection combined with nalmefene hydrochloride injection in treatment of acute hepatocyte functional injury after severe thoracoabdominal injuries. MethodsClinal data of 169 patients with severe thoracoabdominal injuries who treated in The 253th Hospital of PLA between January 2009 and June 2013 were collected retrospectively. The trauma indexes of the 169 patients were all higher than 17 scores. Patients were divided into the intervention group (n=112) and the control group (n=57) according to their receptive treatment:patients of control group underwent traditional treatments such as antishock, hemostasis, and so on; but patients of intervention group received xuebijing for injection combined with nalmefene hydrochloride injection (intravenous infusion). Patients of intervention group were tested at the time of arriving at and leaving the emergency department to the inpatient department with alanine aminotransferase (ALT), aspartate aminotransferase (AST), tumor necrosis factor-α (TNF-α), lipopolysaccharide (LPS), and interleukin-6 (IL-6); the patients of control group were just tested at the time of leaving the emergency department to the inpatient department, then comparison of the indexes between the 2 groups was performed. ResultsIn intervention group, the levels of ALT, AST, TNF-α, LPS, and IL-6 after the interventive treatment were all lower than those of before interventive treatment (P<0.05). Compared with control group, there was no significant difference in the levels of ALT, AST, TNF-α, LPS, and IL-6 before the interventive treatment of intervention group (P>0.05); but the levels ALT, AST, TNF-α, LPS, and IL-6 were all lower after the interven-tive treatment (P<0.05). ConclusionsXuebijing for injection combined with nalmefene hydrochloride injection can evidently improve the acute hepatocyte functional injury after severe thoracoabdominal injuries, and improve the prognosis.
ObjectiveTo construct large block of engineered liver tissue by co-culture of fibroblasts and hepatocytes on collagen hydrogels in vitro and do in vivo implantation research. MethodsSilastic mould was prepared using three-dimensional printing technology. The collagen hydrogel scaffold was prepared by collagen hydrogel gel in the silicone mould and was removed. Sprague Dawley rat lung fibroblasts were co-cultured with primary hepatocytes at a ratio of 0.4:1 on the collagen hydrogel scaffold to construct large block of engineered liver tissue in vitro (group B), and primary hepatocytes cultured on the collagen hydrogel scaffold served as control group (group A). The cell morphology was observed, and the liver function was tested at 1, 3, 7, 14, and 21 days after culture. The rat model (n=24) of hepatic cirrhosis was made by subcutaneous injection of carbon tetrachloride. And in vivo implantation study was carried in cirrhosis rat model. The phenotypic characteristics and functional expression of hepatocytes were evaluated at 3, 7, 14, 21, and 28 days after implantation. ResultsIn vitro results indicated that hepatocytes in group B exhibited compact polyhedral cells with round nuclei and high expression of liver function. Moreover, cells aggregated to the most at 7 days. Album production and urea synthesis incresed significantly when compared with group A (P<0.05). In vivo results showed hepatocytes in group B survived for 28 days, and albumin production and urea synthesis were significantly increased. In addition, hepatocytes showed an aggregated distribution and cord-like structures, which was similar to normal liver tissue. ConclusionThe large block of engineered liver tissue constructed by co-cultured cells can form tissue similar to normal liver tissue in vivo, and survive for a long time, laying foundations for building more complete engineered liver tissue in the future.
Objective To investigate the effect of porcine small intestinal submucosa extracellular matrix (PSISM) on the vitality and gene regulation of hepatocyte so as to lay the experimental foundation for the application of PSISM in liver tissue engineering. Methods The experiment was divided into two parts: ① BRL cells were cultured with 50, 100, and 200 μg/mL PSISM-medium which were prepared by adding PSISM into the H-DMEM-medium containing 10%FBS in groups A1, B1, and C1, and simple H-DMEM-medium served as a control (group D1); ② BRL cells were seeded on 1%, 2%, and 3% PSISM hydrogel which were prepared by dissolving PSISM in sterile PBS solution containing 0.1 mol/L NaOH in groups A2, B2, and C2, and collagen type I gel served as a control (group D2). At 1, 3, and 5 days after culture, the morphology and survival of liver cells were detected by the Live/Dead fluorescent staining. The cell vitality was tested by cell counting kit-8 (CCK-8) assay. And the relative expressions of albumin (ALB), cytokeratin 18 (CK18), and alpha-fetoprotein (AFP) in hepatocytes were determined by real-time fluorescent quantitative PCR (RT-qPCR). Results The Live/Dead fluorescent staining showed the cells survived well in all groups. CCK-8 results displayed that the absorbance (A) value of group C1 was significantly higher than that of group D1 at 5 days after culture with PSISM-medium, and there was no significant difference between groups at other time points (P>0.05). After cultured with PSISM hydrogels, theA values of groups A2, B2, and C2 were significantly higher than those of group D2 at 3 and 5 days (P<0.05), theA value of group A2 was significantly higher than that of groups B2 and C2 at 5 days (P<0.05), but there was no significant difference between groups at other time points (P>0.05). RT-qPCR showed that the relative expressions of ALB and CK18 mRNA significantly increased and the relative expression of AFP mRNA significantly decreased in groups A1, B1, and C1 when compared with group D1 (P<0.05). The relative expression of CK18 mRNA in group C1 was significantly lower than that in groups A1 and B1 (P<0.05). The relative expressions of ALB and CK18 mRNA were significantly higher and the relative expression of AFP mRNA was significantly lower in groups A2, B2, and C2 than group D2 (P<0.05); the relative expression of CK18 mRNA in group A2 was significantly higher than that in group B2 (P<0.05), and the relative expression of AFP mRNA in group A2 was significantly lower than that in group C2 (P<0.05), but no significant difference was found between other groups (P>0.05). Conclusion PSISM has good compatibility with hepatocyte and can promote the vitality and functional gene expression of hepatocyte. PSISM is expected to be used as culture medium supplement or cell carrier for liver tissue engineering.
ObjectiveTo discuss the possibility of constructing injectable tissue engineered adipose tissue, and to provide a new approach for repairing soft tissue defects.MethodsHuman adipose-derived stem cells (hADSCs) were extracted from the lipid part of human liposuction aspirate by enzymatic digestion and identified by morphological observation, flow cytometry, and adipogenic induction. The hADSCs underwent transfection by lentivirus vector expressing hepatocyte growth factor and green fluorescent protein (HGF-GFP-LVs) of different multiplicity of infection (MOI, 10, 30, 50, and 100), the transfection efficiency was calculated to determine the optimum MOI. The hADSCs transfected by HGF-GFP-LVs of optimal MOI and being adipogenic inducted were combined with injectable fibrin glue scaffold, and were injected subcutaneously into the right side of the low back of 10 T-cell deficiency BALB/c female nude mice (transfected group); non-HGF-GFP-LVs transfected hADSCs (being adipogenic inducted) combined with injectable fibrin glue scaffold were injected subcutaneously into the left side of the low back (untransfected group); and injectable fibrin glue scaffold were injected subcutaneously into the middle part of the neck (blank control group); 0.4 mL at each point. Twelve weeks later the mice were killed and the implants were taken out. Gross observation, wet weight measurement, HE staining, GFP fluorescence labeling, and immunofluorescence staining were performed to assess the in vivo adipogenic ability of the seed cells and the neovascularization of the grafts.ResultsThe cultured cells were identified as hADSCs. Poor transfection efficiency was observed in MOI of 10 and 30, the transfection efficiency of MOI of 50 and 100 was more than 80%, so the optimum MOI was 50. Adipose tissue-like new-born tissues were found in the injection sites of the transfected and untransfected groups after 12 weeks of injection, and no new-born tissues was found in the blank control group. The wet-weight of new-born tissue in the transfected group [(32.30±4.06) mg] was significantly heavier than that of the untransfected group [(25.27±3.94) mg] (t=3.929, P=0.001). The mature adipose cells in the transfected group [(126.93±5.36) cells/field] were significantly more than that in the untransfected group [(71.36±4.52) cells/field] (t=30.700, P=0.000). Under fluorescence microscopy, some of the single cell adipocytes showed a network of green fluorescence, indicating the presence of GFP labeled exogenous hADSCs in the tissue. The vascular density of new-born tissue of the transfected group [(16.37±2.76)/field] was significantly higher than that of the untransfected group [(9.13±1.68)/field] (t=8.678, P=0.000).ConclusionThe hADSCs extracted from the lipid part after liposuction can be used as seed cells. After HGF-GFP-LVs transfection and adipose induction, the hADSCs combined with injectable fibrin glue scaffold can construct mature adipose tissue in vivo, which may stimulate angiogenesis, and improve retention rate of new-born tissue.
Objective To investigate effect of hepatocyte growth factor (HGF) after lentivirus-mediated RNA interference (RNAi) targeting c-Met on invasion of colonic carcinoma cell line SW480. Methods The experiment was assigned into 3 groups: NC group, the normal cells were infected by the shRNA negative control virus (the NC-20 andNC-40 represented the negative group which were added 20 ng/mL and 40 ng/mL respectively HGF after being infected); KD group, the normal cells were infected by the shRNA-c-Met target virus (the KD-20 and KD-40 represented the interfered group which were added 20 ng/mL and 40 ng/mL HGF respectively after being infected; KD1, KD2, KD3, and KD4 represented the different RNAi targets for the purpose gene); CON group, the normal cells were not infected by any virus. The lentiviral vector shRNA-c-Met was constructed and verified by polymerase chain reaction (PCR) and DNA sequencing. The SW480 cells were infected with the shRNA-c-Met after packed with lentivirus plasmid. Fourty-eight hours transfection later, the c-Met mRNA of the transfected SW480 cell was detected by real time PCR and the c-Met protein was examined by Western blot. Seventy-two hours after transfection, the cell apoptosis was detected by flow cytometry and the invasions in the different cells with stable transfection were detected by Transwell test. Results The RNAi sequence targeting c-Met gene was successfully inserted into the lentiviral vector. The shRNA-c-Met transfection resulted in an obviously reduced expression of c-Met mRNA in the SW480 cells. The efficency of gene knock down of the KD4 (the cells with No.4 target spot knocked down) was 81.4%. The shRNA-c-Met tansfection resulted in an obviously reduced expression of c-Met protein in the SW480 cells. After transfection, the apoptosis rate of the KD group was significantly higher than that in the NC group (P<0.001) or the CON group (P<0.001). The invasion ratios in the NC group, NC-20 group, and NC-40 group were significantly higher than those in the KD group (P<0.001), KD-20 group (P=0.015), and KD-40 group (P=0.017), respectively; which in the NC-20 group and NC-40 group were increased as compared with the NC group (P<0.001,P<0.001), and in the NC-40 group was increased as compared with the NC-20 group (P=0.005). The invasion ratios in the KD-20 group and KD-40 group were increased as compared with the KD group (P<0.001,P<0.001), and in the KD-40 group was increased as compared with the KD-20 group (P=0.014). Conclusion Lentivirus-mediated RNAi targeting c-Met could effectively suppress expression of c-Met in SW480 cells and could reduce invasion of HGF on SW480 cells with knocked down c-Met.
Bioartificial liver support system (BALSS) provides a new way to treat liver failure and leaves more time for patients who are waiting for liver transplantation. It has detoxification function as well as the human liver, at the same time it can provide nutrition and improve the internal environment inside human body. Bioreactors and hepatocytes with good biological activity are the cores of BALSS which determine the treatment effect. However, in the course of prolonged treatment, the function and activity of hepatocytes might be greatly changed which could influence the efficacy. Therefore, it is very important to detect the status of the hepatocytes in BALSS. This paper presents some common indicators of cell activity, detoxification and synthetic functions, and also introduces the commonly detection methods corresponding to each indicator. Finally, we summarize the application of detection methods of the hepatocyte status in BALSS and discuss its development trend.