Objective
To investigate the effect of hepatocyte growth factor (HGF) on the barrier function of retinal peigment epithelium (RPE) and to detect the pathological mechanism of retinal detachment (RD) induced by over expression of HGF in RPE.
Methods
Sub-retina injection of E1/E3deleted adenoviral vectors encoding HGF (Ad CMV.HGF) and green fluorescent protein (Ad CMV.GFP) in adult pigmented rabbits [5times;104 plaque-forming units (pfu)/eye] to set up the model of retinal detachment. The ocular fundus and pathological changes were observed 3, 7, 14, and 28 days after injection. The expression level of HGF in retina and vitreous body was detected by immunohistochemistry and enzyme linked immunosorbent assay (ELISA).
Results
In the control eyes injected with AdCMV.GFP, expression of GFP only detected in RPE monolayer. The eyes injected with AdCMV.HGF had b HGF immune positive action in RPE cells at the injection site. The expression level of HGF in vitreous body reached the peak 7 days after injection and decreased to the basic level 28 days after injection. Chronic RD and chronic choroidal inflammation were found in the eyes injected with AdCMV.HGF within the time frame of HGF expression. Proliferative RPE cells were found in subretinal space in the region of RD, and multilayered cellular membranes developed in some eyes.
Conclusion
Over expression of HGF in RPE may induce chronic serous RD with subretinal proliferation of RPE, which suggests that HGF should be further studied as a target for therapeutic intervention in RD.
(Chin J Ocul Fundus Dis, 2007, 23: 193-197)
【Abstract】Objective To investigate the effects of ethanol on polarization of cultured hepatocytes.Methods Sandwich hepatocytes were used to study the effects of ethanol on polarization of shortperiod cultured hepatocytes. Expression of albumin mRNA and P450-b mRNA,specific distribution of plasma membrance protein and the morphology were observed before and after ethanol was given. Results After treated ethanol, hepatocytes became flat and polynuclear. Its cellular plasma became watery and anatomical networks like board structure disappeared. Immunohistochemical method proved that the distribution of specific plasma membrane protein was damaged, and in situ hybridization studies demonstrated that the levels of albumin mRNA, P450-b mRNA expression decreased after ethanol was given.Conclusion Ethanol induces significant disturbance of cell polarization in cultured hepatocytes to result in injury. The current study is important for exploring the mechanism of alcohol liver disease from the cellular plane to provide a model.
【Abstract】ObjectiveTo establish an efficient, effective hepatocyte isolation technique in order to increase cell production and decrease the prime cost. Methods The inferior vena cava below diaphragm was dissected and ligatured, and the inferior vena cava below liver was separated. Subsequently, the liver was perfused with EGTA through the portal vein while the inferior vena cava below liver was opened, and then the liver was harvested. The liver tissue was cut into 1 mm×1 mm×1 mm and digested at 37 ℃ water bath with Ⅳ collagenase for 30-40 minutes, then the hepatocytes were purified and cultured in CO2 incubator. The production and function of hepatocytes were assessed. ResultsThe isolated hepatocytes using this technique were more than 95% among the all isolated cells. No statistic difference was found in cell production and cell function comparing with traditional technique. But this technique was simplified and more economically. ConclusionThis modified hepatocyte isolation technique is efficient and effective. It can ensure the amount of production and purity of hepatocytes.
Objective To investigate the immunological rejection after hepatocyte transplantation for acute liver failure （ALF） in mice．Methods The hepatocytes were isolated from pig，BALB/c and C57BL/6 mice livers were conducted and then transplanted into C57BL/6 mice．CCl4 was used to make ALF mice model．The experimental animals were randomly divided into three groups， including syngenic group，allogeneic group，and xenogenic group．The survival statuses of all the mice were recorded. The alteration of T lymphocyte subsets，immune globulin，and cytokine were determined．Results ①The survival ratio was 8/10，6/10， and 3/10 in the syngenic group， allogeneic group， and xenogenic group， respectively．The survival ratio in the syngenic group was significantly higher than that in the other two groups （P＜0.05）．②The CD4+ and CD8+ T cells of the peripheral blood in the syngenic group did not change significantly on week one after transplantation．The CD4+ T cells in the allogeneic group reached the peak on day 3 after hepatocyte transplantation （P＜0.05）， while CD8+ T cells did not change much in one week．The CD4+ and CD8+ T cells in the xenogenic group increased and reached the peak on day 3 after transplantation （P＜0.05）．③There were no significantly differences of IgM and IgG in the syngenic group among 0.5， 1， and 3 d after transplantation. IgM of the allogeneic group and xenogenic group reached the peak on day 1 （P＜0.05） and IgG reached the peak on day 3 （P＜0.05） after transplantation．④The concentrations of IFN-γ， TNF-ɑ， and IL-2 in the allogeneic group and xenogenic group were significantly higher than those in the syngenic group （P＜0.05）．The concentration of IL-6 of the xenogenic group was higher than that of the other two groups （P＜0.05）. Conclusions CD4+ and CD8+ T cells play an important role in immune response to both allogeneic and xenogenic hepatocyte transplantation， as well as induce humoral immune response early after hepatocyte transplantation．
ObjectiveTo study the protective effect of hepatocyte growth factor(HGF) on grafted mucous membrane of transplanted small bowel.MethodsTotal small bowel transplantation was made in inbred Wistar (RT1k) rats heterotopically,either total parenteral nutrition (control group,n=10) or hepatocyte growth factor supplemented TPN (HGF group,n=10) was given to the recipients from the 2nd day to the 10th day postoperatively. Morphological parameters of the transplanted intestinal mucosa, such as mucosal villous height, villous width, crypt depth, mucosal thickness and villous surface area were observed. Variation of ultrastructure of transplanted epithelial cells was observed. Composition of mucosal protein and DNA content were tested. ResultsComparison between HGF group and the control group were as follows. Mucosal villous height (298.79±22.31） μm vs （176.45±14.62） μm, P=0.001, villous width (107.46±12.34） μm vs （74.20±16.85） μm, P=0.004, crypt depth (104.56±11.17） μm vs （74.45±8.34） μm, P=0.000 5, mucosal thickness (409.53±35.83） μm vs （259.38±24.65） μm, P=0.003, and villous surface area (0.101±0.011) mm2 vs (0.041±0.005) mm2, P=0.001 were found significantly increased in HGF group compared with control group, there were no obvious difference decrease as compared to pretransplant parameters.Mucosal protein composition was higher in HGF group than that in control group （89.65±9.28) mg/g wet wt vs (53.73±11.45) mg/g wet wt, P=0.012, baseline (92.64±10.52) mg/g wet wt, nearly equal to baseline; DNA composition was also high in HGF group （0.89±0.09) mg/g wet wt vs (0.51±0.06) mg/g wet wt, P=0.008, baseline (0.91±0.09) mg/g wet wt. Nearly normal ultrastructure of the graft was maintained in HGF group, atrophied microvilli and broken mitochondrial crista were observed in control group.ConclusionHepatocyte growth factor can improve mucosal structure, preserve enterocyte ultrastructure of isograft after small bowel transplantation in rat.
Objective To evaluate the effect of hepatocyte growth factor(HGF) on intestinal permeability and bacterial translocation after small bowel transplantation in rats. Methods Twenty Wistar rats were as recptors and twenty SD rats as donors. After heterotopic intestinal grafting, cyclosporine A was administered at 6mg/kg·day intramuscularly for inhibiting rejection. The SD rats were divided into 2 groups(n=10). HGF was administered at 150 μg/kg·day (HGF group) and normal saline was administered at 150 μg/kg·day (controlgroup). Intestinal permeability and bacterial translocation to the mesenteric lymph nodes and portal vein were assessed at the 8th postoperative day. Results The lactulose and lactulose/ mannitol of control group (0.0931%±0.008 5% and 0.132± 0.021) were higher than those of normal reference value (0.015 0%±0.002 0% and 0.020±0.005)（Plt;0.05）. The lactulose and lactulose/ mannitol of HGF group (0.039 6%±0.009 0% and 0.056±0.013) were also higher than those of normal reference value（Plt;0.05）.The bacterial culture positive proportion of lymphaden in HGF group and control group were 10% and 60%, showing statistically significant difference（Plt;0.05）. The bacterial culture positive proportion of portal vein in HGF group and control group were 10% and 20% respectively（P＞0.05）. Conclusion HGF can decrease intestinal permeability and bacterial translocation from the lumen of the graft to the mesenteric lymph nodes,thus improve gut barrier function, may be of help to reduce the incidence of septic complications after intestinal grafting.
Objective To investigate the effects of human recombinant hepatocyte growth factor(rh-HGF) on the expression of c-Met in intima of allograft vessels after cardiac transplantation in rats. Methods Heterotopic heart transplantation were established in abdominal cavity with eighty Wistar rats and forty SD rats. Donors’ cardiac grafts from Wistar rats were transplanted to SD rats(allograft) or Wistar rats(isograft).Sixty recipient rats were divided into three groups, control group：20 Wistar rats were injected with normal saline 1ml/kg·d intraperitoneally after transplantation; cyclosporine A (CsA) group:20 SD rats were injected with CsA 5mg/kg·d intraperitoneally on operation day; rhHGF group:20 SD rats were injected with rh-HGF 500μg/kg·d and CsA 5mg/kg·d intraperitoneally on operation day. The cardiac grafts were harvested at the 15th day and 60th day after transplantation. The crosssection of vascular tissues were used for immunohistochemistrical staining of c-Met, and investigated the expression of c-Met messenger ribonucleic acid (mRNA ) in intima of allograft vessels by reverse transcriptionpolymerase chain reaction(RT-PCR). The pathologic changes of allograft coronary vessels were observed with histopathological method. Results The allograft coronary arteries showed minimal intimal thickening, the endothelium and internal elastic lamina remained almost intact in rh-HGF group after transplantation.The expression of c-Met and c-Met mRNA in intima of allograft vessels after transplantation in rhHGF group were significantly higher than those in CsA group and control group（expression of c-Met at 60d: 1.85±0.26 vs. 0.96±0.10, t=8.491,P=0.000;1.85±0.26 vs. 0.58±0.03, t=13.725,P=0.000; expression of c-Met mRNA at 60d: 192±0.22 vs. 0.88±0.07, t=11.940,P=0.000;1.92±0.22 vs. 0.42±0.02,t=19.206,P=0.000）. Conclusion rh-HGF may prevent the progression of cardiac allograft vasculopathy through upregulating the expression of c-Met to stimulate endothelial cell repair and growth.
Objective To evaluate the transfection efficiency and expression level of hepatocyte growth factor (HGF) by transfecting a recombinant adenovirus carrying HGF gene (Ad-HGF) into bone marrow mesenchymal stem cells (BMSCs) and to explore the effect of the expression supernatant on BMSCs in vitro so as to lay a foundation for the manufacture of gene medicine which expresses efficient cell factors. Methods Rat BMSCs were isolated using Percoll density gradient method and cultured according to the adherent property of BMSCs. The expression of c-Met was detected by immunohistochemical examination. BMSCs were infected with a recombinant adenovirus carrying green fluorescent protein gene (Ad-GFP) at multipl icity of infection (MOI, 0, 25, 50, 100, and 200 pfu/cell). To select an optimal MOI, the transfection efficiency and the degree of cell damage were assayed by flow cytometry and MTT, respectively, at 48 hours after transfecting. The expression of HGF in BMSCs transfected with optimal MOI Ad-HGF was measured with ELISA assay. MTT method was used to evaluate the prol iferation effect of HGF expression supernatant on BMSCs. Results Immunohistochemical staining showed that BMSCs expressed c-Met receptor for HGF. At 48 hours after transfecting with different MOI Ad-GFP (0, 25, 50, 100, and 200 pfu/cell), the transfection efficiencies were 0.34% ± 0.04%, 40.72% ± 0.81%, 61.72% ± 1.04%, 85.33% ± 0.83%, and 17.91% ± 0.63%, respectively; and the highest transfection efficiency was observed at 100 pfu/cell MOI. The cell damage was obviously observed when MOI was 200 pfu/cell. The expression of HGF in BMSCs reached the highest level after being transfected with 100 pfu/cell MOI Ad-HGF for 48 hours. The expression product could stimulate the prol iferation of BMSCs. The prol iferation of BMSCs gradually rose with the increase of HGF protein, and reached the highest level at 10% (320 pg). Conclusion BMSCs can be transfected efficiently with Ad-HGF and express HGF protein, which stimulates the prol iferation of BMSCs. It suggests that BMSCs is an ideal repair cells with gene vector.
Objective To investigate the effect of combined delivery of hepatocyte growth factor (HGF) and insulinlike growth factor-1 (IGF-1) on the development of bone mesenchymal stem cells (BMSCs) differentiation by expression of GATA-4，and to supply some evidence for clinical BMSCs transplantation therapy. Methods BMSCs were isolated from the femurs and tibias of the randomly assigned rabbits and cocultured with myocytes in a ratio of 1∶1. Myocytes were obtained from neonatal rabbits ventricles. 150 ng/ml HGF and 200 ng/ml IGF-1 were added into 4 culture bottles of 8 bottles and the other 4 bottles were not. After BMSCs were cocultured with myocytes for 1 day, 3 days, 1 week, and till 6 weeks， differentiated BMSCs were targeted and microdissected with a laser capture microdissection system, and then ribonucleic acid (RNA) was extracted and isolated. The differentiation of BMSCs in coculture was confirmed by immunohistochemistry, electron microscopy, and reverse transcriptionpolymerase chain reaction (RT-PCR). And expression of GATA-4 in BMSCs was detected by semiquantitative RT-PCR. Results Before coculturing, the BMSCs were negative for α-actinin and exhibited a nucleus with many nucleoli. After coculture with myocytes, some BMSCs became αactininpositive and showed a cardiomyocytelike ultrastructure, including sarcomeres, endoplasmic reticulum, and mitochondria. BMSCs cocultured with myocytes expressed cardiac transcription factor GATA-4. IGF-1 and HGF delivery can significantly increased expression of GATA-4 for the differentiated BMSCs as compared with cells of no delivery of HGF and IGF-1. The expression level of GATA-4 in captured BMSCs began to increase at the 1st day, reach the peak at the 2nd week and kept high expression level after the 2nd week. Conclusion BMSCs can transdifferentiate into cells with a cardiac phenotype when cocultured with myocytes. Differentiated myocytes express cardiac transcription factors GATA-4. Administration of HGF and IGF-1 promoted the development of BMSCs transdifferentiate into cardiac phenotype, which is associated with the increase in expression level of GATA-4.
【Abstract】Objective To investigate the injury of calcium on the liver. Methods By using collagenase-containing solution to perfuse the rat livers, the rat liver suspension with different viability was prepared, preserved hypothermically, and the cytosolic calcium comcentration was detected with Fura2/AM. Results ① The concentration of cytosolic calcium after 2-hour storage: Experiment group 1(viability 5%) (1055.0±30.79) nmol/L, experiment group 2(viability 10%) (853.0±20.42) nmol/L, experiment group 3(viability 30%) (616.0±13.20) nmol/L, experiment group 4(viability 50%) (562.0 ±26.06) nmol/L, experiment group 5(viability 70%-80%) (318.0±13.01) nmol/L, experiment group 6(viability 90%) (114.6±6.11) nmol/L. ②The concentration of cytosolic calcium after 24hour storage: Experiment group A(viability 10%) (1704.0±70.11) nmol/L, experiment group B(viability 50%) (1125.0±23.22) nmol/L. The results showed that the lower was the viability, the higher was the cytosolic calcium concentration.With the same viability the cytosolic calcium concentration elevated more than two times higher than the original concentration with the time lengthened. Conclusion Calcium overload is one of the main factors which attribute to the ischemiareperfusion injury of the hepatocytes.
