Hepatocyte Isolation, Culture, and Its Clinical Applications / 한양의대학술지

Hepatocytes, parenchymal cells of the liver, are specially differentiated cells to perform most of the body metabolisms. Many clinicians are interested in utilizing hepatocytes as cell therapeutics. A great number of investigators have been harvesting hepatocytes using two-step portal vein perfusion method, in which Ca2+-free EDTA-containing buffer and Ca2+-enriched collagenase solution are pumped into liver in sequence. Among various attempts for long-term culture of hepatocytes, collagen gel sandwich configuration is recognized to be the most effective technique. In the biomedical field, hepatocytes have been used in three methods of applications. First is hepatocyte transplantation for the treatment of acute, chronic liver failure and metabolic diseases. Donated livers not suitable for organ transplantation are rare, which is the major human hepatocyte source. This shortage of human hepatocyte source is expected to be resolved by virtue of rapid progressing stem cell technologies. The second application is biological components of bioartificial liver (BAL) system for acute liver failure patients. Due to the lack of functional activity of clinically studied BAL systems and difficulty of establishing a manufacturing system for ready-to-use, additional research activities are stagnated. The third utilization of hepatocytes is in vitro drug screening studies such as drug metabolism, transport, biliary excretion, and toxicity tests. If cell therapeutic treatments using hepatocytes are clinically valuable to some types of liver diseases, the demand for liver transplantation would be significantly diminished.

Progress of treating hepatic failure with induced pluripotent stem cell-derived hepatocytes / 国际生物医学工程杂志

Hepatocyte transplantation is effective in the treatment of liver failure caused by a variety of factors.In pace with the progress of the study of induced pluripotent stem cells and its differentiation technology,a new method has arisen to obtain a great number of safe hepatocytes with biological function,which are suitable for seed cells of hepatocyte transplantation.In this article,we review the latest research progress about induced pluripotent stem cell-derived hepatocytes being transplanted to treat liver failure.

Evaluation of acute liver injury in mice model with different does of CCl 4 / 重庆医学

Objective To establish a simple ,stable acute liver injury model induced by CCl4 to observe effects of hepatocyte transplantation .Methods CCl4 plant oil with different concentration of 20% and 50% was used in mice by intraperitoneal injection , of which the dose was 2 mL/kg ,and then materials were taken at different time points respectively .Mice survival rate ,alanine amin-otransferase (ALT) ,aspartate aminotransferase (AST) and the pathological changes of the liver were detected .Results Mice sur-vival rate in 20% CCl4 intraperitoneal injection was significantly higher than that of 50% .ALT and AST in experiment group were significantly higher than that of control group ,but there was no significant difference between two experimental groups .Pathologi-cal examination showed that mice liver cells showed typical cytoplasmic ,ballooning ,scattered punctate ,piecemeal necrosis and in-flammatory cell infiltration in 20% CCl4 intraperitoneal injection ;while in 50% CCl4 ,there was obvious fibrosis ,in addition to the mentioned heavier lesions .Conclusion 20% -50% CCl4 intraperitoneal injection in 2 mL/kg dose can induce different degrees of relatively stable liver injury ,and its concentration determines the degree of liver injury .Acute liver injury induced by 20% -50%CCl4 was an ideal model for hepatocyte transplantation experiment .

Effect of Liver Cell Transplantation on Acute Hepatic Failure Induced by Massive Liver Resection in the Rat

PURPOSE: This study is designed to ascertain the most effective quantity and injection route of hepatocytes in an acute liver failure model induced by massive liver resection in rats. METHODS: Rats weighing 450 to 650 gm underwent partial hepatectomy that was 80% of their liver weight, resulting in acute liver failure. Hepatocytes were obtained by perfusing collagenase (Wako, Japan) solution through portal vein into liver of the allogenic rat. These hepatocytes were injected into different places with different dosage. The experimental groups were divided into the Control group, Splenic group I (2x10(6) cells into splenic capsule), Splenic group II (2x10(7) cells into splenic capsule), Portal vein group (2x10(7) cells into portal vein), Subperitoneal group (2x10(7) cells into subperitoneum). The experimental animals were observed carefully for 5 days for assessment of survival and regeneration of liver. Liver function tests including serum alanine aminotransferase (ALT), total bilirubin, gamma-glutamyl transferase (gamma-GTP) on postoperative 1, 2, 3, 5th days and histologic examinations of specimens obtained from each respective groups on postoperative 5th day were performed. RESULTS: Serum ALT level on postoperative day 1 peaked and then gradually normalized showing statistical significance (p=0.035). Study groups showing statistically significant difference under repeated anova analysis were between the Splenic group II and Control (p=0.035), and between the Splenic group II and Portal vein group (p=0.001) with respect to serum ALT levels. Also, progression of each study group showed statistical significance. (p=0.02). Serum total bilirubin and r-GTP did not show any significant difference. CONCLUSION: Hepatocyte transplantation of 2x10(7) cells into spleen showed the best results in the acute hepatic failure rat.

Hepatocyte Transplantation / 대한이식학회지

Since the first hepatocyte transplantation (HT) was done by Matas and colleagues 3 decades ago in Gunn rats, there has been much development in the field of HT and clinical application to over 70 humans has been performed worldwide ever since. HT has many advantages compared to the conventional liver transplantation (LT) such as lower cost, lower morbidity, possibility of genetic modification, availability of cryopreservation, utilization of inadequate livers for LT and possibility to transplant to multiple recipients from a single donor. HT has been applied as a definitive treatment option in metabolic liver disease or fulminant hepatic failure with marginal results. It has also been used as a bridge to LT in severely ill patients awaiting LT, but it has not yet been widely accepted due to the shortcoming of clinical efficacy compared to LT. Much effort was made over the past several years to improve these results, and we hope that with the help of future research, HT will soon have a clear clinical role in the management of liver diseases. This review article will cover various animal models used for researching HT and methods used for transplanting hepatocytes. It will also briefly introduce the worldwide clinical results in human trials as well as our experience on a successful case with glycogen storage disease and its long term result.

The Factors Affecting Transplanted Hepatocytes Repopulation in Rats with Liver Fibrosis

PURPOSE: Cell therapy for various diseases has gained wide acceptance. Because most patients with chronic liver failure have mild-to-severe liver cirrhosis, there are many limitations to clinical applications. We analyzed how to increase cell engraftment in rats with liver fibrosis. METHODS: We used analbuminemic SD rats (NARs) as recipients of syngeneic CAG-EGFP SD hepatocytes obtained by the 2 perfusion method. Hepatic fibrosis was induced with thioacetamide in drinking water for 6 weeks in the recipient NARs. NARs were pre-treated with gadolinium, doxorubicin, and gliotoxin before hepatocyte transplantation. We evaluated the degree of cell engraftment by RT-PCR and immunofluorescent staining for GFP and albumin. The transplanted cells were detected by immunostaining for albumin, and serum albumin was also measured. RESULTS: Although detection of GFP by RT-PCR was variable, albumin was detected in all groups 4 wks after hepatocyte transplantation. GFP and albumin were also detected by immunofluorescent staining 1 and 4 wks after cell transplantation. In control rats, albumin production was maximal at 3 wks, and after that it rapidly decreased. In the gadolinium and doxorubicin-treated group, albumin production was increased up to 4 wks. Albumin production in the gadolinium-treated group was superior to that of the doxorubicin-treated group. CONCLUSION: Kupffer cells play the most important role in cell engraftment in hepatic fibrosis. Therefore, perturbation of kupffer cells in hepatic fibrosis is needed to increase cell engraftment.

The Factors Affecting Transplanted Hepatocytes Repopulation in Rats with Liver Fibrosis

PURPOSE: Cell therapy for various diseases has gained wide acceptance. Because most patients with chronic liver failure have mild-to-severe liver cirrhosis, there are many limitations to clinical applications. We analyzed how to increase cell engraftment in rats with liver fibrosis. METHODS: We used analbuminemic SD rats (NARs) as recipients of syngeneic CAG-EGFP SD hepatocytes obtained by the 2 perfusion method. Hepatic fibrosis was induced with thioacetamide in drinking water for 6 weeks in the recipient NARs. NARs were pre-treated with gadolinium, doxorubicin, and gliotoxin before hepatocyte transplantation. We evaluated the degree of cell engraftment by RT-PCR and immunofluorescent staining for GFP and albumin. The transplanted cells were detected by immunostaining for albumin, and serum albumin was also measured. RESULTS: Although detection of GFP by RT-PCR was variable, albumin was detected in all groups 4 wks after hepatocyte transplantation. GFP and albumin were also detected by immunofluorescent staining 1 and 4 wks after cell transplantation. In control rats, albumin production was maximal at 3 wks, and after that it rapidly decreased. In the gadolinium and doxorubicin-treated group, albumin production was increased up to 4 wks. Albumin production in the gadolinium-treated group was superior to that of the doxorubicin-treated group. CONCLUSION: Kupffer cells play the most important role in cell engraftment in hepatic fibrosis. Therefore, perturbation of kupffer cells in hepatic fibrosis is needed to increase cell engraftment.

Application of Fah knockout mice in the hepatocyte transplantation and the pathological changes / 中国药科大学学报

Aim: To investigate the pathological changes in NTBC[2-(2-nitro-4-trifluoro-methyl-benzoyl) -1,3 cy-clohexanedione]-induced hepatic injury in mice and in the repopulation of adult hepatocytes in Fah~(-/-) mouse. Methods: Autogenous hepatic injuries in Fah~(-/-) mice were induced by the treatment of NTBC. Injection of hepatocytes obtained from wild-type mice to spleen were transplanted into the Fah~(-/-) mice. Then, changes to body weight and the likelihood of the transplanted Fah~(-/-) mice, and hepatic immunohistochemistry were ob-served. In addition, pathological changes to liver damage induced by NTBC treatment were analyzed under HE-staining microscopy and electron microscopy. Results: The surviving Fah~(-/-) mice subjected to hepatocyte trans-plantation were found to be healthy and in stable body weight. liver repopulation reached to 90% in the 8th week. Repopulating hepatocytes caused no alteration to histological structure of the recipient liver, and subacute hepatic injury occurred in the Fah~(-/-) mice after NTBC treatment. Electronic microscopy observations indicated that necrosis in the hepatocytes occurred at early stage and that apoptosis gradually appeared. It was also shown both necrosis and apoptosis co-existed in the same samples of interest at the following stages of the induced liver injury. Conclusion: Transplanted hepatocytes proliferated in Fah~(-/-) mice allow 90% of the hepatocytic repopula- tion. Repopulation renders normal hepatic function and structure in the recipient Fah~(-/-) mice, as a model of liver repopulation, could be applicable in study of stem cell derived hepatic cells in transplantation assay.

Smac/DIABLO and cytochrome c mRNA levels in liver tissue of rats with acute hepatic failure treated by microencapsulated hepatocytes / 中华临床感染病杂志

Objective To investigate Smac/DIABLO and cytochrome c(cyt-c)mRNA levels in liver tissue of rats with acute hepatic failure treated by microencapsulated hepatocyte.Methods Acute hepatic failure were induced by intraperitoneal injection of D-galactosamine in rats.and the rats were treated with microencapsulated hepatocytes,free hepatocytes and physical saline(contr01),respectively.Smac/DIABLO and cyt-c mRNA in liver tissue was detected by RT-PCR and the mRNA expression levels among three groups were compared.Results Smac/DIABLO and cyt-c mRNA levels in liver tissues of rats with acute hepatic failure were higher than those of normal rata(F=4.345,14.821,47.565,42.178 and 62.961,P<0.05).The peak values of Smac/DIABLO and cyt-C mRNA expressions in free hepatocytes and control groups were at 48 h.while that in microencapsulated hepatoeytes group was at 24 h.Conclusion Smac/DIABLO and cyt-c mRNA expression is an indicator of apoptosis of hepatocytes.

In vitro Differentiation of Human Mesenchymal Stem Cells into Hepatocytes / 대한이식학회지

PURPOSE: Whole liver transplantation has limitation including donor shortage and fatal surgical complications. Hepatocyte transplantation, which is simpler and less expensive than whole liver transplantation, allows the use of living related donors, permits the use of a single donor organ for multiple recipients, and makes possible the cryopreser-vation of hepatocytes for future use. However, hepatocytes have limitation of proliferation and lose their property during culture period. To over come this problems, here we performed differentiation of bone marrow derived mesenchymal stem cells into hepatocytes. METHODS: Human bone marrow cells were harvested from posterior iliac spine of male and then mononuclear cells were obtained by Ficoll-Paque density-gradient centrifuge and plated in tissue culture flasks. For hepatogenic differentiation, we used modified Kuan-Der Lee's method. After differentiation, hepatocytes were collected and RT-PCR and PAS stain analysis were performed. RESULTS: After 5 weeks of cultivation period, mesenchymal stem cells showed cuboidal morphology and contained abundant granules in the cytoplasm. RT-PCR analysis showed increased expression of hepatocyte-specific marker genes (albumin,CK18, PERCK, CPS). Undifferentiated MSCs were not stained with PAS and differentiated hepatocytes from human MSCs stained with PAS indicating that hepatocytes contained glycogen in the cytoplasm. CONCLUSION: Hepatocyte transplantation could be one of the most effective treatments for chronic liver disease. However, hepatocyte has several disadvantages and problems. For alternative cell therapy sources, human bone marrow derived MSCs are considered as transplantable cells. Human MSCs are able to differentiate into functional hepatocytes in vitro and can be a possible cell transplantation source for chronic liver disease patients. Further studies should be done for differentiating human MSCs to hepatocytes in vivo condition.

"Seed cells" for the therapy of liver failure with hepatocyte transplantation / 国际生物医学工程杂志

At present, the survival of patients with liver failure mainly depends on orthotopic liver transplantation(OLT), howerer, the clinical use of OLT therapy has been limited because of many practical difficulties. Although the emergence of hepatocyte transplantation offered assist great or substitute for OLT owing to its unique advantages. Finding a best sources of hepatocyte become an pressing problem we are facing. The cells used in present studies include bone marrow stem cells(BSC) , embryonic stem cells (ESC) and umbrilical stem cells (USC), et al. This review summarized progresses on the differentiation of these stem cells into hepatocyte and we hope these basic concepts may benefit a better understanding for ″seed cells″ choice in hepatocyte transplantation.

Hepatic Stem Cell Transplantation in Liver Failure

Whole or partial liver transplantation has become one of the main treatment strategies for hepatic failure. The availability of a donor liver is the single most limiting factor in liver transplantation. In cell therapy, liver cells have been used clinically to bridge patients to whole organ transplantation and/or as an alternative to whole organ transplants. The crucial property that defines a stem cell is its ability to give rise to a large family of descendants, and at least some hepatocytes do exhibit this pleuripotency. Thus hepatocyte-like cells derived from the bone marrow, embryonal stem cells, or placental sources can also be used. Stem cells of many types have been prodded and cajoled by combinations of growth factors, matrix and culture modifications to identify conditions that favor differentiation or transdifferentiation of stem/progenitor cells into hepatocytes. Additional studies are needed to determine whether hepatocyte-like cells derived from the bone marrow or other sources express the full complement of hepatic functions. If a sufficient number of differentiated hepatocytes can be produced from these stem cells, they could be useful in clinical transplantation programs.

Hepatocyte Transplantation Using Fibrin Gel / 대한이식학회지

PURPOSE: Whole liver transplantation, an effective therapy for many inherited and acquired hepatic disorders, has limitations including donor shortage and fatal surgical complications. Hepatocyte transplantation, which is simpler and less expensive than whole liver transplantation, allows the use of living related donors, permits the use of a single donor organ for multiple recipients, and makes possible the cryopreservation of hepatocytes for future use. However, choosing a proper scaffold for hepatocyte transplantation hampers wide use of hepatocyte transplantation. We performed hepatocyte transplantation using fibrin gel, as a cell transplantation scaffold and evaluated their effectiveness. METHODS: Female, five week old FVB mice, were prepared for donors, and two male, five week old nude mice, were used as recipients. Liver cells were isolated from FVB donors. The cell viability exceeded 95% as assessed by the trypan blue exclusion method. For three nude mice, 5x10(6) cells resuspended in 500microliter of fibrinogen were mixed with 500microliter thrombin, and were injected into the peritoneal cavity of each mouse. One nude mouse was transplanted with 5x10(6) cells resuspended in 500 microliter medium, which served as a negative control. Specimens were retrieved at one week, and histological and immunohistochemical analyses wereperformed. RESULTS: In the negative control, all transplanted hepatocytes disappeared at one week. In mice transplanted both fibrin gel and hepatocytes, conglomerates containing hepatocytes were observed on the intestinal mesentery. The hepatocytes were identified by H & E staining and immunohistochemistry using anti-hepatocyte antibody. Functional activity was evaluated with PAS staining. CONCLUSION: In this preliminary study, stable hepatocyte engraftment was achieved in hepatocyte transplantation with fibrin gel, but not in hepatocyte transplantation without scaffold. More studies on comparison between fibrin gel and injectable scaffolds would be necessary. Improvement on both initial vascularization and proliferation of transplanted hepatocytes is a target of our future work.

Current Research Advancement of Hepatocyte Transplantation / 中国普外基础与临床杂志

Objective To investigate the application and advancement of hepatocyte transplantation (HCT). Methods Literatures about the advancement of HCT were reviewed and analyzed. Results There have been many novel technologies and advancements in the application of HCT. For example, gene modified cell can be used as seed cell, subcutaneous transplantation can be taken when combined with giant molecule material and the encapsulation preconditioning technique can also carried before operation to improve the rate of survival. Conclusion With more and deeper understanding of hepatocyte transplantation and the development of advanced techniques such as the application of giant molecule, HCT will be extensively used in the clinical treatment of acute and chronic hepatic diseases.

Stable Hepatocyte Engraftment after Hepatocyte Transplantation Using Biodegradable Injectable Polymer / 대한이식학회지

PURPOSE: Whole liver transplantation, an effective therapy for many inherited and acquired hepatic disorders, has limitations including donor shortage and fatal surgical complications. Hepatocyte transplantation, which is simpler and less expensive than whole liver transplantation, allows the use of living related donors, permits the use of a single donor organ for multiple recipients, and makes possible the cryopreservation of hepatocytes for future use. However, choosing a proper scaffold for hepatocytes hampers wide use of hepatocyte transplantation. We performed hepatocyte transplantation using biodegradable injectable polymers, fabricated form poly (lactide-co-glycolide) (PLGA) microspheres, as scaffolds to evaluate their effectiveness. METHODS: Female, five week old FVB mice, were prepared for donors, and four male, five week old nude mice, were used for recipients. Liver cells were isolated from FVB donors. The cell viability exceeded 95% as assessed by trypan blue exclusion method. For three nude mice, 2X10(6) cells resuspended in 200micro liter medium were mixed with 200micro liter PLGA microspheres, and were injected into the peritoneal cavity of each mouse. One nude mouse was transplanted with 2X10(6) cells resuspended in 200micro liter medium only, and it served as a negative control. Specimens were retrieved at one week, and histological and immunohistochemical analyses were performed. RESULTS: In the negative control, all transplanted hepatocytes disappeared at one week. In mice transplanted both microspheres and hepatocytes, conglomerates, which contained hepatocytes, were observed in the peritoneal cavity, The hepatocytes were identified by H and E staining and immunohistochemistry using anti- hepatocyte antibody. CONCLUSION: In this preliminary study, stable hepatocyte engraftment was achieved in hepatocyte transplantation with PLGA microspheres, but not in hepatocyte transplantation only. More studies on comparison between sponge-type scaffolds and injectable scaffolds would be necessary. Improvement on both initial vascularization and proliferation of transplanted hepatocytes is a target of our future work.

Application of ESC-derived hepatic stem cells in therapeutic liver repopulation / 中国病理生理杂志

0.05).No teratoma was formed in the experimental group,while a large teratoma was observed in control group in 6 weeks post-transplantation.CONCLUSION:The ESC-derived hepatic stem cells are normally incorporated into mouse liver parenchymal structure,proliferate and differentiate further in vivo and possess some hepatic functions without forming teratomas.

The Effects of Intrasplenic Transplantation of Hepatocytes on Rats with Acute Liver Failure Induced by a 90% Hepatectomy

BACKGROUND: Acute liver failure either after liver resection or as part of underlying liver disease is still associated with high mortality. Various treatments have been tried to improve liver function during acute liver failure, including metabolic and nutritional support, hemodialysis, hemoperfusion, plasmapheresis, and hepatocyte and liver transplantation. Hepatocyte transplantation in various forms has attracted attention recently. We investigated whether allogeneic isolated hepatocytes transplanted in the spleen would prolong survival, facilitate liver regeneration, and improve biochemical parameters in rats with acute liver failure induced by a 90% hepatectomy. METHODS: Allogeneic male Sprague-Dawley rats were used. Group I rats (n=26) received an intrasplenic injection of 2 107 hepatocytes in 0.3 ml of Dulbecco's modified Eagle's medium (DMEM), and 1 days later acute liver failure was induced. Group II acute-liver-failure rats (n=24) received an intrasplenic injection of DMEM. The survival time was determined for 22 rats in Group I and for 20 rats in Group II. The remaining 8 (4/each group) acute liver failure rats were used to assess the liver function and regeneration. RESULTS: The survival was longer and the number of long-term survivors was greater for Group I rats than for the Group II controls. At 24 hour after the hepatectomy, Group I rats had lower ammonia, lower total bilirubin, lower activities of liver enzymes, and higher glucose levels than did Group II rats. In Group I, there was significant increase in the ratio of the weight of the remnant liver lobes to the body weight. CONCLUSION: Compared with the Control group intrasplenic hepatocyte transplantation in acute liver failure rats acts as a bridge to support experimental rats in going from acute or fulminant liver failureto liver regeneration or compensation, prolongs survival in rats with acute liver failure induced by a 90% hepatectomy, and improves the biochemical parameters, except for the albumin levels and prothrombin time. Transplantation of hepatocytes may be beneficial in supporting a liver which has been acutely devastated by a 90% hepatectomy.

Effect of Intrasplenic Transplantation of Cryopreserved Hepatocytes into Partially Hepatectomized Rats / 대한간학회지

BACKGROUND/AIMS: Various techniques of hepatocyte transplantation were actively studied as an alternative to liver transplantation, because of the difficulty of obtaining donor organ, technical difficulties, and high cost. Isolated hepatocytes could be appropriately banked and distributed on demand. We tried to investigate the effect of intrasplenic transplantation of allogenic cryopreserved hepatocytes, into spleen prior to 90% partial hepatectomy in rats, on the survival rate. METHODS: Cryopreserved hepatocytes, isolated by collagenase perfusion of the liver via the portal vein, were thawed and transplanted into the spleen of rats prior to induction of acute hepatic failure by resection of all lobes except caudate lobe (2.0x107 hepatocytes/rat). RESULTS: 1. The viability of freshly isolated hepatocyte was 70-5%, but cell viability after cryopreservation 30-0%. 2. Difference of survival in control and transplant group is not statistically significant. but the survival rate, 48 hours after 90% partial hepatectomy, for control (7) and transplanted group (11) were 0% and 18%, respectively. 3. Although the glucose reduction gradient was not significantly different between two groups, it was more prominent in the control group than in the transplanted group. 4. Engraftment and survival of transplanted hepatocytes were noted in the spleen 2 days after transplantation. CONCLUSIONS: We could not observe statistically significant improvement of survival with intrasplenic transplantation of cryopreserved hepatocytes in rats with 90% partial hepatectomy-nduced acute liver failure. However, 18% survival after 90% partial hepatectomy was noted in the transplanted group, compared to no survival in the control group. This suggests that intrasplenic transplantation of cryopreserved hepatocytes might be effective in the treatment of acute liver failure.

Biodistribution of Intrasplenically Transplanted Hepatocytes in the Rat

Liver failure still constitutes a major cause of morbidity and death in patients with liver disease. Various potential treatments have been discussed to reduce liver failure-induced deaths. Recently, interest in hepatocyte transplantation(HT) has markedly increased, and HT has been tried for the treatment of liver failure. Both experimental and clinical data indicate that HT may be beneficial both for the support of an acutely devastated liver and for the correction of genetic disorders resulting in states of metabolic deficiency. For this purpose, the location of the HT has been suggested as a critical point. Therefore, we tried to study the biodistribution of hepatocytes in acute and chronic liver failure model in rats at 2 hours and 24 hours after the injection of 125I-labeled hepatocytes into the spleen. A ninety percent partial hepatectomy model and a dimethylnitrosamine-induced liver-cirrhosis model were used as the acute and chronic liver-failure models respectively. An SV-40 T-transfected immotilized cell line named L2A2 was transplanted intrasplenically. The biodistribution of the transplanted hepatocytes was similar in both the acute and the chronic liver-failure groups. In both groups, the biodistribution studies at 2 hours and 24 hours after intrasplenic transplantation demonstrated that the hepatocytes were localized predominantly in the spleen. However, intrasplenic retention of the transplanted hepatocytes progressively decreased with time. We conclude that methods for preventing the progressive loss of intrasplenic transplanted hepatocytes should be investigated.

Construction of recombinant adenoviruses carrying human hepatocyte growth factor and investigation of its effect on human fetal hepatocytes in vitro / 中华消化杂志

Objective To construct the replication deficient recombinant adenoviruses (AdHGF) of human hepatocyte growth factor (HGF) and green fluorescent protein (GFP) cDNA derived from CMV promoter using homologous recombination in bacteria provided by AdEasy system and to further investigate the effect of HGF on human fetal hepatocyte growth. Methods The HGF cDNA was obtained from the plasmid-pUCHGF by digestion, and the shuttle plasmid-pAdTrack-CMV-HGF in which the HGF cDNA was inserted into the downstream of CMV promoter was established by ligation. Then the linearized shuttle plasmid was co-transformed into bacteria with backbone vector AdEasy-1 to obtain the recombinant adenoviral plasmid-pAdHGF by homologous recombination. After packed in 293 cells, the recombinant adenoviruses-AdHGF was generated. The expression of HGF in human fetal hepatocyte was detected by RT-PCR and the effect of AdHGF on the cell cycle of fetal hepatocyte was examined by flow cytometry. Results The recombinant plasmid pAdHGF was established by homologous recombination and confirmed by restriction endonuclease digestion and sequencing. GFP expression could be observed on the third day after packing of the linearized pAdHGF in 293 cells and 4?10 10 efu/ml titer of AdHGF was obtained by CsCl gradient purification. Three days after the human fetal hepatocytes were infected by the viruses, expression of HGF in hepatocytes increased significantly and most of the hepatocytes in G 0/G 1 stages was changed to that in S and G 2/M stages.Conclusions AdHGF can be simply and rapidly generated using AdEasy system. The infection of human fetal hepatocytes by AdHGF could result in the high expression of HGF and promote the cell regeneration. AdHGF may serve as a new tool for hepatocyte transplantation and gene therapy of hepatic fibrosis.