Differentiation Of Stem Cells And Therapy Of Transplanted Stem Cells Derived From Marrow For The Acute Liver Disfunction Of Rats

Human health has been threatened badly by liver disease. Liver transplantation is presently the sole curative therapy for hepatic failure. But it is highly costly complex and limited by the availability of donor livers. As an alternative , hepatocyte transplantation has been considered with the hope of assisting hepatic regeneration in acute failure,but the short life of isolated hepatocytes and limits to the transplantable cell numbers as well as the same donor problem are serious obstacles to apply approach to therapy. Hepatic oval cells are the adult hepatic stem cells,whose activation, proliferation, and differentiation have been observed under physiological conditions, mainly when the proliferation of existing hepatocytes has been inhibited by severe hepatic injury. It is very important to study the proliferation and differentiation of oval cells as well as to research regulation of proliferation and differentiation and the relationship among the cytokines. Recently, many studies have demonstrated that bone marrow cells have the potential to differentiate into hepatocytes.The isolation, purification and cultureing conditions of bone marrow stem cells were different in various studies. There are a number of factors that can induce marrow stem cells differentistion into hepatocyte. The effection of differentiation is very different, so it in necessary to optimize the differentiation conditions. Therapy of transplanted stem cells derived from marrow of mammals for acute liver injury has developed. But it is still inconsistent about the fate and function of stem cells in recipient, how can the stem cells infuse into liver parenchyma? Whether there is relationship between injection way and the fate of transplanted cells? It is still unclear about the distribution of transplanted cells in the recipient rats. Therefore, we will investigate followings in this study. 1. Studying the regulation of the cytokines for proliferation and differentiation on WB F-344 in vitro and the relationship among the cytokines; 2. Isolating mouse marrow mesenchymal stem cells and culturing for 4 weeks; optimizing the cultureing conditions; 3. Rats were treated with 2-AAF and partial hepatectomy were operated for acute liver injury; Mesenchymal stem cells were labeled with DAPI and transplanted them via the portal vein or intrahepatic; Evaluating curative effect by detecting the sera level of aminotransferase and albumin; Studying the distribution of transplanted cells in the recipient. This study aims at 1. Investigating characteristic of hepatic oval cells and their differentiation mechanisms; 2. Searching for simple diffrentiation conditions of inducing mesenchymal stem cells into hepatocytes in viro; 3. Researching the pathway about transplantation of mesenchymal stem cells and the feasibility of such therapy for acute liver injury. Methods 1. WB F-344 cells were treated with various concentrations of cytokines such as HGF , EGF , FGF, TGF-? ,IL-6 and TNF . The proliferation was detected after 24 hours by [3H] thymidine incorporation test. To detect the receptor proteins of HGF,EGF,FGF, TGF-?, IL-6,TNF by western blooting the cytoplasmic protein of WB F-344 cells were prepared. 2. WB F-344 cells differentiated into hepatocytes. WB F-344 cells were cultured in the special medium(DMEM,10%Fcs, HGF 10ng~50ng/ml, EGF 20ng/ml, Isulin 1ug/ml, dexamethasone 1μM) , at the time point of differentiation, the total RNA of cells were extracted ,and then the ALB mRNA and AFP mRNA were detected by RT-PCR. 3. Isolation and culture of mesenchymal stem cells. Bone marrow was harvested from male mice and mononuclear cells were isolated by 1.073 percoll(2000g,25min).The cells were resuspended in DMEM with 10% FBS and planted in culture plates at the concentration of 104 /ml and incubated at 37℃5%CO2. Removed culture medium after 48h and incubated continued with medium refreshed every 3-4 days. When cells proliferated and completed to 80% of plate they were passaged at the rate of 1:3. 4. Mesenchymal stem cells differentiation into hepatocyte in vitro. Cells were seeded at concentration of 5×104 in 6-well culture plates and incubated in DMEM supplemented with 10%FBS and 100ng/ml HGF. The half of medium was changed every 3 days and the cytomorphology were observed every day, and establishing controls that incubated in the same medium but without HGF. The total RNA was prepared at different time points. Detected the expression of ALB and AFP mRNA by semi-quantification RT-PCR and analyzed the expression of ALB,AFP and CK19 protein by immunohistochemistry 5. Detected glucose -6-phosphatase activity of differentiated cells The cells have been induced for 3 weeks were washed twice and added incubating solution (0.125% glucose -6-potassium phosphate, 0.2 mol/L Tris maleic acid , 2% plumbum nitrate),incubated for next 20 minutes at 37℃,washed cells twice with distilled water, added 1% (NH4)2S, whose colour were observed after 1 minute. 6. The mesenchymal stem cells were isolated from Wistar Rat and labeled with DAPI Marrow mononuclear cells were isolated by 1.073 percoll(2000g,25min)and were purified by incubation and labeled with DAPI . 7.Acute liver injury rat model were established and transplanting MSC from male donor into femal rats treated with 2-AAF or performed partial hepatectomy operation.106 mesenchymal stem cells labeled with DAPI were transplanted by portal vein or introhepatic injected into recipient. Negative controls were carried out using injected same dose of saline. 8. Blood chemistry Collected blood sample at different time points after MSC transplantation. The level of aminotransferase (ALT and AST) and albumin in the sera were measured with an autoanalyzer according to standard procedures.9. Fluorescent microscopy Liver,lung and spleen specimens were collected at different time points after transplantion and cut into 5um thick frozen sections. DAPI positive cells were observed under Fluorescent microscopy and analysised. 10. Detected SRY DNA by Nestle-PCR Genomic DNA from recipients were prepared at different time points after transplantation. SRY DNA was detected by nestle-PCR with SRY-specific primers. 11. Immunostaining 5-um frozen sections from different tissues were prepared . SRY protein was detected by immunohistochemistry.. 12. Histopathology The liver tissues were harvested at different time points and fixed in formaldehyde solution for 7 h, paraffin embedded and sectioned at 7-um, dyed with HE and were observed under microscope. Results 1.Our results show that there are HGF,EGF,FGF, TGF-? receptors expressed on the WB F-344 cells. Mitogenic responsiveness was founded to a wide variety of cytokines. HGF,EGF,FGF promoted proliferation of WB F-344 cell and the IL-6,TNF were not and TGF-? inhibited. We also found that HGF caused a dissociation of the colonies and there was a remarkable scattering of the cells. 2. Differentiation of WB F-344 cells into hepatocytes. When WB F-344 cells were cultured in the specific medium (DMEM,10%Fcs,HGF 10ng~50ng/ml,EGF 20ng/ml,Isulin 1ug/ml,Dex 1μM) , the cells could differentiated into hepatocytes. 3. Remove suspending cells from marrow mononuclear cells after incubated for 72 h. There are a few of small spindly cells adhered on bottom at this time, cells proliferated and appeared long spindly cytomorphology at 10 days. Subculture was performed 4 times .The number of cells increased 100 times and did not show any changes in cytomorphology. Cell arranged gyratly after induced for 2 weeks, but HGF groups were as same as no HGF group in morphocytology. 2 weeks later, gyrate frame more clear and appeared round-shape cells and clonized in HGF groupes, butappeared bone frame in no HGF group. After 3 weeks, round-shape cells apoptosised and total cells decreased in HGF groups, and bone frame was remarkable in no HGF groups. 4. Semi-quantification RT-PCR results indicated freshly isolated mononuclear marrow cells expressed a small quantity of AFP mRNA. In HGF groups, ALB mRNA can be detected at day 7, and increased at day 14, the maximum at day 21, reduced in following days .If induced with HGF for 2 weeks and then removed HGF , ALB mRNA was negative at 4 weeks,but with HGF for 3 weeks and then removed it , ALB mRNA was positive at 4 weeks.The AFP mRNA was positive at all times ,however it tended decrease after day 14 in HGF groups. ALB mRNA can not be detected in this study without HGF .The expression of AFP mRNA was reduced distinctly at day 14,and can not be detdcted after day 21. 5. The cells were positive for ALB and AFP immunostaining , but was negative for CK19 in HGF groups at day 21. Induced for 3 weeks with HGF, the cells expressed glucose -6-phosphatase, but did not without HGF. 6. The rats treated with 2-AAF for 10 days weight decreased remarkably and appeared hemorrhage trend. 7. In groups that were treated with 2-AAF , albuim level in serum increased in transplanted cells groups than in controls after 3 weeks and the results were resemble in portal vein transplantation and introhepatic injection .The transferase were higher in introhepatic injection groups than portal vein injection 48 hours after transplantation . In groups that were treated with 2-AAF , albuim level in sera after 3 weeks is higher by portal vein transplantation than introhepatic injection , this were not happened in partial hepatectomy groups. 8. A,DAPI positive cells were observed with fluorescent microscopy .The results indicated that relationship were not found between distribute characteristic of transplanted cells in recipient and transplantation ways. 1 week after transplantation, DAPI positive cells dispersed in liver, lung and spleen. But only found positivecells clusters in damaged liver after 3 weeks , and can not find out positive cells in other tissues.These positive cells in damaged liver resembles hepatocyte in morphology, was polygon cells ,oval-shaped nucleus . B,SRY protein positive cells can be detected using immunohistochemistry in liver, lung and spleen 1week after transplantation ,and the rate of positive cells had not significant difference in health liver and damaged liver ,but were more than in lungs and spleens.The positive cells decreased and there were fews of positive cells in health livers, lungs and spleens 3 weeks later . In damaged livers there were still positive cells and in groups treated with 2-AAF, the positive cells arranged at 3-6 cells and at 1 or 2-4 cells in hepatectomy groups 3 weeks after transplanted. C,We detected SRY DNA in livers,lungs and spleens 1 week after transplantation by nestal –PCR , but 3 weeks later only detected SRY DNA in damaged liver. The results were similar in portal vein injection and introhepatic injection. 9. Histopathology indecated 2-AAF could result in liver injury, observed punctate necrosis , cells swelled in liver tissues. Partial hepatectomy led other hepatocyte swell, fatten and fews of lymphocytes infusing. 3 week after cells transplantation histopathologic injury had mended than before. Conclusion 1. WB F-344 cells express receptors of HGF, EGF, FGF, TGF-? ; The cytokines of HGF,EGF,FGF show a proliferative responses to WB F-344 cell and TGF-? show a inhibited response to the hepatic stem like cells; 2. WB F-344 cells could differentiated into hepatocytes when the cells were cultured in the differential system(DMEM,10%Fcs,HGF 10ng~50ng/ml,EGF 20ng/ml,Isulin 1ug/ml,dexamethasone 1μM). 3. Mouses mesenchymal stem cells can be induced differentiated into cells like hepatocytes. They can expressed ALB , AFP and not CK19 when induced with HGF 100ng/ml. ALB mRNA was maximum at 3 weeks and AFP mRNA at 2 weeks4. The differentiation of mesenchymal stem cells was not reversible. It had some of hepatic functions such as glucose -6-phosphatase activity at 3 weeks. The optimal differentiation time was 2-3 weeks. 5. The rats liver injury was established when treated with 2-AAF (20mg/d /kg) for 10 days . There was a few of curative effection when donor mesenchymal stem cells were transplanted into recipient rat treated with 2-AAF, and injected by introhepatic may be better than via portal vein. 6. The curative effection was not remarkable in hepatectomy rats when mesenchymal stem cells were transplanted. 7. 1week after mesenchymal stem cells transplanted ,there were cells derived from donor in livers ,lungs and spleens of recipient., and most of transplanted cells homing 3 weeks after transplantion ,only a little of cells derived from donors located in damaged liver , some of the cells was bigger and was resemble hepatocyte in morphocytology.