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In Vivo Induced Differentiation of Bone Mesenchymal Stem Cells into Hepatocytes and the Influence on Hepatic Function after Transplantation

Author: LuoYanBo
Tutor: ChenBin
School: Southern Medical University,
Course: Surgery
Keywords: Bone mesenchymal stem cells Biological characteristics Cell culture Bone mesenchymal stem cells Lentiviral vector Transfection Cellular transplantation Hepatocyte Differentiation Lentivirus
CLC: R329
Type: Master's thesis
Year: 2009
Downloads: 122
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Abstract

Bone mesenchymal stem cells' dissertation">Bone mesenchymal stem cells are thought to be pluripotent cells that can be differentiated into a variety of cell or tissue types and can be ideal resources of transplantation therapy.Recently accumulating evidence indicates that bone mesenchymal cells that can diferentiate into hepatocyte.It is a very good prospects that BMSCs transplantation be used in the treatment of acute and chronic liver failure. However,little is known about the mechanism of bone mesenchymal stem cells diferentiation into hepatocytes.It is important to determine suitable culture conditions in which bone mesenchymal stem cells will be differentiated into hepatocytes not only for understanding diferentiation mechanisms but also for eficient amplification of hepatocyte-progenitor cells of bone marrow origin,this being a prerequisite for potential therapeutic use.Recently accumulating evidence indicates that bone mesenchymal cells that can diferentiate into hepatocyte under the condition of hepatocyte growth factor in vitro,and can release the various hepatocyte factors.But whether transplanted bone mesenchymal stem cells can repopulate hepatic and biliary duct epithelia in vivo is rarely reported.For this reason,in the present,we study to observe whether bone mesenchymal stem cells have the potential to transdifferentiate into functional hepatocyte-like cells in the special niche as well as the therapeutic feasibility to repair damaged liver after transplantation in rats. PartⅠApproaches to isolate,culture and identify the bone marrowderived mesenchymal stem cells of ratObjective To establish a method for isolation and cultivation of bone mesenchymal stem cells from rat in vitro and explore their biological properties in order to provide an experimental basis for applying BMSCs to achieve the therapeutic benefit as seed cells in clinical diseases.Methods BMSCs were isolated from femur in the rat by the density gradient centrifugation with Percoll solution and purified by the characteristic of the tissue culture plastic.Cell surface antigens were detected through flow cytometer.The configuration was observed under phase - contrast microscope consecutively and the growth curve of cells was drew.Results BMSCs were very purified by density gradient centrifugation and adherent culture.The subcultured cells showed active proliferative ability.These cultured cells showed immunoreactivity to CD29 and CD44 but not CD34 and CD45.Conclusion BMSCs can be cultured and purified in vitro by density gradient centrifugation and adherent culture.BMSCs showed stable and rapid growth in vitro. All the characteristics make it possible to BMSCs to be the seed cells for tissue engineering.PartⅡLentiviral transfection of mesenchymal stem cells derived from rat’s bone marrow and its biological characteristicsObjective To isolate and culture bone mesenchymal stern cells derived from rat’s bone marrow and transfected the cells by lentiviral vectors.To provide an experimental basis which will play a role in researching on the function of BMSCs in gene engineering.Methods BMSCs were isolated from femur in the rat by the density gradient centrifugation with Percoll solution and purified by the characteristic of the tissue culture plastic.The BMSCs were transfected by lentiviral vectors using polybrene. Results 72h after transfection,the fluorescent intensity of GFP was found in the third generation bone mesenchymal stem cells under confocal microscopy,96h after transfection,the higher fluorescent intensity of GFP was found.One month after transfection,we can still see fluorescent intensity of GFP,but it is weaken.In certain viral plural number scope,the lentiviral virus advances to the bone mesenchymal stem cell transfection efficiency along with the viral plural number increase.When the multiply of infection is five,the transfection efficiency will reach to 80%.when the viral plural number surpasses certain scope,the transfection efficiency decreases.The growth curve have not obvious changed between transfected and not transfected bone mesenchymal stem cells.Conclusion Lentivirus/GFP was transfected into bone mesenchymal stem cells of rats,the expression of GFP was efficiency and stable,which will play a role in researching on the function of BMSCs in gene engineering.PartⅢTransplanted bone mesenchymal stem cells influence on hepatic functionObjective To observe whether bone mesenchymal stem cells have the potential to transdifferentiate into functional hepatocyte-like cells in the special niche as well as the therapeutic feasibility to repair damaged liver after transplantation in rats.Methods BMSCs were isolated from femur in the rat by the density gradient centrifugation with Percoll solution and purified by the characteristic of the tissue culture plastic.The third generation of BMSCs,after transfected by lentiviral vectors, were injected into portal vein of liver resection of rats.At the 7th,14th and 28th day after transplantation,liver function indexes were inspected by automated biochemical analyzer.At the 28th day after transplantation,liver samples were collected.The distribution of BMSCs in the liver were observed by fluorescence.Results After continuously feeded with 2-AAF 6 day,rats appeared to the energetic dispirited,lose of appetite,few activitied,deep yellow urine.The liver was hyperemia and enlarger,the edge of liver was obtuse,small particles was seen on the surface of liver,seen the small bleeding point in the partner.The hepatocyte was swelling,the cytoplasm was osteoporosis.All experimental animals survived after hepatectomy.The hepatic function gradually restored with the time.After 28 days,the BMSCs transplanted group’s liver function restored normally,but the DMEM/F 12 transplanted group did not.The BMSCs were more effective to promote the restoration of hepatic function than the DMEM/F12.BMSCs can successfully reside and multiplied in the damage of liver.BMSCs mainly collected around the district deparment of the surrouding,also distributed in the parenchyma of liver.The hepatic lobule structure is normal,hepatic cord arrangemented rule in the BMSCs transplanted group with HE stain.The hepatic cord arrangemented disorder,hepatic sinusoid expanded,narrowed or disappesr in the DMEM/F12 transplanted group with HE stain.Conclusion BMSCs transplanted through portal vein can successfully reside and multiplied in the damage of liver.Transplantation of BMSCs might amend the damaged tissue of host liver to a certain extent.Through above the experimental study,we’ve grasped experimental skills,such as cell culture,abstraction and separation stem cell.BMSCs have the biological characteristics of extremely self-renew and multiplex differentiation potential;Lentivirus/GFP was transfected into bone mesenchymal stem cells,the expression of GFP was efficiency and stable,which will play a role in researching on the function of BMSCs in gene engineering.BMSCs transplanted through portal vein can successfully reside and multiplied in the damage of liver,transplantation of BMSCs might amend the damaged tissue of host liver to a certain extent.

In Vivo Induced Differentiation of Bone Mesenchymal Stem Cells into Hepatocytes and the Influence on Hepatic Function after Transplantation

Abstract

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