| The first section:the expression of CXCR4 on the surface of rat bone marrow mesenchymal stem cellsBackground:Bone marrow mesenchymal stem cells (MSCs) are attractive candidates for cellular therapy. The SDF-1α/CXCR4 biological axis consisted of the chemokine SDF-1αand its unique receptor CXCR4 plays a critical role in mediating the homing of MSCs to injured tissues.In the prevenient research we have found that :1. In the patients who have developed chronic liver function failure, increased expression of SDF-1αin the local injured liver environment along with decreased SDF-1αin the peripheral blood may create a homing gradient, which facilitates the recruitment of stem cells from the bone marrow into the circulation and then into the liver.2. The enhanced expression of CXCR4 on the surface of MSCs has been found in the patients with chronic liver failure. Therefore,to up-regulate the expression of CXCR4 on the surface of MSCs can facilitate the homing of MSCs to the injured liver,which may be a new way to treat liver failure.Objective:First,separate and cultivate the MSCs from rat bone marrow.Then, Study the influence of some intervention factors to the expression of CXCR4 on the surface of MSCs. Methods:MSCs were isolated from rat bone marrow and cultured with cell culture technique,then adipogenic differentiation identification was carried out. MSCs were stimulated with plasma from rats with acute liver failure, IL-6, LPS or TNF-alpha respectively for 48 hours. Cell-surface expression of CXCR4 was assessed by flow cytometry.Results: Rat MSCs looks fusiform under light microscope. The expression of CXCR4 of MSCs in groups stimulated with plasma from acute liver failure,TNF-α,IL-6 and LPS is 59.46±1.014%,56.34±0.89%,51.34±0.85%and 44.66±0.36%,respectively.While in common culture medium, the expression of CXCR4 is 26.76± 0.27%,which has statistical difference between four stimulated groups and control group (P<0.05). The expression of CXCR4 on the surface of MSCs stimulated with plasma from healthy rat is 27.13±0.67%. There is no statistical difference compared with control group (P>0.05).Conclusions: Rat MSCs can be succesfully isolated and cultured by whole bone marrow adherent culture. The enhanced expression of CXCR4 on the surface of MSCs has been found in the rat with acute liver failure,TNF-α,IL-6 and LPS may be important stimulating factors in this procedure.The second section:Transplantation of mesenchymal stem cell to treat rat acute liver failureBackground: Now the research of mesenchymal stem cells transplantation provides a new alternative method to treat myocardial infarction,central nerve injury,diabetes mellitus and many kinds of liver diseases. The homing efficiency of mesenchymal stem cells transplantation is a key point in the therapy. The SDF-1α/CXCR4 biological axis consisted of the chemokine SDF-1αand its unique receptor CXCR4 plays a critical role in mediating the homing of MSCs to injured tissues. However, the functionally active CXCR4 is low expressed on the surface of MSCs under the normal situation.In the first part we have found that plasma from the rat with acute liver failure can stimulated the expression of CXCR4 on Cell-surfaces.Therefore,to up-regulate the expression of CXCR4 on the surface of MSCs can facilitate the homing of MSCs to the injured liver,which may be a new way to improve the therapeutic effect of mesenchymal stem cells transplantation.Objective: First cultivate the MSCs that expressed high level of CXCR4 by the plasma from the rat with acute liver failure , and transplant the cells into the rat model of acute liver failure induced by D-gal,then compare the liver function, survival rate and the homing efficiency among the control group and the normal MSCs transplantation group . Finally,study the relationship between CXCR4 with MSCs transplantation and the homing efficiency,to improve the curative effect of MSCs transplantation to treat severe hepatitis.Methods: Establish the rat model of acute liver failure induced by D-gal.MSCs cultivated in vitro as the first part and were labeled by Brdu.38 rats with liver failure induced by D-gal were divided into three groups: Group A (MSCs with CXCR4 high expressed transplantation group):Before transplantion,MSCs was labeled by Brdu and stimulated with plasma from acute liver failure,then suspended the MSCs into 1.5ml saline at a concentration of 1×106 cells and transplanted by portal vein injection ( n=10). Group B (MSCs transplantation group): labeled bone marrow stem cells were suspended in 1.5ml saline at a concentration of 1×106 cells and transplanted by portal vein injection ( n=10). Group C( control roup):1.5ml saline were injected by portal vein(n=8). Blood samples were taken from the rats on 1d, 3d and 7d after transplantation.8 day after transplantation, survival rats were executed and the liver tissues were sectioned into slices,then used immunohistochemistry analysis to detect the BMSCs in the liver parenchyma.Results: About 92±2.3% of the cells were labeled by Brdu. Liver failure was observed in the rats induced by D-gal.The liver failure was characterized with decreaed activity,downcast,hypnody,exanimation,low response to stimulus and urinary incontinence,liver function was severely damaged,and the histological examination shows hepatocyte necrosis along with the inflammatory cell infiltrate. All the above have aproved that the establishment of the rat model of acute liver failure induced by D-gal is successful.The survival rate was about 40%.There were significantly decrease in the ALT,AST levels on 3d and 7d after transplantation in group A and B when compared to group C.Group A has a better improvement of liver function ,but with no raise of survival rate. Both A and B group could find Brdu+ cells,and the number of the disposition of Brdu+ cells in group A was much more than group B. Furthermore,the homing MSCs of group B were centered in portal vein,header area,or detained in vascellum;while the disposition of the cells of group A was more extensive,which could be found not only in portal vein,header area,but also in liver parenchyma.Conclusion: MSCs have the capacity of homing to the liver of rat with acture liver failure, which can hasten the recovery of liver function and relieve the pathological changes of hepatic tissue. The expression of CXCR4 on the surface of MSCs can be stimulated by plasma from rats with acute liver failure ,and have a stronger homing efficiency and therapeutical effect .
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