| ObjectivLiver fibrosis is the common stage of end-stage liver disease, which if not treated effectively,it will eventually developed to cirrhosis of the liver,and even lead to the death of patients.For end-stage liver disease, orthotopic liver transplantation (OLT)should be the most ideal treatment option theoretically, but the donor shortage,immune rejection after OLT and the high costs of treatment became a new social problems. Even if the updated heterotopic liver transplantation and orthotopic liver transplantation also have the same clinical problems,therefore it must seek other possible solutions.Under certain conditions, the stem cells can differentiate into hepatocyte-like cells,reverse and delay the process of liver fibrosis in a certain extent, and added liver cells which injuried in the liver fibrosis without immune rejection, provides a new ideas for the clinical end-stage liver disease treatment.In view of this,this experiment is on the basis of BMSCs isolation,culture in vitro and animal models of cirrhosis of the liver,infusion BMSCs through the tail vein to detect whether they can improved liver function, and to probe the best transplant period,provide a experimental basis for the clinical treatment of liver BMSCs.Methods1.Sixty SD rats weighing about 200～250g,set up as normal control group (n=10),give common food and potable water;2.Subcutaneous injection with 40% CCl4 solution 0.3ml/100g,take 10% alcohol as the sole drinking water,induce rat liver cirrhosis model;3.Take normal,healthy 3-weeks-old rat bone marrow,cultured MSCs followling the way of closed to the wall,passaged and amplified to obtain sufficient amount of MSC suspension for transplant;4.MSC which received from the healthy SD rats marked with DAPI,inject 1×106 MSCs through tail vein respectively,in 6 weeks,10 weeks of the liver cirrhosis modeling;5.Evaluation the survival state of the transplantation group and control group after transplanted 4 weeks later,observe the general changes in the liver and liver weight,do hepatic function tests (ALB.ALT.TBIL.),do pathological examination, observe under fluorescence microscope,to understand the integration situation in the liver of BM-MSCs;6.Collect, record data, make statistical analysis.Results 1. Successfully gainded a BM-MSCS, under the phase contrast microscope, the freshly isolated bone marrow mononuclear cell is brightness, different sizes, suspended in culture medium, the first three days after the exchange solution, we can see a small amount of short spindle-shaped, star-shaped cells, scattered adherent growth, after the adherent,cells began to divide and proliferation,then volume increased, have a appearance of fibroblast spindle-shaped;2. After 8 weeks of Subcutaneous injection of 40% CCl4 solution of the oil, rats spirit become poor,eat little, body weight decreased significantly (283±19 vs 335±16g), after Caesarean section out from the abdominal cavity find about 5ml ascites, liver slightly enlarged(13±1.69 vs 10.02±1.06g),appear dark red, the surface shows a large number of point-like necrosis. Biochemical markers compared with normal control group:ALT (336.25±6.25 vs 37.67±14.35IU/L, P<0.01), ALB (23.13±1.56 vs 33.13±1.51g/L, P<0.01), TBIL (16.14±1.28 vs 0.93+0.55μmol/L, P<0.01).HE staining showed:normal hepatic lobule structure disappeared and was replaced by pseudo-lobular, hepatic cells have a different degrees of fatty infiltration, necrosisa and have a inflammatory cell infiltration;3.MSCs which transplanted through the tail vein to the liver cirrhosis rats,6 weeks transplanted group,10 weeks transplantated group s liver function were significantly improved than the control group.ALT(97±17.35,180±19.68 vs 336.25±6.25IU/L,P<0.01),ALB(30.81±1.30,28.8±1.82 vs 23.13±1.56g/L,P<0.01), TBIL(13.42±0.82,14.91±1.18 vs 16.14±1.28μmol/L,P<0.01) (P <0.05) Compared with the control group was statistically significant, of which 6 weeks transplanted group compared with a 10-week transplantation group is better (F= 2.86, P<0.05), body weight also increased (324±17 vs 302±21g).Pathologic examination results showed:in 6 weeks group the hyperplasia of collagen fibers in the liver is reduced significantly, although pseudolobule still exist,the surrounding lobular and interlobular collagen fibers was reduction,in 10 weeks group,liver restor partially, but damage cells,hemorrhage and inflammatory infiltration wsa still visible.Conclusion1.Whole bone marrow adherent culture method is suitable for the cultivation of rat bone marrow MSCs, simple, economic,efficiently,the third-generation cells in vitro is still have a strong value-added capability to meet the migration requirements;2.Adopt to a joint of alcohol and CCl4 complex method, CCl4 and bean oil mixed into a 40% concentration, subcutaneous inject twice a week, in the first 8 weeks, in wide area of rat liver observed pseudolobule, and with a low mortality, simple, fast, efficient, etc,is suitable for the mass production;3.DAPI used to tag and track cell growth, integration is feasible;4. Bone marrow MSCs immune have a tolerance to allogeneic transplantation without immune rejection;5.MSCs transplantation can improve liver cell synthesis,metabolism,improving the biochemical characteristics of rats blood and liver histological structure, and in the early stage of liver fibrosis transplanted induced a better results. |
PDF Full Text Request