| Backgrounds and aimsLiver cirrhosis is a common disease and one of main death causes in clinic. At present there are no effective therapy for the disease. And it is very important for liver dysfunctional patients to improve their liver function, deal with symptoms and complications. Although orthotopic liver transplantation can almost cure liver cirrhosis, it can not be widely applied in clinic due to shortage of donor-derived liver, high expense and long period use of immunosuppressor medicines. Artificial liver just has some limited effect on improving liver cirrhosis symptoms during the terminal stage. Because physical artificial liver doesn't break away from hemodialysis and has some faultinesses in replacing liver synthesis and biotransformation, it can not acquire expected curative goal. Bioartificial liver can not be widely practiced in the clinic and hepatocytes transplantation are stagnated because of hepatocytes shortage. It is urgent to find a highly effective means to treat liver diseases or solve the problem of hepatocytes shortage now.Stem cells which are of self-renew, intrinsic versatility and its transdifferentiation provide a novel method to deal with the disease. There are two types of stem cells in bone marrow, one is hematopoietic stem cells (HSCs), another is mesenchymal stem cells (MSCs). MSCs derives from mesoblast, and has strong ability of self-renew and plasticity. Under proper conditions they can transdifferentiate into multilineage cells, including osteoblasts, neurocytes, hepatocytes. MSCs have two advantages. Firstly, it is convenient to acquire and the process do no harm to body. Secondly, autologous mesenchymal stem cells have noimmune rejection reaction. Therefore, researches of MSCs are increasingly becoming a hot issue. Achievements of stem cells in biomedical field provide a new way to treat liver diseases. Transdifferentiation of MSCs into hepatocyte-like cells which performing hepatocyte function such as systhesis and secretion is demonstrated in vivo and vitro by domestic and overseas scientists. The aim of the research is to investigate conformity of bone marrow mesenchymal cells into liver in vivo and changes of liver biochemical and fibrosis indexes in liver cirrhosis rats model by transfusing male bone marrow MSCs into female rats, and provide a new curative means for liver diseases during terminal stage or a new cell source for bioartificial liver.Materials and methods1 > Bone marrow cells were collected sterilely from healthy male Wistar rats aged about 6~8 weeks. Then MSCs were cultivated, purified and proliferated in DMEM-LG medium containing 10%FSB by adherence wall cell culture technique. At passage 3, BrdU was added into the medium,and co-cultured for 72 hours, MSCs were harvested from the culture bottle wall, analysed viability via trypan blue exclusion and transfused into female liver cirrhosis rats by tail vein.2> Healthy female Wistar rats were randomly divided into three groups: Normal group (N) which were injected with liquid paraffine intra-abdominally for 8 weeks, 0.75ml/kg, 3 times a week. Control group (C) and Experimental group (E) which were all administrated intra-abdominally with 15% carbon tetrachloride liquid paraffine for 8 weeks, 0.75ml/kg, 3 times a week.3 -, Male Wistar rats bone marrow MSCs labelled with BrdU were transfused into rats of E, N groups by tail vein, and 0.9% physiological saline was transfused into rats of C group by tail vein. Five female Wistar rats were killed by exsanguinating abdominal arteria at l/2h, 1 wk, 2wk, 3wk, 4wk after bone marrow MSCs transplantation in C, E group.4> SRY gene derived from male donor was detected by PCR amplification and BrdU antigen in liver of recipients were detected by immunohistochemistry. Serum ALT, AST > ALB and GLO were inspected by whole-automated biochemical analyzer, and AFP and liver fibrosis indexes (HA^ PCIV\ LN, PCIII) were inspected by radioimmunoassay.5, Statistic analysis: completely random design analysis of variance and the least significant difference t test were adopted to compare every index values at different time among different group at different time by SPSS 10.0 software. And significance level a is 0.05.Results1 -. Freshly isolated bone marrow MSCs had uniform size and form, and transparency was very good.2n MSCs adhered to the culture flask wall within 24 hours and adherence cell number increased in clonal manner as time went on. Spindle-or-triangle-shaped cells appeared at day 3 and reached logarithmic stage by day 10. At passage 3, MSCs fused into single layer and arranged in distinct directions so that they looked like swirl, shoal, reticulation. Vital force is more than 95% via trypan blue exclusion.3 - Liver specimens were randomly taken out after injected 15% CCU liquid paraffine intra-abdominally. They looked brown or taupe. Their size was smaller than that of normal liver and they were tougher than those of normal liver. There were diffuse asymmetrical protuberances and dent region in the surface of cirrhosis liver. Liver lobules were disappeared or destroyed and replaced by fake lobule. Hepatocytes were varicose, denaturalized, regenerated and flake necrosis, and were soaked by fatty. There were inflammatory cells in liver. Because of conjection tissue hyperplasia, collection tube region became wider significantly. There were no fibrosis in normal livers.4 ^ No SRY-positive was detected in the N, C group all the time and the E group after transfusion at l/2h. 2 of 5 cases were SRY-positive in the E group at lwk; 3 of 5 cases were SRY-positive in the E group at 2wk; and all were SRY-positive in the E group at 3wk and 4wk.5 > ALT and AST values in E group were significantly lower than those in C group at 4wk (28.0 + 3.74,84.6+36.38 VS 56. 0 + 26.66,161.2 + 55.43 , respectively P<0.05) , but werenot significantly different from those in N group. ALB values in E group were significantly higher than those in C group(30.3±2.18 VS 34.4±2.49, PO.05), but were not significantly different from those in N group. GLO values between C and E group were not significantlydifferent, but were significantly higher than those in N group. Although AFP values were not significantly different from those in N group and between the E and C group, there was a ascending trend.6^ The liver fibrosis indexes were not significantly different between C and E groups ateach time, but were significantly different between N and C, E groups.Conclusion1 > Adherence wall culture was suitable for rat bone marrow MSCs and cells obtained wereof higher viability and purity. 2^ DMEM low glucose medium containing 10% fetal bovine serum was suitable for cultureand proliferation of rat bone marrow MSCs in vitro.3 ^ It was feasible to trace growth and conformity of MSCs by labeling BrdU. 4n Liver cirrhosis model in rats could be established by injecting 15% CCI4 liquid paraffineintra-abdominally and the death rate is low (about 15%).5 > MSCs could engraft into liver after transfusion as early as 7d and localized in collectiontube region and liver sinus.6 -> The engrafted MSCs could improve liver function, but in short term it could hardly slowthe fibrosis process.
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