Preliminary Study Of Combine Treatment To Acute Liver Failure With Traditional Chinese Medicine Effected On “clean-heat, Cool The Blood, Disintoxication And Removing Blood Stasis” And Bone Marrow Mesenchymal Stem Cells

Chapter I: Separation, purification, identification of rats` Bone marrow mesenchymal stem cellsObjective: Separation, cultivating, identifying of Bone marrow mesenchymal stem cells, measure the purity with flow cytometry.Methods: 1. Using whole marrow culture way to cultivate Bone marrow mesenchymal stem cells, BMSCs were gained from thighbones and tibias of 3-5 week aged SPF SD rats under aseptic condition. After 4min centrifuged with 1200r/min, bone marrow cells were cultured in plastic culture dishes by L-DMEM added with 10% FBS. Generationpassage was be done when 70%-80% basal areal filled with cells, passage were ended until the 4th generation of cells. 2. Chose 4th generation cells with good grow condition, digest with trypsin, collect cells and marked with monoclonal antibodies(Mc Ab), then, specific cell-surface marker expressions(CD29, CD44, CD34, CD45) were be detected by flow cytometry, which helped to identify Bone marrow mesenchymal stem cells.Results: 1. Adherent growth cells were successfully screened out, fusiform cells and polygonal cells with or without pseudopodium after passage to 4th generation, the distribution of cells was gyrate or flame-like. 2. Identification result by flow cytometry: positive rate of CD29 99.2%, positive rate of CD44 99.4%, positive rate of CD34 4.5%, positive rate of CD 45 16.9%, which matched the characteristic of BMSCs.Conclusions: Bone marrow mesenchymal stem cells can be successfully screened out by whole marrow cultivate.Chapter II: Observe the curative effect of acute liver failure rats with Combined treatment of traditional Chinese medicine and BMSCs transplantationObjective: Treat acute liver failure rats with traditional Chinese medicine combined with BMSCs transplantation, compare with only BMSCs transplantation, evaluating the therapeutic effect by liver function test, albumin, AFP, TNF-α and IL-10.Methods: 1. Mark BMSCs gained by chapter I with CM-Dil. 2. Establish acute liver failure rats model, randomly grouping to 4 groups(group A: treat with traditional Chinese medicine & BMSCs transplantation, group B: only BMSCs therapy, group C: only traditional Chinese medicine therapy, group D: the model control group), the step of stem cells transplantation surgery has did as follow:(1). Narcotize rats, shaving, disinfection and draping(2). Made a longitudinal incision on middle of epigastrium which length about 2cm, transplant BMSCs(the number of cells were about1*107 every rat) via portal vein, suture the incision and cover with sterile gauze after finished transplantation*. *Group A and B was injected BMSCs, group C and D was injected equivalent BMSCs culture medium. 3. Group A and C was treated with tradition Chinese drugs pharmaceutics “Yin Chen Si Ling” every day by intragastric administration, while group B and C was gavaged equivalent distilled water. 4. All laboratory animal were taken blood samples on 3rd and 7th day of gavage, execute rats and fetch liver tissue after 2 hours of gavage on 7th days, evaluating recovery of hepatocyte and liver function by TBil, ALT, TNF-α, IL-10, albumin, AFP and pathological section.Results: 1. Acute liver failure model were established after 48 h of D-Gal N injection, 13 rats died within 48 hours while the total amount was 60. 2. Observation of survival condition of laboratory animal: Group A: 1 died within 1 day after surgery, 1 died within 2 day after surgery; Group B: 2 died within 1day, 1died on 2nd day after surgery; Group C: 2 died within 1day, 2died on 2nd day and 2 died on 3rd day after surgery; Group D: 3 died within 1 day, 1 died on 2nd day and 1died on 3rd day.3. Serological indicator: Effective improvement can be shown on serological indicator of group A compared with other 3 groups, which also can be seen by comparison between group B and D, all comparison results have statistical significance, lesser improvement can be shown between group C and D, but which also has statistical significance. 4. Pathological section: Group A and B shown better recovery of iver structure and hepatocytes, group A was better than group B, tiny recovery can be seen in group C but did not have distinctly different compared with group D. 5. Fluorescence microscope observe results: A certain amount of marked cells can be seen in group A and B but less rate compared with sum total of all kind of cells.Conclusions: 1. Acute Liver failure rats model can be successfully created by intraperitoneal injection 1.4g/kg D-Gal N. 2. Tradition Chinese drugs pharmaceutics “Yin Chen Si Ling” and BMSCs transplantation can promote liver function recover and liver regeneration of acute liver failure rats, combined treatment of the two therapy better than only using one therapy.3. The two therapy-Traditional Chinese Medicine and BMSCs transplantation may has synergistic effect, it may achieved by adjust immune system though both two influence factor. 4. BMSCs can help liver regeneration, it may achieved by differentiation to hepatocytes or activate liver stem cells. 5. The short period curative effect of BMSCs treatment on ALF may not related with differentiation to hepatocytes to achieve the effect of liver regeneration and recover of liver function.