| Background:Acute liver failure is a clinical syndrome which seriously threats against life and health. Today liver transplantation still represents the only effective treatment for liver failure. However, it is not able to be widespreadly applicated in clinic due to critical shortage of donor organs and high cost issues. So it is urgent to explore a new effective feasible treatment for acute liver failure. Recent study found that bone marrow mesenchymal stem cell (BMSC) can be induced and differentiated into hepatocytes in vivo and vitro. BMSC transplantation could provide liver function support and stimulate the regeneration of hepatocytes by proliferation and differentiation. So some people applied BMSC transplantation to treat acute liver failure, which demonstrated significant therapeutic efficacy. The study on the mechanism of BMSC transplantation for acute liver failure focused on the potential differentiation ability of stem cell. The differentiation of BMSC into hepatocytes needs 6～12 days in vivo and 4～5 days in vitro. However, BMSC transplantation showed obvious effects during the early stage when BMSCs were far from differentiation into hepatocytes or hepatocyte-like cells. The theory of stem cell differentiation is still insufficient to explain the early response to acute liver failure.What's the mechanism of BMSC transplantation for early treatment of acute liver failure? Studies found that BMSC had anti-inflammatory and immune regulation functions. Dose BMSC transplantation effect in the early stage of transplantation by the function? In addition, many clinical studies showed that patients with acute liver failure with intestinal endotoxemia (IETM), which play an important role in the occurrence and development of acute liver failure. Endotoxin can directly damage hepatocytes, activate the inflammatory cascade, lead to apoptosis and necrosis of hepatocytes, and then liver failure. Professor Han DW proposed that endotoxin was common material basis of liver failure due to any causes, which has been confirmed by many experimental and clinical studies. What were the effects of BMSC on IETM due to acute liver failure and its mechanism? These were the focus of the article.This study first developed a simple, quick and effective method to culture BMSC. Then we observed the effect of BMSC transplantation on the treatment of acute liver failure and explored the possible mechanism in vivo and vitro. Our study may provide new ideas for the clinical treatment of acute liver failure.This thesis is composed of three parts:Part 1:Study of the isolation, culture and biological properties of rat bone marrow Part 1:Study of the isolation, culture and biological properties of rat bone marrow mesenchymal stem cellObjective To develop a quick and effective method of isolating and culturing rat bone marrow mesenchymal stem cell (BMSC) in vitro and to initially identificate the cultured cells.Methods The bone marrow was obtained from the four limbs of the rats under the aseptic condition and cultured in DMEM/F12 contained 20% bovine serum with conventional method. The change of morphology was observed with invert microscope. The cell member antigens CD34,CD44,CD45 were examined with immunocytochemical technique. Cell cycle was detected with flowing cytometry. The cells were induced in adipogenic inductive medium for 14 days, stained with Oil Red O after 14 days and observe the differentiation of BMSC.Results The isolated and cultured cells were attach to culture flask after 2～3 days and proliferated rapidly. The morphology of P3 BMSC was identical fibroblast-like cell. The results of immunocytochemicstry of BMSC showed that CD44 was positive and CD34,CD45 negative. (80.13±1.24)% P3 BMSC were in G1 phase. The cells appeared orange-red lipid droplets by Oil Red O staining after BMSC were cultured in adipogenic inductive medium for 14 daysConclusion BMSC can be conveniently and quickly gotten by the way of bone marrow adherent culture. The cultured cells can be induced into adipocyte and show general stem cell biological properties. The bone marrow adherent culture method might be an ideal method for isolation and cultivation of BMSC in vitro.Part 2:Effect of BMSC transplantation on IETM due to the acute liver failure ratsExperiment One:A rat model of the acute liver failure followed with Intestinal Endotoxemia induced by ThioacetamideObjective To investigate the correlation between dose and effect of thioacetamide (TAA) on rat models of the acute liver failure with Intestinal Endotoxemia.Methods The models of Intestinal Endotoxemia were induced by three different doses of TAA by twice gavage administration of TAA 200,400,600mg/(kg·d) of bodyweight respectively at same time for two days. Each group included 10 rats. The control group rats were administrated 2ml 0.9% NaCl saline gavage. The mortality of the rats in 24 hour and 48 hour were observed. The abdominal aorta blood of the living rats was taken. The serum ALT, AST, plasma endotoxin levels were examined. The liver and ileum tissue were taken out and stained by hematoxylin-eosin. The histopathological changes of liver and ileum were examined under light microscope.Results Compared with the control group, the levels of liver injure score, serum ALT, AST and plasma endotoxin in the different TAA dose groups were significantly increased (P＜0.05),which showed significantly correlation between dose and effect.200mg/kg TAA group showed little necrosis in liver histology, while 400mg/kg TAA group showed obvious necrosis. Compared with the 200mg/kg TAA group, the liver injure score,serum ALT, AST and plasma endotoxin were significantly increased in 400mg/kg TAA group (P<0.05).Conclusion The liver injure induced by TAA has obvious dose dependence.400 mg/(kg·d)TAA is an appropriate dose to manufacture acute liver failure rat models followed with IETM.Experiment Two:Effects of bone marrow mesenchymal stem cells thansplantation on endotoxin and cytokines of acute liver failure ratsObjective To evaluate the effects of BMSC thansplantation on endotoxin and cytokines of acute liver failure rats induced by TAA.Methods Thirty female Wistar rats, weighed 250+10g, were randomly assigned to control group, thioacetamide group(TAA) and TAA+BMSC group. Each group included 10 rats. The model of acute liver failure rats was induced by the gavage administration of TAA 400mg/kg in two consecutive days. Control group were administrated 2ml Saline at the same time. After the second gavage, the TAA+BMSC group were injected BMSC (1×106/rat) by tail vein while the control and TAA group were injected Sterile PBS. All rats were put to death at 72 hour after BMSC transplantation. The serum ALT, AST and plasma endotoxin (ET) levels were measured. The contents of TNF-αand IL-10 in plasma and liver tissue were measured by ELISA. The liver and ileum tissues were collected for the pathohistological evaluation.Results Compared with the control group, the levels of ALT, AST, ET, TNF-αand IL-10 were significantly increased in the TAA group (P<0.05), TNF-α/IL-10 were significantly increased (P＜0.05). Compared with the TAA group, the levels of ALT,AST,ET were significantly decreased, the level of IL-10 in plasma and liver tissue was increased, TNF-α/IL-10 were significantly decreased in the TAA+BMSC group (P<0.05). Compared with the control group, the TNF-α/IL-10 have no significant difference in the TAA+BMSC group (P>0.05). The liver tissues pathological examination showed less inflammatory damage in TAA+BMSC group than that in the TAA group. The ileal tissues pathological examination showed similar damage in TAA+BMSC group and the TAA group.Conclusion The acute liver failure rats induced by TAA followed with IETM. BMSC transplantation can protect the injured liver in the early stage of transplantation and adjust anti-inflammatory and pro-inflammatory cytokines to reach a new equilibrium. It might be one of the mechanisms of BMSC transplantation on the treatment of acute liver failure.Part 3:Mechanisms of BMSC transplantation on the acute liver failure rats with Intestinal EndotoxemiaExperiment One:Effects of BMSC on cytokines secration from Kuffer cells stimulated by endotoxinObjective To explore the effect of BMSC on TNF-αsecration from Kupffer cells (KC) stimulated by LPS in vitro.Methods Rat BMSCs were cultured by the way of bone marrow adherent culture. KC were isolated from liver by in situ perfusion with pronase and collagenase and density gradient centrifugation with Percoll, and then were cultured. The KC cultured for 48 hours were used in the experiment. The KC were stimulated by 1μg/ml LPS. The co-culture system was established by planting BMSC on the KC. Four groups were established according to the following arrangement:①KC groups;②KC+LPS;③KC+LPS+BMSC;④KC+LPS+supernatants from BMSC. The supernatants were collected for TNF-αassay after stimulating KC with LPS for 24h,48h,72h.Results There was small amounts of TNF-αin KC supernatants without stimulating by LPS. The concentration of TNF-αincreased significantly in the supernatant after KC were actived by LPS. When BMSC acted on co-cultured system, the concentration of TNF-αdramatically decreased. When BMSC culture supernatants acted on co-cultured system, the concentration of TNF-αdramatically decreased after LPS stimulation compared with KC+LPS group. However, the effect was lower than that of BMSC.Conclusion BMSC can inhabit the activation of rat KC stimulated by LPS and decrease the production of TNF-α. BMSC culture supernatants have weaker effect than BMSC.Experiment Two:Effects of BMSC transplantation on CD14, NF-κin acute liver failure ratsObjective To observe the effects of BMSC transplantation on liver tissue CD14,NF-κB in acute liver failure rats. Methods Thirty female Wistar rats, weighed 250±10g, were randomly assigned to control group, thioacetamide group(TAA) and BMSC treated group. Each group included 10 rats. All rats were put to death at 72 hour after BMSC transplantation. The liver tissues were collected for the evaluation after the abdominal aorta blood was taken. The expression of liver tissue CD14,NF-κB were measured by SABC immunohistochemistry.Results There were some expression of CD 14,NF-κB in the control group rat liver tissue. After BMSC treatment, the expression of CD14,NF-κB in the BMSC group liver tissue were significantly decreased (P＜0.05). Compared with the control group, the expression of CD14,NF-κwere significantly increased in BMSC group (P<0.05).Conclusion BMSC transplantation can inhibit the expression of CD14 and NF-κB in liver tissue. BMSC transplantation may counter the effects of endotoxin by inhibiting CD 14 and NF-κB expression. |
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