【作者】 郝彦琴；

【导师】 赵龙凤；

【作者基本信息】 山西医科大学 ， 内科学， 2010， 博士

【摘要】 研究背景：急性肝衰竭(acute liver failure, ALF)是一种严重危害人类生命健康的临床综合征,肝移植是目前最有效的治疗肝衰竭的方法,但是,肝移植由于供体短缺及费用昂贵等问题限制了其广泛应用。因此,迫切需要探索新的治疗方法以挽救肝衰竭患者的生命。近年来许多动物及临床研究发现,骨髓间充质干细胞(bone marrow mesenchymal stem cell, BMSC)可以在体内外诱导分化为肝细胞,BMSC移植可以在体内增殖、分化为肝细胞或肝样细胞,替代受损的肝细胞,促进肝功能恢复,因此,有人已将BMSC移植用于治疗ALF并显示出具有显著的疗效。目前,对于BMSC治疗肝衰竭作用机制的研究主要集中在BMSC的分化潜能方面。然而实验研究发现,BMSC在体外诱导分化为肝细胞需要6-12天,体内分化也需要4-5天,而BMSC在移植的早期未分化为肝细胞或肝样细胞时即显示出具有显著的疗效,用干细胞分化机制不足以解释BMSC移植的这种早期应答。那么,BMSC移植早期是通过何种机制发挥作用?有研究提出,BMSC具有抗炎和免疫调节作用,这种作用是否在BMSC移植早期发挥其效应?另外,大量临床研究结果表明,ALF患者伴有的肠源性内毒素血症(Intestinal Endotoxemia, IETM)在肝衰竭的发生发展过程中起重要作用,内毒素可以直接损伤肝细胞,还可以激活下游炎症级联反应,引起大量肝细胞凋亡及坏死,导致肝衰竭。韩德五教授提出了IETM是各种原因导致肝衰竭发生的唯一的共同物质基础,并且这一理论在实验和临床研究中已经得以证实。那么：BMSC对ALF大鼠IETM究竟有何作用?其作用机制如何?是本文的研究重点。本课题首先建立一种简便、快捷的体外分离培养大鼠BMSC的方法,在此基础上观察BMSC移植治疗ALF大鼠的作用,从细胞水平及在体水平探讨其可能的机制,为临床治疗ALF提供新的思路。本研究共分三个部分：第一部分骨髓间充质干细胞的分离培养及其生物学特性的研究第二部分骨髓间充质干细胞移植对急性肝衰竭大鼠肠源性内毒素血症的影响第三部分骨髓间充质干细胞移植对急性肝衰竭大鼠肠源性内毒素血症治疗作用机制的研究第一部分骨髓间充质干细胞的分离培养及其生物学特性的研究目的建立一种简便、快速、有效的体外分离培养大鼠BMSC的方法并进行初步鉴定。方法无菌条件下取大鼠四肢长骨骨髓,在含20%胎牛血清的DMEM/F12中贴壁培养,倒置显微镜下观察细胞的形态变化；采用免疫细胞化学方法检测细胞膜抗原CD34、CD44及CD45的表达；采用流式细胞术检测细胞周期；细胞加成脂诱导剂体外培养14天后,行油红O染色,观察细胞分化结果。结果分离培养的细胞2-3天后呈多形性贴壁生长并开始大量增殖,3代以后的细胞呈均一的成纤维细胞样形态。免疫细胞化学染色显示,细胞表面分子CD44阳性,CD34、CD45阴性。细胞周期检测结果显示,(80.13±1.24)%的第3代BMSC处于G1期。细胞加成脂诱导剂后14天经油红O染色可见胞质内有橙红色的脂滴。结论采用全骨髓贴壁培养法可以方便、快捷地获得BMSC,并且在体外培养条件下能大量增殖,经诱导后可以向脂肪细胞分化,具有干细胞的生物学特性,是一种比较理想的体外培养方法。第二部分骨髓间充质干细胞移植对急性肝衰竭大鼠肠源性内毒素血症的影响实验一急性肝衰竭大鼠伴肠源性内毒素血症模型的建立目的探讨用不同剂量硫代乙酰胺(Thioacetamide, TAA)制作ALF大鼠伴IETM模型的量-效关系。方法设立3个TAA剂量组,每组10只,分别以200、400、600 mg/(kg·d)剂量的TAA灌胃,24h后相同剂量TAA重复灌胃一次,建立不同剂量TAA致大鼠IETM的动物模型,并设立对照组,以等体积0.9%氯化钠溶液灌胃。观察造模后24h、48h大鼠的死亡率,48h后剩余大鼠腹主动脉采血检测血清中丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)及血浆内毒素含量,取部分肝脏及回肠组织作常规HE染色,光镜下观察肝脏及回肠组织的病理变化。结果不同剂量TAA组大鼠在肝损伤评分、ALT、AST、内毒素水平方面与对照组相比差异均有统计学意义(P<0.05)。TAA剂量越大,死亡率越高,肝损评分、ALT、AST及内毒素水平也越高。200mg/kg TAA模型组肝坏死不明显,400mg/kg TAA模型组与600mg/kg TAA模型组有明显肝坏死,ALT、AST及内毒素水平与200mg/kg TAA组相比明显升高,差异有统计学意义(P<0.05)。不同剂量TAA模型组有不同程度的肠粘膜损伤。结论TAA致肝损伤具有明显的剂量依赖性,其中400 mg/(kg·d)TAA剂量为制备ALF伴IETM模型的适宜剂量。实验二骨髓间充质干细胞移植对急性肝衰竭大鼠内毒素及细胞因子的影响目的探讨BMSC移植对TAA所致ALF大鼠内毒素及TNF-α、IL-10的影响。方法雌性Wistar大鼠30只,体重250±10g,随机分为对照组、TAA模型组和TAA+BMSC治疗组,每组10只。采用TAA 400mg/kg灌胃,24h后重复灌胃建立ALF大鼠模型,对照组给予0.9%氯化钠溶液2m1灌胃；第二次灌胃后将BMSC(1×106个细胞/只)经尾静脉移植到TAA+BMSC治疗组大鼠体内,对照组和TAA模型组大鼠经尾静脉给予等量无菌磷酸盐缓冲液(PBS)。BMSC治疗72h后将大鼠麻醉状态下腹主动脉采血,检测血清中ALT、AST以及血浆内毒素含量,ELISA法检测血浆及肝组织匀浆中TNF-α、IL-10含量。取部分肝组织及回肠组织作常规病理检查。结果TAA模型组与对照组比较,大鼠血清ALT、AST、血浆内毒素、血浆与肝组织TNF-α、IL-10水平均显著上升(P<0.05), TNF-α/IL-10的比值明显升高。TAA+BMSC治疗组与TAA模型组相比大鼠血清ALT、AST以及血浆内毒素水平均显著降低,血浆与肝组织IL-10水平明显升高,TNF-α/IL-10的比值明显降低,差异有统计学意义(P<0.05)。TAA+BMSC治疗组与对照组相比,血浆与肝组织TNF-α/IL-10的比值无明显差异(P>0.05)。病理切片显示BMSC能够明显减轻TAA对肝组织的损害,TAA模型组及TAA+BMSC治疗组回肠组织病理显示均有不同程度损害。结论TAA造模形成的ALF大鼠伴有IETM, BMSC尾静脉移植对ALF大鼠IETM有一定的保护作用,可以调节促炎与抗炎因子达到新的平衡,这可能是BMSC移植治疗ALF的作用机制之一。第三部分骨髓间充质干细胞移植对急性肝衰竭大鼠肠源性内毒素血症治疗作用机制的研究实验一骨髓间充质干细胞对脂多糖刺激枯否细胞分泌细胞因子的影响目的探讨在体外条件下BMSC对脂多糖(LPS)刺激后大鼠肝脏枯否细胞(KC)分泌TNF-α的影响。方法采用全骨髓贴壁培养法分离、纯化大鼠BMSC,原位灌流、Percoll密度梯度离心法原代分离培养肝KC,取体外培养48h后的KC用于实验,用LPS(终浓度1μg/ml)刺激KC,BMSC加入KC中建立BMSC共培养体系,实验分为四组：①KC组；②KC+LPS组；③KC+LPS+BMSC组；④KC+LPS+BMSC培养上清组。LPS作用24h、48h、72h后收集培养上清检测培养体系中肿瘤坏死因子-α(TNF-α)的水平。结果无LPS刺激时,正常肝脏KC培养上清中可检测到少量TNF-α,经LPS刺激后KC培养上清中TNF-α含量明显上升；而与BMSC共培养时,TNF-α含量显著减少(P<0.05)；KC与BMSC培养上清共培养时,TNF-α含量与②组相比明显减少,与③组相比TNF-α含量较高,差异均有统计学意义(P<0.05)。结论BMSC可以抑制LPS刺激后大鼠肝脏KC的活化,减少TNF-α的分泌,BMSC培养上清同样可以抑制KC的活化,但作用比BMSC弱。实验二骨髓间充质干细胞移植对急性肝衰竭大鼠CD14、NF-κB的影响目的观察BMSC移植对ALF大鼠肝组织中内毒素受体CD14及核因子NF-κB表达的影响。方法30只雌性Wistar大鼠,体重250±10g,随机分为对照组、TAA模型组和TAA+BMSC治疗组,每组10只,BMSC移植治疗72h后将各组存活大鼠麻醉,腹主动脉采血后取部分肝组织备检。SABC免疫组化法检测肝组织中内毒素受体CD14及核因子NF-κB的表达。结果对照组肝组织可见少量CD14、NF-κB表达阳性的细胞。TAA模型组与对照组相比,CD14、NF-κB表达明显升高,差异有统计学意义(P<0.05)。经BMSC治疗后,CD14、NF-κB表达比TAA模型组明显降低,差异有统计学意义(P<0.05),与对照组相比,BMSC治疗组CD14、NF-κB仍然很高,差异也有统计学意义(P<0.05)。结论BMSC移植可以抑制肝组织CD14及NF-κB的表达。BMSC移植可能通过抑制内毒素受体CD14及核因子NF-κB的表达来阻断内毒素在体内发挥效应。更多还原

【Abstract】 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.更多还原