【作者】 陈露；

【导师】 项春生；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2017， 博士

【摘要】 宫血干细胞(Menstrual blood derived mesenchymal stem cell,MenSCs)是一种新型来源的成体间充质干细胞,它来源于女性的月宫血,拥有获得方便,安全无伦理问题等优点。外泌体(exosomes)是一类可由不同细胞产生的直径在30-100nm之间的双层脂质的囊胞,其中包含有蛋白质和RNA等物质。本研究重点阐述MenSCs来源的exosomes在体内和体外对D-GalN/LPS诱导的急性肝衰竭的治疗效果,以及小鼠肝脏细胞AML12条件培养上清(AML12-CM)体外诱导MenSCs分化为肝脏样细胞的研究。首先,我们在体内和体外证实了 MenSCs来源的exosomes对D-GalN/LP诱导的急性肝衰竭的治疗效果。在成功提取MenSCs来源的exosomes后,鉴定其表达exosomes相关标记蛋白如CD63和TSG101。在体内和体外示踪实验中,exosomes经染色后,可以在小鼠模型肝脏内或共培养的肝脏细胞中观察到。MenSCs和MenSCs来源的exosomes的抗体芯片结果显示:exosomes含有较高含量的ICAM-1,angiopoietin-2,Axl,angiogenin,IGFBP-6,osteoprotegerin,IL-6 和 IL-8 等蛋白。在细胞实验中,MenSCs来源的exosomes可以抑制由D-GalN/LPS诱导的小鼠肝细胞系AML12的凋亡。体内实验结果显示MenSCs来源的exosomes注射到D-GalN/LPS诱导的急性肝衰竭小鼠模型体内后,可以显著抑制小鼠血清中ALT和AST的升高、抑制炎症因子(IL-6,IL-1β和TNF-a)的升高,并抑制肝脏细胞的凋亡。我们推测并证实其机制是通过抑制凋亡因子(caspase-3)的表达,以及阻止小鼠肝脏中炎症细胞(NK细胞和CD11b+细胞)在肝脏中聚集而发挥作用。其次,我们研究了肝细胞系AML12细胞培养上清体外诱导MenSCs分化为肝细胞样细胞。诱导分化21天后,细胞逐渐发生胞质浓缩形成上皮样细胞,表达ALB、AFP、CK-18、CK-19、HNF-4a和CAT-1等肝细胞的特异性基因,以及表达ALB和AFP肝细胞特异性蛋白。功能实验显示诱导分化后的肝细胞样细胞具有糖原储存,ICG摄取和分泌以及尿素合成能力。综上,我们通过D-GalN/LPS诱导的急性肝衰竭小鼠模型和体外共培养实验初步证实了 MenSCs来源的exosomes可以用于治疗急性肝衰竭。此外,MenSCs在条件培养诱导下可以分化为具有成熟肝细胞功能的肝细胞样细胞,可用于急性肝衰竭缓解期的后续治疗。总而言之,MenSCs和其来源的exosomes可以作为一种治疗急性肝衰竭的可选性方案,为临床应用提高理论依据。更多还原

【Abstract】 Human menstrual blood-derived mesenchymal stem cells(MenSCs)are a novel source of MSCs that provide the advantage of being easy to collect,safety and no ethical concerns.Exosomes are bi-lipid membrane vesicles that have a diameter of 30-100 nm and are secreted by various cell types.Exosomes carry a complex cargo load of proteins and RNAs.This paper aimed to investigate the therapeutic potential of MenSCs derived exosomes on D-GalN/LPS-induced AML12 cells(in vitro)and fulminant hepatic failure mice(in vivo),and MenSCs were induced to transform into hepatic in vitro under induction of the conditioned medium of AML12(AML12-CM).First,we explored the therapeutic effects of MenSCs derived exosomes in vivo and in vitro on D-GalN/LP-induced fulminant hepatic failure.We found that MenSCs derived exosomes were round membranous vesicles with diameters between 30-100 nm,expressing exosome-related marker proteins such as CD63 and TSG101.The tracing of the exosomes showed that exosomes can be entered into the liver cells in vivo and in vitro.Cytokine arrays have shown that MenSCs derived exosomes expressed cytokines,including ICAM-1,angiopoietin-2,Ax1,angiogenin,IGFBP-6,osteoprotegerin,IL-6 and IL-8.MenSCs derived exosomes were found to inhibit the apoptosis of AML12 which were induced by D-GalN/LPS in vitro.The results of in vivo experiments showed that the injection of MenSCs derived exosomes could significantly decrease the levels of ALT and AST in serum and inhibit the expression of IL-6,IL-1β And TNF-α of the liver in the D-GalN/LPS-induced fulminant hepatic failure mice,which finally inhibited the apoptosis of liver cells.Inhibition of the expression of apoptotic factors(caspase-3)and to prevent the accumulation of inflammatory cells(NK and CDllb+)in the liver of the D-GalN/LPS-induced fulminant hepatic failure mice might be the mechanism.It is concluded that the MenSCs derived exosomes can play a therapeutic role by inhibiting the apoptosis of liver cells induced by D-GalN/LPS in fulminant hepatic failure.Second,we studied the differentiation of MenSCs into human hepatocellular cells in vitro under induction of AML12-CM.The cells after induction could expresse ALB,AFP,CK-18,CK-19,HNF-4a and CAT-1,the specific genes expressed in liver cells,which were significantly higher than those in the control group.Immunofluorescence of the differentiated cells showed that they can express the specific protein of liver cells,such as ALB and AFP.Functional experiments showed that the differentiated cells had the ability of glycogen storage,ICG uptake and secretion,and urea synthesis.This suggests that AML12-CM can induce MenSCs into hepatocellular-like cells.In summary,we demonstrate that MenSCs derived exosomes has the effect of treating D-GalN/LPS-induced fulminant hepatic failure model in vivo and in vitro.MenSCs can be differentiated into hepatocellular-like cells which have the function of mature hepatocellular cells under the induction of AML12-CM.In summary,MenSCs derived exosomes can be used as an alternative method to treat fulminant hepatic failure,laying the foundation for future clinical applications of MenSCs derived exosomes in the fulminant hepatic failure.更多还原