骨髓间充质干细胞来源exosomes对大鼠肝脏缺血再灌注损伤修复作用的实验研究

发布时间：2018-05-18 05:10

本文选题：骨髓间充质干细胞 + exosomes　；参考：《四川医科大学》2015年硕士论文

【摘要】：目的：肝脏缺血再灌注损伤(hepatic ischemia reperfusion injury,HIRI)是指肝脏因缺血、缺氧而造成功能障碍和结构损伤,恢复血供后,功能障碍和结构损伤不但不能完全恢复,反而损伤进一步加重,甚至发生不可逆性损伤的现象。HIRI常见于肝脏外科、肝移植以及低血容量性休克等过程中,尤其肝移植过程中难以避免。目前,HIRI在临床上受到广泛的关注,但对其防治还没有理想的措施。近年来研究表明骨髓间充质干细胞来源exosomes(bone-marrow mesenchymal stem cell-derived exosomes, BMSCs-exosomes)能通过传递具有生物学活性的蛋白质、脂类、mRNA及mircoRNA等物质,激活受损伤组织细胞的自我保护与内源性再生机制,同时还可调控机体免疫应答反应,参与受损伤组织细胞的修复以及对器官功能的保护作用。本研究旨在探讨BMSCs-exosomes对大鼠HIRI后肝脏组织的修复作用及可能的分子机制,为深入研究BMSCs-exosomes临床防治HIRI提供理论依据。方法：(1)无菌条件下获取大鼠骨髓间充质干细胞,进行原代细胞培养并传代纯化至3～6代,得到较为稳定的骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)。(2)收集骨髓间充质干细胞培养的上清液,采用ExoQuick-TC试剂盒分离提取培养上清液中微囊体颗粒,即BMSCs-exosomes,并采用负染透射电镜进行形态学鉴定。(3)将32只清洁级健康雄性SD大鼠随机平均分为4组(即假手术组、PBS组、BMSCs组和exosomes组),通过建立SD大鼠HIRI模型,观察各实验组大鼠肝功能指标ALT、炎症因子TNF-α、 IL-1β和IL-10的表达水平及肝组织病理学、肝细胞的凋亡情况和肝组织中凋亡基因相关蛋白bcl-2、bax表达水平的变化。结果：(1) BMSCs原代细胞经培养48 h后,镜下可见细胞逐渐贴壁,9～10 d后可见细胞形态呈梭形或多边形,14 d左右细胞贴满整个培养瓶,经传代培养3代后,其形态逐渐表现为相对均一的梭形。(2)透射电子显微镜下可见BMSCs-exosomes呈具有明显异质性的圆形或椭圆形小囊泡,直径30-100nm,包膜完整,且囊内含有低电子密度物质。(3)成功建立SD大鼠HIRI模型,各组相关指标改变如下：①血清ALT水平PBS组大鼠血清ALT显著高于假手术组,其差异有统计学意义(P0.05), BMSCs组和exosomes组血清ALT均显著低于PBS组(P0.05),而BMSCs组与exosomes组间的血清ALT的差异无统计学意义(P0.05)。②血清促炎因子TNF-α、DL-1β和抑炎因子IL-10表达水平PBS组血清TNF-α和IL-1β表达水平显著高于假手术组(P0.01), BMSCs组和exosomes组血清TNF-α和IL-1β表达水平显著低于PBS组(P0.05),而BMSCs组与exosomes组间TNF-α和IL-1p表达水平的差异无统计学意义(P0.05)；假手术组和PBS组血清IL-10水平均较低,BMSCs组和exosomes组血清IL-10水平均显著高于PBS组(P0.05),而BMSCs组与exosomes组间血清IL-10水平的差异无统计学意义(P0.05)。③肝脏组织病理学PBS组可见明显的肝细胞肿胀、部分肝细胞胞浆空泡化、肝细胞点状坏死、肝窦充血和炎性细胞浸润；而与PBS组相比,BMSCs组和exosomes组肝脏的病理损伤明显减轻,而二者之间肝脏损伤情况无明显差异。④肝细胞凋亡指数肝细胞呈现棕褐色者为TUNEL阳性细胞。PBS组肝细胞凋亡指数显著高于假手术组(P0.05)；BMSCs组和exosomes组肝细胞凋亡指数均显著低于PBS组(P0.05)；但BMSCs组和exosomes组间肝细胞凋亡指数无明显差异(P0.05)。⑤肝组织中bcl-2、bax表达水平PBS组肝组织中bcl-2的表达水平显著低于假手术组(P0.05),bax表达水平显著高于假手术组(P0.05),BMSCs组和exosomes组肝组织中bcl-2表达水平显著高于PBS组(P0.05),bax表达水平显著低于PBS组(P0.05),而BMSCs组与exosomes组间肝组织bcl-2、bax表达水平的差异无统计学意义(P0.05)。结论：本结果表明BMSCs-exosomes可通过抗凋亡机制,抑制HIRI时肝细胞凋亡；同时通过调控促炎因子与抑炎因子的平衡,减轻炎症对肝组织细胞的损伤,对大鼠肝脏缺血再灌注损伤发挥修复作用。还发现BMSCs和BMSCs-exosomes在大鼠HIRI中的修复功能是等效的,这为BMSCs-exosomes可完全替代BMSCs移植防治HIRI提供坚实的理论基础
[Abstract]:Objective: liver ischemia reperfusion injury (hepatic ischemia reperfusion injury (HIRI)) refers to the liver dysfunction and structural damage caused by ischemia and hypoxia. After the recovery of blood supply, the dysfunction and structural damage can not be completely recovered, but the damage is further aggravated, even the phenomenon of irreversible damage is common in the liver. In the process of surgery, liver transplantation and hypovolemic shock, especially liver transplantation, it is difficult to avoid it. At present, HIRI has been widely concerned in clinic, but there are no ideal measures for its prevention and control. In recent years, the study showed that bone marrow mesenchymal stem cells (exosomes) derived from bone-marrow mesenchymal stem cell-derived exosomes, BMSCs-exo Somes) can activate the mechanism of self protection and endogenous regeneration of damaged tissue cells by transferring biological active proteins, lipids, mRNA and mircoRNA, and also regulate the immune response response, repair the damaged tissue cells and protect the function of organs. This study aims to explore BMSCs-ex. The effect of osomes on the repair and possible molecular mechanism of liver tissue after HIRI in rats provides a theoretical basis for the in-depth study of BMSCs-exosomes clinical prevention and control of HIRI. Methods: (1) the rat bone marrow mesenchymal stem cells were obtained under aseptic conditions, and the primary cells were cultured and purified to 3~6 generations to obtain more stable mesenchymal stem cells (MSCs). Bone marrow mesenchymal stem cells, BMSCs). (2) collect the supernatant of bone marrow mesenchymal stem cells culture, use ExoQuick-TC reagent box to separate and extract microcapsule particles in the supernatant, that is BMSCs-exosomes, and use negative transmission electron microscope for morphological identification. (3) 32 healthy male SD rats were randomly divided into 4 groups. That is, sham operation group, group PBS, group BMSCs and group exosomes), by establishing HIRI model of SD rats, the expression level of hepatic function index ALT, inflammatory factor TNF- alpha, the expression level of IL-1 beta and IL-10, the liver histopathology, the apoptosis of hepatocytes and the changes of Bcl-2, Bax expression of apoptosis gene related proteins in liver tissues were observed. (1) After 48 h cells were cultured for 48 h, the cells were gradually adhered to the wall. After 9~10 D, the cell morphology was found to be shuttle or polygon, and 14 d cells were filled with the whole culture bottle. After the passage culture, the morphology gradually showed a relatively homogeneous shuttle shape. (2) the BMSCs-exosomes showed obvious heterogeneity under the transmission electron microscopy. Round or oval vesicles with a diameter of 30-100nm, a complete capsule and a low electron density substance in the capsule. (3) the HIRI model of SD rats was successfully established. The changes of the correlation indices of each group were as follows: (1) the serum ALT of the serum ALT level in the PBS group was significantly higher than that of the sham operation group (P0.05), and the serum ALT of the BMSCs group and the exosomes group showed a significant difference. There was no significant difference in serum ALT between group BMSCs and group exosomes (P0.05). (P0.05), there was no significant difference in serum ALT between group BMSCs and exosomes group (P0.05). The expression level of TNF- alpha and IL-1 beta in serum of serum proinflammatory factor TNF- a, DL-1 beta and anti inflammatory factors was significantly higher than that in sham operation group. There was no significant difference in the level of TNF- alpha and IL-1p expression between group BMSCs and exosomes group (P0.05), but the level of serum IL-10 in the sham operation group and the PBS group was lower than that in the BMSCs group and the exosomes group. The level of serum IL-10 in the BMSCs and exosomes groups was significantly higher than that in the BMSCs group and exosomes group, but there was no significant difference in the level of serum levels between the group and the group. (P0.05). (3) liver histopathological group PBS group showed obvious liver cell swelling, partial hepatocyte vacuolation, hepatic cell punctate necrosis, hepatic sinusoidal hyperemia and inflammatory cell infiltration, but compared with group PBS, the pathological damage of liver in group BMSCs and exosomes group was obviously reduced, and there was no significant difference between the two groups. 4. The apoptosis index of hepatocyte in group.PBS of dead index hepatocyte and TUNEL positive cell was significantly higher than that in sham operation group (P0.05), and the apoptosis index of hepatocyte in group BMSCs and exosomes group was significantly lower than that in group PBS (P0.05), but there was no significant difference between BMSCs group and exosomes group (P0.05). The expression level of Bcl-2 in the liver tissue of group PBS was significantly lower than that in sham operation group (P0.05), and the expression level of Bax was significantly higher than that of sham operation group (P0.05). The level of bcl-2 expression in the liver tissues of group BMSCs and exosomes was significantly higher than that in the PBS group (P0.05), and the level of Bax expression was significantly lower than that in the PBS group. The difference was not statistically significant (P0.05). Conclusion: this result shows that BMSCs-exosomes can inhibit the apoptosis of liver cells by anti apoptosis mechanism and reduce the damage of inflammation to liver tissue cells by regulating the balance of proinflammatory factors and anti inflammatory factors, and remediates the liver ischemia-reperfusion injury in rats. Also, BMSCs and BMSCs- are also found. The repair function of exosomes in rat HIRI is equivalent, which provides a solid theoretical foundation for BMSCs-exosomes to completely replace BMSCs transplantation to prevent HIRI.
【学位授予单位】：四川医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R657.3

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