乙醇诱导大鼠骨髓间充质干细胞凋亡机制的研究

发布时间：2018-11-18 21:01

【摘要】：目的:在体外建立一种SD大鼠骨髓间充质干细胞分离纯化、培养扩增、诱导分化及鉴定的方法,为下一步研究提供细胞基础。方法:应用全骨髓细胞贴壁分离培养法分离培养SD大鼠骨髓间充质干细胞,BMSCs细胞表面抗原CD44、CD90、CD105、CD34、CD45鉴定;BMSCs成脂诱导,油红O染色鉴定;BMSCs成骨诱导,碱性磷酸酶活性检测和硝酸银染色鉴定。结果:应用全骨髓培养法分离培养出纯度较高SD大鼠骨髓间充质干细胞,分离后细胞活性均大于95%。流式细胞仪检测大鼠骨髓间充质干细胞的CD44、CD90、CD105阳性细胞表达分别为99.34%、99.34%、99.35%,CD34、CD45阴性表达分别为1.47%、1.56%;BMSCc成脂诱导9天,细胞形态学观察及诱导油红O染色均可见细胞内有大量脂滴形成,BMSCc成骨诱导12天,细胞形态学观察可见细胞团块聚集,有不透光致密影,碱性磷酸酶染色和硝酸银染色均表达阳性。结论:本实验采用的全骨髓细胞贴壁培养法是比较理想的骨髓间充实干细胞培养法,培养的骨髓MSCs纯度高,细胞活力强,生物学性状稳定,其具有在体外自我更新和增殖、多向分化潜能的能力。背景:骨髓间充质干细胞成骨能力下降及成脂分化能力增加在酒精性股骨头坏死中发挥重要作用,有研究表明乙醇诱导骨髓间充质干细胞凋亡并引起成骨细胞和破骨细胞数量的减少,或者是破坏两者之间动态平衡可能是导致骨质疏松甚至股骨头坏死的重要原因之一。但乙醇对骨髓间充质干细胞凋亡的影响以及作用机制目前尚不十分清楚。目的:通过观察乙醇对大鼠骨髓间充质干细胞凋亡、线粒体功能的影响及Bcl-2、Caspase-3表达、超氧化物歧化酶和微量丙二醛的变化了解其凋亡机制。方法:应用全骨髓培养法分离培养SD大鼠骨髓间质干细胞;置于0,100,200,300,400,500,600,700,800,900 mmol/L乙醇中作用24h,MTT法进行细胞毒性药物实验;置于0,100,200,300,400,500 mmol/L乙醇中作用6,12,24h,AnnexinV/ PI双标记法流式细胞仪检测细胞凋亡和线粒体膜电位变化情况;置于0 ,427mmol/L乙醇中作用24h,RT-PCR法检测与凋亡有关的基因Bcl-2和Caspase-3mRNA表达水平,同时进行超氧化物歧化酶和微量丙二醛的测定。结果与结论:MTT检测结果显示427 mmol/L是乙醇对大鼠骨髓间充质干细胞生长的半数抑制浓度;AnnexinV/ PI检测结果表明,与0mmol/L组比较,随作用时间的延长与乙醇浓度的增加,骨髓间充质干细胞凋亡率及线粒体跨膜电位的破坏水平明显升高(P0.05)。与0mmol/L组比较,乙醇作用24 h后427 mmol/L组Bcl-2 mRNA表达水平下降,Caspase-3 mRNA表达水平增加,超氧化物歧化酶降低和微量丙二醛升高(P0.05)。说明乙醇能诱导大鼠骨髓间充质干细胞凋亡,凋亡的发生可能与线粒体膜电位破坏、线粒体功能障碍、Bcl-2和Caspase-3激活、超氧化物歧化酶降低和微量丙二醛升高有关。
[Abstract]:Aim: to establish a method of isolation, culture, amplification, differentiation and identification of SD rat bone marrow mesenchymal stem cells in vitro. Methods: bone marrow mesenchymal stem cells (BMSCs) of SD rats were isolated and cultured by the method of whole bone marrow adherent isolation and culture, the surface antigen of BMSCs cells was identified by CD44,CD90,CD105,CD34,CD45, lipogenic induction by BMSCs, and identification by oil red O staining. BMSCs osteogenic induction, alkaline phosphatase activity and silver nitrate staining. Results: bone marrow mesenchymal stem cells of high purity SD rats were isolated by whole bone marrow culture method. The expression of CD44,CD90,CD105 positive cells in rat bone marrow mesenchymal stem cells was 99.34 ~ 99.34 and 99.35 ~ 99.35 respectively. The negative expression of CD34 + CD45 in bone marrow mesenchymal stem cells was 1.4747 ~ 1.56 respectively. After 9 days of adipogenic induction by BMSCc, a large number of lipid droplets were observed in cells by morphological observation and oil red O staining. After 12 days of osteogenesis induced by BMSCc, the aggregation of cell mass and opacity and dense shadow were observed in cell morphology. Alkaline phosphatase staining and silver nitrate staining were positive. Conclusion: the whole bone marrow cell adherent culture method is an ideal method for bone marrow stem cell culture. The cultured bone marrow MSCs has high purity, strong cell viability, stable biological properties, and has self-renewal and proliferation in vitro. The ability to differentiate potential in multiple ways. Background: bone marrow mesenchymal stem cells (BMSCs) play an important role in alcoholic osteonecrosis of femoral head. Some studies have shown that ethanol induces apoptosis of bone marrow mesenchymal stem cells and reduces the number of osteoblasts and osteoclasts or may be one of the important reasons leading to osteoporosis and even osteonecrosis of femoral head. However, the effect of ethanol on the apoptosis of bone marrow mesenchymal stem cells and its mechanism are still unclear. Aim: to investigate the effect of ethanol on apoptosis, mitochondrial function and Bcl-2,Caspase-3 expression of rat bone marrow mesenchymal stem cells (BMSCs), and the changes of superoxide dismutase (SOD) and malondialdehyde (MDA) in rat bone marrow mesenchymal stem cells (BMSCs). Methods: bone marrow mesenchymal stem cells of SD rats were isolated and cultured by whole bone marrow culture method. The apoptosis and mitochondrial membrane potential were detected by flow cytometry with Annexin V / PI double labeling method in 0100200300400500 mmol/L ethanol. The expression levels of Bcl-2 and Caspase-3mRNA, superoxide dismutase (SOD) and malondialdehyde (MDA) were detected by RT-PCR in 0, 427mmol/L ethanol for 24 h. Results and conclusion: the results of MTT showed that 427 mmol/L was the half inhibitory concentration of ethanol on the growth of rat bone marrow mesenchymal stem cells. The results of AnnexinV/ PI showed that the apoptosis rate of bone marrow mesenchymal stem cells and the damage level of mitochondrial transmembrane potential were significantly increased with the prolongation of time and the increase of ethanol concentration compared with 0mmol/L group (P0.05). Compared with 0mmol/L group, the Bcl-2 mRNA expression, Caspase-3 mRNA expression, superoxide dismutase (SOD) and malondialdehyde (MDA) increased in 427 mmol/L group after ethanol treatment for 24 h (P0.05). The results suggested that ethanol could induce apoptosis of rat bone marrow mesenchymal stem cells, which might be related to mitochondrial membrane potential damage, mitochondrial dysfunction, activation of Bcl-2 and Caspase-3, decrease of superoxide dismutase (SOD) and increase of malondialdehyde (MDA).
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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