【摘要】：目的:在體外建立一種SD大鼠骨髓間充質(zhì)干細(xì)胞分離純化、培養(yǎng)擴(kuò)增、誘導(dǎo)分化及鑒定的方法,為下一步研究提供細(xì)胞基礎(chǔ)。 方法:應(yīng)用全骨髓細(xì)胞貼壁分離培養(yǎng)法分離培養(yǎng)SD大鼠骨髓間充質(zhì)干細(xì)胞,BMSCs細(xì)胞表面抗原CD44、CD90、CD105、CD34、CD45鑒定;BMSCs成脂誘導(dǎo),油紅O染色鑒定;BMSCs成骨誘導(dǎo),堿性磷酸酶活性檢測和硝酸銀染色鑒定。 結(jié)果:應(yīng)用全骨髓培養(yǎng)法分離培養(yǎng)出純度較高SD大鼠骨髓間充質(zhì)干細(xì)胞,分離后細(xì)胞活性均大于95%。流式細(xì)胞儀檢測大鼠骨髓間充質(zhì)干細(xì)胞的CD44、CD90、CD105陽性細(xì)胞表達(dá)分別為99.34%、99.34%、99.35%,CD34、CD45陰性表達(dá)分別為1.47%、1.56%;BMSCc成脂誘導(dǎo)9天,細(xì)胞形態(tài)學(xué)觀察及誘導(dǎo)油紅O染色均可見細(xì)胞內(nèi)有大量脂滴形成,BMSCc成骨誘導(dǎo)12天,細(xì)胞形態(tài)學(xué)觀察可見細(xì)胞團(tuán)塊聚集,有不透光致密影,堿性磷酸酶染色和硝酸銀染色均表達(dá)陽性。 結(jié)論:本實(shí)驗(yàn)采用的全骨髓細(xì)胞貼壁培養(yǎng)法是比較理想的骨髓間充實(shí)干細(xì)胞培養(yǎng)法,培養(yǎng)的骨髓MSCs純度高,細(xì)胞活力強(qiáng),生物學(xué)性狀穩(wěn)定,其具有在體外自我更新和增殖、多向分化潛能的能力。 背景:骨髓間充質(zhì)干細(xì)胞成骨能力下降及成脂分化能力增加在酒精性股骨頭壞死中發(fā)揮重要作用,有研究表明乙醇誘導(dǎo)骨髓間充質(zhì)干細(xì)胞凋亡并引起成骨細(xì)胞和破骨細(xì)胞數(shù)量的減少,或者是破壞兩者之間動(dòng)態(tài)平衡可能是導(dǎo)致骨質(zhì)疏松甚至股骨頭壞死的重要原因之一。但乙醇對骨髓間充質(zhì)干細(xì)胞凋亡的影響以及作用機(jī)制目前尚不十分清楚。 目的:通過觀察乙醇對大鼠骨髓間充質(zhì)干細(xì)胞凋亡、線粒體功能的影響及Bcl-2、Caspase-3表達(dá)、超氧化物歧化酶和微量丙二醛的變化了解其凋亡機(jī)制。 方法:應(yīng)用全骨髓培養(yǎng)法分離培養(yǎng)SD大鼠骨髓間質(zhì)干細(xì)胞;置于0,100,200,300,400,500,600,700,800,900 mmol/L乙醇中作用24h,MTT法進(jìn)行細(xì)胞毒性藥物實(shí)驗(yàn);置于0,100,200,300,400,500 mmol/L乙醇中作用6,12,24h,AnnexinV/ PI雙標(biāo)記法流式細(xì)胞儀檢測細(xì)胞凋亡和線粒體膜電位變化情況;置于0 ,427mmol/L乙醇中作用24h,RT-PCR法檢測與凋亡有關(guān)的基因Bcl-2和Caspase-3mRNA表達(dá)水平,同時(shí)進(jìn)行超氧化物歧化酶和微量丙二醛的測定。 結(jié)果與結(jié)論:MTT檢測結(jié)果顯示427 mmol/L是乙醇對大鼠骨髓間充質(zhì)干細(xì)胞生長的半數(shù)抑制濃度;AnnexinV/ PI檢測結(jié)果表明,與0mmol/L組比較,隨作用時(shí)間的延長與乙醇濃度的增加,骨髓間充質(zhì)干細(xì)胞凋亡率及線粒體跨膜電位的破壞水平明顯升高(P0.05)。與0mmol/L組比較,乙醇作用24 h后427 mmol/L組Bcl-2 mRNA表達(dá)水平下降,Caspase-3 mRNA表達(dá)水平增加,超氧化物歧化酶降低和微量丙二醛升高(P0.05)。說明乙醇能誘導(dǎo)大鼠骨髓間充質(zhì)干細(xì)胞凋亡,凋亡的發(fā)生可能與線粒體膜電位破壞、線粒體功能障礙、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關(guān)。
[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).
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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