發(fā)布時(shí)間：2018-03-30 21:36

  本文選題：骨髓間充質(zhì)干細(xì)胞　切入點(diǎn)：膠質(zhì)瘤C6細(xì)胞　出處：《西南醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:探討SD大鼠骨髓間充質(zhì)干細(xì)胞對膠質(zhì)瘤C6細(xì)胞增殖、遷移和侵襲特性的影響,為深入研究SD大鼠骨髓間充質(zhì)干細(xì)胞作為膠質(zhì)瘤基因治療載體的生物安全性提供實(shí)驗(yàn)依據(jù)。方法:1.采用全骨髓貼壁法獲取SD大鼠骨髓間充質(zhì)干細(xì)胞并進(jìn)行原代、傳代培養(yǎng)和形態(tài)學(xué)觀察。2.通過成脂誘導(dǎo)分化和成骨誘導(dǎo)分化鑒定SD大鼠骨髓間充質(zhì)干細(xì)胞多向分化潛能,采用流式細(xì)胞儀鑒定SD大鼠骨髓間充質(zhì)干細(xì)胞表面標(biāo)記物。3.通過SD大鼠骨髓間充質(zhì)干細(xì)胞和膠質(zhì)瘤C6細(xì)胞Transwell非接觸共培養(yǎng)獲得Con、A1-3、A2-3、A3-3四組膠質(zhì)瘤C6細(xì)胞用于后續(xù)實(shí)驗(yàn)。4.通過CCK-8試驗(yàn)檢測OD值和平板克隆實(shí)驗(yàn)檢測克隆數(shù)明確膠質(zhì)瘤C6細(xì)胞增殖特性的改變;通過劃痕實(shí)驗(yàn)檢測遷移距離明確膠質(zhì)瘤C6細(xì)胞遷移特性的改變;通過Transwell侵襲實(shí)驗(yàn)明確膠質(zhì)瘤C6細(xì)胞侵襲特性的改變。5.通過裸鼠體內(nèi)成瘤實(shí)驗(yàn)進(jìn)一步驗(yàn)證SD大鼠骨髓間充質(zhì)干細(xì)胞對膠質(zhì)瘤C6細(xì)胞增殖特性的影響。結(jié)果:1.第三代SD大鼠骨髓間充質(zhì)干細(xì)胞形態(tài)均勻,呈梭形,旋渦狀生長。2.第三代SD大鼠骨髓間充質(zhì)干細(xì)胞成脂誘導(dǎo)21天,用油紅O染色脂滴呈鮮紅色,圓形,位于胞漿,大的脂滴排列成串、小的脂滴排列簇。成骨誘導(dǎo)分化21天礦化鈣結(jié)節(jié)經(jīng)茜素紅染色后呈橘紅色。3.第三代SD大鼠骨髓間充質(zhì)干流式細(xì)胞儀檢測結(jié)果提示:CD29,CD90高表達(dá),陽性率分別為95.32%和99.97%;CD34,CD45低表達(dá),陽性率分別為0.08%和0.32%,由此驗(yàn)證提取細(xì)胞是SD大鼠骨髓間充質(zhì)干細(xì)胞,且純度較高。4.CCK-8實(shí)驗(yàn)提示在24h、48h、72h、96h Con、A1-3、A2-3、A3-3四組膠質(zhì)瘤C6細(xì)胞增殖活力呈依次遞減趨勢(P0.001)。平板克隆實(shí)驗(yàn)提示Con、A1-3、A2-3、A3-3四組膠質(zhì)瘤C6細(xì)胞平板克隆14天GIMSA染色后計(jì)數(shù),克隆形成數(shù)呈依次遞減(F=190.218,P0.001)。劃痕實(shí)驗(yàn)提示Con、A1-3、A2-3、A3-3四組膠質(zhì)瘤C6細(xì)胞24h遷移距離(um)呈依次遞增(F=1010.726,P0.001)。Transwell侵襲實(shí)驗(yàn)提示Con、A1-3、A2-3、A3-3四組膠質(zhì)瘤C6細(xì)胞48h侵襲率呈依次遞增(F=5150.74,P0.001)。5.裸鼠體內(nèi)成瘤14天提示Con、A1-3、A2-3、A3-3四組細(xì)胞形成腫瘤體積依次減小(F=90.46,P0.001)。結(jié)論:1.采用全骨髓貼壁法可獲取純度較高的SD大鼠骨髓間充質(zhì)干細(xì)胞。2.SD大鼠骨髓間充質(zhì)干細(xì)胞能抑制膠質(zhì)瘤C6細(xì)胞體外和體內(nèi)增殖能力。3.SD大鼠骨髓間充質(zhì)干細(xì)胞能促進(jìn)膠質(zhì)瘤C6細(xì)胞的侵襲和遷移能力。
[Abstract]:Aim: to investigate the effects of bone marrow mesenchymal stem cells (BMSCs) on the proliferation, migration and invasion of glioma C6 cells in SD rats, and to provide experimental evidence for the further study of the biological safety of SD rat bone marrow mesenchymal stem cells (BMSCs) as gene therapy vectors for glioma.Method 1: 1.Bone marrow mesenchymal stem cells (BMSCs) of SD rats were obtained by whole bone marrow adherent method.The multidirectional differentiation potential of bone marrow mesenchymal stem cells of SD rats was identified by adipogenic differentiation and osteogenic differentiation. The surface marker of bone marrow mesenchymal stem cells of SD rats was identified by flow cytometry.Four groups of C6 glioma C6 cells were obtained by Transwell non-contact co-culture of SD rat bone marrow mesenchymal stem cells and glioma C6 cells.The OD value of glioma C6 cells was detected by CCK-8 assay and the number of clones was detected by plate cloning assay, and the change of migration distance of C6 glioma cells was determined by scratch test.The change of invasion characteristics of glioma C6 cells was determined by Transwell invasion assay.The effect of bone marrow mesenchymal stem cells (BMSCs) on the proliferation of glioma C6 cells was further verified by tumorigenesis in nude mice.The result is 1: 1.The third generation SD rat bone marrow mesenchymal stem cells were uniform in shape, spindle-shaped and vortex growth.The third generation SD rat bone marrow mesenchymal stem cells (BMSCs) were induced to lipids for 21 days. The lipid droplets stained by oil red O were bright red and round, located in the cytoplasm. The large lipid droplets were arranged in strings and the small lipid droplets were arranged in clusters.After 21 days of osteogenic differentiation, mineralized calcium nodules were stained with alizarin red.The results of stem flow cytometry of bone marrow mesenchymal mesenchymal cells of the third generation SD rats showed that the positive rates of CD29 and CD90 were 95.32% and 99.97%, respectively, and the positive rates were 0.08% and 0.32%, respectively. It was confirmed that the extracted cells were bone marrow mesenchymal stem cells of SD rats.The results of CCK-8 experiment indicated that the proliferation activity of C6 cells in four groups of C6 glioma cells showed a decreasing trend in turn at 24 h, 48 h, 72 h and 96 h, and the proliferation activity of C6 cells in the four groups showed a decreasing trend in turn (P 0.001).The results of plate cloning showed that the number of clone formation of C6 glioma cells was decreased after 14 days GIMSA staining, and the number of clones was decreased in order of FG 190.218U P0.001C ~ (3). The results showed that C6 glioma cells in four groups of C6 glioma cells were stained with GIMSA for 14 days and the number of clones was decreased.14 days after tumorigenesis in nude mice, it was suggested that the tumor volume of the four groups was decreased in turn.Conclusion 1.High purity SD rat bone marrow mesenchymal stem cells. 2. SD rat bone marrow mesenchymal stem cells can inhibit the proliferation of glioma C6 cells in vitro and in vivo. 3. SD rat bone marrow mesenchymal stem cells can promoteInvasion and migration of glioma C6 cells.
【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.41

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