大鼠骨髓间充质干细胞对胶质瘤C6细胞增殖、迁移和侵袭特性的影响

发布时间：2018-03-30 21:36

本文选题：骨髓间充质干细胞　切入点：胶质瘤C6细胞　出处：《西南医科大学》2017年硕士论文

【摘要】：目的:探讨SD大鼠骨髓间充质干细胞对胶质瘤C6细胞增殖、迁移和侵袭特性的影响,为深入研究SD大鼠骨髓间充质干细胞作为胶质瘤基因治疗载体的生物安全性提供实验依据。方法:1.采用全骨髓贴壁法获取SD大鼠骨髓间充质干细胞并进行原代、传代培养和形态学观察。2.通过成脂诱导分化和成骨诱导分化鉴定SD大鼠骨髓间充质干细胞多向分化潜能,采用流式细胞仪鉴定SD大鼠骨髓间充质干细胞表面标记物。3.通过SD大鼠骨髓间充质干细胞和胶质瘤C6细胞Transwell非接触共培养获得Con、A1-3、A2-3、A3-3四组胶质瘤C6细胞用于后续实验。4.通过CCK-8试验检测OD值和平板克隆实验检测克隆数明确胶质瘤C6细胞增殖特性的改变;通过划痕实验检测迁移距离明确胶质瘤C6细胞迁移特性的改变;通过Transwell侵袭实验明确胶质瘤C6细胞侵袭特性的改变。5.通过裸鼠体内成瘤实验进一步验证SD大鼠骨髓间充质干细胞对胶质瘤C6细胞增殖特性的影响。结果:1.第三代SD大鼠骨髓间充质干细胞形态均匀,呈梭形,旋涡状生长。2.第三代SD大鼠骨髓间充质干细胞成脂诱导21天,用油红O染色脂滴呈鲜红色,圆形,位于胞浆,大的脂滴排列成串、小的脂滴排列簇。成骨诱导分化21天矿化钙结节经茜素红染色后呈橘红色。3.第三代SD大鼠骨髓间充质干流式细胞仪检测结果提示:CD29,CD90高表达,阳性率分别为95.32%和99.97%;CD34,CD45低表达,阳性率分别为0.08%和0.32%,由此验证提取细胞是SD大鼠骨髓间充质干细胞,且纯度较高。4.CCK-8实验提示在24h、48h、72h、96h Con、A1-3、A2-3、A3-3四组胶质瘤C6细胞增殖活力呈依次递减趋势(P0.001)。平板克隆实验提示Con、A1-3、A2-3、A3-3四组胶质瘤C6细胞平板克隆14天GIMSA染色后计数,克隆形成数呈依次递减(F=190.218,P0.001)。划痕实验提示Con、A1-3、A2-3、A3-3四组胶质瘤C6细胞24h迁移距离(um)呈依次递增(F=1010.726,P0.001)。Transwell侵袭实验提示Con、A1-3、A2-3、A3-3四组胶质瘤C6细胞48h侵袭率呈依次递增(F=5150.74,P0.001)。5.裸鼠体内成瘤14天提示Con、A1-3、A2-3、A3-3四组细胞形成肿瘤体积依次减小(F=90.46,P0.001)。结论:1.采用全骨髓贴壁法可获取纯度较高的SD大鼠骨髓间充质干细胞。2.SD大鼠骨髓间充质干细胞能抑制胶质瘤C6细胞体外和体内增殖能力。3.SD大鼠骨髓间充质干细胞能促进胶质瘤C6细胞的侵袭和迁移能力。
[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.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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