DADS对RhoGDI2高表达MGC803细胞迁移侵袭的影响

发布时间：2018-11-10 08:02

【摘要】：目的:我们前期实验表明,二烯丙基二硫(diallyl disulfide,DADS)通过阻断Rac1-Pak1/Rock通路下调LIMK1抑制人胃癌MGC803细胞的迁移与侵袭。近来蛋白组学发现,DADS可下调Rho GDI2蛋白。本研究在前期研究基础上,通过构建Rho GDI2稳定高表达MGC803细胞系,探讨DADS对Rho GDI2高表达MGC803细胞生物学行为的影响。方法:将p IRES2-EGFP-Rho GDI2真核表达载体转染MGC803细胞,构建Rho GDI2稳定高表达MGC803细胞系。Western blot验证Rho GDI2蛋白的表达水平。划痕实验与Transwell侵袭实验检测DADS对Rho GDI2高表达MGC803细胞迁移侵袭的影响。裸鼠成瘤实验检测DADS对Rho GDI2高表达MGC803细胞移植瘤生长的影响。结果:1.成功建立Rho GDI2稳定高表达的MGC803细胞系。两组转染细胞(Rho GDI2/MGC803与p IRES2-EGFP/MGC803)均可见绿色荧光。Western blot显示,Rho GDI2/MGC803细胞中Rho GDI2的蛋白表达量均明显高于p IRES2-EGFP/MGC803及MGC803细胞,证明成功构建Rho GDI2稳定高表达的MGC803细胞系。2.DADS对Rho GDI2高表达MGC803细胞迁移的影响。划痕实验显示,Rho GDI2/MGC803组的划痕距离小于p IRES2-EGFP/MGC803组和MGC803组(P0.05),表明Rho GDI2高表达促进MGC803细胞的迁移。DADS处理24h的MGC803组、空载体组、Rho GDI2高表达组的划痕距离明显分别大于相对应的DADS未处理组(P0.05),表明DADS抑制MGC803、Rho GDI2/MGC803的迁移。3.DADS对Rho GDI2高表达MGC803细胞侵袭的影响。侵袭实验显示,Rho GDI2高表达组穿膜细胞数显著多于MGC803组和空载体组(P0.05),表明Rho GDI2高表达促进MGC803细胞的侵袭。DADS处理24h后的MGC803组、空载体组、Rho GDI2高表达组的穿膜细胞数明显小于DADS未处理的相对应各组(P0.05),表明DADS抑制MGC803、Rho GDI2/MGC803的侵袭。4.DADS对Rho GDI2高表达MGC803细胞成瘤能力的影响。裸鼠成瘤实验显示,MGC803组移植瘤生长速度慢于Rho GDI2高表达组(P0.05),Rho GDI2高表达可促进MGC803细胞移植瘤的生长能力。DADS处理组的移植瘤生长速度显著慢于未处理组(P0.05),表明DADS可抑制MGC803组与Rho GDI2高表达组移植瘤生长。移植瘤免疫组化显示,DADS处理组较相应的未处理组,E-cadherin表达量增多,Vimentin、Ki-67、CD34的表达量减少;Rho GDI2高表达组较MGC803组的Vimentin、Ki-67、CD34的表达量增多,E-cadherin的表达量减少。结论:1.DADS可抑制Rho GDI2高表达MGC803细胞迁移侵袭与成瘤能力。2.Rho GDI2高表达促进MGC803细胞迁移侵袭及成瘤能力。
[Abstract]:Aim: our previous study showed that diallyl disulfide (diallyl disulfide,DADS) inhibited the migration and invasion of human gastric cancer MGC803 cells by blocking the Rac1-Pak1/Rock pathway. Recently, proteomics found that DADS can down-regulate Rho GDI2 protein. On the basis of previous studies, we studied the effects of DADS on the biological behavior of Rho GDI2 overexpression MGC803 cells by constructing MGC803 cell lines with stable and high expression of Rho GDI2. Methods: the eukaryotic expression vector of p IRES2-EGFP-Rho GDI2 was transfected into MGC803 cells and the stable and high expression MGC803 cell line. Western blot was constructed to verify the expression of Rho GDI2 protein. The effect of DADS on the migration and invasion of Rho GDI2 overexpression MGC803 cells was detected by scratch test and Transwell invasion assay. The effect of DADS on the growth of transplanted MGC803 cells with high expression of Rho GDI2 was detected by nude mice tumorigenesis test. The result is 1: 1. MGC803 cell lines with stable and high expression of Rho GDI2 were successfully established. Green fluorescent. Western blot showed that the expression of Rho GDI2 protein in Rho GDI2/MGC803 cells was significantly higher than that in p IRES2-EGFP/MGC803 and MGC803 cells. It was proved that MGC803 cell line with stable and high expression of Rho GDI2 was successfully constructed. The effect of 2.DADS on the migration of MGC803 cells with high expression of Rho GDI2 was studied. Scratch test showed that the scratch distance of Rho GDI2/MGC803 group was smaller than that of p IRES2-EGFP/MGC803 group and MGC803 group (P0.05), indicating that the high expression of Rho GDI2 promoted the migration of MGC803 cells. The scratch distance of high expression group of Rho GDI2 was significantly larger than that of untreated group of DADS (P0.05), indicating that DADS inhibited the migration of MGC803,Rho GDI2/MGC803. 3.DADS had an effect on the invasion of MGC803 cells with high expression of Rho GDI2. Invasion assay showed that the number of perforated cells in Rho GDI2 high expression group was significantly higher than that in MGC803 group and empty vector group (P0.05), indicating that high expression of Rho GDI2 promoted the invasion of MGC803 cells. The number of perforated cells in the high expression group of Rho GDI2 was significantly lower than that in the untreated group of DADS (P0.05), indicating that DADS inhibited the invasion of MGC803,Rho GDI2/MGC803 and the effect of 4.DADS on the tumorigenic ability of Rho GDI2 overexpression MGC803 cells. In nude mice, the tumor growth rate of MGC803 group was slower than that of Rho GDI2 high expression group (P0.05). The high expression of Rho GDI2 could promote the growth ability of MGC803 cell transplantation tumor. The growth rate of transplanted tumor in DADS treatment group was significantly slower than that in untreated group (P0.05), which indicated that DADS could inhibit the growth of transplanted tumor in MGC803 group and Rho GDI2 high expression group. Immunohistochemical staining showed that the expression of E-cadherin increased and the expression of Vimentin,Ki-67,CD34 decreased in DADS group compared with the corresponding untreated group. The expression of Vimentin,Ki-67,CD34 in the high expression group of Rho GDI2 was higher than that in the group of MGC803, and the expression of E-cadherin was decreased in the group of high expression of Rho GDI2. Conclusion: 1.DADS can inhibit the migration, invasion and tumorigenesis of MGC803 cells with high expression of Rho GDI2, and the high expression of 2.Rho GDI2 can promote the migration, invasion and tumorigenesis of MGC803 cells.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.2

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