【摘要】：目的:我們前期實(shí)驗(yàn)表明,二烯丙基二硫(diallyl disulfide,DADS)通過阻斷Rac1-Pak1/Rock通路下調(diào)LIMK1抑制人胃癌MGC803細(xì)胞的遷移與侵襲。近來蛋白組學(xué)發(fā)現(xiàn),DADS可下調(diào)Rho GDI2蛋白。本研究在前期研究基礎(chǔ)上,通過構(gòu)建Rho GDI2穩(wěn)定高表達(dá)MGC803細(xì)胞系,探討DADS對(duì)Rho GDI2高表達(dá)MGC803細(xì)胞生物學(xué)行為的影響。方法:將p IRES2-EGFP-Rho GDI2真核表達(dá)載體轉(zhuǎn)染MGC803細(xì)胞,構(gòu)建Rho GDI2穩(wěn)定高表達(dá)MGC803細(xì)胞系。Western blot驗(yàn)證Rho GDI2蛋白的表達(dá)水平。劃痕實(shí)驗(yàn)與Transwell侵襲實(shí)驗(yàn)檢測DADS對(duì)Rho GDI2高表達(dá)MGC803細(xì)胞遷移侵襲的影響。裸鼠成瘤實(shí)驗(yàn)檢測DADS對(duì)Rho GDI2高表達(dá)MGC803細(xì)胞移植瘤生長的影響。結(jié)果:1.成功建立Rho GDI2穩(wěn)定高表達(dá)的MGC803細(xì)胞系。兩組轉(zhuǎn)染細(xì)胞(Rho GDI2/MGC803與p IRES2-EGFP/MGC803)均可見綠色熒光。Western blot顯示,Rho GDI2/MGC803細(xì)胞中Rho GDI2的蛋白表達(dá)量均明顯高于p IRES2-EGFP/MGC803及MGC803細(xì)胞,證明成功構(gòu)建Rho GDI2穩(wěn)定高表達(dá)的MGC803細(xì)胞系。2.DADS對(duì)Rho GDI2高表達(dá)MGC803細(xì)胞遷移的影響。劃痕實(shí)驗(yàn)顯示,Rho GDI2/MGC803組的劃痕距離小于p IRES2-EGFP/MGC803組和MGC803組(P0.05),表明Rho GDI2高表達(dá)促進(jìn)MGC803細(xì)胞的遷移。DADS處理24h的MGC803組、空載體組、Rho GDI2高表達(dá)組的劃痕距離明顯分別大于相對(duì)應(yīng)的DADS未處理組(P0.05),表明DADS抑制MGC803、Rho GDI2/MGC803的遷移。3.DADS對(duì)Rho GDI2高表達(dá)MGC803細(xì)胞侵襲的影響。侵襲實(shí)驗(yàn)顯示,Rho GDI2高表達(dá)組穿膜細(xì)胞數(shù)顯著多于MGC803組和空載體組(P0.05),表明Rho GDI2高表達(dá)促進(jìn)MGC803細(xì)胞的侵襲。DADS處理24h后的MGC803組、空載體組、Rho GDI2高表達(dá)組的穿膜細(xì)胞數(shù)明顯小于DADS未處理的相對(duì)應(yīng)各組(P0.05),表明DADS抑制MGC803、Rho GDI2/MGC803的侵襲。4.DADS對(duì)Rho GDI2高表達(dá)MGC803細(xì)胞成瘤能力的影響。裸鼠成瘤實(shí)驗(yàn)顯示,MGC803組移植瘤生長速度慢于Rho GDI2高表達(dá)組(P0.05),Rho GDI2高表達(dá)可促進(jìn)MGC803細(xì)胞移植瘤的生長能力。DADS處理組的移植瘤生長速度顯著慢于未處理組(P0.05),表明DADS可抑制MGC803組與Rho GDI2高表達(dá)組移植瘤生長。移植瘤免疫組化顯示,DADS處理組較相應(yīng)的未處理組,E-cadherin表達(dá)量增多,Vimentin、Ki-67、CD34的表達(dá)量減少;Rho GDI2高表達(dá)組較MGC803組的Vimentin、Ki-67、CD34的表達(dá)量增多,E-cadherin的表達(dá)量減少。結(jié)論:1.DADS可抑制Rho GDI2高表達(dá)MGC803細(xì)胞遷移侵襲與成瘤能力。2.Rho GDI2高表達(dá)促進(jìn)MGC803細(xì)胞遷移侵襲及成瘤能力。
[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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.2

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