【摘要】：目的探究光甘草定(glabridin)對(duì)人結(jié)直腸癌細(xì)胞RKO和HCT116細(xì)胞的遷移和凋亡的影響,并探討其可能的分子機(jī)制。方法不同濃度的光甘草定、MLCK抑制劑ML-7、ROCK抑制劑H-1152以及p38通路抑制劑SB203580、JNK抑制劑SP600125和PKC激活劑PMA處理RKO和(或)HCT116細(xì)胞;采用MTT比色法檢測(cè)光甘草定對(duì)RKO和HCT116細(xì)胞活力的影響,平板克隆實(shí)驗(yàn)檢測(cè)光甘草定對(duì)結(jié)直腸癌細(xì)胞克隆形成能力的影響,通過Hoechst 33258染色檢測(cè)光甘草定對(duì)RKO細(xì)胞凋亡的影響;通過細(xì)胞劃痕和Transwell實(shí)驗(yàn)觀察光甘草定對(duì)RKO和HCT116細(xì)胞遷移和侵襲能力的影響;免疫熒光檢測(cè)光甘草定處理結(jié)直腸癌細(xì)胞后E-cadherin、N-cadherin以及HCT116細(xì)胞內(nèi)緊密連接蛋白Occludin和ZO-1的變化。Western blot檢測(cè)RKO細(xì)胞中凋亡相關(guān)蛋白(BCL-2、Bax、Caspase家族),HCT116細(xì)胞中緊密連接蛋白ZO-1、occludin,RKO和HCT116細(xì)胞中E-cadherin、N-cadherin以及遷移相關(guān)蛋白(Rho A、ROCK、MLCK、MLCP、p-MLC)以及MAPK信號(hào)轉(zhuǎn)導(dǎo)通路相關(guān)蛋白的表達(dá)情況,并加入抑制劑和激活劑分析其可能的機(jī)制。結(jié)果光甘草定作用于RKO和HCT116細(xì)胞后,對(duì)細(xì)胞的增殖、克隆形成能力和遷移、侵襲有明顯抑制作用。光甘草定誘導(dǎo)RKO細(xì)胞凋亡,并下調(diào)抗凋亡蛋白Bcl-2、Procaspase 9表達(dá)水平和上調(diào)Bax、Caspase 3蛋白表達(dá),而對(duì)Caspase 8蛋白表達(dá)影響不明顯。光甘草定處理后,RKO和HCT116細(xì)胞遷移相關(guān)蛋白MYPT1磷酸化水平及MLC磷酸化水平下調(diào),RKO細(xì)胞內(nèi)Rho A、ROCK1、ROCK2表達(dá)水平均明顯下調(diào),而HCT116細(xì)胞內(nèi)Rho A、ROCK1、ROCK2蛋白無明顯變化;為進(jìn)一步的驗(yàn)證,我們采用MLCK的特異性抑制劑ML-7或ROCK的特異性抑制劑H-1152處理RKO和(或)HCT116細(xì)胞,觀察到二者對(duì)RKO和(或)HCT116細(xì)胞的遷移存在抑制作用且能下調(diào)MLCK、ROCK1或p-MYPT1蛋白的表達(dá),提示光甘草定可能通過抑制MLCK和ROCK活性而抑制RKO和(或)HCT116細(xì)胞的遷移能力。光甘草定在不同時(shí)間點(diǎn)處理結(jié)直腸癌細(xì)胞后,兩株細(xì)胞中ERK無明顯變化,RKO細(xì)胞內(nèi)JNK磷酸化水平先上升后下降,p38通路上調(diào),而HCT116細(xì)胞中JNK通路下調(diào)后恢復(fù),p38通路上調(diào)。加入JNK和p38/MAPK通路抑制劑后能下調(diào)遷移相關(guān)蛋白MYPT1和MLC的磷酸化水平,表明光甘草定可能部分通過JNK信號(hào)通路,下調(diào)MLC磷酸化水平,從而導(dǎo)致細(xì)胞運(yùn)動(dòng)能力降低而抑制遷移。光甘草定能抑制RKO和HCT116細(xì)胞上皮間質(zhì)轉(zhuǎn)化過程,上調(diào)上皮細(xì)胞標(biāo)志物E-cadherin,下調(diào)間葉細(xì)胞標(biāo)志物N-cadherin;并且上調(diào)HCT116細(xì)胞膜上緊密連接蛋白Occludin和ZO-1的蛋白水平。結(jié)論光甘草定抑制結(jié)直腸癌RKO和HCT116細(xì)胞的增殖與遷移,并誘導(dǎo)RKO細(xì)胞凋亡;其抑制遷移的機(jī)制可能是通過JNK信號(hào)通路下調(diào)MLC磷酸化水平,增加E-cadherin表達(dá),降低N-cadherin表達(dá),逆轉(zhuǎn)上皮間質(zhì)轉(zhuǎn)化,并上調(diào)緊密連接蛋白ZO-1和Occludin的表達(dá)抑制結(jié)直腸癌細(xì)胞的遷移。
[Abstract]:Objective to investigate the effects of (glabridin) on the migration and apoptosis of RKO and HCT116 cells, and to explore its possible molecular mechanism. Methods RKO and / or HCT116 cells were treated with different concentrations of Glycyrrhizin, MLCK inhibitor ML-7,ROCK inhibitor H-1152, p38 pathway inhibitor SB203580,JNK inhibitor SP600125 and PKC activator PMA. Plate cloning assay was used to detect the effect of Gangliandrine on the clone forming ability of colorectal cancer cells, and the effect of GG on the apoptosis of RKO cells was detected by Hoechst 33258 staining. The effects of Glycyrrhizin on the migration and invasion of RKO and HCT116 cells were observed by cell scratch and Transwell assay. Changes of E-cadherin and Occludin and ZO-1 in HCT116 cells after treatment of Colorectal Cancer cells with Light-Glycyrrhizin by immunofluorescence Detection of apoptosis-related proteins (BCL-2,Bax,Caspase family) in RKO cells and ZO-1,occludin,RKO and HCT116 cells in HCT116 cells by. Western blot The expression of E-cadherin, Rho, MLCP p-MLC and MAPK signal transduction pathway related proteins were observed. The possible mechanisms were analyzed by adding inhibitors and activators. Results after treated with RKO and HCT116 cells, Guanglianding inhibited cell proliferation, clone formation, migration and invasion. Glycyrrhizin induced apoptosis of RKO cells, down-regulated the expression of anti-apoptotic protein Bcl-2,Procaspase 9 and up-regulated the expression of Bax,Caspase 3 protein, but had no significant effect on the expression of Caspase 8 protein. The level of MYPT1 phosphorylation and MLC phosphorylation of migration related protein in RKO and HCT116 cells were down-regulated, and the expression level of Rho AroCK1 and ROCK2 in RKO cells were significantly down-regulated, while Rho AroCK1 and ROCK2 protein in HCT116 cells had no significant changes after treatment with Ligandrine. We treated RKO and / or HCT116 cells with ML-7, a specific inhibitor of MLCK, or H-1152, a specific inhibitor of ROCK, and observed that both of them could inhibit the migration of RKO and / or HCT116 cells and down-regulate the expression of MLCK,ROCK1 or p-MYPT1 protein. These results suggest that Licorrhizine inhibits the migration of RKO and / or HCT116 cells by inhibiting the activities of MLCK and ROCK. There was no significant change of ERK in the two cell lines after treated with licorice at different time points. The level of JNK phosphorylation in RKO cells increased at first, then decreased, the p38 pathway increased, while the JNK pathway in HCT116 cells was down-regulated, and the p38 pathway was up-regulated. The addition of JNK and p38/MAPK pathway inhibitors could down-regulate the phosphorylation levels of MYPT1 and MLC, suggesting that Glycyrrhizin might down-regulate the phosphorylation level of MLC through JNK signaling pathway, resulting in the decrease of cell motility and inhibition of migration. Glycyrrhizin inhibited the process of epithelial interstitial transformation of RKO and HCT116 cells, up-regulated E-cadherin, down-regulated N-cadherin, and up-regulated the protein levels of Occludin and ZO-1 on HCT116 cell membrane. Conclusion Gliangandrine inhibits the proliferation and migration of RKO and HCT116 cells and induces apoptosis of RKO cells in colorectal cancer, and its mechanism may be to down-regulate the level of MLC phosphorylation, increase E-cadherin expression and decrease N-cadherin expression through JNK signaling pathway. Reverse epithelial interstitial transformation and up-regulate the expression of ZO-1 and Occludin to inhibit the migration of colorectal cancer cells.
【學(xué)位授予單位】：安徽醫(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)】：R735.34

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 周庚寅;蔡永萍;;上皮間葉轉(zhuǎn)化的研究進(jìn)展[J];鄭州大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2010年03期

2 陳功;萬德森;;基于循證醫(yī)學(xué)依據(jù)的結(jié)腸癌輔助化療[J];中國(guó)癌癥雜志;2009年01期

3 賽力曼·哈得爾;李宏智;木合布力·阿布力孜;巴哈爾古麗·卡哈爾;;新疆甘草黃酮類成分光甘草定的制備工藝改進(jìn)[J];亞太傳統(tǒng)醫(yī)藥;2008年09期

相關(guān)碩士學(xué)位論文 前1條

1 石月;光甘草定規(guī)�；苽渑c穩(wěn)定性影響因素研究[D];華中科技大學(xué);2011年

，

本文編號(hào)：2289599