姜黄素对HCT116细胞上皮间质转化的影响及其相关机制的研究

发布时间：2018-01-10 22:10

本文关键词：姜黄素对HCT116细胞上皮间质转化的影响及其相关机制的研究　出处：《广西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:研究姜黄素(Curcumin)对人结肠癌HCT116细胞株的生长、增殖以及迁移的影响,初步探究姜黄素对肿瘤坏死因子-?(Tumor necrosis factor-?,TNF-?)诱导的人结肠癌上皮间质转化(Epithelial mesenchymal transition,EMT)、迁移的影响及其可能的机制,为姜黄素的抗肿瘤治疗提供理论依据。方法:1.利用MTT法检测不同时间、不同浓度的姜黄素对人结肠癌HCT116细胞株的毒性作用:分别使用浓度为0?mol/L、5?mol/L、10?mol/L、15?mol/L、20?mol/L、25?mol/L及30?mol/L的姜黄素作用于人结肠癌HCT116细胞株,并于12h、24h、36h及48h后,用MTT比色法检测HCT116细胞的存活率。2.探索姜黄素对人结肠癌HCT116细胞株上皮间质转化、迁移的影响及其可能的机制:根据MTT的实验结果选择适合的姜黄素药物干预浓度,将HCT116细胞分为对照组、TNF-?组和姜黄素+TNF-?组。使用倒置显微镜观察各组细胞的形态变化;利用蛋白印迹(Western Blot)法检测各组细胞EMT相关标记物E-钙黏蛋白(E-cadherin)和波形蛋白(Vimentin)的表达水平及NF-?B信号通路上p65蛋白的表达水平;用细胞划痕实验检测经处理后各组细胞的迁移能力。结果:1.当姜黄素浓度小于20?mol/L时,细胞存活率在浓度及时间效应上的差异均无统计学意义(P0.05)。当姜黄素浓度大于20?mol/L时,作用于人结肠癌HCT116细胞株12h后,能明显抑制细胞的存活,并且随着药物浓度的增大,作用时间越长,HCT116细胞的存活率越低;细胞存活率在浓度和时间效应上的差异均存在统计学意义(P0.05)。2.Western Blot法检测对照组和TNF-?组E-cadherin蛋白的相对表达量分别为(0.1278±0.0082)和(0.0921±0.0076)(P0.05),Vimentin蛋白的相对表达量分别为(0.1353±0.0140)和(0.2864±0.0096)(P0.05),NF-?Bp65蛋白的相对表达量分别为(0.6516±0.0268)和(0.8407±0.0307)(P0.05),结果显示TNF-?作用于HCT116细胞后,上皮表型蛋白E-cadherin表达下调,间质表型蛋白Vimentin表达上调,同时NF-?Bp65蛋白的表达也上调。此外,TNF-?组人结肠癌HCT116细胞多见长梭形、纺锤状细胞,且多数细胞长出细长的丝状伪足,细胞与细胞之间的排列较对照组疏松,细胞间隙明显增宽,呈间质细胞形态改变;细胞划痕实验中对照组和TNF-?组细胞48h平均迁移距离分别为(144.07±11.31)?m和(319.84±20.93)?m(P0.05),表明TNF-?能通过调节NF-?Bp65诱导HCT(16)(16)(21)细胞发生EMT,并增强其迁移能力。3.使用姜黄素干预TNF-?诱导EMT的HCT116细胞模型48h后,Western Blot法检测TNF-?组和姜黄素+TNF-?组E-cadherin蛋白的相对表达量分别为(0.0921±0.0076)和(0.1185±0.0033)(P0.05),Vimentin蛋白的相对表达量分别为(0.2864±0.0096)和(0.2043±0.0238)(P0.05),NF-?Bp65蛋白的相对表达量分别为(0.8407±0.0307)和(0.5406±0.0224)(P0.05),结果显示姜黄素+TNF-?组E-cadherin表达上调,Vimentin表达下调,同时NF-?Bp65蛋白的表达也下调。此外,姜黄素+TNF-?组细胞中见长梭形细胞与凋亡的类圆形细胞并存,且细胞的丝状伪足明显减少,细胞之间的排列较单纯TNF-?组紧密,细胞划痕实验中对照组和TNF-?组细胞48h平均迁移距离分别为(319.84±20.93)?m和(90.70±7.25)?m(P0.05),表明姜黄素通过下调NF-?Bp65抑制由TNF-?诱导的人结肠癌HCT116细胞的EMT和转移。结论:姜黄素通过下调NF-?Bp65抑制TNF-?诱导的人结肠癌HCT116细胞的EMT和转移。
[Abstract]:Objective: To investigate the effects of curcumin (Curcumin) on human colon cancer HCT116 cells growth, proliferation and migration effects on tumor necrosis factor - (Tumor necrosis factor- curcumin??, TNF-?) induced human colon epithelial mesenchymal transition (Epithelial mesenchymal, transition, EMT), the impact of migration and its possible the mechanism, to provide theoretical basis for anti-tumor treatment of curcumin. Methods: 1. using MTT assay at different time, the toxic effects of different concentrations of curcumin on human colon cancer cell line HCT116 were used for concentration of 0? Mol/L, 5? Mol/L, 10? Mol/L, 15? Mol/L, 20? Mol/L, 25? Mol/L and 30? Mol/L of curcumin on human colon cancer cell line HCT116 and 12h, 24h, 36h and 48h, with MTT HCT116 cells assay the survival rate of.2. to explore the effect of curcumin on human colon cancer cell line HCT116 in epithelial mesenchymal transition, migration and its possible effects The mechanism: according to the selection of suitable MTT concentration of curcumin intervention experiments, HCT116 cells were divided into control group, TNF- group and curcumin group? +TNF-?. the morphological changes of cells were observed using inverted microscope; using Western blot method (Western Blot) was detected by EMT cell related markers of E-cadherin (E- E-cadherin) and vimentin (Vimentin) and the expression level of NF-? B signaling pathway on the expression level of p65 protein; migration by cell scratch assay after treatment of cells in each group. Results: 1. when the concentration of curcumin is less than 20? Mol/L, the cell survival rate in different concentration and time effect were not statistically significant (P0.05). When the concentration of curcumin is greater than 20? Mol/L, the effect on human colon cancer cell line HCT116 after 12h can significantly inhibit cell survival, and with the increase of drug concentration, the longer the time, HCT116 cells The lower the survival rate; survival rate in different concentration and time effect on cells were statistically significant (P0.05) detection method of Blot.2.Western group and TNF- control group? The relative expression of E-cadherin protein were (0.1278 + 0.0082) and (0.0921 + 0.0076) (P0.05), the relative expression level of Vimentin protein respectively. (0.1353 + 0.0140) and (0.2864 + 0.0096), NF- (P0.05)? The relative expression level of Bp65 protein were (0.6516 + 0.0268) and (0.8407 + 0.0307) (P0.05), the results show that TNF-? On HCT116 cell, epithelial surface protein E-cadherin expression, expression of mesenchymal phenotype at the same time also increased NF- protein Vimentin? Bp65 protein expression. In addition, TNF-? Group HCT116 human colon cancer cells is more fusiform, spindle shaped cells, and most cells grow slender filopodia, between cells and cells arranged in loose control group, cell gap widened significantly, a Changing between interstitial cell morphology; cell scratch test in control group and TNF- group of cells? The average migration distance of 48h were (144.07 + 11.31)? M and (319.84 + 20.93)? M (P0.05), showed that TNF-? By regulating NF-? Bp65 induced by HCT (16) (16) (21) cells EMT, and enhance the migration ability of.3. TNF- 48h HCT116 using curcumin? Cell model induced by EMT, TNF- Western Blot detection method? Group and curcumin +TNF- group? The relative expression level of E-cadherin protein were (0.0921 + 0.0076) and (0.1185 + 0.0033) (P0.05), the relative expression level of Vimentin protein respectively. For (0.2864 + 0.0096) and (0.2043 + 0.0238), NF- (P0.05)? The relative expression level of Bp65 protein were (0.8407 + 0.0307) and (0.5406 + 0.0224) (P0.05), the results showed that curcumin group +TNF-? E-cadherin expression and Vimentin expression, and NF- expression of Bp65 protein was also down regulated?. in addition, curcumin +TNF-? Round cells in long fusiform cells and apoptotic cells coexist, and filopodia reduced obviously, cells arranged between the relatively simple TNF-? Group closely, cell scratch test in control group and TNF- group of cells? The average migration distance of 48h were (319.84 + 20.93)? M and (90.70 + 7.25) m? (P0.05), showed that curcumin through down-regulation of NF-? Bp65 inhibited by TNF-? Induced human colon cancer HCT116 cells EMT and metastasis. Conclusion: Curcumin through down-regulation of NF- induced inhibition of TNF-? Bp65? HCT116 human colon cancer cells EMT and metastasis.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285

【参考文献】