本文關(guān)鍵詞：姜黃素對HCT116細(xì)胞上皮間質(zhì)轉(zhuǎn)化的影響及其相關(guān)機(jī)制的研究　出處：《廣西醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 結(jié)腸癌 姜黃素 上皮間質(zhì)轉(zhuǎn)化 核轉(zhuǎn)錄因子?B

【摘要】：目的:研究姜黃素(Curcumin)對人結(jié)腸癌HCT116細(xì)胞株的生長、增殖以及遷移的影響,初步探究姜黃素對腫瘤壞死因子-?(Tumor necrosis factor-?,TNF-?)誘導(dǎo)的人結(jié)腸癌上皮間質(zhì)轉(zhuǎn)化(Epithelial mesenchymal transition,EMT)、遷移的影響及其可能的機(jī)制,為姜黃素的抗腫瘤治療提供理論依據(jù)。方法:1.利用MTT法檢測不同時(shí)間、不同濃度的姜黃素對人結(jié)腸癌HCT116細(xì)胞株的毒性作用:分別使用濃度為0?mol/L、5?mol/L、10?mol/L、15?mol/L、20?mol/L、25?mol/L及30?mol/L的姜黃素作用于人結(jié)腸癌HCT116細(xì)胞株,并于12h、24h、36h及48h后,用MTT比色法檢測HCT116細(xì)胞的存活率。2.探索姜黃素對人結(jié)腸癌HCT116細(xì)胞株上皮間質(zhì)轉(zhuǎn)化、遷移的影響及其可能的機(jī)制:根據(jù)MTT的實(shí)驗(yàn)結(jié)果選擇適合的姜黃素藥物干預(yù)濃度,將HCT116細(xì)胞分為對照組、TNF-?組和姜黃素+TNF-?組。使用倒置顯微鏡觀察各組細(xì)胞的形態(tài)變化;利用蛋白印跡(Western Blot)法檢測各組細(xì)胞EMT相關(guān)標(biāo)記物E-鈣黏蛋白(E-cadherin)和波形蛋白(Vimentin)的表達(dá)水平及NF-?B信號(hào)通路上p65蛋白的表達(dá)水平;用細(xì)胞劃痕實(shí)驗(yàn)檢測經(jīng)處理后各組細(xì)胞的遷移能力。結(jié)果:1.當(dāng)姜黃素濃度小于20?mol/L時(shí),細(xì)胞存活率在濃度及時(shí)間效應(yīng)上的差異均無統(tǒng)計(jì)學(xué)意義(P0.05)。當(dāng)姜黃素濃度大于20?mol/L時(shí),作用于人結(jié)腸癌HCT116細(xì)胞株12h后,能明顯抑制細(xì)胞的存活,并且隨著藥物濃度的增大,作用時(shí)間越長,HCT116細(xì)胞的存活率越低;細(xì)胞存活率在濃度和時(shí)間效應(yīng)上的差異均存在統(tǒng)計(jì)學(xué)意義(P0.05)。2.Western Blot法檢測對照組和TNF-?組E-cadherin蛋白的相對表達(dá)量分別為(0.1278±0.0082)和(0.0921±0.0076)(P0.05),Vimentin蛋白的相對表達(dá)量分別為(0.1353±0.0140)和(0.2864±0.0096)(P0.05),NF-?Bp65蛋白的相對表達(dá)量分別為(0.6516±0.0268)和(0.8407±0.0307)(P0.05),結(jié)果顯示TNF-?作用于HCT116細(xì)胞后,上皮表型蛋白E-cadherin表達(dá)下調(diào),間質(zhì)表型蛋白Vimentin表達(dá)上調(diào),同時(shí)NF-?Bp65蛋白的表達(dá)也上調(diào)。此外,TNF-?組人結(jié)腸癌HCT116細(xì)胞多見長梭形、紡錘狀細(xì)胞,且多數(shù)細(xì)胞長出細(xì)長的絲狀偽足,細(xì)胞與細(xì)胞之間的排列較對照組疏松,細(xì)胞間隙明顯增寬,呈間質(zhì)細(xì)胞形態(tài)改變;細(xì)胞劃痕實(shí)驗(yàn)中對照組和TNF-?組細(xì)胞48h平均遷移距離分別為(144.07±11.31)?m和(319.84±20.93)?m(P0.05),表明TNF-?能通過調(diào)節(jié)NF-?Bp65誘導(dǎo)HCT(16)(16)(21)細(xì)胞發(fā)生EMT,并增強(qiáng)其遷移能力。3.使用姜黃素干預(yù)TNF-?誘導(dǎo)EMT的HCT116細(xì)胞模型48h后,Western Blot法檢測TNF-?組和姜黃素+TNF-?組E-cadherin蛋白的相對表達(dá)量分別為(0.0921±0.0076)和(0.1185±0.0033)(P0.05),Vimentin蛋白的相對表達(dá)量分別為(0.2864±0.0096)和(0.2043±0.0238)(P0.05),NF-?Bp65蛋白的相對表達(dá)量分別為(0.8407±0.0307)和(0.5406±0.0224)(P0.05),結(jié)果顯示姜黃素+TNF-?組E-cadherin表達(dá)上調(diào),Vimentin表達(dá)下調(diào),同時(shí)NF-?Bp65蛋白的表達(dá)也下調(diào)。此外,姜黃素+TNF-?組細(xì)胞中見長梭形細(xì)胞與凋亡的類圓形細(xì)胞并存,且細(xì)胞的絲狀偽足明顯減少,細(xì)胞之間的排列較單純TNF-?組緊密,細(xì)胞劃痕實(shí)驗(yàn)中對照組和TNF-?組細(xì)胞48h平均遷移距離分別為(319.84±20.93)?m和(90.70±7.25)?m(P0.05),表明姜黃素通過下調(diào)NF-?Bp65抑制由TNF-?誘導(dǎo)的人結(jié)腸癌HCT116細(xì)胞的EMT和轉(zhuǎn)移。結(jié)論:姜黃素通過下調(diào)NF-?Bp65抑制TNF-?誘導(dǎo)的人結(jié)腸癌HCT116細(xì)胞的EMT和轉(zhuǎn)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.

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285

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