本文選題：Smac類(lèi)似物　切入點(diǎn)：LCL161　出處：《蚌埠醫(yī)學(xué)院》2016年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的:1.觀察Smac類(lèi)似物L(fēng)CL161對(duì)結(jié)腸癌細(xì)胞株增殖的影響。2.研究LCL161是否通過(guò)誘導(dǎo)結(jié)腸癌細(xì)胞凋亡發(fā)揮對(duì)其增殖的影響。3.探討LCL161誘導(dǎo)凋亡的可能機(jī)制。方法:1.MTT法檢測(cè)LCL161對(duì)結(jié)腸癌細(xì)胞SW480及HT-29存活率的影響。2.克隆形成實(shí)驗(yàn)檢測(cè)LCL161對(duì)結(jié)腸癌細(xì)胞克隆形成的影響。3.JC-1染色法檢測(cè)LCL161對(duì)結(jié)腸癌SW480及HT-29細(xì)胞內(nèi)ATP水平的影響。4.DAPI染色法檢測(cè)LCL161對(duì)人結(jié)腸癌SW480及HT-29細(xì)胞株細(xì)胞核的影響。5.溴化丙啶(propidium iodide,PI)單染法檢測(cè)LCL161對(duì)結(jié)腸SW480及HT-29細(xì)胞株凋亡的影響。6.電鏡觀察LCL161處理結(jié)腸癌細(xì)胞后細(xì)胞亞顯微結(jié)構(gòu)的變化。7.Western blot法檢測(cè)LCL161處理人結(jié)腸癌SW480及HT-29細(xì)胞株對(duì)IAPs(XIAP,c IAP1/2)及TNF-α表達(dá)的影響。結(jié)果:1 LCL161抑制人結(jié)腸癌SW480和HT-29細(xì)胞株的增殖1.1 MTT實(shí)驗(yàn)結(jié)果顯示不同濃度LCL161(0,0.2,1,5,10mmol/L)對(duì)人結(jié)腸癌SW480及HT-29細(xì)胞株均抑制細(xì)胞存活率的作用,且隨著LCL161濃度的增加和作用時(shí)間的延長(zhǎng),對(duì)細(xì)胞存活率的抑制逐漸增強(qiáng),高濃度LCL161抑制作用較明顯。在濃度為10μmol/L時(shí),觀察LCL161作用24 h時(shí)細(xì)胞存活率分別為44.6%,78.6%。1.2克隆形成實(shí)驗(yàn)顯示LCL161可以抑制人結(jié)腸癌SW480及HT-29細(xì)胞株的克隆增殖,且隨著LCL161濃度的增加,對(duì)克隆的抑制逐漸增強(qiáng)。2 LCL161誘導(dǎo)人結(jié)腸癌SW480和HT-29細(xì)胞株的細(xì)胞凋亡2.1 LCL161可以降低JC-1在線粒體膜上的紅色熒光強(qiáng)度,提高其綠色熒光強(qiáng)度,說(shuō)明LCL161可以降低兩株細(xì)胞的線粒體膜電位。2.2 DAPI染色實(shí)驗(yàn)結(jié)果顯示,LCL161可導(dǎo)致兩株乳腺癌細(xì)胞內(nèi)的細(xì)胞核濃染,核分裂增多,并和LCL161濃度相關(guān),表明LCL161可引起結(jié)腸癌細(xì)胞核固縮,分裂。2.3不同濃度的LCL161(0mmol/L,5mmol/L,10mmol/L)處理人結(jié)腸癌細(xì)胞SW480和HT-29 24 h后,PI單染法檢測(cè)結(jié)果顯示:隨著LCL161濃度的增加,SW480和HT-29細(xì)胞的死亡率逐漸增多,凋亡細(xì)胞比例分別為1.6%、30.1%、49.1%和5.8%、14.7%、26.1%。說(shuō)明LCL161可以誘導(dǎo)結(jié)腸癌細(xì)胞凋亡。2.4透射電鏡(TEM)觀察結(jié)腸癌細(xì)胞受LCL161刺激后的亞細(xì)胞顯微結(jié)構(gòu)的改變。結(jié)果示:相比較空白對(duì)照組,實(shí)驗(yàn)組(10mmol/L)的較多結(jié)腸癌細(xì)胞均出現(xiàn)細(xì)胞膜皺縮,染色質(zhì)邊集,細(xì)胞核固縮及凋亡小體形成等典型凋亡形態(tài)學(xué)特征。3 LCL161誘導(dǎo)c IAP1降解,促進(jìn)TNF-α合成,介導(dǎo)結(jié)腸癌細(xì)胞的凋亡3.1 Western-blot法分別檢測(cè)LCL161處理后各組結(jié)腸癌SW480和HT-29兩種細(xì)胞株細(xì)胞凋亡抑制蛋白(c IAP1、c IAP2、XIAP)的表達(dá)水平。從實(shí)驗(yàn)的結(jié)果可以觀察出:1.隨著藥物濃度的增加,c IAP1的蛋白的表達(dá)在兩個(gè)細(xì)胞株中都存在逐漸減少的趨勢(shì)。2.同樣的方法和時(shí)間點(diǎn),但是觀察到c IAP2、XIAP的蛋白的表達(dá)變化不明顯。3.2隨著LCL161濃度增高,兩株細(xì)胞內(nèi)的TNF-α在PVDF膜上顯示出條帶逐漸濃深。說(shuō)明LCL161可以促進(jìn)外源性細(xì)胞凋亡通路配體TNF-α的表達(dá)。結(jié)論:1 Smac類(lèi)似物L(fēng)CL161能夠顯著地抑制人結(jié)腸癌SW480和HT-29細(xì)胞株的增殖能力,作為臨床治療結(jié)腸癌的新藥物具有一定的潛在研究?jī)r(jià)值。2 Smac類(lèi)似物L(fēng)CL161引起的人結(jié)腸癌細(xì)胞的增殖抑制的可能機(jī)制是通過(guò)誘導(dǎo)結(jié)腸癌細(xì)胞發(fā)生細(xì)胞凋亡實(shí)現(xiàn)的。3 Smac類(lèi)似物L(fēng)CL161可能通過(guò)結(jié)合并降解c IAP1以及刺激細(xì)胞TNF-α的合成和分泌,進(jìn)而調(diào)控結(jié)腸癌細(xì)胞凋亡。
[Abstract]:Objective: To observe the 1. Smac analogs of LCL161 effects on the proliferation of colon cancer cell line.2. by investigating whether LCL161 induced apoptosis of human colon cancer cells exert its effects on proliferation of.3. and explore the possible mechanism of apoptosis induced by LCL161. Methods: LCL161 1.MTT method.2. effect on the rate of survival of cloned SW480 and HT-29 colon cancer cell formation assay LCL161 the formation of colon cancer cell clones.3.JC-1 staining was used to detect the effect of LCL161 on the level of ATP SW480 and HT-29 colon cancer cells.4.DAPI staining was used to detect LCL161 on human colon cancer cell lines SW480 and HT-29 were affected.5. bromide propidium (propidium iodide, PI) single staining was used to detect the effect of LCL161 on apoptosis of colon SW480 HT-29 and.6. cells was observed by electron microscope.7.Western blot SUBMICROSTRUCTURE LCL161 colon cancer cell LCL161 of human colon cancer SW480 and HT-29 cells IAPs on cell lines (XIAP, C IAP1/2) and TNF- expression. Results: 1 LCL161 inhibited SW480 human colon cancer cell line HT-29 proliferation and 1.1 MTT experimental results showed that different concentrations of LCL161 (0,0.2,1,5,10mmol/L) on human colon cancer SW480 and HT-29 cell lines inhibited cell survival, and with the increase of LCL161 concentration and action time, the inhibition of cell survival rate gradually increased, the high concentration of LCL161 inhibition is more obvious. At the concentration of 10 mol/L, we observe the effect of LCL161 24 h cell survival rate were 44.6%, 78.6%.1.2 clone formation assay showed that the cloned LCL161 can inhibit the proliferation of human colon cancer cell lines SW480 and HT-29 and, with the increasing concentration of LCL161, inhibition of cloning gradually increased.2 cell apoptosis induced by LCL161 in human colon cancer cell lines SW480 and HT-29 2.1 LCL161 can reduce JC-1 in the mitochondrial membrane of red fluorescence Strength, increase the intensity of green fluorescence, indicating that LCL161 can reduce the mitochondrial membrane potential of.2.2 DAPI two cell staining results showed that LCL161 can lead to two strains of breast cancer cells hyperchromatic nuclei, nuclear fission and increased, and the concentration of LCL161 showed that LCL161 can lead to colon cancer cell nuclear pyknosis, split.2.3 the concentration of LCL161 (0mmol/L, 5mmol/L, 10mmol/L) SW480 and HT-29 in human colon cancer cells after 24 h, the PI single staining showed that with the increasing concentration of LCL161, SW480 and HT-29 cell death rate gradually increased, the proportion of apoptotic cells were 1.6%, 30.1%, 49.1% and 5.8%, 14.7%, 26.1%. LCL161 can induce the apoptosis of human colon cancer cells.2.4 transmission electron microscope (TEM) subcellular microstructure observation of colon cancer cells stimulated by LCL161. The results showed: the change compared to the blank control group, experimental group (10mmol/L) of colon Cancer cells showed cell membrane shrinkage, chromatin margination, karyopyknosis and apoptotic bodies of the typical morphological features of apoptosis induced by LCL161.3 C IAP1 TNF- alpha degradation, promote the synthesis of LCL161 were detected after treatment of colon cancer SW480 and HT-29 two cell lines apoptosis inhibitory protein mediated apoptosis of colon cancer cells 3.1 Western-blot (C IAP1, C IAP2, XIAP) expression level. From the experimental results can be observed: 1. with the increase of drug concentration, the expression of C IAP1 protein existed in the method and time point decreasing trend.2. the same in the two cell lines, but C IAP2 expression was observed. The change of XIAP protein of.3.2 is not obvious with the increasing of LCL161 concentration, TNF- alpha two cell lines in PVDF membrane showed bands gradually deep. The results showed that LCL161 can promote the apoptosis pathway of exogenous alpha ligand TNF- expression. Conclusion: 1 Sm AC analogue LCL161 can significantly inhibit human colon cancer SW480 and HT-29 cell proliferation ability, new drugs for clinical treatment of colon cancer with the possible mechanism of certain potential value of.2 Smac analogues LCL161 induced human colon cancer cell proliferation inhibition by inducing apoptosis of colon cancer cells to achieve the.3 Smac analogues by LCL161 might bind and degrade C and IAP1 synthesis of TNF- alpha and stimulate cell secretion, regulating apoptosis of colon cancer cells.

【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R735.35

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