Smac类似物LCL161对结肠癌细胞株增殖与凋亡的影响及其机制的初步探讨

发布时间：2018-03-06 04:15

本文选题：Smac类似物　切入点：LCL161　出处：《蚌埠医学院》2016年硕士论文　论文类型：学位论文

【摘要】：目的:1.观察Smac类似物LCL161对结肠癌细胞株增殖的影响。2.研究LCL161是否通过诱导结肠癌细胞凋亡发挥对其增殖的影响。3.探讨LCL161诱导凋亡的可能机制。方法:1.MTT法检测LCL161对结肠癌细胞SW480及HT-29存活率的影响。2.克隆形成实验检测LCL161对结肠癌细胞克隆形成的影响。3.JC-1染色法检测LCL161对结肠癌SW480及HT-29细胞内ATP水平的影响。4.DAPI染色法检测LCL161对人结肠癌SW480及HT-29细胞株细胞核的影响。5.溴化丙啶(propidium iodide,PI)单染法检测LCL161对结肠SW480及HT-29细胞株凋亡的影响。6.电镜观察LCL161处理结肠癌细胞后细胞亚显微结构的变化。7.Western blot法检测LCL161处理人结肠癌SW480及HT-29细胞株对IAPs(XIAP,c IAP1/2)及TNF-α表达的影响。结果:1 LCL161抑制人结肠癌SW480和HT-29细胞株的增殖1.1 MTT实验结果显示不同浓度LCL161(0,0.2,1,5,10mmol/L)对人结肠癌SW480及HT-29细胞株均抑制细胞存活率的作用,且随着LCL161浓度的增加和作用时间的延长,对细胞存活率的抑制逐渐增强,高浓度LCL161抑制作用较明显。在浓度为10μmol/L时,观察LCL161作用24 h时细胞存活率分别为44.6%,78.6%。1.2克隆形成实验显示LCL161可以抑制人结肠癌SW480及HT-29细胞株的克隆增殖,且随着LCL161浓度的增加,对克隆的抑制逐渐增强。2 LCL161诱导人结肠癌SW480和HT-29细胞株的细胞凋亡2.1 LCL161可以降低JC-1在线粒体膜上的红色荧光强度,提高其绿色荧光强度,说明LCL161可以降低两株细胞的线粒体膜电位。2.2 DAPI染色实验结果显示,LCL161可导致两株乳腺癌细胞内的细胞核浓染,核分裂增多,并和LCL161浓度相关,表明LCL161可引起结肠癌细胞核固缩,分裂。2.3不同浓度的LCL161(0mmol/L,5mmol/L,10mmol/L)处理人结肠癌细胞SW480和HT-29 24 h后,PI单染法检测结果显示:随着LCL161浓度的增加,SW480和HT-29细胞的死亡率逐渐增多,凋亡细胞比例分别为1.6%、30.1%、49.1%和5.8%、14.7%、26.1%。说明LCL161可以诱导结肠癌细胞凋亡。2.4透射电镜(TEM)观察结肠癌细胞受LCL161刺激后的亚细胞显微结构的改变。结果示:相比较空白对照组,实验组(10mmol/L)的较多结肠癌细胞均出现细胞膜皱缩,染色质边集,细胞核固缩及凋亡小体形成等典型凋亡形态学特征。3 LCL161诱导c IAP1降解,促进TNF-α合成,介导结肠癌细胞的凋亡3.1 Western-blot法分别检测LCL161处理后各组结肠癌SW480和HT-29两种细胞株细胞凋亡抑制蛋白(c IAP1、c IAP2、XIAP)的表达水平。从实验的结果可以观察出:1.随着药物浓度的增加,c IAP1的蛋白的表达在两个细胞株中都存在逐渐减少的趋势。2.同样的方法和时间点,但是观察到c IAP2、XIAP的蛋白的表达变化不明显。3.2随着LCL161浓度增高,两株细胞内的TNF-α在PVDF膜上显示出条带逐渐浓深。说明LCL161可以促进外源性细胞凋亡通路配体TNF-α的表达。结论:1 Smac类似物LCL161能够显著地抑制人结肠癌SW480和HT-29细胞株的增殖能力,作为临床治疗结肠癌的新药物具有一定的潜在研究价值。2 Smac类似物LCL161引起的人结肠癌细胞的增殖抑制的可能机制是通过诱导结肠癌细胞发生细胞凋亡实现的。3 Smac类似物LCL161可能通过结合并降解c IAP1以及刺激细胞TNF-α的合成和分泌,进而调控结肠癌细胞凋亡。
[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.

【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.35

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