人胃癌耐药细胞株的建立和新型β-内酰胺类化合物抗肿瘤耐药研究

发布时间：2018-08-01 16:19

【摘要】：第一部分:人胃癌耐紫杉醇细胞株MGC803/PTX的建立及机制研究采用紫杉醇浓度梯度递增法诱导人胃癌MGC803细胞建立耐药细胞株MGC803/PTX,探讨人胃癌耐药细胞MGC803/PTX与亲本细胞MGC803之间的差异。方法:用紫杉醇以浓度梯度递增诱导法构建人胃癌耐药细胞株MGC803/PTX;倒置显微镜下观察紫杉醇诱导过程中细胞形态学变化;CCK8法检测MGC803/PTX细胞株的耐药指数及其稳定性;CCK8法检测MGC803/PTX细胞株对5-FU和ADR的耐药性;细胞计数法绘制MGC803和MGC803/PTX细胞株的生长曲线;克隆形成实验测定MGC803和MGC803/PTX细胞株的克隆形成率;荧光显微镜下观察紫杉醇作用后细胞和细胞核形态学变化;流式细胞仪分析细胞凋亡和周期分布情况。Western blot实验检测耐药细胞株与亲本细胞株中P-gp、Bcl-2、Bax、PARP、β-catenin、p-GSK-3β等蛋白的表达,比较两株细胞蛋白表达的差异,分析MGC803/PTX细胞株可能的耐药机制。结果:历时10个月成功构建了人胃癌耐药细胞株MGC803/PTX,对紫杉醇的耐药指数为9.3,诱导过程中细胞由长梭形逐步变成短多边形,体积变小,诱导结束后3个月内紫杉醇对MGC803/PTX细胞IC50变化不大。MGC803/PTX对5-FU和ADR这两种化疗药物存在一定的交叉耐药性,RI分别为4.38和1.4;耐药细胞MGC803/PTX的生长速度比亲本细胞MGC803慢,群体倍增时间延长,分别为24.55h和22.5h;MGC803和MGC803/PTX细胞克隆形成率有明显差异(p0.001),耐药细胞MGC803/PTX克隆形成能力明显高于亲本细胞MGC803;PTX作用两株细胞,Hoechst 33258染色后细胞核的染色增强,细胞核染色质浓缩且有凋亡小体生成,但MGC803/PTX细胞凋亡率明显低于MGC803细胞;流式细胞仪检测结果同样显示耐药细胞株MGC803/PTX凋亡率低于MGC803细胞,20nMPTX作用后凋亡率分别为1.8%和24.2%。MGC803/PTX细胞周期分布中G0/G1期占71.95%、S期占14.8%、G2期占10.15%,亲本细胞MGC803中G1期占62.4%、S期占11.275%、G2期占20.47%,耐药株中G0/G1期明显增多。耐药细胞株MGC803/PTX与亲本细胞相比,P-gp、Bcl-2、PARP蛋白表达上调,Bax、β-catenin和p-GSK-3β蛋白的表达下调。结论:1首次成功建立了稳定的人胃癌耐药细胞株MGC803/PTX;2耐药细胞株MGC803/PTX的生物学特性包括细胞形态,细胞增殖率,细胞凋亡率,周期分布等均发生明显变化;3与亲本细胞MGC803比较,P-gp明显上调,抗凋亡蛋白Bax明显下调,同时促凋亡蛋白Bcl-2,PARP等也有一定程度的上调,这是其耐药的部分原因。同时耐药细胞MGC803/PTX中,β-catenin和p-GSK-3β蛋白的表达与MGC803细胞比较也有差异,说明其耐药机制与Wnt/β-catenin相关通路有关。第二部分:化合物20对MGC803/PTX的作用研究探讨新型化合物20对耐药细胞株MGC803/PTX的作用,以及初步的机制研究。方法:CCK8法检测不同作用时间化合物20对MGC803/PTX的抗肿瘤活性;克隆形成实验检测化合物20对单个细胞增殖的作用;Hoechst 33258染色法观察化合物20作用后MGC803/PTX细胞核形态变化;高内涵分析技术结合PI染色法检测化合物20对耐药细胞周期阻滞作用;Western blot检测化合物20作用后P-gp、Bcl-2、Bax、Pro-PARP1、Cleaved caspase-3等蛋白的表达情况。结果:CCK8实验结果表明化合物20对MGC803和MGC803/PTX细胞均有很好的活性;化合物20作用后MGC803/PTX细胞出现明显的染色质皱缩,核碎裂等细胞凋亡形态;化合物20作用后MGC803/PTX细胞周期分布发生变化,G2/M期占的比例明显增多;化合物20作用后P-gp、Bcl-2、Pro-PARP1蛋白下调,Bax,Cleaved caspase-3上调。结论:1化合物20对MGC803/PTX细胞有很强的增殖抑制作用2化合物20能将MGC803/PTX细胞阻滞在G2/M期3化合物20的作用机制与细胞线粒体凋亡通路有关,能上调Bax、Cleaved caspase-3等促凋亡蛋白,下调Bcl-2、Pro-PARP1蛋白的表达,同时化合物20还能下调P-gp,从而导致MGC803/PTX细胞凋亡。4新型β-内酰胺类化合物20对肿瘤耐药细胞MGC803/PTX作用明显,可以考虑下一步开发相关制剂,深入研究其机制。
[Abstract]:The first part: the establishment and mechanism of human gastric cancer resistant paclitaxel cell line MGC803/PTX and its mechanism study using paclitaxel concentration gradient increase method to induce human gastric cancer MGC803 cells to establish drug resistant cell line MGC803/PTX, to explore the difference between human gastric cancer resistant cell MGC803/PTX and parental cell MGC803. A human gastric cancer resistant cell line, MGC803/PTX, was built, and the cell morphological changes in the induction of taxol were observed under the inverted microscope; the resistance index and stability of MGC803/PTX cell lines were detected by CCK8; the drug resistance of MGC803/PTX cell lines to 5-FU and ADR was detected by CCK8; and the growth curves of MGC803 and MGC803/PTX cell lines were plotted by cell counting method; The clone formation rate of MGC803 and MGC803/PTX cells was measured by the experiment of lung formation; the morphological changes of cells and nuclei were observed under the action of taxol under the fluorescence microscope; the flow cytometry was used to analyze the cell apoptosis and the distribution of cell cycle. The.Western blot test was used to detect P-gp, Bcl-2, Bax, PARP, beta -catenin, p-GSK-3 beta in the parent cell lines. The expression of protein and the difference of the expression of two cell proteins were compared and the possible mechanism of drug resistance of MGC803/PTX cell lines was analyzed. Results: the resistance index of human gastric cancer cell line MGC803/PTX was successfully constructed for 10 months. The resistance index of paclitaxel was 9.3. The cells gradually changed from long spindle shape to short polygon in the induction process, and the volume became smaller, and 3 after the induction. The changes of paclitaxel to MGC803/PTX cell IC50 in MGC803/PTX cells were not changed in a month, and there was a certain cross resistance to 5-FU and ADR, and RI was 4.38 and 1.4, respectively. The growth rate of MGC803/PTX in drug resistant cells was slower than that of parent cell MGC803, and the time of population multiplication was prolonged, respectively, 24.55h and 22.5h, MGC803 and MGC803/PTX cell clones were formed. There was a significant difference in rate (p0.001). The ability of MGC803/PTX clone formation in drug-resistant cells was significantly higher than that of parent cell MGC803; two cells with PTX action, Hoechst 33258 stained nuclei, chromatin concentration and apoptotic body formation, but the apoptosis rate of MGC803/PTX cells was significantly lower than that of MGC803 cells; flow cytometry showed the same results. The apoptotic rate of MGC803/PTX was lower than that of MGC803 cells. After 20nMPTX, the apoptosis rate was 1.8% and 24.2%.MGC803/PTX cells were 71.95% in G0/G1 stage, 14.8% in S stage, 10.15% in G2 stage, 62.4% in MGC803 in MGC803, 11.275% in S, 20.47% in G2 stage, and significantly increased in drug-resistant strains. Compared with parental cells, the expression of P-gp, Bcl-2, PARP protein was up regulation, Bax, beta -catenin and p-GSK-3 beta protein expression down regulated. Conclusion: 1 the stable human gastric cancer cell line MGC803/PTX was successfully established for the first time. The biological characteristics of the 2 drug resistant cell strain MGC803/PTX include cell morphology, cell proliferation rate, apoptosis rate, and periodic distribution. 1 3 compared with the parent cell MGC803, P-gp was obviously up-regulated, the anti apoptotic protein Bax was obviously down, and the apoptotic protein Bcl-2, PARP and so on were also up regulated to a certain extent, which was partly responsible for its resistance. Meanwhile, the expression of beta -catenin and p-GSK-3 beta protein was also different from MGC803 cells in the drug resistant cell MGC803/PTX. The mechanism of drug resistance is related to the Wnt/ beta -catenin related pathway. Second part: the effect of compound 20 on MGC803/PTX, the effect of new compound 20 on drug resistant cell line MGC803/PTX, and preliminary mechanism study. Method: CCK8 method was used to detect the anti-tumor activity of MGC803/PTX in different time compounds, and the cloning and formation of experimental examination. The effect of compound 20 on the proliferation of single cell was measured; Hoechst 33258 staining was used to observe the morphological changes of MGC803/PTX nucleus after compound 20; high intension analysis technique combined with PI staining method to detect the cycle block effect of compound 20 on drug resistant cells; Western blot was used to detect P-gp, Bcl-2, Bax, Pro-PARP1, Cleaved caspase-3 after the 20 action of compounds. Results of protein expression. Results: CCK8 experimental results showed that compound 20 had good activity for MGC803 and MGC803/PTX cells. After compound 20, MGC803/PTX cells appeared obvious chromatin crumbling, nuclear fragmentation and other cell apoptosis morphology. After the compound 20, the MGC803/PTX cell cycle distribution changed, and the proportion of G2/M period increased significantly. P-gp, Bcl-2, Pro-PARP1 protein down regulation after compound 20, Bax, Cleaved caspase-3 up regulation. Conclusion: 1 compound 20 on MGC803/PTX cells has strong proliferation inhibition effect 2 compound 20 can block MGC803/PTX cells in G2/M phase 3 compound 20 mechanism related to the cell line granular apoptosis pathway, can up Bax, Cleaved caspase-3 Equal apoptotic protein, down regulation of Bcl-2, Pro-PARP1 protein expression, and compound 20 can also reduce P-gp, thus leading to MGC803/PTX cell apoptosis,.4 new beta lactam compound 20 has obvious effect on tumor resistant cell MGC803/PTX. We can consider the next step to develop the related preparations and further study the mechanism.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.2

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