人胃癌耐藥細(xì)胞株的建立和新型β-內(nèi)酰胺類化合物抗腫瘤耐藥研究
發(fā)布時(shí)間：2018-08-01 16:19
【摘要】：第一部分:人胃癌耐紫杉醇細(xì)胞株MGC803/PTX的建立及機(jī)制研究采用紫杉醇濃度梯度遞增法誘導(dǎo)人胃癌MGC803細(xì)胞建立耐藥細(xì)胞株MGC803/PTX,探討人胃癌耐藥細(xì)胞MGC803/PTX與親本細(xì)胞MGC803之間的差異。方法:用紫杉醇以濃度梯度遞增誘導(dǎo)法構(gòu)建人胃癌耐藥細(xì)胞株MGC803/PTX;倒置顯微鏡下觀察紫杉醇誘導(dǎo)過程中細(xì)胞形態(tài)學(xué)變化;CCK8法檢測MGC803/PTX細(xì)胞株的耐藥指數(shù)及其穩(wěn)定性;CCK8法檢測MGC803/PTX細(xì)胞株對5-FU和ADR的耐藥性;細(xì)胞計(jì)數(shù)法繪制MGC803和MGC803/PTX細(xì)胞株的生長曲線;克隆形成實(shí)驗(yàn)測定MGC803和MGC803/PTX細(xì)胞株的克隆形成率;熒光顯微鏡下觀察紫杉醇作用后細(xì)胞和細(xì)胞核形態(tài)學(xué)變化;流式細(xì)胞儀分析細(xì)胞凋亡和周期分布情況。Western blot實(shí)驗(yàn)檢測耐藥細(xì)胞株與親本細(xì)胞株中P-gp、Bcl-2、Bax、PARP、β-catenin、p-GSK-3β等蛋白的表達(dá),比較兩株細(xì)胞蛋白表達(dá)的差異,分析MGC803/PTX細(xì)胞株可能的耐藥機(jī)制。結(jié)果:歷時(shí)10個(gè)月成功構(gòu)建了人胃癌耐藥細(xì)胞株MGC803/PTX,對紫杉醇的耐藥指數(shù)為9.3,誘導(dǎo)過程中細(xì)胞由長梭形逐步變成短多邊形,體積變小,誘導(dǎo)結(jié)束后3個(gè)月內(nèi)紫杉醇對MGC803/PTX細(xì)胞IC50變化不大。MGC803/PTX對5-FU和ADR這兩種化療藥物存在一定的交叉耐藥性,RI分別為4.38和1.4;耐藥細(xì)胞MGC803/PTX的生長速度比親本細(xì)胞MGC803慢,群體倍增時(shí)間延長,分別為24.55h和22.5h;MGC803和MGC803/PTX細(xì)胞克隆形成率有明顯差異(p0.001),耐藥細(xì)胞MGC803/PTX克隆形成能力明顯高于親本細(xì)胞MGC803;PTX作用兩株細(xì)胞,Hoechst 33258染色后細(xì)胞核的染色增強(qiáng),細(xì)胞核染色質(zhì)濃縮且有凋亡小體生成,但MGC803/PTX細(xì)胞凋亡率明顯低于MGC803細(xì)胞;流式細(xì)胞儀檢測結(jié)果同樣顯示耐藥細(xì)胞株MGC803/PTX凋亡率低于MGC803細(xì)胞,20nMPTX作用后凋亡率分別為1.8%和24.2%。MGC803/PTX細(xì)胞周期分布中G0/G1期占71.95%、S期占14.8%、G2期占10.15%,親本細(xì)胞MGC803中G1期占62.4%、S期占11.275%、G2期占20.47%,耐藥株中G0/G1期明顯增多。耐藥細(xì)胞株MGC803/PTX與親本細(xì)胞相比,P-gp、Bcl-2、PARP蛋白表達(dá)上調(diào),Bax、β-catenin和p-GSK-3β蛋白的表達(dá)下調(diào)。結(jié)論:1首次成功建立了穩(wěn)定的人胃癌耐藥細(xì)胞株MGC803/PTX;2耐藥細(xì)胞株MGC803/PTX的生物學(xué)特性包括細(xì)胞形態(tài),細(xì)胞增殖率,細(xì)胞凋亡率,周期分布等均發(fā)生明顯變化;3與親本細(xì)胞MGC803比較,P-gp明顯上調(diào),抗凋亡蛋白Bax明顯下調(diào),同時(shí)促凋亡蛋白Bcl-2,PARP等也有一定程度的上調(diào),這是其耐藥的部分原因。同時(shí)耐藥細(xì)胞MGC803/PTX中,β-catenin和p-GSK-3β蛋白的表達(dá)與MGC803細(xì)胞比較也有差異,說明其耐藥機(jī)制與Wnt/β-catenin相關(guān)通路有關(guān)。第二部分:化合物20對MGC803/PTX的作用研究探討新型化合物20對耐藥細(xì)胞株MGC803/PTX的作用,以及初步的機(jī)制研究。方法:CCK8法檢測不同作用時(shí)間化合物20對MGC803/PTX的抗腫瘤活性;克隆形成實(shí)驗(yàn)檢測化合物20對單個(gè)細(xì)胞增殖的作用;Hoechst 33258染色法觀察化合物20作用后MGC803/PTX細(xì)胞核形態(tài)變化;高內(nèi)涵分析技術(shù)結(jié)合PI染色法檢測化合物20對耐藥細(xì)胞周期阻滯作用;Western blot檢測化合物20作用后P-gp、Bcl-2、Bax、Pro-PARP1、Cleaved caspase-3等蛋白的表達(dá)情況。結(jié)果:CCK8實(shí)驗(yàn)結(jié)果表明化合物20對MGC803和MGC803/PTX細(xì)胞均有很好的活性;化合物20作用后MGC803/PTX細(xì)胞出現(xiàn)明顯的染色質(zhì)皺縮,核碎裂等細(xì)胞凋亡形態(tài);化合物20作用后MGC803/PTX細(xì)胞周期分布發(fā)生變化,G2/M期占的比例明顯增多;化合物20作用后P-gp、Bcl-2、Pro-PARP1蛋白下調(diào),Bax,Cleaved caspase-3上調(diào)。結(jié)論:1化合物20對MGC803/PTX細(xì)胞有很強(qiáng)的增殖抑制作用2化合物20能將MGC803/PTX細(xì)胞阻滯在G2/M期3化合物20的作用機(jī)制與細(xì)胞線粒體凋亡通路有關(guān),能上調(diào)Bax、Cleaved caspase-3等促凋亡蛋白,下調(diào)Bcl-2、Pro-PARP1蛋白的表達(dá),同時(shí)化合物20還能下調(diào)P-gp,從而導(dǎo)致MGC803/PTX細(xì)胞凋亡。4新型β-內(nèi)酰胺類化合物20對腫瘤耐藥細(xì)胞MGC803/PTX作用明顯,可以考慮下一步開發(fā)相關(guān)制劑,深入研究其機(jī)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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R735.2
本文編號：2158143
[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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R735.2
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