人胃癌体外耐药模型的建立及新型甾体联苯类化合物抗肿瘤活性研究

发布时间：2018-05-04 00:06

本文选题：肿瘤耐药 + 5-氟尿嘧啶　；参考：《郑州大学》2017年硕士论文

【摘要】：首先,我们采用浓度递增法刺激人胃癌细胞MGC803,建立了人胃癌体外耐药细胞模型MGC803/5-FU,并初步探讨了MGC803和MGC803/5-FU的生物学性质及耐药株MGC803/5-FU的耐药机制。在这部分的研究中,我们以人胃癌细胞MGC803为亲本细胞株,以化疗药物5-氟尿嘧啶低浓度持续刺激,并逐渐加大刺激浓度,建立人胃癌体外耐药细胞模型MGC803/5-FU。耐药细胞株建立后,我们采用CCK8法检测了亲本细胞株及耐药细胞株对5-氟尿嘧啶、顺铂、紫杉醇、阿霉素的耐药性及耐药细胞株撤药6个月内的耐药稳定性。同时,5-氟尿嘧啶作用亲本细胞与耐药细胞后,经DAPI染色和Annexin V/PI染色进一步验证耐药株MGC803/5-FU对5-FU的耐药性;然后,我们通过PI染色、平板克隆和绘制生长曲线等检测了MGC803和MGC803/5-FU生物学性质的差异。最后,通过Western blot检测亲本细胞和耐药细胞P-gp、TS、β-catenin等蛋白表达量的差异,分析可能的耐药机制。经过8个月的诱导,成功建立了人胃癌体外耐药细胞模型MGC803/5-FU,耐药指数为13.02,但交叉耐药现象不明显;同时,耐药细胞株撤药六个月内耐药指数能够保持在9到13之间,耐药稳定性较好。5-氟尿嘧啶作用后,相同浓度下,耐药细胞的凋亡率明显低于亲本细胞。与亲本细胞相比,耐药细胞G0/G1期比例增加,S期比例减少;耐药细胞生长速度减慢,群体倍增时间增大,克隆形成率降低。耐药细胞中TS蛋白表达量增加,同时Wnt/β-catenin通路相关蛋白发生改变。在耐药模型的基础上,我们接着研究了新型甾体联苯类化合物ZYL-263对胃癌细胞MGC803及其耐药细胞MGC803/5-FU的抗肿瘤活性及其机制。我们采用CCK8法和平板克隆法检测化合物ZYL-263对MGC803及MGC803/5-FU细胞生长增殖的影响。DAPI染色和Annexin V/PI染色检测ZYL-263对两株细胞细胞凋亡的影响;同时,DCFH-DA染色检测ZYL-263对两株细胞活性氧的影响。最后,ZYL-263作用两株细胞后,Western blot检测Bcl-2、Bax、Total-Caspase-7、Cleaved-Caspase-7、Total-PARP-1、Cleaved-PARP-1、TS等蛋白表达量的变化。结果显示,ZYL-263对MGC803及MGC803/5-FU有较好的生长抑制作用,同时MGC803/5-FU的IC50低于其对MGC803的的IC50;同时,ZYL-263能够抑制两株细胞的克隆形成。ZYL-263能够使两株细胞的细胞核发生皱缩和明显的凋亡,且MGC803/5-FU的凋亡率高于MGC803的凋亡率。ZYL-263作用两株细胞后,胞内活性氧水平明显增高。ZYL-263能够降低两株细胞内的Bcl-2、Total-Caspase-7、Total-PARP-1蛋白表达量,同时增加两株细胞内的Bax、Cleaved-Caspase-7、Cleaved-PARP-1蛋白表达量;另外,ZYL-263显著下调耐药株MGC803/5-FU的TS蛋白表达量,而对MGC803的TS蛋白表达量没有明显影响。综上所述,我们成功建立了稳定的人胃癌体外耐药模型MGC803/5-FU,检测了其相关生物学特性及其可能的耐药机制,为进一步研究耐药相关原因及寻找新型抗肿瘤化合物提供了稳定的体外模型。新型化合物ZYL-263能够明显促进亲本细胞MGC803及其耐药细胞MGC803/5-FU的凋亡,且对MGC803/5-FU的促凋亡作用更明显,为寻找新型抗肿瘤化合物研究提供一定的思路。
[Abstract]:First, we used the concentration increase method to stimulate human gastric cancer cell MGC803, and established the human gastric cancer cell model MGC803/5-FU, and preliminarily discussed the biological properties of MGC803 and MGC803/5-FU and the resistance mechanism of the drug resistant strain MGC803/5-FU. In this part, we use the human gastric cancer cell MGC803 as the parent cell line and the chemotherapeutic drug. After the low concentration of 5- fluorouracil was sustained and the concentration was gradually increased, the drug resistant cell line of human gastric cancer was established, and the drug resistance of MGC803/5-FU., cisplatin, paclitaxel and amamycin and the withdrawal of drug resistant cell lines were detected by CCK8 method within 6 months. Resistance stability. At the same time, after 5- fluorouracil acted on the parent and drug-resistant cells, the resistance of MGC803/5-FU to 5-FU was further verified by DAPI staining and Annexin V/PI staining. Then, we detected the differences in the biological properties of MGC803 and MGC803/5-FU by PI staining, flat clones and plotting growth curves. Finally, we passed Weste. RN blot detected the difference in the expression of P-gp, TS, and beta -catenin in the parent and drug-resistant cells, and analyzed the possible mechanism of drug resistance. After 8 months of induction, the drug resistance cell model of human gastric carcinoma was successfully established, and the resistance index was 13.02, but the cross resistance was not obvious. At the same time, drug resistant cell lines were withdrawn for six months. At the same concentration, the apoptosis rate of drug resistant cells was significantly lower than that of parental cells at the same concentration of 9 to 13, with the same resistance and stability of.5- fluorouracil. Compared with the parent cells, the proportion of G0/G1 phase increased, the proportion of S phase decreased, the growth rate of drug-resistant cells decreased, the population doubling time increased, and the clone formation rate decreased. The expression of TS protein in the cells increased and the Wnt/ beta -catenin pathway related proteins changed. On the basis of the drug resistance model, we then studied the anti-tumor activity and mechanism of the new steroidal biphenyl compound ZYL-263 on the gastric cancer cell MGC803 and its drug-resistant cell MGC803/5-FU. We used the CCK8 method and the plate cloning method to detect the antitumor activity. The effect of ZYL-263 on the growth and proliferation of MGC803 and MGC803/5-FU cells,.DAPI staining and Annexin V/PI staining were used to detect the effect of ZYL-263 on cell apoptosis of two cells. At the same time, DCFH-DA staining was used to detect the effect of ZYL-263 on the reactive oxygen species of two cells. Finally, ZYL-263 action two cells, Western blot -Caspase-7, Total-PARP-1, Cleaved-PARP-1, TS and other protein expression changes. The results showed that ZYL-263 had better growth inhibition on MGC803 and MGC803/5-FU, while MGC803/5-FU's IC50 was lower than the IC50 on MGC803. At the same time, ZYL-263 could inhibit the formation of two cells, which could cause the cells of two cells to hair wrinkle. Contraction and obvious apoptosis, and MGC803/5-FU apoptosis rate is higher than the apoptosis rate of MGC803.ZYL-263 two cells, the intracellular active oxygen level is obviously increased,.ZYL-263 can reduce the Bcl-2, Total-Caspase-7, Total-PARP-1 protein expression in two cells, and increase the Bax, Cleaved-Caspase-7, Cleaved-PARP-1 protein table in two cells. In addition, ZYL-263 significantly downregulated the TS protein expression of the drug resistant strain MGC803/5-FU, but had no significant influence on the TS protein expression of MGC803. In summary, we successfully established a stable human gastric cancer drug resistance model MGC803/5-FU, which detected its related biological characteristics and its possible mechanism of resistance to further study of drug resistance. The cause and the search for new antitumor compounds provide a stable in vitro model. The new compound, ZYL-263, can obviously promote the apoptosis of the parent cell MGC803 and its drug-resistant MGC803/5-FU cells, and the apoptosis promoting effect on MGC803/5-FU is more obvious, which provides a certain way of thinking for the study of new antitumor compounds.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

【相似文献】