【作者】 李倩；

【导师】 潘超；

【作者基本信息】 厦门大学 ， 临床病理学， 2021， 硕士

【摘要】 目的:1、探讨OCTN2是否参与了 HCT15结直肠癌细胞株耐药性的形成。2、明确HCT15结直肠癌耐药细胞株与亲本细胞株中OCTN2与Cav1之间的关系。3、探讨OCTN2在敲减Cav1后逆转耐药性的作用。4、探讨敲减Cav1后,肿瘤耐药性逆转的机制。方法:1、阿霉素诱导恢复本实验室前期已构建的结直肠癌HCT15/ADM的耐药细胞株,使其达到高度耐药的标准,CCK8检测其耐药性。2、探讨Cav1与OCTN2在HCT15细胞株耐药形成过程中的作用及关系,Western Blot检测HCT15亲本细胞株与HCT15/ADM耐药细胞株中Cav1与OCTN2蛋白表达量。3、构建shRNA-Cav1干扰质粒,使用293T细胞包装质粒,转染HCT15/ADM耐药细胞株,在HCT15/ADM结直肠癌耐药细胞株中构建敲减shCav1稳转细胞株;RT-PCR和western blot检测敲减效果。4、CCK8检测敲减Cav1后,HCT15/ADM耐药细胞株的耐药性的变化,并计算逆转耐药的倍数;western blot检测OCTN2蛋白表达量的变化。5、CCK8比较shCav1细胞与空载细胞中使用OCTN2抑制剂meldonium的前后细胞耐药性的变化情况;western blot 比较各组细胞之间Cav1与OCTN2蛋白表达量的变化。结果:1、HCT15/ADM结直肠癌耐药细胞株在0-128ug/ml浓度梯度的阿霉素的作用下半数抑制率(IC50)显著高于HCT15亲本株,其耐药指数(RI)为22.75>15,符合高度耐药;2、Cav1在HCT15/ADM耐药株中表达量高于HCT15亲本株,OCTN2(SLC22A5)在HCT15/ADM耐药株中表达量低于HCT15亲本株。3、HCT1 5/ADM-shCav1细胞株在0-64ug/ml浓度梯度的阿霉素作用下半数抑制率(IC50)显著低于HCT1 5/ADM-Vector细胞株,其耐药性逆转3.72倍;HCT15/ADM-shCav1细胞株中OCTN2(SLC22A5)的蛋白表达量上调;在阿霉素持续作用24H后,HCT15亲本、HCT15/ADM、空载细胞株、shCav1细胞株中Cav1的表达量均上调,OCTN2(SLC22A5)的表达被抑制。4、在HCT15/ADM-shCav1细胞株中,加入OCTN2(SLC22A5)竞争性抑制剂meldonium后,HCT15/ADM-shCav1细胞株对阿霉素的敏感性降低,耐药性部分恢复;比较HCT15亲本株、HCT15/ADM耐药株、HCT15/ADM-Vector细胞株、HCT15/ADM-shCav1细胞株中分别加入OCTN2(SLC22A5)竞争性抑制剂meldonium前后,Cav1的表达量未发生明显的变化。结论:1、在HCT15/ADM结直肠癌耐药细胞株中,OCTN2蛋白表达量降低,参与其耐药性的形成过程。2、Cav1在HCT15/ADM耐药细胞株中表达量上调,OCTN2在HCT15/ADM耐药细胞株中表达量下调,二者具有负相关性。3、敲减Cav1后,肿瘤细胞耐药性逆转,OCTN2表达量上调。4、敲减Cav1是通过OCTN2(SLC22A5)逆转HCT15/ADM耐药细胞耐药性。更多还原

【Abstract】 Objective:1.To explore whether OCTN2 is involved in the process of drug resistance in colorectal cancer HCT15 cell line.2.Identify the relationship between OCTN2 and Cavl in drug-resistant cell lines and parent cell lines.3.Explore the role of OCTN2 in reversing drug resistance after Cavl is knocked down.4.Explore the mechanism of tumor drug resistance reversal after Cav1 is knocked down.Method:1.ADM induces recovery in the colon cancer HCT15/ADM resistant cell line that has been constructed in our laboratory in the early stage,so that it reaches the standard of high drug resistance,and CCK8 detects its drug resistance.2.Western Blot was used to detect the expression of Cavl and OCTN2 protein between HCT15 parent strain and MDR strain,and the result clear wether Cav1 and OCTN2 are involved in the mechanism of drug resistance in HCT15 cell line.3.There were three pairs of interference primers and one pair of negative control primer were designed.And then the interference plasmid of ShRNA-cav1 was constructed and the plamids were transfected into 293T cells,the supernatant was used to infect HCT15/ADM cells.The silencing efficiency of HCT15/ADM-shRNA3 was highest which were screened by RT-PCR and western blot.4.CCK8 was used to detect the proliferation inhibition rates before and after Cav1 knock-down,and calculates the multiple of reversal of drug resistance,at the same time western blot detect the expression of OCTN2 protein.5.CCK8 was used to detect and compare the optical density of HCT15/ADM cells under deferent concentrate of ADM incubation before and after meldonium treatment in Cav1 knock-down cells and control cells,and western blot detected the expression of Cavl and OCTN2 protein respectively.Result:1.The half maximal inhibitory concentration(IC50)under deferent concentrations gradient of ADM from 0 to 128ug/ml in HCT15/ADM MDR strains were significantly higher than the HCT15 parent strains,and the resistance index(RI)was 22.75>15.The expreesion of Cav1 in HCT15/ADM resistant strains were higher than HCT15 parent strains,and OCTN2(SLC22A5)in HCT15/ADM resistant strains ware lower than HCT15 parent strains.2.After knocking down the Cav1,the half maximal inhibitory concentration(IC50)was significantly lower than before with the reversal fold of 3.72,the expression of OCTN2(SLC22A5)protein was increased.The expression of Cav1 protein was increased respectively in HCT15 parent strains,HCT15/ADM MDR strains,HCT15/ADM-Vector cells,HCT15/ADM-shCav1 cells,in contrast the expression of OCTN2(SLC22A5)was reduced.3.The sensitivity to the ADM was reduced before and after treating with meldonium which was the competitive inhibitor of OCTN2 protein in HCT15/ADM-shCav1 cells,however the half maximal inhibitory concentration(IC50)was still lower than HCT15/ADM MDR stains.The expression of Cav1 was not affected.Conclusion:1.In the HCT15 colorectal cancer drug-resistant cell line,the expression of OCTN2 transporter is reduced,which is involved in the formation of its drug resistance.2.Cav1 was up-regulated in HCT15/ADM cell lines,and OCTN2 was down-regulated in HCT15/ADM cell lines.There is a negative correlation between the two proteins.3.After Cav1 knocking down,the drug resistance of tumor cells is reversed,and the expression of OCTN2 is up-regulated.4.The drug resistance was reversed by knocking down in HCT15/ADM cell lines through OCTN2(SLC22A5).更多还原