RNA干扰MRP1的基因表达逆转NSCLC化疗耐药的研究

发布时间：2018-05-12 07:47

本文选题：非小细胞肺癌（NSCLC） + 多药耐药蛋白　；参考：《大连医科大学》2012年硕士论文

【摘要】：目的：为了研究多药耐药基因MRP1在非小细胞肺癌中表达的差异，以及观察siRNA干扰技术是否能有效抑制非小细胞肺癌耐药细胞株中M RP1的表达，探讨体外高效和特异逆转肺癌耐药的策略。方法：运用实时荧光定量PCR、免疫组化法检测30例NSCLC组织及癌旁组织标本中耐药基因MRP1mRNA及其蛋白的表达水平，并且以MRP1为靶标设计合成siRNA，直接转染入A549/DDP，转染前后用实时荧光定量PCR检测MRP1mRNA表达水平，用Western blot检测MRP1蛋白的表达，MTT法检测A549/DDP对顺铂耐药性的变化。结果：1.在30例病理组织中，MRP1蛋白在肺癌组织中的阳性率为70%（21/30），癌旁组织中的阳性率为16.67%（5/30），表达有统计学差异(P0.05)；对于癌组织，MRP1蛋白在已经化疗的病理组织中阳性率为90.9%(10/11)，在未化疗的病理组织中阳性率为57.89%(11/19)，，计算两者有统计学差异(P0.05)，说明MRP1在已化疗的组织中高表达。而且PCR结果显示MRP1mRNA在癌组织中的表达也高于癌旁组织，经过统计学分析有明显差异(P0.01)。 2.体外实验中A549/DDP转染MRP1siRNA后,MRP1mRNA表达明显降低，和转染前相比较有显著性差异(P0.05)，抑制率达到了78%。转染后MRP1蛋白相对表达强度从之前的0.820±0.037降低为0.435±0.042有明显差异(P0.05)；MTT结果显示转染后A549/DDP对顺铂的敏感性增加了1.43倍。结论：1.多药耐药基因MRP1在肺癌组织中的高表达,且MRP1蛋白在化疗的非小细胞肺癌组织中表达高于未化疗组织，说明在非小细胞肺癌患者中存在原发性耐药。 2.体外实验证实了在耐药细胞转染MRP1siRNA后MRP1的转录和翻译都受到了不同程度的抑制，也使其对顺铂的药物敏感性增强，从一定程度上说明了逆转耐药。
[Abstract]:Objective: to study the difference of multidrug resistance gene MRP1 expression in non-small cell lung cancer (NSCLC) and whether siRNA interference can effectively inhibit the expression of M RP1 in NSCLC cell lines. Objective: to study the strategy of reversing drug resistance of lung cancer in vitro. Methods: the expression of drug resistance gene MRP1mRNA and its protein in 30 cases of NSCLC and its adjacent tissues were detected by real-time fluorescence quantitative PCR and immunohistochemistry. SiRNAs were designed and synthesized with MRP1 as target and transfected directly into A549 / DDP. The expression of MRP1mRNA was detected by real-time quantitative PCR before and after transfection, and the expression of MRP1 protein was detected by Western blot. The change of cisplatin resistance of A549/DDP to cisplatin was detected by Western blot assay. The result is 1: 1. The positive rate of MRP1 protein was 70% in lung cancer tissue and 16.67% 5 / 30% in paracancerous tissue, and the positive rate of MRP1 protein was 90.9% / 1111% in cancer tissues which had already been treated with chemotherapy, and the positive rate of MRP1 protein was 16.67% 5 / 30% in adjacent tissues, and the positive rate of MRP1 protein in cancer tissues was 90.9% / 1111% in chemotherapeutic pathological tissues, and the positive rate of MRP1 protein was 16.67% 5 / 30% in adjacent tissues of lung cancer. The positive rate of MRP1 was 57.89% and 11 / 19% in pathological tissues. There was a statistical difference between the two groups (P 0.05), which indicated that MRP1 was highly expressed in the tissues that had been treated with chemotherapy. PCR results showed that the expression of MRP1mRNA in cancer tissues was higher than that in paracancerous tissues, and the difference was statistically significant (P 0.01). 2. The expression of MRP1 mRNA in MRP1siRNA transfected with A549/DDP was significantly lower than that before transfection, and the inhibitory rate was 78%. The relative expression intensity of MRP1 protein decreased from 0.820 卤0.037 to 0.435 卤0.042 after transfection. The results showed that the sensitivity of A549/DDP to cisplatin increased by 1.43 times after transfection. Conclusion 1. The high expression of multidrug resistance gene MRP1 in lung cancer tissues and the expression of MRP1 protein in non-small cell lung cancer tissues were higher than those in non-small cell lung cancer tissues, indicating that there was primary drug resistance in patients with non-small cell lung cancer. 2. In vitro experiments confirmed that the transcription and translation of MRP1 were inhibited in different degrees after transfection of drug resistant cells into MRP1siRNA, which also enhanced the drug sensitivity to cisplatin, which indicated that the drug resistance was reversed to some extent.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R734.2;R3416

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