论文部分内容阅读
目的 观察有丝分裂阻滞缺陷蛋白 2 (mitoticarrestdeficientprotein 2 ,MAD2 )基因的选择性剪接体MAD2 β在人胃癌多药耐药性形成过程中的作用机制。 方法 从耐阿霉素人胃癌细胞系SGC790 1/ADR中提取总RNA ,通过RT PCR获得MAD2 β全长编码cDNA ,并克隆至pUCm T载体 ,正向亚克隆插入真核表达载体pcDNA3.1中。以脂质体介导pcDNA3.1/MAD2 β真核表达载体转染胃腺癌细胞SGC790 1。以体外药敏试验检测转染MAD2 β的胃癌细胞及其对照组细胞对化疗药物的敏感性。通过流式细胞仪检测MAD2 β基因转染组和对照组胃癌细胞在细胞周期中的分布和细胞内阿霉素的荧光强度。结果 RT PCR扩增获得约 0 .5 3kb片段 ,DNA序列测定证实为MAD2的一种选择性剪接形式 ,命名为MAD2 β。重组正义真核表达载体pcDNA3.1/MAD2 β瞬时转染SGC790 1细胞 ,经MTT法证实 ,转染细胞SGC790 1/MAD2 β对化疗药物阿霉素、长春新碱和丝裂霉素的耐药性增强。与SGC790 1/pcDNA3.1比较 ,阿霉素大剂量短期作用后 ,SGC790 1/MAD2 β细胞内的平均荧光强度比对照组低 (P <0 .0 5 )。结论 MAD2 β基因在一定程度上增强了人亲本胃癌细胞SGC790 1对化疗药物的耐受性 ,其可能的机制为肿瘤细胞在化疗药物作用下 ,有丝分裂出现异常 ,耐药细胞通过调控MAD2?
Objective To investigate the mechanism of MAD2β, an alternative splice variant of MAD2, in the development of multidrug resistance in human gastric cancer. Methods Total RNA was extracted from human adriamycin resistant human gastric cancer cell line SGC790 1 / ADR. The full length MAD2 cDNA was obtained by RT PCR and cloned into pUCm T vector and inserted into eukaryotic expression vector pcDNA3.1 . Transfection of gastric cancer cell line SGC790 with liposome - mediated pcDNA3.1 / MAD2 β eukaryotic expression vector. In vitro susceptibility tests were performed to detect the sensitivity of chemotherapeutic drugs to MAD2 β-transfected gastric cancer cells and their control cells. The distribution of gastric cancer cells in the MAD2 β gene transfection group and the control group in the cell cycle and the fluorescence intensity of intracellular doxorubicin were detected by flow cytometry. Results Approximately 0.53 kb fragment was obtained by RT-PCR. The DNA sequence was confirmed to be an alternative spliced form of MAD2, named as MAD2 β. The recombinant sense eukaryotic expression vector pcDNA3.1 / MAD2 β was transfected into SGC7901 cells transiently. MTT assay confirmed that the transfected cells SGC790 1 / MAD2 β were resistant to doxorubicin, vincristine and mitomycin Sexual enhancement. Compared with SGC790 1 / pcDNA3.1, the average fluorescence intensity of SGC790 1 / MAD2 β cells was lower than that of the control group after high dose and short-term dose of doxorubicin (P <0.05). Conclusions MAD2 β gene enhances the tolerance of human parental gastric cancer cell SGC790 1 to chemotherapeutic drugs to a certain extent. The possible mechanism is that the mitosis of tumor cells under the action of chemotherapeutic drugs is abnormal, and the resistant cells regulate MAD2?