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目的研究转染survivin反义mRNA对Jurkat淋巴瘤细胞生长的影响以及转染后淋巴瘤细胞对化疗药物的敏感性。方法构建survivin反义mRNA真核表达质粒pcDNA3.1反义(As)survivin;利用脂质体转染法将其转入高表达survivinmRNAT淋巴母细胞淋巴瘤Jurkat细胞系,用逆转录聚合酶链反应(RTPCR)、免疫组织化学SP法、Western印迹法检测细胞中survivin表达;用细胞计数、流式细胞术(FCM)检测其细胞生长曲线、细胞凋亡指数,并进行光镜、电镜形态学观察;并对转染pcDNA3.1Assurvivin前后Jurkat细胞分别加入4羟基环磷酰胺(CTX)、甲氨蝶呤(MTX)72h后,常规MTT检测细胞存活率。结果RTPCR检测转染pcDNA3.1Assurvivin后48h、5和6周Jurkat细胞survivinmRNA表达,发现survivinmRNA表达皆低于对照组;转染后survivin蛋白表达也明显降低。转染pcDNA3.1Assurvivin后Jurkat细胞生长倍增时间(52h)明显延长;用FCM检测细胞凋亡发现,转染pcDNA3.1Assurvivin后Jurkat细胞凋亡指数[20.2%(48h)]明显高于对照组(转染空质粒和未转染组,2.1%和1.3%);5和6周为6.2%和6.8%,明显高于未转染细胞(1.3%和1.0%)。光镜、电镜观察见转染细胞出现较多凋亡细胞及一些变性肿胀细胞;MTT检测结果显示Jurkat细胞转染前后,经化疗药物4羟基环磷酰胺和甲氨蝶呤作用,转染细胞的抑制率明显大于未转染组,差异有统计学意义(P<0.05)。结论survivin基因对Jurkat细胞系的生长起着重要的作用,抑制survivin基因表达在T淋巴母细胞淋巴瘤治疗中可能有重要的意义,该基因似可能作为治疗的靶点。
Objective To investigate the effect of transfection of antisense survivin mRNA on the growth of Jurkat lymphoma cells and the sensitivity of lymphoma cells to chemotherapeutic drugs after transfection. Methods The survivin antisense mRNA eukaryotic expression plasmid pcDNA3.1 antisense (survivin) was constructed and transfected into Jurkat cell line with high expression of survivinmRNAT lymphoblastic lymphoma by lipofection. The expression of survivin gene was detected by reverse transcriptase polymerase chain reaction (RTPCR), immunohistochemistry (SP) and Western blotting were used to detect the expression of survivin. Cell growth curve and apoptosis index were detected by cell counting and flow cytometry (FCM) The survival rate of Jurkat cells was detected by conventional MTT assay after adding CTX and MTX to the Jurkat cells transfected with pcDNA3.1Assurvivin. Results RTPCR detected survivin mRNA expression in Jurkat cells 48h, 5 and 6 weeks after transfection with pcDNA3.1Assurvivin, and found that survivin mRNA expression was lower than that of the control group; Survivin protein expression was also significantly decreased after transfection. The doubling time (52h) of Jurkat cells was significantly prolonged after transfection with pcDNA3.1Assurvivin. The apoptotic index of Jurkat cells detected by FCM was significantly higher than that of the control group (20.2%, 48h) after transfection with pcDNA3.1Assurvivin (2.1% and 1.3%, respectively); 6.2% and 6.8% at 5 and 6 weeks respectively, which was significantly higher than that of untransfected cells (1.3% and 1.0%). Light microscope and electron microscopy showed that more apoptotic cells and some degenerative cells were found in transfected cells. MTT assay showed that Jurkat cells transfected by 4-hydroxycyclophosphamide and methotrexate before and after transfection The inhibition rate was significantly higher than that of the untransfected group (P <0.05). Conclusion The survivin gene plays an important role in the growth of Jurkat cell line. Suppression of survivin gene expression may play an important role in the treatment of T lymphoblastic lymphoma. The gene may serve as a therapeutic target.