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作 者:高美华[1] 李华侨[1] 李冰[2] 王忠[1] 丛蓓蓓[1] 王冰[3] GAO Mei-Hua LI Hua-Qiao LI Bing WANG Zhong CONG Bei-Bei WANG Bing(Department of Immunology Medical College, Qingdao University, Qingdao 266071, China)
机构地区:[1]青岛大学医学院免疫学教研室,青岛266071 [2]青岛大学医学院遗传学教研室,青岛266071 [3]青岛大学医学院科研处,青岛266071
出 处:《中国免疫学杂志》2017年第5期693-696,共4页Chinese Journal of Immunology
基 金:国家自然科学基金(No.81273206)资助
摘 要:目的:探讨下调CD59对急性T系白血病细胞株Jurkat凋亡相关分子的影响。方法:运用RNAi慢病毒为载体下调急性T系白血病Jurkat细胞株CD59的表达;激光共聚焦观察转染效率及CD59分子的定位;Real-time-PCR、Western blot筛选下调效果最好的一组细胞,用其做后续的分子生物学水平的实验;Western blot检测Bcl-2、Bax、Caspase-3、Survivin蛋白表达量的变化;ELISA检测各组的细胞培养上清中IL-3、TNF-β的表达。结果:激光共聚焦观察到转染各组的转染效率在90%以上,CD59分子主要位于细胞膜;Real-time-PCR筛选得出下调A组的转染效果最好;Western blot结果显示下调A组的CD59蛋白的表达量减少最明显,将RNAi-CD59-A定义为RNAi-CD59作为实验组进行后续实验;实验组能增强Bax、Caspase-3蛋白的表达(P<0.05),抑制Bcl-2、Survivin蛋白的表达(P<0.05);ELISA结果显示下调组IL-3表达水平降低(P<0.05),TNF-β的表达水平升高(P<0.05)。结论:下调D59基因表达可使急性T系白血病细胞株Jurkat的促凋亡分子表达增高,促增殖分子表达降低。Objective:To study the change of related molecular about apoptosis we reduce the expression of CD59 on acute T lymphocyte Jurkat cell lines.Methods:RNA interference(RNAi) was used to reduce the expression of CD59 gene by lentivirus,confocal was applyed to observe the transfection efficiency and the location of CD59 molecular then Real-time-PCR and Western blot were used to select the most effective group to do the rest experiment;Western blot was used to detect the change of expression about Bcl-2,Bax,Caspase-3 and Survivin;ELISA was used to investigate the expression of IL-3 and TNF-β.Results:Confocal observed each group's transfection efficiency over 90%,CD59 molecules were mainly located in cell membrane;Real-time-PCR and Western blot showed group A had the best down-regulation efficiency;we defined RNAi-CD59-A as experimental group for subsequent experiments named RNAi-CD59;the experimental group can enhance the expression of Bax,caspase-3(P〈0.05),inhibit the expression of Bcl-2 and Survivin(P〈0.05);ELISA showed that the expression of IL-3 in the down-regulation group increase(P〈0.05),the expression of TNF-βdecrease(P〈0.05).Conclusion:Down-regulation CD59 can promote the expression of apoptosis molecular in acute leukemia Jurkat cell lines restrain the expression of proliferation molecular.
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