摘要
该实验旨在探讨磷脂酰肌醇特异性磷脂酶Cε(phospholipase Cε,PLCε)是否在白介素2(interleukin 2,IL-2)治疗肾癌的过程中发挥一定的作用。采用sh-PLCε慢病毒转染肾癌细胞株786-o。MTT法检测IL-2处理肾癌细胞最适浓度,使用最适浓度IL-2处理细胞。流式细胞术检测细胞凋亡。DAPI染色观察细胞中凋亡小体。q-PCR、Western blot检测Fas/FasL以及Fas下游相关分子在基因水平以及蛋白水平的表达变化。将肿瘤细胞与淋巴细胞进行共培养,流式细胞术检测细胞凋亡情况。结果显示,敲低PLCε后,Fas/FasL表达降低,而IL-2处理后,会逆转这种效果。检测Fas下游相关通路后发现,IL-2处理阴性对照(negative control,NC)组后,Fas下游凋亡抑制信号通路FADD/cFlip/Traf2启动,IL-2处理sh-PLCε组后,Fas下游凋亡信号通路FADD/Caspase8/Caspase3启动。共培养实验后流式细胞术结果显示,IL-2处理与sh-PLCε联用组细胞凋亡比例最高。该实验证明,sh-PLCε可以通过启动Fas下游凋亡信号通路,抑制凋亡抑制信号通路来提高IL-2治疗肾癌的疗效。
The purpose of this study was to investigate whether PLCε(phospholipase Cε)plays a role in the treatment of renal cell carcinoma with IL-2(interleukin 2).We first built sh-PLCεcell line by transfecting LVsh-PLCεinto renal caner cell 786-o.Cells were treated with the optimum concentration of IL-2 detected by MTT.Flow cytometry was used to detect apoptotic cells.DAPI staining was used to observe apoptotic bodies in cells.qPCR and Western blot were used to detect Fas/FasL and Fas downstream molecules at mRNA level and protein level.Tumor cells and lymphocytes were co-cultured,then apoptotic cells were detected by flow cytometry.The results showed that the expression of Fas/FasL was decreased after knocking down PLCε,but the effect was reversed after IL-2 treatment.Detecting the downstream pathway of Fas,we found that FADD/cFlip/Traf2 signaling pathway was initiated in NC(negative control)group treated with IL-2 and the Fas downstream signaling pathway FADD/Caspase8/Caspase3 was initiated in sh-PLCεgroup after IL-2 treatment.The results of flow cytometry showed that the apoptotic rate was the highest in the group treated with IL-2 and sh-PLCε.This study demonstrated that sh-PLCεcould improve the therapeutic effect of IL-2 on renal cell carcinoma by activating the downstream apoptotic signaling pathway of Fas and inhibiting the apoptotic inhibition signaling pathway.
作者
杨锦潇
段李梅
范佳鑫
李婷
范砚茹
袁鸿玲
吴小侯
罗春丽
YANG Jinxiao;DUAN Limei;FAN Jiaxin;LI Ting;FAN Yanru;YUAN Hongling;WU Xiaohou;LUO Chunli(Key Laboratory of Diagnostics Medicine of Ministry of Education,Chongqing Medical University,Chongqing 400016,China;Department of Urinary Surgery,First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China)
出处
《中国细胞生物学学报》
CAS
CSCD
2019年第12期2351-2359,共9页
Chinese Journal of Cell Biology
基金
国家自然科学基金(批准号:81072086)资助的课题.
作者简介
通讯作者:罗春丽,Tel:023-68584890,E-mail:luochunli79@126.com
