shRNA干扰蛋白激酶D—2基因提高Tca8113对化疗药物的敏感性研究.docVIP

shRNA干扰蛋白激酶D—2基因提高Tca8113对化疗药物的敏感性研究 　　[摘要] 目的 探讨蛋白激酶D（PKD）-2基因沉默对Tca8113细胞增殖、细胞程序性死亡及对化疗药物敏感性的影响。方法 构建针对pkd-2基因的shRNA干扰质粒及空载体对照组质粒，建立pkd-2基因沉默的稳定细胞株；采用甲基噻唑基四唑（MTT）方法检测shRNA干扰后细胞株的增殖情况及对化疗药物的半数抑制质量浓度（IC50）；采用流式细胞术检测pkd-2基因沉默前后细胞程序性死亡率及对化疗药物的敏感性；采用免疫组化方法检测pkd-2沉默后细胞中P糖蛋白（P-gp）的表达。结果 筛选并建立pkd-2基因沉默的稳定细胞株；经shRNA干扰后Tca8113细胞的增殖速度与野生型细胞相比差异无统计学意义，但IC50明显降低；pkd-2沉默后Tca8113细胞程序性死亡率明显上升，且对化疗药物的敏感性显著提高；与野生型对照组Tca8113相比较，shRNA-pkd-2基因沉默的Tca8113经化疗药物诱导后P-gp表达明显下降。结论 shRNA干扰技术特异性沉默Tca8113中的pkd-2基因，促进了肿瘤细胞的程序性死亡，降低对化疗药物的IC50，并明显提高肿瘤细胞对化疗药物的敏感性，同时降低了P-gp的表达。 　　[关键词] 蛋白激酶D； shRNA干扰； 细胞程序性死亡 　　[中图分类号] Q 78 [文献标志码] A [doi] 10.7518/hxkq.2014.01.019 　　The shRNA-mediated downregulation of protein kinase D-2 enhanced chemosensitivity of Tca8113 Dai Lina1， Zhang Ping2， Su Qin2. （ 1. Dept. of Children’s Dental Central， The Affiliated Hospital of Stomatology， Chongqing Medical Univer-sity， Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences， Chongqing 400015， China； 2. State Key Laboratory 　　of Oral Diseases， West China Hospital of Stomatology， Sichuan University， Chengdu 610041， China） 　　[Abstract] Objective To explore the effect of silencing protein kinase D （PKD）-2 on Tca8113 cell proliferation， pro-grammed cell death， and chemosensitivity. Methods The stable cell lines of pkd-2 gene silencing and empty vector plasmid group were established. The proliferation and 50% inhibitory concentration （IC50） of shRNA-mediated Tca8113 chemotherapy drugs were detected through methyl thiazolyl tetrazolium assay （MTT）. The programmed cell death rate and sensitivity to Tca8113 chemotherapy drugs before or after pkd-2 gene silencing were measured through flow cytometry. P-glycoprotein （P-gp） expression of pkd-2 silencing cells was identified by immunohistochemical methods. Results Stable cell lines of pkd-2 gene silencing were established. Compared with parental cells， the proliferation of shRNA-mediated Tca8113 was not significantly different， but its IC50 was lower. Meanwhile