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

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。

The shRNA-mediated downregulation of protein kinase D-2 enhanced chemosensitivity of Tca8113

 Objective  To explore the effect of silencing protein kinase D (PKD)-2 on Tca8113 cell proliferation, programmed cell death,and chemosensitivity. Methods  The stable celllines 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,cell programmed death rate and sensitivity to chemotherapeutic drugs of shRNA-mediated Tca8113 significantly increased. Compared with wild group Tca8113, a significant decrease in P-gp ex-pression was induced by chemotherapy drugs with shRNA-pkd-2 gene silencing. Conclusion  The pkd-2 gene of shRNA interference silencing Tca8113 promotes programmed cell death of Tca8113, reduces the IC50 of the chemotherapy drugs, and significantly improves the sensitivity of Tca8113 to chemotherapeutic drugs while reducing the expression of P-gp.