存活蛋白对胶质瘤细胞替莫唑胺敏感性的影响

Knockdown of survivin expression increases chemosensitivity of glioma cells to temozolomide

目的探讨存活蛋白(survivin)对人脑胶质瘤细胞替莫唑胺(TMZ)敏感性的影响及其可能机制。方法构建过表达survivin的U87/sur胶质瘤细胞,以及抑制survivin表达的U87/sur si胶质瘤细胞,经100μmol/L TMZ处理后,采用MTT法检测细胞增殖情况,克隆形成实验检测细胞的克隆形成能力,异硫氰酸荧光素标记的膜联素Ⅴ/碘化丙啶(annexinⅤ-FITC/PI)双标记联合流式细胞术检测细胞凋亡情况,Western blotting检测活化caspase-3表达水平。结果经TMZ处理后,与亲代细胞相比,U87/sur si细胞的增殖活性明显下降,克隆形成率明显降低,凋亡率明显增加,而U87/sur细胞的增殖活性明显增强,克隆形成率明显增高,凋亡率明显下降。经TMZ处理后,与亲代细胞相比,U87/sur si细胞中活化的caspase-3蛋白水平升高,而U87/sur细胞中活化的caspase-3蛋白水平下降。结论抑制survivin可明显提高胶质瘤细胞对TMZ的敏感性,其机制可能与survivin促进细胞凋亡有关。

Objective To study the effect of survivin on temozolomide（TMZ） resistance of glioma cells and possible mechanism. Methods Survivin RNA and survivin sh RNA plasmids were transfected into U87 cells, respectively. The cell growth curve drawn by MTT was used to test the changes in the proliferation of glioma cells. Colony forming assay was used to calculate cell surviving fraction. The Annexin V-fluorescein isothiocyanate（FITC）/propidium iodide（PI） staining were applied to detect the apoptosis rate by flow cytometer（FCM）. Western blotting was used to detect the expression of activated caspase-3. ResultsThe cell proliferation and cell surviving fraction were inhibited by TMZ, and it was more significantly inhibited in TMZ plus downregulation of survivin. Compared with parental cells, U87/sur si cells, treated by TMZ, showed a decrease in proliferation and colony forming efficiency and an increase in apoptosis rate, whereas U87/sur cells, treated by TMZ, showed an increase in proliferation and colony forming efficiency and a decrease in apoptosis rate. Western blotting showed that compared with TMZ alone, the expression of activated caspase-3 protein was promoted in TMZ plus down-regulation of survivin. Conclusion Down-regulation of survivin could enhance the sensitivity of glioma cells to TMZ, which may be related to survivin promoting cell apoptosis.