Girdin蛋白调控恶性胶质瘤细胞凋亡的研究  

Effect of Girdin on apoptosis of human glioblastoma cells

作　　者：王静[1] 丁婷[2] 刘晓丽[1] 李文良[3] 谷峰[2] 马勇杰[1] 

机构地区：[1]乳腺癌防治教育部重点实验室天津市肿瘤防治重点实验室天津医科大学附属肿瘤医院中心实验室,天津300060 [2]乳腺癌防治教育部重点实验室天津市肿瘤防治重点实验室天津医科大学附属肿瘤医院乳腺病理研究室,天津300060 [3]乳腺癌防治教育部重点实验室天津市肿瘤防治重点实验室天津医科大学附属肿瘤医院颅脑肿瘤科,天津300060

出　　处：《中华实验外科杂志》2011年第5期733-736,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30700253、30800355）;教育部长江学者与创新团队发展计划（IRT0734）

摘　　要：目的观察抑制恶性胶质瘤LN229细胞中Girdin蛋白的表达对细胞凋亡的影响，并探讨其分子机制。方法利用小RNA干扰（siRNA）技术，沉默LN229细胞中Girdin的表达，得到实验组克隆（siardin／LN229），阴性对照组命名为scr／LN229。采用Westemblot技术检测Girdin、细胞色素c（Cyt—C）及Bad蛋白水平的变化。利用增殖实验、噻唑蓝（MTT）比色法检测LN229细胞的存活率；应用流式细胞术检测线粒体释放的Cyt—C量的变化。建立成瘤模型（每组20只），观察Girdin降表达对移植瘤生长情况的影响。结果siRNA技术有效沉默了LN229细胞中Girdin的表达。增殖实验显示siGirdin／LN229细胞增殖能力下降（P〈0．05），至第6天时实验组细胞数为（15．08±1．17）×10^4；对照组为（53．93±6．26）×10^4。MTr实验发现siGirdin／LN229细胞的存活率明显下降（P〈0．05），第5天时实验组为（323．15±57．01）％；对照组为（640．67±59．66）％。流式检测显示siGirdin／LN229细胞Cyt—C含量增加，平均荧光强度为12531．00±1412．98，对照组为183．67±41．55（P〈0．01）。分析Westernblot结果显示，siGirdin／LN229细胞Cyt—C、Bad的相对灰度值分别为3．57±0．43和4．78±1．03，均高于对照组Cyt—C、Bad的表达水平（相对灰度值分别为1．13±0．11、1．78±0．20）。体内实验证实Girdin降表达组肿瘤体积明显小于对照组（P〈0．05），至第7．5周时，siGirdiir／LN229组肿瘤体积为（36．42±2．00）mm3，对照组为（262．42±24．12）mm3。结论Girdin能够调控胶质瘤细胞LN229的凋亡，其调节细胞凋亡的机制可能与Cyt—C从线粒体释放及Bad的表达有关。Objective To investigate the effects of Girdin on apoptosis of LN229 glioblastoma cells by Girdin small RNA interference （RNAi） technology and the molecular mechanisms. Methods Girdin expression was knocked down in LN229 glioblastoma cells by Girdin small interfering RNA （siRNA）. The expression levels of Girdin, cytoehrome C （ Cyt-C ） and Bad proteins in glioblastoma LN229 cells were detected by Western blotting. Stable clones were used to measure cells survival ratio by proliferation assay and methyl thiazol tetrazolium （MTT） assay. Flow Cytometry was used to examine the content of Cyt-C in siGirdin/LN229 and scr/LN229. 20 male athymic Nu/Nu mice were subcutaneously injected with siGirdin/LN229 or scr/LN229 cells respectively to study the growth of tumor in each group. Results Western blotting showed that the expression of Girdin was decreased significantly. The relative intensity of Cyt-C and Bad in siGirdin/LN229 cells was 3.57 ± 0. 43 and 4. 78 ± 1.03 respectively, and the relative intensity of Cyt-C and Bad in scr/LN229 was 1.13 ±0. 11,1.78 ±0. 20 respectively （P 〈0.05）. The mean fluorescence density of Cyt-C in siGirdin/LN229 and ser/LN229 by flow eytometry was 12 531.00 ± 1412. 98 and 183.67 ± 41.55 respectively. Meanwhile, the number of siGirdin/LN229 cells was obviously decreased as compared with control group in proliferation assay. The number of siGirdin/LN229 cells on the 6th day was （ 15.08 ± 1.17） ×10^4, and that of ser/LN229 cells was （53.93 ±6. 26） × 10^4. MTT assay revealed the survival rate of siGirdin/LN229 on the 5th day was （323.15 ±57.01 ）%, and that of scr/LN229 was （ 640.67 ± 59. 66 ） % , P 〈 0. 05. Additionally, the volume of xenograft tumors in siGirdin/LN229 group was decreased significantly as compared with control group. The volume of xenograft tumors in siGirdin/LN229 group was （ 36.42 ± 2. 00） mm3 , and that in scr/LN229 group was （262. 42 ±24. 12） mm3 at the 7.5th week. Conclusion Girdin plays a critical role in apoptosi

关 键 词：胶质瘤 Girdin 脱噬作用 

分 类 号：R739.4[医药卫生—肿瘤]