【作者】 曾飞跃；

【导师】 方加胜；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 目的：分析IDH1R132基因突变在人脑胶质瘤中的分布特征。方法：90例不同级别的胶质瘤活体标本及其血液标本,苯酚—氯仿抽提法提取DNA,设计相应引物,PCR及直接测序。结果：90例胶质瘤标本中共发现42例IDH1基因突变,IDH1基因突变为R132H。其中在Ⅱ、Ⅲ级胶质瘤中发现突变38例,其突变率高达55%(突变平均年龄为38.6±2.2岁；非突变平均年龄为47.5±2.9岁),两组间年龄比较有显著性差异。而在Ⅰ级胶质瘤中未发现；Ⅳ级胶质瘤中发现突变4例,突变率为21.1%(突变组患者平均年龄41.5±2.3岁,非突变组53.3±3.6岁),两组间年龄比较有显著性差异。而所有患者其相应血液标本中未发现该突变。结论：在国内率先报道了IDH1R132基因在中国人群胶质瘤中的突变频率。并且发现突变组平均年龄较非突变组小。目的：构建稳定表达IDH1 wild-pCDNA3.1及IDH1 R132H-pCDNA3.1的Hela及U251细胞株。方法：将IDHlwild和R132H型分别转染Hela和U251细胞,然后运用RT-PCR、Western-Blotting进行验证。结果：获得了稳定转染IDH1 wild-pCDNA3.1及IDH1 R132H-pCDNA3.1的Hela及U251细胞株。结论：获得了稳定转染IDH1 wild-pCDNA3.1及IDH1 R132H-pCDNA3.1的Hela及U251细胞株。目的：在稳定转染IDH1 wild及R132H的Hela及U251细胞株中,观察IDH1R132H对细胞功能的影响。方法：(1)通过MTT法检测比较转染细胞活性并绘制生长曲线,并用流式细胞仪检测转染后细胞周期的变化及细胞凋亡的情况；(2)同时对IDH1酶活力进行检测；(3)免疫组化检测同级别胶质瘤中突变标本和非突变标本中HIF-1α及VEGF的表达情况。结果：(1)转染野生型基因的Hela或U251细胞生长增殖速度与空转组、未转染、突变转染组相比均显著降低,突变型与空转组及未转染组相比均无统计学差异。流式细胞仪检测细胞周期发现：空转、野生和突变转染组Hela细胞处于G1期比例分别为：57.75±4.15%、61.10±3.22%、47.41±2.17%,S期的细胞比例分别为：30.54±2.11%、29.81±2.92%、38.84±1.92%,G2期的细胞比例分别：11.70±3.17%、7.21±2.41%、13.74±3.01%。突变转染组细胞出现G2+S期细胞比例高于空转和野生转染组,野生转染组和空转组比较无统计学差异。U251细胞空转、野生和突变转染细胞G2+S比例分别为34.12±3.56%、36.45±4.73%、43.68±3.16%,突变转染组细胞均出现G2+S期细胞比例高于空转和野生转染组,而野生转染组和空转组比较无统计学差异。(2)转染后,酶的活力测定结果表明：与野生转染组相比,Hela突变转染组酶的活力下降了64.09%,空转组下降了72.44%。U251突变转染组酶的活力较野生转染组下降了39.79%,空转组下降了48.9%。野生转染组与突变转染及空转组相比酶活力增加(P<0.05),空转组及突变转染组比较无统计学差异。(3)HIF-1α、VEGF阳性反应物分别定位于细胞核、胞浆,染色为棕黄色,两者在突变胶质瘤中阳性表达率显著高于野生组(P<0.05)。结论：在两种转染的细胞株中均发现IDH1R132H组生长增殖活跃、而转染野生型后IDH酶活力明显增强。IDH1基因可能是抑癌基因。同时研究发现在同级别胶质瘤突变组中HIF-1α及VEGF表达高于非突变组。目的：在转染的U251细胞株中,检测IDH1 R132H突变对化疗药物敏感性。方法：观察不同浓度顺铂对于U251细胞株在不同时间点的细胞抑制率。从而选择合适的药物干预浓度及时间点。在转染之野生和突变U251细胞株分别加入顺铂,流式细胞仪检测细胞凋亡及Western-Blotting观察Caspase-3蛋白的表达情况。结果：突变转染组较野生转染及空转组细胞凋亡明显增加(P<0.05)；野生转染组与空转组无明显差异。突变组其表达Caspase-3蛋白(P<0.05)高于野生转染组及空转组。野生转染与空转组无明显差异。结论：IDH1基因突变后增强了U251细胞对于化疗药物敏感性。更多还原

【Abstract】 Objective To investigate isocitrate dehydrogenase 1R132(IDH1R132) mutation in human gliomas. Methods 90 tumor samples and matched norma blood lymphocytes samples were collected for DNA extraction. Exon 4 of IDH1 gene was amplified with the use of a polymerase-chain-reaction(PCR) assay and sequenced in DNA from the tumor and lymphocytes from each patient. Results IDH1R132 mutation was more than 55% in WHO gradeⅡandⅢgliomas(patients with mutation had a median age of 38.6±2.2 years,while wild-type 47.5±2.9 years), none in WHO grade,21.1% in in WHO gradeⅣ(patients with mutation had a median age of 41.5±2.3 years, wild-type 53.3±3.6 years). None mutation of IDH1R132 was found in lymphocytes. Conclusion Mutations of the IDH1R132were frequently found in several types of grade II and III gliomas with a younger age compared with wild type.Objective To establish Hela and U251 cell line stably expressing IDH1 wild-pCDNA3.1 and IDH1 R132H-pCDNA3.1. Methods After Hela and U251 cell line stably expressing IDH1 wild-pCDNA3.1 and IDH1 R132H-pCDNA3.1 were established, RT-PCR、Western-Blotting were used for confirmation. Results We established Hela and U251 cell line stably expressing IDH1 wild-pCDNA3.1 and IDH1 R132H-pCDNA3.1. Conclusion Hela and U251 cell line stably expressing DH1 wild-pCDNA3.1 and IDH1 R132H-pCDNA3.1 were obtained. Objective To investigate the functional change of IDH1R132H mutation gene. Methods (1)The proliferation ability of transfected cells was examined by MTT assay, and then draw cell growth curve. The change of cell cycle and cell apoptosis were analyzed by flow cytometry; (2)The enzymatic activity of wild and IDH1R132H transfected types was assessed; (3)We compared HIF-la and VEGF expression in mutation and wild tumor samples using immunohistochemistry. Results (1)MTT assay was used to measure the proliferative index. Compared with the empty vector, untransfection cells and IDH132H transfected groups, the expression of wild transfected group could effectively inhibit the proliferation in Hela and U251.While no statistics difference between mutant IDH1R132H and empty vector, untransfected groups.The cell cycle was analyzed by flow cytometry.The percentage in G1 phase of Hela-Vector, Hela-Vector-WT and Hela-Vector-IDHR132H were 57.75±4.15%、61.10±3.22%、47.41±2.17% respectively, and S phase were 30.54±2.11%、29.81±2.92%、38.84±1.92%. Compared with wild type and empty vector, G2+S phase in IDH132H increased. While in U251-Vector, U251-Vector-WT-IDH1, and U251-Vector-IDHR132H, the percentage of G2+S were 34.12±3.56%、36.45±4.7%,43.68±3.16%, respectively. The results showed that the percentage of cells(Hela& U251)-Vector-IDH1R132H in G2+S phase all increased, compared with their matched wild type and empty vector. While compared wild type and empty vector, there was no statistics difference. (2)Compared with wild transfected group type,the enzymatic activity of IDH1R132H transfected group decreased 64.09%(Hela,P<0.05),39.79%(U251, P<0.05) respectively, empty-vector decreased 2.44%(Hela, P<0.05),48.9% (U251 P<0.05),while there was no significance between Vector-IDHR132H and empty-vector. (3)The HIF-1α、VEGF positive staining were brown-yellow.They were located in the cell nucleus and cytoplasm respectively. The both positive expression rates in IDHR132H cases increased significantly compared with wild cases(P<0.01). Conclusion After transfection, IDHR132H-vector group had a more proliferative capability,while a higher enzymatic activity in wild-vector group. Two positive(HIF-1α&VEGF) expression rates in mutations cases increased significantly compared with wild cases.Objective To identify Chemosensitivity of IDH1R132H mutation to Chemotherapy agents in U251 cell line. Methods Different concentration DDP was added into U251 cells, and its inhibitive rate was obtained at different time. DDP were added into three kinds of U251 cells. Cell apoptosis was obtained by FCM, and its Caspase-3 protein was expressed by Western-Blotting. Results Compared with empty vector and wild transfected group, IDH1R132H had a obvious apoptosis. The same as cleaved Caspase-3 protein. Conclusion Chemosensitivity was enhanced after IDH1R132H mutation.更多还原