【作者】 王均；

【导师】 杨小朋； 郑勇；

【作者基本信息】 石河子大学 ， 外科学， 2014， 硕士

【摘要】 目的：检测人胶质瘤标本的IDH1变异情况，同时建立稳定的人胶质瘤动物模型，不同时间点通过对肿瘤大小的观察、微血管测定、抗原Ki-67检测来初步探讨胶质瘤IDH1基因变异对胶质瘤大小及肿瘤细胞增殖的影响，为进一步的临床应用打下坚实的基础。方法：收集新疆维吾尔自治区人民医院神经外科经外科手术切除并病理报告确诊胶质瘤的肿瘤标本，PCR方法检测IDH1基因变异及免疫组化法检测微血管测定（MVD）、抗原Ki-67表达情况，变异及未变异的胶质瘤标本细胞培养传代，采用立体定向仪，将自行培养的胶质瘤细胞接种于成年雄性Wistar大鼠脑内的方法，制作鼠人脑胶质瘤模型。采用随机数表将120只雄性wistar大鼠随机分为手术组60只、假手术组30只、空白组30只，手术组又分为两组各30只，分别移植变异型人脑胶质瘤细胞和野生型人脑胶质瘤细胞，假手术组移植等量生理盐水，空白对照组给予常规饲养。小鼠均正常饲养，造模后的1W、2W、3W行影像学检查并记录肿瘤大小，3周后将各组大鼠处死取脑，行常规H E染色、胶质纤维酸性蛋白(GFAP)免疫荧光染色、微血管测定（MVD）及抗原Ki-67检测。结果：1.动物实验表明变异型胶质瘤组肿瘤大小与未变异组比较无统计学差异，分别为197.55±18.84和205.51±20.84，且生长速度也无差异；IDH1基因变异型胶质瘤组Ki-67蛋白表达较未变异组胶质瘤降低，差异有统计学意义（P<0.05），IDH1基因变异型胶质瘤血管密度计数（MVD）较未变异组减低（P<0.05），表明IDH1基因变异可降低肿瘤的增殖活性。2.人体标本实验表明IDH1基因变异型胶质瘤Ki-67蛋白表达较野生型胶质瘤降低，差异有统计学意义（t=2.134，P=0.039），IDH1变异型与野生型胶质瘤Ki-67标记指数分别为（14.44%±8.23%）和（21.19%±11.49%）；IDH1基因变异型胶质瘤血管密度计数（MVD）较野生型减低（t=2.328，P=0.025），其血管密度计数分别为31.11±13.47和40.54±12.11；低级别胶质瘤Ki-67及微血管密度（MVD）表达较高级别胶质瘤低（t=9.138，P<0.001；t=-8.369，P<0.001），低级别胶质瘤（WHOI级、II级）和高级别胶质瘤（WHO III级、IV级）Ki-67表达分别是8.36%±2.84%和25.55%±7.53%，其血管密度计数分别为23.94±8.03和45.54±8.19，两者表达随胶质瘤级别增高而增高，动物实验与人体肿瘤标本实验检测结果基本一致。结论：胶质瘤IDH1基因变异对肿瘤生长及大小无明显影响，但其对肿瘤细胞的增殖有影响。因为IDH1基因变异影响肿瘤的能量代谢，而能量代谢受阻降低细胞的增殖活性，使大量细胞处于休眠期，导致新生血管内皮细胞分裂缓慢，新生肿瘤血管数量减低，恶变和转移受到抑制，为IDH1基因变异延长患者的生存期的机制提供新的理论依据。更多还原

【Abstract】 Objective: IDH1mutation detection of human glioma specimens, and establish astable animal model of human glioma, different time points observe tumor size,microangiopathy, and Ki-67antigen detection to explore how glioma IDH1genemutation impact the size of glioma tumor and cell proliferation, lay the foundation forclinical applicationsMethods: Collecting the Xinjiang Uygur Autonomous Region people’s Hospital ofNeurosurgery surgically excised and the pathology report confirmed glioma tumorsamples,using PCR method detected IDH1gene mutation, and immunohistochemicaldetected microvessel density (MVD), Ki-67antigen expression, mutation and nonmutation cell in glioma specimens was cell culture, using stereotactic, theself-cultured glioma cells were seeded in Adult male Wistar rat brain, making ratglioma model. The120male wistar rats were randomly divided into surgicalgroup(N=60), the sham group (N=30,) control group (N=30). surgical group wasdivided into two groups(N=30),were transplanted human glioma mutant and wild-typecells, sham group were transplanted saline, and the control group receivedconventional breeding.The wistar rats were normal feeding, imaging examinationafter1W,2W,3W modeling and recording tumor size, after three weeks later take therats brain, HE staining, glial fibrillary acidic protein (GFAP) immunofluorescencestaining, microvessel density (MVD) and Ki-67antigen detection.Results:1. Animal experiments show that the mutant group glioma tumor size was nosignificant difference between the wild-type group, the tumor size were197.55±18.84and205.51±20.84,and no significant difference in the growth rate. The antigen Ki-67expression of IDH1mutation group were significantly lower than those wild-typeglioma, the difference was statistically significant (P<0.05), the microvessel densityin IDH1mutation group were significantly lower than those wild-type glioma（P<0.05）, showed the gene mutation IDH1could reduced the cell’s proliferativeactivity. The antigen Ki-67expression in IDH1mutation group were significantlylower than those wild-type glioma, the difference was statistically significant(t=2.134，P=0.039).the Ki-67labeling index in the mutation group and wild-typeglioma group were21.19%±11.49%and14.44%±8.23%. The microvessel density inIDH1mutation group were significantly lower than those wild-type glioma (t=2.328，P=0.025), the microvessel density were40.54±12.11and31.11±13.47. low-gradegliomas Ki-67and microvessel density (MVD) expression were significantly lowerthan high-grade gliomas (t=9.138, P <0.001; t=-8.369, P <0.001), low gradegliomas (WHO I grade, II grade) and high-grade gliomas (WHO III grade, IV grade) Ki-67expression were8.36%±2.84%and25.55%±7.53%,and its vascular densitycounts23.94±8.03and45.54±8.19,Both expression increased with the grade ofgliomas, animal experiments consistent with human tumor samples assay results.Conclusions: Glioma,s IDH1gene mutation had no significant effect on tumor growthand size, but its impact on the proliferation of tumor cells. The mutation affecttumor’s energy metabolism, and energy metabolism decreased cell proliferationactivity, so that a large number of cells in a dormant stage, resulting in neovascularendothelial cells divide slowly and reduce the number of neoplastic blood vessels,malignancy and metastasis was inhibited, suppressed for IDH1gene mutation prolongsurvival mechanism provides a new theoretical basis.更多还原