胶质瘤IDH1基因变异与胶质瘤大小及肿瘤细胞增殖的研究

发布时间：2018-01-19 13:25

本文关键词： 胶质瘤 IDH1突变 Ki-67 微血管密度（MVD）　出处：《石河子大学》2014年硕士论文　论文类型：学位论文

【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（P0.05），IDH1基因变异型胶质瘤血管密度计数（MVD）较未变异组减低（P0.05），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，P0.001；t=-8.369，P0.001），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。
[Abstract]:Objective: to detect the variation of IDH1 in human glioma specimens and to establish a stable animal model of human glioma. The effect of IDH1 gene variation on glioma size and tumor cell proliferation was preliminarily studied by Ki-67 assay, which laid a solid foundation for further clinical application. Methods: the specimens of glioma were collected from neurosurgery department of Xinjiang Uygur Autonomous region people's Hospital after surgical resection and pathological report. PCR method was used to detect the variation of IDH1 gene and immunohistochemical method was used to detect the expression of Ki-67 and the expression of Ki-67. The self-cultured glioma cells were inoculated into the brain of adult male Wistar rats by stereotactic orientation instrument. Using random number table, 120 male wistar rats were randomly divided into operation group (n = 60), sham operation group (n = 30) and blank group (n = 30). The operation group was divided into two groups: 30 rats in each group were transplanted with variant human glioma cells and wild-type glioma cells respectively, and the sham-operation group were transplanted with the same amount of normal saline. The rats in the blank control group were given routine feeding. The mice were fed normally. The rats in each group were killed and brain samples were taken by routine H _ E staining after imaging examination and recording of tumor size 3 weeks after the model was made. Glial fibrillary acidic protein (GFAP) immunofluorescence staining, microvessel determination and antigen Ki-67 detection. Results 1. There was no significant difference in tumor size between variant glioma group and non-variant group (197.55 卤18.84 and 205.51 卤20.84, respectively). There was no difference in growth rate. The expression of Ki-67 protein in IDH1 gene variant gliomas was significantly lower than that in non-variant gliomas (P 0.05). The vascular density count of IDH1 gene variant glioma was significantly lower than that of no mutation group (P 0.05). The results showed that IDH1 gene mutation could reduce the proliferative activity of tumor. 2. The human specimens showed that the Ki-67 protein expression of IDH1 gene variant glioma was lower than that of wild-type glioma. The difference was statistically significant (2.134P 0.039). The Ki-67 labeling index of IDH1 variant gliomas and wild-type gliomas were 14.44% 卤8.23% and 21.19% 卤11.49%, respectively. The vascular density count of IDH1 gene variant glioma was lower than that of wild-type glioma. The blood vessel density counts were 31.11 卤13.47 and 40.54 卤12.11, respectively. The expression of Ki-67 and MVD in low grade gliomas was higher than that in high grade gliomas. TV-8.369 (P0.001), low grade glioma (WHOI class II) and high grade glioma (III grade). The expression of Ki-67 in grade IV was 8.36% 卤2.84% and 25.55% 卤7.53%, respectively. The blood vessel density counts were 23.94 卤8.03 and 45.54 卤8.19, respectively. The results of animal experiments and human tumor specimens were basically consistent. Conclusion: IDH1 gene mutation has no significant effect on tumor growth and size, but it has an effect on tumor cell proliferation, because IDH1 gene mutation affects tumor energy metabolism. But the energy metabolism block reduces the cell proliferation activity, causes the large number of cells to be in the dormant phase, causes the neovascularization endothelial cell division slowly, the neovascularization blood vessel quantity reduces, the malignant transformation and the metastasis are inhibited. To provide a new theoretical basis for prolonging the survival of patients with IDH1 gene mutation.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.4

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