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目的通过免疫组化S-P法及RT-PCR法测定HIF-1α、MGMT、CD133在胶质母细胞瘤细胞中的表达,探讨三者在胶质母细胞瘤中的表达相关性。方法收集自2010年12月至2012年12月手术切除病理证实的胶质母细胞瘤(WHOⅣ级)33例,同期收集低级别胶质瘤25例(WHOⅠ~Ⅱ级)作为对照组,收集脑深部肿瘤皮层造瘘正常脑组织5例,作为阴性对照组。采用免疫组织化学S-P法检测HIF-1α、MGMT、CD133在人脑胶质母细胞瘤、低级别胶质瘤及正常脑组织中的表达;应用RT-PCR法测定HIF-1α、MGMT、CD133在胶质母细胞瘤细胞、低级别胶质瘤中的mRNA的表达,统计软件选用SPSS 13.0,计量资料采用卡方检验。相关统计分析采用Spearman等级相关分析。以α=0.05为检验水准,当P<0.05时具有统计学意义。结果免疫组织化学S-P法检测33例胶质母细胞瘤,其中HIF-1α阳性表达32例(96.7%),MGMT阳性表达23例(69.7%),CD133阳性表达33例(100%),均阳性表达23例(69.7%)。对照组25例低级别胶质瘤中HIF-1α阳性表达9例(36.0%),MGMT阳性表达8例(32.0%),CD133阳性表达8例(32.0%),均阳性表达4例(16.0%)。在正常脑组织中HIF-1α、MGMT、CD133均阴性表达。可见随肿瘤级别升高,HIF-1α、MGMT、CD133表达显著升高,差异具有统计学意义(P<0.05)。RT-PCR法测定HIF-1α、MGMT、CD133在低级别胶质瘤及胶质母细胞瘤中mRNA的表达,其中HIF-1αmRNA在低级别胶质瘤中相对定量0.23±0.05,在胶质母细胞瘤中相对定量0.87±0.12;MGMT mRNA在低级别胶质瘤中相对定量0.27±0.07,在胶质母细胞瘤中相对定量0.67±0.09;CD133 mRNA在低级别胶质瘤中相对定量0.16±0.05,在胶质母细胞瘤中相对定量0.57±0.08。相关性分析发现HIF-1α与MGMT的r=0.91、P<0.05,HIF-1α与CD133的r=0.85、P<0.05,MGMT与CD133的r=0.802、P<0.05,提示三者均存在正相关性。结论随肿瘤级别升高,HIF-1α、MGMT、CD133阳性表达率升高。三者的相关性研究提示HIF-1α与MGMT、HIF-1α与CD133、MGMT与CD133均存在正相关性。提示在胶质母细胞瘤的发生、发展、复发、预后中三者存在协同作用,通过干预不同因子的表达,为胶质母细胞瘤的治疗提供新的思路及方向。
Objective To investigate the expression of HIF-1α, MGMT and CD133 in glioblastoma cells by immunohistochemical S-P method and RT-PCR, and to explore the correlation between them in glioblastoma. Methods Thirty-three cases of glioblastoma (WHO Ⅳ grade) confirmed by pathology from December 2010 to December 2012 were collected. 25 cases of low grade gliomas (WHOⅠ ~ Ⅱ grade) were collected as control group, Deep tumor cortical fistula normal brain tissue in 5 cases, as a negative control group. The expression of HIF-1α, MGMT and CD133 in human glioblastoma, low-grade glioma and normal brain tissues were detected by immunohistochemical SP method. The expressions of HIF-1α, MGMT and CD133 were detected by RT-PCR Glioblastoma cells, low-grade glioma mRNA expression, statistical software selection SPSS 13.0, measurement data using the chi-square test. Relevant statistical analysis using Spearman rank correlation analysis. Α = 0.05 for the test level, when P <0.05 was statistically significant. Results Immunohistochemical SP method was used to detect 33 cases of glioblastoma, including 32 cases (96.7%) positive for HIF-1α, 23 cases (69.7%) for MGMT positive and 33 cases positive for CD133 23 cases were expressed (69.7%). The positive expression of HIF-1α in 25 cases of low grade gliomas in control group was 9 (36.0%), MGMT positive in 8 cases (32.0%) and CD133 positive in 8 cases (32.0%), all of which were positive in 4 cases ). In normal brain tissue, HIF-1α, MGMT and CD133 were all negatively expressed. Shows that with the tumor grade increased, HIF-1α, MGMT, CD133 expression was significantly increased, the difference was statistically significant (P <0.05). The mRNA expression of HIF-1α, MGMT and CD133 in low grade glioma and glioblastoma was determined by RT-PCR. The relative quantification of HIF-1α mRNA in low grade glioma was 0.23 ± 0.05, The relative quantification was 0.87 ± 0.12 in the oncocytoma, 0.27 ± 0.07 in the low grade glioma and 0.67 ± 0.09 in the glioblastoma, while the relative quantification of CD133 mRNA in the low grade glioma was 0.16 ± 0.05, and the relative quantification in glioblastoma was 0.57 ± 0.08. Correlation analysis showed that there was r = 0.91, P <0.05 for HIF-1αand MGMT, r = 0.85 for HIF-1αand CD133, P <0.05, r = 0.802 for MGMT and CD133, P < Correlation. Conclusion With the increase of tumor grade, the positive rate of HIF-1α, MGMT and CD133 increased. The correlation between the three studies suggest that there is a positive correlation between HIF-1α and MGMT, HIF-1α and CD133, MGMT and CD133. These findings suggest that there is synergistic effect among the occurrence, development, recurrence and prognosis of glioblastoma, and provide new ideas and directions for the treatment of glioblastoma by intervention of different factors.