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目的:观察电压-门控钠离子通道(voltage-gated sodium channel,VGSC)亚型nNav1.5在人脑胶质瘤组织中的表达,并探讨其对脑胶质瘤U251细胞迁移及侵袭的影响。方法:收集中国医科大学附属第一医院神经外科于2011年10月至2012年10月手术切除并经病理证实的脑胶质瘤组织标本68例,应用免疫组织化学S-P法检测脑胶质瘤组织中nNav1.5的表达。设计并化学合成nNav1.5基因特异性小干扰RNA(nNav1.5-siRNA),用脂质体介导转染胶质瘤U251细胞,应用Real-time PCR和Western blotting法分别检测U251细胞中nNav1.5 mRNA和蛋白的表达水平,并采用细胞划痕实验和Transwell侵袭实验检测U251细胞迁移和侵袭能力的变化。结果:nNav1.5在人脑胶质瘤组织中表达的阳性率显著高于正常组织(72.6%vs23.0%,P<0.01),并且其在高级别胶质瘤(WHOⅢ~Ⅳ级)组织中的阳性率明显高于低级别胶质瘤(WHOⅠ~Ⅱ级)组织(85.8%vs 52.9%,P<0.01)。nNav1.5-siRNA转染可显著抑制U251细胞中nNav1.5 mRNA和蛋白的表达(P<0.01);转染后U251细胞的迁移距离明显小于未转染细胞[(0.019±0.015)vs(0.223±0.031)mm,P<0.01],且其侵袭指数明显低于未转染细胞[(2.99±0.15)%vs(6.77±0.26)%,P<0.01]。结论:nNav1.5在人脑胶质瘤组织中高表达,干扰nNav1.5表达可显著抑制胶质瘤细胞的迁移和侵袭能力,nNav1.5是胶质瘤恶性侵袭的调控因子并有望成为胶质瘤的新标志物和治疗靶点。
OBJECTIVE: To observe the expression of voltage-gated sodium channel (VGSC) subtype nNav1.5 in human glioma and to investigate its effect on the migration and invasion of glioma U251 cells . Methods: 68 cases of glioma tissue specimens surgically removed from the Department of Neurosurgery, the First Affiliated Hospital of China Medical University from October 2011 to October 2012 were collected. Immunohistochemical SP method was used to detect the expression of glioma tissue In nNav1.5 expression. Design and synthesis of nNav1.5 gene-specific small interfering RNA (nNav1.5-siRNA), liposome-mediated transfection of glioma U251 cells were detected by Real-time PCR and Western blotting were detected in U251 cells nNav1 .5 mRNA and protein expression level, and the cell migration and invasion ability of U251 cells were detected by cell scratch assay and Transwell invasion assay. Results: The positive rate of nNav1.5 expression in human glioma tissues was significantly higher than that in normal tissues (72.6% vs 23.0%, P <0.01), and the positive expression rate of nNav1.5 in gliomas of high grade (WHO grade Ⅲ ~ Ⅳ) (85.8% vs 52.9%, P <0.01), which was significantly higher than that of low grade gliomas (WHO grade Ⅰ ~ Ⅱ). The transfection of nNav1.5-siRNA could significantly inhibit the expression of nNav1.5 mRNA and protein in U251 cells (P <0.01). The translocation distance of U251 cells in transfected U251 cells was significantly lower than that in untransfected cells [(0.019 ± 0.015) vs (0.223 ± 0.031) mm, P <0.01], and the invasive index was significantly lower than that of untransfected cells [(2.99 ± 0.15)% vs (6.77 ± 0.26)%, P <0.01]. Conclusion: nNav1.5 is overexpressed in human glioma tissues and the expression of nNav1.5 may be significantly inhibited by inhibiting the migration and invasion of glioma cells. NNav1.5 is a regulator of malignant invasion of glioma and is expected to become glial New markers of tumors and therapeutic targets.