慢病毒载体介导RNAi稳定抑制VASH1表达对人脑胶质瘤细胞增殖、凋亡的影响
目的探讨运用慢病毒载体介导的RNA干扰技术抑制血管生成抑制因子1(VASH1)的表达对人脑胶质瘤U-87MG细胞增殖和凋亡的影响。方法应用p GCL-GFP质粒构建针对VASH1的慢病毒sh RNA载体,转染入工具细胞293T,筛选出适合浓度的慢病毒转染人脑胶质瘤U-87MG细胞;通过RT-PCR和Western blot评价其对U-87MG细胞内VASH1表达的影响;用四甲基偶氮唑蓝(MTT)法观察U-87MG细胞增殖活性的改变;用流式细胞仪(FCM)检测U-87MG细胞凋亡的变化。结果与转染阴性对照组和空白组相比,转染p GCL-GFP-VASH1慢病毒载体的U-87MG细胞VASH1的m...
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Published in | 肿瘤防治研究 Vol. 43; no. 3; pp. 193 - 196 |
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Main Author | |
Format | Journal Article |
Language | Chinese |
Published |
2016
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Subjects | |
Online Access | Get full text |
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Summary: | 目的探讨运用慢病毒载体介导的RNA干扰技术抑制血管生成抑制因子1(VASH1)的表达对人脑胶质瘤U-87MG细胞增殖和凋亡的影响。方法应用p GCL-GFP质粒构建针对VASH1的慢病毒sh RNA载体,转染入工具细胞293T,筛选出适合浓度的慢病毒转染人脑胶质瘤U-87MG细胞;通过RT-PCR和Western blot评价其对U-87MG细胞内VASH1表达的影响;用四甲基偶氮唑蓝(MTT)法观察U-87MG细胞增殖活性的改变;用流式细胞仪(FCM)检测U-87MG细胞凋亡的变化。结果与转染阴性对照组和空白组相比,转染p GCL-GFP-VASH1慢病毒载体的U-87MG细胞VASH1的m RNA和蛋白表达水平明显降低(P〈0.01);细胞的增殖活性显著上调(P〈0.01),细胞凋亡明显减少(P〈0.01)。结论 VASH1 sh RNA慢病毒载体可抑制VASH1在人脑胶质瘤U-87MG细胞中的表达,并增加肿瘤细胞的增殖活性,抑制细胞凋亡。 |
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Bibliography: | 42-1241/R Objective To investigate the effect of lentiviral vector-mediated RNA interference inhibiting VASH1 gene expression on the proliferation and apoptosis of human glioma cells U-87 MG. Methods A lentiviral vector carrying short hairpin RNA(sh RNA) of human VASH1 gene(p GCL-GFP-VASH1) was constructed and used to transfect 293 T cells. The transfection effi ciency was evaluated and then the lentiviral vector with suitable concentration was transfected into the human glioma cells U-87MG; RT-PCR and Western blot were used to detect the expression of VASH1 in U-87 MG cells. MTT assay was used to observe the inhibion ratio of U-87 MG cells growth. FCM analysis was used to observe the apoptosis. Results RT-PCR and Western blot analyses demonstrated that p GCL-GFP-VASH1 could signifi cantly inhibit the expression of VASH1 m RNA and protein in U-87 MG cells(P〈0.01); MTT results showed that it could increase the growth of U-87 MG cells(P〈0.01). FCM results showed that the occurrence of apoptosis was suppressed si |
ISSN: | 1000-8578 |