| 斯妍娜韩流程浩吕云落蔡朦朦谢欣怡鲍红光△.STAT3 小干扰RNA的构建及对缺氧复氧人肾小管
上皮细胞凋亡的影响*[J].,2014,14(15):2858-2862 |
| STAT3 小干扰RNA的构建及对缺氧复氧人肾小管
上皮细胞凋亡的影响* |
| Construction of the Vector Expressing Small Interference RNA TargetingSTAT3 Gene and its Effect on Apoptosis of Human Tubular EpithelialCells Induced by Hypoxia-Reoxygenation Injury* |
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| DOI： |
| 中文关键词: 信号转导与转录因子3 小干扰RNA 缺氧复氧 肾小管上皮细胞 |
| 英文关键词: Signal transducer and activator of transcription 3 Small interference RNA Hypoxia-Reoxygenation Tubular Epithelial
Cell |
| 基金项目:南京市医学重点科技发展项目（201108028）;
南京市医学科技发展项目（YKK12085） |
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| 中文摘要: |
| 摘要目的：构建信号转导与转录因子3（STAT3）小干扰RNA（siRNA）表达载体，并观察其对缺氧复氧后人肾小管上皮细胞
（HKC）凋亡的影响。方法：设计3 对人STAT3 siRNA靶序列，用DNA重组技术克隆至质粒pRNAT-U6.1/neo 中，构建重组质粒
pRNAT-U6.1-STAT3 siRNA，检测并筛选出最佳抑制效率的siRNA质粒载体。重组质粒转染至缺氧复氧后HKC细胞，Western
blotting和Real Time-PCR测定STAT3 蛋白和mRNA 表达量，流式细胞仪测定细胞凋亡，间接荧光法测定Bcl-2 和Bax表达的变
化。结果：靶向STAT3 基因表达的质粒载体构建成功，并筛选出抑制效率最佳的重组质粒。缺氧复氧后HKC 细胞STAT3表达、
凋亡率和Bax/Bcl-2 比值增加；缺氧复氧后HKC 细胞转染重组质粒后STAT3 表达、凋亡率和Bax/Bcl-2 比值明显降低。结论：成
功构建并筛选最佳抑制效率的靶向STAT3 的重组质粒载体。该载体可有效抑制缺氧复氧后HKC细胞中STAT3 信号转导通路
的活化，并进一步通过上调Bcl-2、下调Bax 蛋白的表达，从而抑制细胞凋亡。 |
| 英文摘要: |
| ABSTRACT Objective：To construct the vector expressing small interference RNA targeting signal transducer and activator of
transcription 3 (STAT3) gene and to investigate its effect on apoptosis of human tubular epithelial cells (HKC) induced by
hypoxia-reoxygenation injury. Methods:Design 3 pairs of small interference RNA sequences targeting STAT3 gene and clone into
plasmid pRNAT-U6.1/neo to construct recombinant plasmid pRNAT-U6.1-STAT3 siRNA within which the best suppressive efficiency
plasmid was determined and elected. Recombinant plasmid was transfected into HKC induced by hypoxia-reoxygenation injury. The
expression of STAT3 protein and mRNA were determined by western blotting and RT-PCR. The apoptosis was measured by flow
cytometry (FCM). The expression of Bcl-2 and Bax was determined by indirect fluorescence method.Results: The recombinant plasmid
was constructed successfully. Select the best recombinant plasmid which produced the best suppression effect. The expression of STAT3,
the apoptosis rate and the ratio of Bax/Bcl-2 of HKC induced by hypoxia-reoxygenation injury was significantly increased. The
expression of STAT3, the apoptosis rate and the ratio of Bax/Bcl-2 was significantly decreased after recombinant plasmid was transfected
into HKC induced by hypoxia-reoxygenation injury. Conclusion:The recombinant plasmid was constructed successfully. This
recombinant plasmid can effectively inhibit the activity of STAT3 pathway of HKC induced by hypoxia-reoxygenation injury, upregulate
the expression of Bcl-2 and downregulate the expression of Bax, which cause the apoptosis of cells. |
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