[抗人肝再生增强因子小干扰RNA表达质粒的构建与鉴定]
[Construction and identification of expressing siRNA plasmid against human augmenter of liver regeneration].
作者信息
Tang Lin, Liu Qi, Sun Hang, Tang Ni, Guo Hui, Deng Jian-Chuan
机构信息
Institute for Viral Hepatitis, Chongqing University of Medical Sciences, Chongqing 400010, China.
出版信息
Zhonghua Gan Zang Bing Za Zhi. 2004 Sep;12(9):534-7.
OBJECTIVES
To detect whether there is an expression of human augmenter of liver regeneration (hALR) in HepG2 cells. To develop a kind of RNAi that specifically targets human augmenter of liver regeneration by synthesizing small interfering RNA (siRNA) in vivo, and to assess the inhibitory effect of this siRNA on hALR expression.
METHODS
The expression of hALR in HepG2 cells was observed with immunocytochemistry. The RNAi plasmid pSIALR-A and the unrelated control plasmid pSIALR-B were transfected into HepG2 cells. Forty-eight hours after transfection, the protein level of hALR was measured with immunocytochemistry; meanwhile, the reverse transcription PCR (RT-PCR) was performed to detect the expression of hALR mRNA.
RESULTS
hALR was expressed by HepG2 cells. siRNA plasmid pSIALR-A, which targets the cDNA of hALR and the unrelated control plasmid pSIALR-B, was successfully constructed. Both immunocytochemistry and RT-PCR showed that pSIALR-A inhibited the expression of hALR in HepG2 cells significantly, compared with that of pSIALR-B.
CONCLUSION
The results showed that the small interfering RNA targeting hALR suppresses the expression of hALR in a sequence-specific manner
目的
检测人肝再生增强因子（hALR）在HepG2细胞中是否表达。通过体内合成小干扰RNA（siRNA），构建一种特异性靶向人肝再生增强因子的RNA干扰，评估该siRNA对hALR表达的抑制作用。
方法
采用免疫细胞化学法观察hALR在HepG2细胞中的表达。将RNA干扰质粒pSIALR-A及无关对照质粒pSIALR-B转染至HepG2细胞。转染48小时后，采用免疫细胞化学法检测hALR蛋白水平；同时，进行逆转录聚合酶链反应（RT-PCR）检测hALR mRNA的表达。
结果
HepG2细胞表达hALR。成功构建靶向hALR cDNA的siRNA质粒pSIALR-A及无关对照质粒pSIALR-B。免疫细胞化学及RT-PCR结果均显示，与pSIALR-B相比，pSIALR-A显著抑制HepG2细胞中hALR的表达。
结论
结果表明，靶向hALR的小干扰RNA以序列特异性方式抑制hALR的表达。
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