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目的构建SIRNA稳定表达载体,抑制B7-H1分子在星形胶质细胞上的表达,为研究大脑内B7-H1分子的功能提供有效实验工具。方法设计合成带有双向启动子具有转录功能性SIRNA载体,转入小鼠星形胶质细胞株CRL-2541内,干扰免疫分子B7-H1的表达,利用荧光载体N1平行观察转染效果,RT-PCR方法检测B7-H1的MRNA表达,免疫组化检测蛋白表达水平,以观察干扰效率。结果星形胶质细胞株CRL-2541经RNAI沉默后B7-H1MRNA表达下调,免疫组化发现其蛋白水平下降。结论成功建立具有双向启动子的SIRNA载体,并在星形胶质细胞中抑制B7-H1的表达。
Objective To construct stable expression vector of SIRNA to inhibit the expression of B7-H1 on astrocytes and to provide an effective experimental tool for studying the function of B7-H1 in brain. Methods Transcriptional SIRNA vector with bidirectional promoter was designed and synthesized and transfected into mouse astrocyte cell line CRL-2541 to interfere with the expression of B7-H1. The transfection efficiency was observed with fluorescent vector N1, RT The expression of B7-H1 MRNA was detected by PCR, and the protein expression was detected by immunohistochemistry to observe the interference efficiency. Results The expression of B7-H1MRNA was down-regulated after RNAi silencing of astrocyte cell line CRL-2541, and the protein level was decreased by immunohistochemistry. Conclusion SIRNA vector with bidirectional promoter was successfully established and the expression of B7-H1 was inhibited in astrocytes.