用腺病毒载体包装不同分化程度平滑肌细胞特异性启动子的策略分离鉴定人前列腺间质细胞亚群

Isolation of Human Prostate Stromal Subpopulations at Different Differentiation Stage with Adenovirus Vectors Containing SM22 or MYH11 Promoter-driven EGFP Reporter

在良性前列腺增生组织间质中,平滑肌细胞收缩表型和合成表型的数量和比例的改变与病理过程密切相关.分离鉴定具有不同平滑肌表型的细胞亚群对研究它们基因表达的差异以及自分泌和旁分泌作用至关重要.PCR扩增得到不同分化程度的人平滑肌细胞特异性基因SM22和MYH11的启动子片段,用荧光素酶活性测定启动子活性.构建基于启动子特异性的绿色荧光蛋白表达质粒;用腺病毒包装系统得到带有平滑肌细胞特异性启动子调控的绿色荧光报道子的病毒颗粒.通过免疫荧光染色,鉴定该启动子序列的特异性.结果表明,用腺病毒包装的带有绿色荧光蛋白的平滑肌细胞标记蛋白SM22和MYH11的启动子具有活性和特异性.腺病毒载体在前列腺正常间质细胞系NAF中总感染效率约为90%,其中SM22阳性表达的细胞约为10%,MYH11阳性表达的细胞约为2%.

The phenotype and the proportion of the smooth muscle cells in the prostatic stroma changed in the benign prostatic hyperplasia tissue.Sorting of the subpopulations at different differentiation stage and analyzing the difference of the gene expression profiles and the reactivity to hormone and growth factors among them,will provide further insights into the pathogenesis of BPH.SM22 promoter and MYH11 promoter were obtained by PCR.Adenovirus vectors containing SM22 or MYH11 promoter-controlled EGFP reporter were obtained through the PDC adenovirus-construct system.The specificity of the promoters was confirmed by immunofluorescence assay.Preliminary experiment results showed that the specific promoter-EGFP reporter can be effectively transfected into prostate stromal cell line NAF,and SM22 promoter was activated in about 10% cells,while MYH11 promoter was activated in about 2% cells.These results indicated that the adenovirus vectors with specific promoters had been constructed successfully.