采用SM 22启动子调节荧光蛋白的策略分选和鉴定人前列腺平滑肌细胞与成纤维细胞

 平滑肌细胞的数量、表型以及在间质细胞中所占的比例在前列腺间质增生的发生和发展中占有重要的地位.获得纯的平滑肌细胞和成纤维细胞，研究它们基因表达的差异，有助于进一步揭示前列腺增生的分子病因学.构建不同长度的 SM 22启动子，测定荧光素酶活性.采用了基于启动子特异性激活红绿色荧光蛋白表达结合流式细胞分选的策略，体外分离纯的平滑肌细胞和成纤维细胞.启动子活性实验结果表明，1 396 bp的人SM22启动子具有平滑肌细胞特异性和较高的相对活性.构建了红绿荧光蛋白的表达载体pDual-color，在此载体中，RFP的表达受1 396bp的SM22启动子调控，GFP的组成型表达受CMV启动子控制.用流式细胞仪分选GFP+/RFP+和GFP+/RFP-细胞，提取总RNA，进行实时定量RT-PCR.结果显示，在分选获得的GFP+/RFP+细胞比GFP+/RFP-细胞的SM22和SMMHC表达水平高10倍以上.提示，基于启动子特异性可以在体外分离纯的平滑肌细胞和成纤维细胞.

Purification and Characterization of Human Prostate Smooth Muscle Cells and Fibroblasts by UsingSM22 Promoter Driven Fluorescent Protein Expression

Smooth muscle cells (SMCs) are critical with regard to stromal function and the pathological changes in the development of benign prostatic hyperplasia (BPH). Purification and characterization of prostatic SMCs and fibroblasts will provide further insights into the pathogenesis of BPH. A promoter-based cell sorting method was selected as the cell purification strategy. SM22 promoter activity and specificity were analyzed and the 1 396 bp SM22 promoter was characterized as a prostatic SMC-specific gene promoter. A dual-color vector was constructed with the expression of the red fluorescent protein (RFP) under the control of the 1 396 bp SMC-specific SM22 promoter, and the expression of the green fluorescent protein (GFP) under constitutively active cytomegalovirus promoter. Fluorescence activated cell sorter (FACS) was used for isolation and enrichment of GFP /RFP and GFP /RFP-cells. SM22 and SMMHC expression was more than ten times higher in FACS-enriched SMCs than in fibroblasts by real-time RT-PCR. These results indicated that the prostatic SMCs and fibroblasts have been successfully isolated from each other.