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Development of a cell‐isolation method for human prostatic smooth muscle cells based on cell type‐specific activation of the SM22 gene promoter
Author(s) -
Wang ChunYu,
Shi JianDang,
Yan ChunHe,
Wu Quan,
Klocker Helmut,
Park Irwin,
Lee Chung,
Zhang Ju
Publication year - 2007
Publication title -
bju international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.773
H-Index - 148
eISSN - 1464-410X
pISSN - 1464-4096
DOI - 10.1111/j.1464-410x.2006.06566.x
Subject(s) - microbiology and biotechnology , green fluorescent protein , cell sorting , transfection , promoter , biology , cell culture , reporter gene , cell , immunofluorescence , stromal cell , gene , gene expression , antibody , flow cytometry , cancer research , immunology , genetics , biochemistry
OBJECTIVE To separate smooth muscle cells (SMCs) from fibroblasts in cultured human prostatic stromal cells (PrSCs) by characterizing the SM22 promoter as a prostatic SMC‐specific gene promoter, and to investigate its use for a promoter‐based cell‐sorting method, as SMCs are critical for stromal function and the pathological changes in the development of benign prostatic hyperplasia. MATERIALS AND METHODS Human PrSCs were cultured in SMC‐selective medium or standard medium, respectively, to obtain typical cultures of SMCs and fibroblasts. SM22 promoter activity and specificity were analysed by luciferase reporter‐gene assay. A dual‐colour vector was constructed with the expression of the red fluorescent protein (RFP) under the control of the 1.4 kb SMC‐specific SM22 promoter, and the expression of the green fluorescent protein (GFP) under cytomegalovirus promoter. Fluorescence‐activated cell sorting (FACS) was used to isolate and enrich GFP+/RFP+ and GFP+/RFP– cells. Cell phenotype was confirmed by reverse transcription‐polymerase chain reaction and immunofluorescence. RESULTS The 1.4 kb SM22 promoter activity was much higher in PrSCs cultured in SMC‐selective medium. Immunofluorescence staining and merged fluorescence microscopy ensured that SM22 promoter‐driven GFP positive cells were SMCs. After transfection of the dual‐colour vector into PrSCs, GFP+/RFP+ cells (SMCs) and GFP+/RFP– cells (fibroblasts) were isolated by FACS. The phenotype of FACS‐enriched SMCs and fibroblasts was confirmed. CONCLUSION These results indicate that the 1.4 kb SM22 promoter is specific for prostatic SMCs. This dual‐colour vector could be a useful tool for separating living SMCs from fibroblasts using FACS.