Development of a cell-isolation method for human prostatic smooth muscle cells based on cell type-specific activation of the SM22 gene promoter.
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.


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.


CONCLUSIONS
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.
Wang
Chun-Yu
CY
Institute for Molecular Biology, Nankai University, Tianjin, China.
Shi
Jian-Dang
JD
Yan
Chun-He
CH
Wu
Quan
Q
Klocker
Helmut
H
Park
Irwin
I
Lee
Chung
C
Zhang
Ju
J
D015153:Blotting, Western; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D005347:Fibroblasts / Q000166:cytology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D008297:Male; D008840:Microfilament Proteins / Q000378:metabolism*; D009124:Muscle Proteins / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011467:Prostate / Q000166:cytology*; D011470:Prostatic Hyperplasia / Q000473:pathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D008840:Microfilament Proteins; D009124:Muscle Proteins; C051615:transgelin