Display Settings:

Format

Send to:

Choose Destination
See comment in PubMed Commons below
BJU Int. 2007 Jan;99(1):183-8. Epub 2006 Oct 11.

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

Author information

  • 1Institute for Molecular Biology, Nankai University, Tianjin, China.

Abstract

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.

PMID:
17034489
[PubMed - indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Blackwell Publishing
    Loading ...
    Write to the Help Desk