Hypertrophic scar tissues and fibroblasts produce more transforming growth factor‐β1 mRNA and protein than normal skin and cells

• DOI: 10.1046/j.1524-475x.2000.00128.x
• Publication Date: 2003-03-12T23:53:22Z
• Authors: Rijian Wang; Aziz Ghahary; Qiong Shen; Paul G Scott; Kenneth Roy; Edward E Tredget
• Keywords: RNA polymerase II

Transforming growth factor‐β1 is a well‐known fibrogenic cytokine produced by many types of cells including dermal fibroblasts. To investigate whether this involved in development hypertrophic scar, transforming gene expression was evaluated small skin samples. Because sufficient quantity normal from patients with scar not readily available, reverse transcription‐polymerase chain reaction technique used. Quantitation difficult partly due to the lack suitable complementary RNA standards. We have established convenient, reliable procedure construct an internal standard for starting specific polymerase product. After digestion product endonuclease, piece cDNA human procollagen α1(I) compatible ends inserted into reaction‐DNA fragment. The recombinant re‐amplified and subcloned plasmid containing bacteriophage T7 T3 promoters. Complementary prepared amplified together tissue or cellular RNA. amplification, products were electrophoresed agarose gel ethidium bromide. bands mRNA scanned, digitized, plotted against amount added reaction. number molecules/cell calculated. examined found that tissues expressed five‐fold more than per unit wet weight. used quantitate 5 pairs fibroblast cultures derived skin. results showed contain significantly molecules (116 ± 6 vs. 97 7, p = 0.017, n 5). These supported Northern analysis enzyme‐linked immunosorbent assay TGF‐β1 protein fibroblast‐conditioned medium. In conclusion, fibroblasts produce factor‐β1, which may be important formation. construction simple useful cells.