Attenuation of alpha1 collagen production with antisense ribonucleic acid in cultured hypertrophic scar fibroblasts.
BACKGROUND
It has been demonstrated that hypertrophic scar fibroblasts (HSFs) overexpress collagen messenger ribonucleic acid (mRNA) and protein, especially alpha1 collagen. Antisense nucleic acids are effective in inhibiting harmful or uncontrolled gene expression, suggesting that antisense ribonucleic acid (RNA) can effectively downregulate the expression of alpha1 collagen gene and attenuate the scars.


OBJECTIVE
This study was conducted to observe the effect of recombinant plasmid pREP9-COL1 on alpha1 collagen expression in HSFs and clarify the prospect of antisense RNA on scar treatment.


METHODS
The alpha1 collagen gene fragment including the region of 5' UTR to exon (229 bp) was cloned in the eukaryotic expression plasmid pREP9 in the antisense orientation relative to the RSV-LTR promoter to reconstruct the pREP9- COL1 plasmid. Then it was transferred into HSFs through lipofectamine. The expression of alpha1 collagen was examined by immunostaining, reverse-transcriptase polymerase chain reaction, and Western blots.


RESULTS
The recombinant plasmid pREP9-COL1 with a correct sequence was constructed successfully; pREP9-COL1 consistently inhibited human alpha1 collagen gene expression at both mRNA and protein levels.


CONCLUSIONS
Antisense RNA was effective in downregulating alpha1 collagen expression of HSFs. Therefore, this approach offered a prospect of scar treatment by attenuation of alpha1 collagen production with antisense RNA.
Ju-Lin
Xie
X
Shao-Hai
Qi
Q
Tian-Zeng
Li
L
Chao-Quan
Luo
L
Hou-Dong
Li
L
Ying-Bin
Xu
X
Xu-Sheng
Liu
L
Bin
Shu
S
Hui-Zhen
Liang
L
Yong
Huang
H
D001706:Biopsy; D015153:Blotting, Western; D002478:Cells, Cultured; D017439:Cicatrix, Hypertrophic / Q000378:metabolism / Q000473:pathology*; D024062:Collagen Type V / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D015870:Gene Expression; D006801:Humans; D008856:Microscopy, Fluorescence; D016372:RNA, Antisense / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
C530725:COL5A1 protein, human; D024062:Collagen Type V; D016372:RNA, Antisense; D012333:RNA, Messenger