ABSTRACT
<p><b>OBJECTIVE</b>To suppress COL1A1 and COL3A1 gene expressions in human skin fibroblasts (HSFs) by means of RNA interference (RNAi).</p><p><b>METHODS</b>Three small interfering RNA (siRNA) expression cassette (SEC) sequences were designed for each of the COL1A1 and COL3A1 gene sequences available in GenBank. The synthesized SECs capable of effective gene suppression were transfected into cultured HSFs, either after cloning into the expression vector or mediated by Lipofectamine 2000, and the suppression of the target genes at both mRNA and protein levels was determined by quantitative fluorescence RT-PCR and Western blotting, respectively.</p><p><b>RESULTS</b>Transfection of the SECs into HSFs resulted in specific depression of COL1A1 and COL3A1 expressions (down to 5.00% and 6.48%, respectively). The expression vector-mediated RNAi established a HSF cell line with persistent gene knockdown for over 30 days (to 25.21% and 22.12%, respectively).</p><p><b>CONCLUSION</b>COL1A1 and COL3A1 gene expressions can be specifically and efficiently inhibited in HSFs by either liposome- or vector-mediated SEC transfection.</p>
Subject(s)
Humans , Blotting, Western , Cells, Cultured , Collagen Type I , Genetics , Collagen Type III , Genetics , Fibroblasts , Cell Biology , Metabolism , RNA Interference , RNA, Small Interfering , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Skin , Cell Biology , Transfection , MethodsABSTRACT
<p><b>OBJECTIVE</b>To identify the localization of hair follicles stem cell (HFSC) in different stages of hair and explore the differentiating capacity of HFSC into epidermis in vitro.</p><p><b>METHODS</b>HFSC were detected by K19 immunostaining in normal human skin. Then, the isolated HFSC through enzyme digestion were seeded on dermal equivalent (DE) and cultured between the air-liquid interfaces for 14 days. Skin-equivalents was harvested and used for evaluation.</p><p><b>RESULTS</b>HFSC mainly located in outer root sheet in hair follicle and human anagen hair follicles containing two distinct reservoirs for K19-positive cells located in the bulge and bulb of the follicle. These two reservoirs fused in line of outer root sheets during the catagen-telogen transition phase and individualized again in the newly forming anagen hair follicle. Based on DE, growing HFSC built a multilayered and confined epidermis.</p><p><b>CONCLUSION</b>HFSC located in outer root sheets can promote hair cycle and differentiate into epidermis in vitro.</p>