title: [Experimental study on vascular endothelial growth factor 165 gene-modified rat hair follicle stem cells mediated by lentiviral vector].
abstract: OBJECTIVE
  To obtain rat hair follicle stem cells (rHFSCs) which can constantly and highly express vascular endothelial growth factor 165 (VEGF165), and to observe the expression of VEGF165 gene in rat HFSCs.
  
  
  METHODS
  The cirri skin of 1-week-old Sprague Dawley rat was harvested and digested by using combination of Dispase and type IV collagenases. The bulge was isolated under microscope. The rHFSCs were cultured by tissue block method. After purified by rapid adhering on collagen type IV, the growth curve of different generations rHFSCs was drawn. The cells were identified by immunofluorescence staining and real time quantitative PCR (RT-qPCR) analysis that tested the expression level of correlated genes. Lentivirus of pLV-internal ribosome entry site (IRES)-VEGF165-enhanced green fluorescent protein (EGFP) (experimental group) and pLV-IRES-EGFP empty vector (control group) was packaged by calcium transfected method and the rHFSCs were transfected. The green fluorescent protein expression was observed by inverted fluorescence microscope, and VEGF165 mRNA and protein expressions were detected using RT-PCR and Western blot.
  
  
  RESULTS
  The rHFSCs which were isolated, cultured, and purified were like the "slabstone", and had strong adhesion ability and colony formation ability. The purified cells were in latent growth phase at 2-3 days; they were in exponential growth phase at 5-6 days. The expressions of cytokeration 15 (CK15), integrin alpha6, and integrin beta1 (markers of HFSCs) were positive by immunocytochemistry. The RT-qPCR analysis showed that CK15, CK19, integrin alpha6, and integrin beta1 expressed highly, but CD34 (a marker of epidermal stem cells) and CK10 (a marker of keratinocyte) expressed lowly. After 14 days, the transfection efficiency was up to 85.76% +/- 1.91%. RT-PCR analysis and Western blot showed that VEGF165 mRNA and protein expressions were positive in experimental group, and were negative in control group.
  
  
  CONCLUSIONS
  The rHFSCs with high purity and strong proliferation ability can be obtained by using microscope combined with tissue cultivation and rapid cell adhesion on collagen type IV. The rHFSCs with high expression of VEGF165 can be successfully obtained by lentiviral transfection. This method provides good seeding cells for tissue engineering to construct artificial hair follicles, blood vessels, and skins.
authors.forename: Xuan
authors.initials: X
authors.lastname: Zheng
authors.forename: Shichao
authors.initials: S
authors.lastname: Xu
authors.forename: Renfu
authors.initials: R
authors.lastname: Quan
mesh_terms: D000818:Animals; D049109:Cell Proliferation; D002478:Cells, Cultured; D005260:Female; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018859:Hair Follicle / Q000166:cytology*; D053569:Keratin-15 / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D008297:Male; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D023822:Tissue Engineering / Q000379:methods*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism
chemical_list: D053569:Keratin-15; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins