title: Biological effects of lentivirus-mediated shRNA targeting collagen type I on the mesangial cells of rats.
abstract: OBJECTIVE
  To investigate the effects of lentivirus-mediated shRNA targeting collagen type I on the mesangial cells of rats and the feasibility of lentivirus-mediated shRNA delivery through renal parenchyma injection.
  
  
  METHODS
  Anti-collagen type I shRNA lentiviral vector was constructed, and rat mesangial cells were transfected with transfection enhancer (control group), blank lentiviral vectors (pSC-GFP group), and pSC-GFP/Col I lentiviral vectors (pSC-GFP/Col I group). Transfection efficiency and cell cycle were determined by flow cytometry. RT-PCR and Western blot were performed to detect the mRNA and protein expressions of Col I. Cell proliferation was evaluated by 3-(4,5)-dimethylthiahiazo-3, 5-di-phenytetrazolium-romide (MTT) assay and direct counting, and apoptosis was detected using AnnexinV/PE staining. The feasibility of renal parenchyma injection of lentiviral vectors was assessed.
  
  
  RESULTS
  The transfection efficiency was 75.42%. The expressions of collagen type I in pSC-GFP/Col I group was markedly decreased when compared with the other two groups. PSC-GFP/Col I group was higher than pSC-GFP group in the inhibition efficiency of mesangial cell after transfection. Results revealed that pSC-GFP/Col I transfection induced apoptosis to a certain extent. The proportion of cells in G2/M phase in pSC-GFP/Col I group and pSC-GFP group was higher than that in control group after of transfection. Moreover, cells arrested in S phase were markedly increased. Our results also revealed renal injection of lentivirus-mediated shRNA was feasible.
  
  
  CONCLUSIONS
  Lentivirus-mediated shRNA targeting collagen type I could stably and efficiently transfect rat mesangial cells and significantly suppressed collagen type I expressions with acceptable safety. Renal injection of Col I lentivirus-mediated shRNA was also feasible.
authors.affiliation: Department of Nephrology, Navy General Hospital, Haidian, Beijing, PR China. zhouch0@163.com
authors.forename: Chunhua
authors.initials: C
authors.lastname: Zhou
authors.forename: Yi
authors.initials: Y
authors.lastname: Shan
authors.forename: Haidan
authors.initials: H
authors.lastname: Zhao
authors.forename: Ping
authors.initials: P
authors.lastname: He
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D024042:Collagen Type I / Q000378:metabolism*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D016086:Lentivirus; D008297:Male; D050527:Mesangial Cells / Q000187:drug effects* / Q000378:metabolism; D009400:Nephrosclerosis / Q000517:prevention & control*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology / Q000627:therapeutic use*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013778:Tetrazolium Salts; D013844:Thiazoles; D014162:Transfection
chemical_list: D024042:Collagen Type I; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013778:Tetrazolium Salts; D013844:Thiazoles; D049452:Green Fluorescent Proteins; C022616:thiazolyl blue