Influence of simvastatin on the proliferation and secretion functions of nucleus pulposus-derived mesenchymal stem cells

English Keywords:Simvastatin  Intervertebral disc degeneration  HIF-1α  Nucleus pulposus mesenchymal stem cells  Proliferation  Secretion  Rat

English Abstract:

  【Abstract】 Objectives: To investigate the effect of simvastatin at different concentrations on the proliferation and secretion functions of nucleus pulposus-derived mesenchymal stem cell(NPMSC) in vitro． Methods: NPMSC was isolated and cultured with collagenase and sequential trypsin digestion from the caudal spinal nucleus pulposus of 8-week-old Sprague-Dawley rats. Cellular morphology was observed and cellular proliferation was detected by MTT assay. The expressions of CD11b, CD45, CD73, CD90 and CD105 were detected by using flow cytometry. The stem cell transcription factor Sox2, Oct-4 and Nanog expression of NPMSC was measured by using RT-PCR and compared with bone marrow derived mesenchymal stem cell. The third generation NPMSC was incubated in various concentrations of simvastatin(0μmol/L, 0.01μmol/L, 0.1μmol/L and 1μmol/L) for 1, 3, 7, 14 and 21 days. The influences of simvastatin on proliferation were assayed with CCK-8 assay, and the type Ⅱ collagen, aggrecan, hypoxia-inducible factor-1α(HIF-1α), glucose transporter 1(GLUT-1) and vascular endothelial growth factor(VEGF) mRNA expressions were detected by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR)． Results: NPMSC isolated from rat nucleus pulposus could form the sunflower-like colonies and exhibit clone-like growth. NPMSC of third passage became homogeneous and exhibited spindle-like morphology. Meanwhile, high expression levels of stem cell-related positive antigen molecules(CD73, CD90, CD105) and low expression levels of negative antigen molecules (CD45, CD11b) were observed. Moreover, NPMSC expressed comparable stem cell-related genes (Sox2, Oct-4, Nanog) with bone marrow mesenchymal stem cells. Simvastatin in a certain range of concentration (0.01μmol/L-0.1μmol/L) significantly promoted proliferation and secretion(HIF-1α, GLUT-1, VEGF) functions(P＜0.05). Simvastatin at 0.1μmol/L concentration reached the maximum effects on the proliferation and secretion functions of NPMSC(P＜0.05), while simvastatin at 1μmol/L concentration showed proliferation and secretion functions(P＜0.05). Simvastatin promoted the mRNA expression of type Ⅱ collagen and aggrecan, in which 0.1μmol/L concentration reached the maximum effects. Conclusions: NPMSCs can be isolated and cultured from the nucleus pulposus of rats, and the cells had the expression of common dry transcription factors. Simvastatin at a certain concentration（0.01μmol/L-0.1μmol/L） could improve proliferation capability and promote mRNA expressions of collagen type Ⅱ and aggrecan of NPMSC.