辛伐他汀对大鼠髓核间充质干细胞增殖及分泌功能的影响

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

目的:观察大鼠髓核间充质干细胞(nucleus pulposus-derived mesenchymal stem cell,NPMSC)的生物学特性和辛伐他汀对髓核间充质干细胞增殖及分泌功能的影响。方法:取8周龄SD大鼠尾椎髓核组织,采用胶原酶及胰酶序贯消化法分离NPMSC并进行体外扩增培养,观察细胞形态并通过噻唑蓝(MTT)法检测细胞增殖情况。通过流式细胞仪检测细胞免疫表型及逆转录PCR(reverse transcription PCR,RT-PCR)检测干细胞基因表达进行NPMCS的鉴定。取第3代NPMSC施加不同浓度辛伐他汀干预(0μmol/L、0.01μmol/L、0.1μmol/L、1μmol/L),在干预1d、3d、7d、14d、21d后通过CCK-8法检测细胞活力,RT-PCR检测蛋白多糖、Ⅱ型胶原、缺氧诱导因子1α(hypoxia-inducible factor-1α,HIF-1α)、葡萄糖转运蛋白1(glucose transporter 1,GLUT-1)及血管内皮生长因子(vascular endothelial growth factor,VEGF)的mRNA表达。结果:原代细胞早期可形成葵花样细胞集落,克隆样生长,传代后细胞生长较原代细胞明显加快,细胞形态以纺锤形为主。第3代细胞高表达干细胞相关阳性表面抗原分子CD73、CD90及CD105,低表达干细胞相关阴性表面抗原分子CD45及CD11b,与骨髓间充质干细胞具有相似的干细胞转录因子Sox2、Oct-4及Nanog表达水平。适当浓度的辛伐他汀(0.01μmol/L^0.1μmol/L)可促进NPMSC增殖及HIF-1α、GLUT-1、VEGF的mRNA表达,且浓度为0.1μmol/L时达到最强,而高浓度辛伐他汀(1μmol/L)对细胞增殖能力及HIF-1α、GLUT-1、VEGF的mRNA表达有明显抑制作用。辛伐他汀可使NPMSC中出现并促进Ⅱ型胶原及蛋白多糖m RNA的表达,0.1μmol/L浓度达到最强。结论:大鼠的髓核组织培养出的NPMSC能够表达骨髓间充质干细胞特异性的干性基因,适当浓度(0.01μmol/L^0.1μmol/L)的辛伐他汀可促进NPMSC增殖和细胞外基质的分泌。

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.