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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。
Objective: To observe the biological characteristics of rat nucleus pulposus-derived mesenchymal stem cells (NPMSCs) and the effect of simvastatin on the proliferation and secretion of nucleus pulposus mesenchymal stem cells. Methods: The nucleus pulposus of 8-week-old Sprague-Dawley rats was taken. NPMSCs were isolated by collagenase and trypsin digestion. The cells were expanded and cultured. The cell morphology was observed and the cell proliferation was detected by MTT assay. The cell phenotypes were detected by flow cytometry and the gene expression of stem cells was detected by reverse transcription PCR (RT-PCR) to identify NPMCS. The third generation of NPMSCs were treated with different concentrations of simvastatin (0μmol / L, 0.01μmol / L, 0.1μmol / L, 1μmol / L) and detected by CCK-8 after 1d, 3d, 7d, Cell viability, proteoglycan, type II collagen, hypoxia-inducible factor-1α (HIF-1α), glucose transporter 1 (GLUT-1) and vascular endothelial growth factor (vascular endothelial growth factor, VEGF) mRNA expression. Results: Primary cells could form sunflower like cell colonies in the early stage, which grew in clonal form. After passage, the growth of the cells was obviously faster than that of the primary cells. The morphology of the cells was spindle-shaped. The third generation of cells highly expressed stem cell associated positive surface antigen molecules CD73, CD90 and CD105, low expression of stem cell associated negative surface antigen molecules CD45 and CD11b, and had similar expression of stem cell transcription factor Sox2, Oct-4 and Nanog with bone marrow mesenchymal stem cells Level. At the appropriate concentration, simvastatin (0.01μmol / L ~ 0.1μmol / L) could promote the proliferation of NPMSCs and the mRNA expression of HIF-1α, GLUT-1 and VEGF at the concentration of 0.1μmol / L, Simvastatin (1μmol / L) significantly inhibited cell proliferation and mRNA expression of HIF-1α, GLUT-1 and VEGF. Simvastatin could promote the expression of type II collagen and proteoglycan mRNA in NPMSCs, and the concentration of 0.1μmol / L was the strongest. CONCLUSION: NPMSC cultured in rat nucleus pulposus can express MSCs specific dry gene. Simvastatin with appropriate concentration (0.01μmol / L ~ 0.1μmol / L) can promote NPMSC proliferation and extracellular Matrix secretion.