【摘要】：目的:觀察大鼠髓核間充質(zhì)干細胞(nucleus pulposus-derived mesenchymal stem cell,NPMSC)的生物學(xué)特性和辛伐他汀對髓核間充質(zhì)干細胞增殖及分泌功能的影響。方法:取8周齡SD大鼠尾椎髓核組織,采用膠原酶及胰酶序貫消化法分離NPMSC并進行體外擴增培養(yǎng),觀察細胞形態(tài)并通過噻唑藍(MTT)法檢測細胞增殖情況。通過流式細胞儀檢測細胞免疫表型及逆轉(zhuǎn)錄PCR(reverse transcription PCR,RT-PCR)檢測干細胞基因表達進行NPMCS的鑒定。取第3代NPMSC施加不同濃度辛伐他汀干預(yù)(0μmol/L、0.01μmol/L、0.1μmol/L、1μmol/L),在干預(yù)1d、3d、7d、14d、21d后通過CCK-8法檢測細胞活力,RT-PCR檢測蛋白多糖、Ⅱ型膠原、缺氧誘導(dǎo)因子1α(hypoxia-inducible factor-1α,HIF-1α)、葡萄糖轉(zhuǎn)運蛋白1(glucose transporter 1,GLUT-1)及血管內(nèi)皮生長因子(vascular endothelial growth factor,VEGF)的mRNA表達。結(jié)果:原代細胞早期可形成葵花樣細胞集落,克隆樣生長,傳代后細胞生長較原代細胞明顯加快,細胞形態(tài)以紡錘形為主。第3代細胞高表達干細胞相關(guān)陽性表面抗原分子CD73、CD90及CD105,低表達干細胞相關(guān)陰性表面抗原分子CD45及CD11b,與骨髓間充質(zhì)干細胞具有相似的干細胞轉(zhuǎn)錄因子Sox2、Oct-4及Nanog表達水平。適當(dāng)濃度的辛伐他汀(0.01μmol/L~0.1μmol/L)可促進NPMSC增殖及HIF-1α、GLUT-1、VEGF的mRNA表達,且濃度為0.1μmol/L時達到最強,而高濃度辛伐他汀(1μmol/L)對細胞增殖能力及HIF-1α、GLUT-1、VEGF的mRNA表達有明顯抑制作用。辛伐他汀可使NPMSC中出現(xiàn)并促進Ⅱ型膠原及蛋白多糖m RNA的表達,0.1μmol/L濃度達到最強。結(jié)論:大鼠的髓核組織培養(yǎng)出的NPMSC能夠表達骨髓間充質(zhì)干細胞特異性的干性基因,適當(dāng)濃度(0.01μmol/L~0.1μmol/L)的辛伐他汀可促進NPMSC增殖和細胞外基質(zhì)的分泌。
[Abstract]:Aim: to observe the biological characteristics of rat nucleus pulposus mesenchymal stem cells (nucleus pulposus-derived mesenchymal stem cell,NPMSC) and the effect of simvastatin on proliferation and secretion of rat nucleus pulposus mesenchymal stem cells. Methods: NPMSC was isolated from the nucleus pulposus of the tail vertebrae of 8-week-old SD rats by collagenase and trypsin digestion. The morphology of the cells was observed and the cell proliferation was detected by thiazolyl blue (MTT) method. The expression of stem cell gene was identified by flow cytometry and reverse transcription PCR (reverse transcription PCR,RT-PCR. The third generation of NPMSC was treated with different concentrations of simvastatin (0 渭 mol/L,0.01 渭 mol/L,0.1 渭 mol/L,1 渭 mol/L). The cell viability was detected by CCK-8 assay and proteoglycan was detected by RT-PCR. The mRNA expression of type 鈪，

本文編號：2411507