【摘要】：目的:觀察大鼠髓核間充質(zhì)干細(xì)胞(nucleus pulposus-derived mesenchymal stem cell,NPMSC)的生物學(xué)特性和辛伐他汀對(duì)髓核間充質(zhì)干細(xì)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(jìn)行體外擴(kuò)增培養(yǎng),觀察細(xì)胞形態(tài)并通過(guò)噻唑藍(lán)(MTT)法檢測(cè)細(xì)胞增殖情況。通過(guò)流式細(xì)胞儀檢測(cè)細(xì)胞免疫表型及逆轉(zhuǎn)錄PCR(reverse transcription PCR,RT-PCR)檢測(cè)干細(xì)胞基因表達(dá)進(jìn)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(guò)CCK-8法檢測(cè)細(xì)胞活力,RT-PCR檢測(cè)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(yùn)蛋白1(glucose transporter 1,GLUT-1)及血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor,VEGF)的mRNA表達(dá)。結(jié)果:原代細(xì)胞早期可形成葵花樣細(xì)胞集落,克隆樣生長(zhǎng),傳代后細(xì)胞生長(zhǎng)較原代細(xì)胞明顯加快,細(xì)胞形態(tài)以紡錘形為主。第3代細(xì)胞高表達(dá)干細(xì)胞相關(guān)陽(yáng)性表面抗原分子CD73、CD90及CD105,低表達(dá)干細(xì)胞相關(guān)陰性表面抗原分子CD45及CD11b,與骨髓間充質(zhì)干細(xì)胞具有相似的干細(xì)胞轉(zhuǎn)錄因子Sox2、Oct-4及Nanog表達(dá)水平。適當(dāng)濃度的辛伐他汀(0.01μmol/L~0.1μmol/L)可促進(jìn)NPMSC增殖及HIF-1α、GLUT-1、VEGF的mRNA表達(dá),且濃度為0.1μmol/L時(shí)達(dá)到最強(qiáng),而高濃度辛伐他汀(1μmol/L)對(duì)細(xì)胞增殖能力及HIF-1α、GLUT-1、VEGF的mRNA表達(dá)有明顯抑制作用。辛伐他汀可使NPMSC中出現(xiàn)并促進(jìn)Ⅱ型膠原及蛋白多糖m RNA的表達(dá),0.1μmol/L濃度達(dá)到最強(qiáng)。結(jié)論:大鼠的髓核組織培養(yǎng)出的NPMSC能夠表達(dá)骨髓間充質(zhì)干細(xì)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(jìn)NPMSC增殖和細(xì)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(hào)：2411507