过氧化氢酶在人脐带间充质干细胞中作用的研究

Effects of Catalase on Human Umbilical Cord Mesenchymal Stem Cells

本研究通过携带过氧化氢酶(catalase,CAT)基因的逆转录病毒载体转染人脐带组织源间充质干细胞(UC-MSC),探讨CAT对人UC-MSC增殖以及多向分化潜能的影响。将携带绿色荧光蛋白(GFP)空载体质粒pMSCV-GFP和携带CAT基因的pMSCV-GFP-CAT逆转录病毒载体分别转染体外培养的人UC-MSC,用流式细胞仪分析并分选获得MSC-GFP、MSC-GFP-CAT两种细胞株;用过氧化氢酶检测试剂盒检测上述细胞株和UC-MSC中过氧化氢酶活力;用cell counting kit-8检测转染后细胞的增殖能力;用von Kossa和油红O染色观察成骨和成脂定向诱导分化。结果表明:转染48小时后即可观察到UC-MSC发出绿色荧光,3天后达到稳定状态,随着细胞的传代荧光强度不会减弱。流式细胞仪检测GFP+转染率显示,MSC-GFP为(25.54±8.65)%,MSC-GFP-CAT为(35.4±18.57)%;体外传代培养后检测UC-MSC、MSC-GFP、MSC-GFP-CAT细胞CAT活性分别为:19.5、20.3、67.2U;MSC-GFP-CAT能被诱导分化为成骨和脂肪细胞;携带CAT基因载体转染对UC-MSC的增殖无显著影响(p>0.05)。结论:成功获得高表达CAT的UC-MSC,其CAT的表达水平为对照组的3.4倍,转染对UC-MSC的增殖和分化能力无显著影响,基因转移使UC-MSC保留了成骨和成脂的能力。

This study was aimed to investigate the growth and multiple differentiation potential of human umbilical cord tissue derived mesenchymal stem cells （UC-MSCs）transfected by a retroviral vector with catalase（CAT） gene. The UC-MSCs cultured in vitro were transfected by using pMSCV carting GFP （pMSCV-GFP） and pMSCV carring CAT （pMSCV-GFP-CAT） respectively, then the MSC-GFP cell line and MSC-GFP-CAT cell line were obtained by sorting of flow cytometry. The GFP expression was observed by a fluorescent microscopy at 48 hours after CAT gene transfection. The GFP ＋ cells were sorted by flow cytometry. The activity of CAT in GFP ~ ceils was detected by catalase assay kit. The proliferative capacity of transfected UC-MSCs was determined by cell counting kit-8. The differentiation ability of gene-transfected GFP ＋ cells into osteogenesis and adipogenesis was observed by von Kossa and oil red O staining. The results indicated that green fluorescence in UC-MSCs was observed at 48 hours after transfection, and the fluorescence gradually enhanced to a steady level on day 3. The percentage of MSCs-GFP was （25.54± 8.65 ）%, while the percentage of MSCs-GFP-CAT was（35.4 ± 18.57）%. The activity of catalase in UC-MSCs, MSCs-GFP, MSCs- GFP-CAT cells were 19. 5, 20. 3, 67. 2 U, respectively. The transfected MSCs-GFP-CAT could be induced into osteoblasts and adipocytes. After 21 days ,von Kossa staining showed induced osteoblasts. Many lipid droplets with high refractivity occurred in cytoplasm of the transfected UC-MSCs, and showed red fat granules in oil red O staining cells. There were no significant differences between transfected and non-transfected UC-MSCs cells （p 〉 0.05 ）. It is concluded that UC-MSCs are successfully transfected by retrovims carrying GFP or CAT gene, the activity of catalase increased by 3. 4-fold. The transfected UC-MSCs maintain proliferation potential and ability of differentiation into osteoblasts and adipocytes.