CAJ | 학술논문

运用慢病毒(Lentivirus)载体构建绿色荧光蛋白(GFP)在小鼠骨髓间充质干细胞(MSCs)中的转染体系,检测转染后 MSCs的表型和增殖能力,并诱导其向成肌细胞分化,检测此转染体系对 MSCs 细胞表型、增殖和分化能力的影响.骨髓细胞悬液破红后贴壁法培养 MSCs,采用流式细胞术鉴定细胞表面标记,鉴定 MSCs 纯度；Lentivirus-GFP以1、10、50和100的感染复数(MOI)转染 MSCs,作用48 h后流式细胞术及免疫荧光法检测转染效率和表达的荧光强度；MTT 法检测转染后 MSCs 的细胞活力.诱导 MSCs-GFP 向成肌细胞分化,蛋白印记法(WB)检测成肌细胞特异性蛋白 desmin 和α-SMA 的表达.流式细胞术检测结果显示,培养的 MSCs 表达 CD44、CD90和CD105,不表达 CD34、CD45和 CD188,符合干细胞特性.MOI 为1、10、50和100的转染效率分别为23.45%、93.51%、95.44%和95.55%,MOI为10时,转染效率较高,且对MSCs活力无明显影响.MSCs-GFP体外经成肌细胞诱导分化后,表达特异性抗原 desmin 和α-SMA.表明本研究成功构建了小鼠骨髓来源 MSCs 的Lentivirus-GFP转染体系,有效标记了 MSCs细胞,且此标记对 MSCs 的表型、增殖和分化能力等细胞生物学特性无明显影响.
운용만병독(Lentivirus)재체구건록색형광단백(GFP)재소서골수간충질간세포(MSCs)중적전염체계,검측전염후 MSCs적표형화증식능력,병유도기향성기세포분화,검측차전염체계대 MSCs 세포표형、증식화분화능력적영향.골수세포현액파홍후첩벽법배양 MSCs,채용류식세포술감정세포표면표기,감정 MSCs 순도；Lentivirus-GFP이1、10、50화100적감염복수(MOI)전염 MSCs,작용48 h후류식세포술급면역형광법검측전염효솔화표체적형광강도；MTT 법검측전염후 MSCs 적세포활력.유도 MSCs-GFP 향성기세포분화,단백인기법(WB)검측성기세포특이성단백 desmin 화α-SMA 적표체.류식세포술검측결과현시,배양적 MSCs 표체 CD44、CD90화CD105,불표체 CD34、CD45화 CD188,부합간세포특성.MOI 위1、10、50화100적전염효솔분별위23.45%、93.51%、95.44%화95.55%,MOI위10시,전염효솔교고,차대MSCs활력무명현영향.MSCs-GFP체외경성기세포유도분화후,표체특이성항원 desmin 화α-SMA.표명본연구성공구건료소서골수래원 MSCs 적Lentivirus-GFP전염체계,유효표기료 MSCs세포,차차표기대 MSCs 적표형、증식화분화능력등세포생물학특성무명현영향.
The obj ect of this study is to construct the green fluorescent protein(GFP)transfection system by lentiviral vector in mouse bone marrow mesenchymal stem cells(MSCs)invitro.We detected the biological characteristics of mesenchymal stem cells (MSCs ),inclouding cell phenotype,proliferation ability and differentiation ability of mesenchymal stem cells after lentiviral-GFP transfection.Then we observed the myoblasts differentiation ability of mesenchymal stem cells(MSCs)after we confirmed the expression of green fluorescent protein.Bone marrow cell suspensions from mice bone were adherent cultured after erythrocyte broken.We detect the cell surface marker (CD44,CD90,CD105,CD34,CD45 and CD188)of MSCs to identify the purity of mesenchymal stem cells by flow cy-tometry analysis.We transfected mesenchymal stem cells (MSCs)with Lentivirus-GFP for 48 hours,with the multiplicity of infection (MOI)of 1,10,50 and 100,respectively.The transfection efficiency and fluorescence expression were detected by both flow cytometry and immunofluorescence.We evaluated the cell viability by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)test after lentiviral-GFP transfection.MSCs-GFP were induced into myoblasts after the transfection were completed,and the expression of myoblast specific protein desmin andα-SMA were detected by western blot(WB)method.The results of flow cytometry analysis are shown:In accordance with the characteristics of stem cells surface markers,our cultured mesenchymal stem cells (MSCs)expressed CD44,CD90 and CD105,but did not express CD34,CD45 and CD188.The transfection efficiency were 23.45%,93.51%,95.44% and 95.55%,while the transfect MOI were 1,10,50 and 100,respectively.At transfect MOI 10,the transfection efficiency of mesenchymal stem cells (MSCs)was the best,and there was no obvious effect on cell activity observed.After myoblast differentiation invitro,the MSCs-GFP could express desmin andα-SMA.In this study,we constructed the green fluorescent protein (GFP )transfection system by lentiviral vector,and this transfect system could labeled the mouse bone marrow mesenchymal stem cells(MSCs)successfully invitro.This transfect system has no obvious effect on cell biological characteristics of mice MSCs,inclouding cell phenotype,proliferation ability and differentiation ability of mesenchymal stem cells.