摘要
目的研究增强型绿色荧光蛋白(eGFP)慢病毒载体标记人牙周膜干细胞(PDLSCs)的理想条件,以获得稳定高表达eGFP的人PDLSCs。方法 eGFP慢病毒载体以25、50、100、200和400的感染复数(MOI)转染人PDLSCs48 h,荧光倒置显微镜及流式细胞术检测各组的转染效率和荧光强度,MTT法评价转染对细胞增殖的影响,检测细胞多向分化能力以及碱性磷酸酶(ALP)的表达状况,从而确定理想的转染条件。结果 eGFP慢病毒作用48 h,不同组的转染效率分别为44.7%、60.9%、71.7%、85.8%、86.9%;除MOI为400时以外,eGFP慢病毒转染对细胞增殖无明显影响;MOI为200时,转染细胞的多向分化能力未受影响,ALP活性与未转染组的差异无统计学意义(P>0.05)。结论 MOI为200作用48 h对细胞的增殖分化无明显影响,是eGFP慢病毒载体标记PDLSCs的理想条件,保持了其基本的生物学特性。
Objective The aim of this study is to optimize conditions for labeling human periodontal ligament stem cells （PDLSCs） using enhanced green fluorescent protein （eGFP） infected by lentivirus vector and to obtain PDLSCs with high stable expressed eGFP. Methods PDLSCs were transfected with eGFP by lentivirus vector for 48 h via different multiplicity of infection （MOI） （25, 50, 100, 200 and 400） and the infection efficiency were analyzed by both fluorescent microscope and flow cytometry. The proliferation rate of infected PDLSCs was evaluated by MTr. The in- fected PDLSCs were further for detection of pluripotent, differentiation ability and alkaline phosphatase （ALP） expression ability. Results The infection efficiency for each group were 44.7%, 60.9%, 71.7%, 85.8% and 86.9% respectively. Proliferation of PDLSCs was not affected when MOI was below 200; however, at MOI 400, the proliferation ability was affected compared with control group. The pluripotent and ALP abilities of PDLSCs were not changed by the infection. Conclusion Infection for 48 h at MOI 200 is optimal for labeling PDLSCs with eGFP using lentivirus vector, and the proliferation and differentiation abilities of PDLSCs are not affected obviously.
出处
《华西口腔医学杂志》
CAS
CSCD
北大核心
2012年第1期82-86,共5页
West China Journal of Stomatology
基金
山东省科技发展计划基金资助项目(2010GSF10268)
山东省自然科学基金资助项目(Y2008C107)
关键词
牙周膜干细胞
慢病毒载体
增强型绿色荧光蛋白
成骨能力
periodontal ligament stem cells
lentivirus vector
enhanced green fluorescent protein
osteogenic ability
