GFP转染对版纳微型猪骨髓间充质干细胞成脂分化的影响  

Effect of GFP transfection on adipogenic differentiation of pBMSC of Banna minipig

作　　者：庞荣清 方伟 王金祥 王强 石琳琳 李自安 于倩倩 李晔 阮光萍 潘兴华 Pang Rongqing;Fang Wei;Wang Jinxiang;Wang Qiang;Shi Linlin;Li Zi'an;Yu Qianqian;Li Ye;Ruan Guangping;Pan Xinghua(Cell Biological Therapy Center of Hospital 920,Joint Logistic Support Force of PLA,National&Local Joint Engineering Laboratory for Stem Cell and Immune Cell Bio-medical Technology,Yunnan Provincial Key Laboratory for Translational Medicine in Cellular Therapy Technology,Stem Cell Engineering Laboratory of Yunnan Province,Kunming,Yunnan,650032,China)

机构地区：[1]解放军920医院细胞生物治疗中心,干细胞与免疫细胞生物医药技术国家地方联合工程实验室,云南省细胞治疗技术转化医学重点实验室,云南省干细胞工程实验室,昆明650032

出　　处：《西南国防医药》2020年第7期624-628,共5页Medical Journal of National Defending Forces in Southwest China

基　　金：国家自然科学基金面上项目(31970515);院内科技计划项目(2019YGA05)。

摘　　要：目的绿色荧光蛋白(GFP)基因转染标记版纳微型猪骨髓间充质干细胞(pBMSC),分析GFP转染对pBMSC增殖和成脂分化的影响。方法以不同剂量携带GFP基因慢病毒共培养的方法转染标记pBMSC,以hUCMSC为对照,流式分析方法检测不同细胞GFP的阳性率,CCK-8法检测转染细胞的增殖活性。观察GFP标记的pBMSC成脂分化诱导后的脂滴形成,异丙醇溶解脂滴,测定各组光密度值并进行比较分析。结果以MOI为50和100的病毒载体剂量共培养72 h,可以成功标记pBMSC,MOI为50时pBMSC的GFP阳性率明显低于hUCMSC的GFP阳性率(P<0.01)。转染GFP的pBMSC生长曲线依然呈现野S冶的分布特征,总体趋势与hUCMSC相似。不同GFP阳性率的pBMSC和hUCMSC成脂分化后脂滴溶解液OD值没有显著差异(P>0.05)。结论GFP转染可以标记近交系版纳微型猪来源的pBMSC,但不会显著影响其增殖和成脂分化。Objective To analyze the effect of green fluorescent protein(GFP) transfection on the proliferation and the adipogenic differentiation of porcine bone marrow mesenchymal stem cells(pBMSCs) by transfecting and labeling the pBMSCs of Banna minipig with GFP gene. Methods pBMSCs were transfected and labeled by co-cultivation of different doses of lentivirus with GFP gene, and human umbilical cord-derived mesenchymal stem cells(hUCMSC) served as control. Flow analysis method was used to detect the positive rate of GFP in different cells, and Cell Counting Kit-8(CCK-8) assay was performed to detect the proliferation activity of transfected cells. The formation of lipid droplet was observed after induction of lipid differentiation of GFP-labeled pBMSC.After the lipid droplet being dissolved with isopropanol, the optical density(OD) values of each group were determined and compared.Results pBMSC could be successfully labeled after being co-cultured with virus vectors with MOI of 50 and 100 for 72 hours. When MOI was 50, the GFP positive rate of pBMSC was significantly lower than that of hUCMSC(P < 0.01). The growth curve of GFPtransfected pBMSC still showed an "S-shaped" distribution feature, with the overall trend similar to that of hUCMSC. The OD values of lipid droplet dissolved indicated no significant difference between pBMSCs and hUCMSCs with different GFP positive rates after adipogenic differentiation(P > 0.05). Conclusion GFP transfection can label pBMSC derived from inbred line of Banna minipig without marked effect on its proliferation and adipogenic differentiation.

关 键 词：猪 骨髓间质干细胞 绿色荧光蛋白 增殖 成脂分化 

分 类 号：R446.61[医药卫生—诊断学]