体外传代对版纳微型猪骨髓间充质干细胞绿色荧光蛋白表达的影响  

Isolation and proliferation of bone marrow mesenchymal stem cells of Banna inbred minipig in vitro

作　　者：余璞[1,2] 龙海[3] 霍金龙[4] 王强[1] 李自安[1] 王金祥[1] 刘菊芬[1] 潘兴华[1] 庞荣清[1] Yu Pu Long Hai Huo Jinlong Wang Qiang Li Zi＇an Wang Jinxiang Lui Jufeng Pan Xinghua Pang Rongqing(the Cell Biological Therapy Center of Kunming General Hospital of Chendu Military Commend, the Integrated Engineering laboratory of Cell Biological Medicine of State and Regions, The Transfer Medicine Key Laboratory of Cell Therapy Technology of Yunan Province, The Stem Cell Engineering laboratory of Yunan Province, Kunming 650032, China Special Branch Consultation of Kunming General Hospital of Chendu Military Commend, Kunming 650032, China Kunming Medical University Kunming General Hospital of Chendu Military Commend, Kunming 650500, China yunnan Agricultural University College of Animal Science and Technology, Kunming 650201, China)

机构地区：[1]成都军区昆明总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干细胞工程实验室,昆明650032 [2]昆明医科大学成都军区昆明总医院临床学院,650500 [3]成都军区昆明总医院特诊科,昆明650032 [4]云南农业大学动物科技学院,昆明650201

出　　处：《中华细胞与干细胞杂志（电子版）》2016年第6期363-368,共6页Chinese Journal of Cell and Stem Cell（Electronic Edition）

基　　金：云南省应用基础研究重点项目(2015FA039)

摘　　要：目的通过分离扩增版纳微型猪骨髓间充质干细胞(BMSCs)观察传代对慢病毒转染的绿色荧光蛋白(GFP)表达的影响。方法采集4月龄版纳微型猪的骨髓,在DMEM/F12培养液中分离间充质干细胞(MSCs),并根据其形态学、抗原标志表达和分化潜能给予鉴定。MSCs与GFP慢病毒载体共培养,荧光显微镜观察GFP的表达,流式细胞仪检测传代后MSCs GFP表达率的变化。标记后传代至1、2、3、4代细胞间比较用非参数检验。结果采用直接培养骨髓,可以分离到高表达CD13、CD29、CD90、CD105的MSCs,并可诱导分化为脂肪、骨和软骨细胞。MSCs与携带GFP的慢病毒载体共培养3天即可观察到GFP的表达,最佳转染复数(MOI)值为50,最佳共培养时间为6 d。传代后MSCs GFP表达率呈下降趋势[1代转染率(42.3±2.25)%、2代转染率(41.6±2.65)%、3代转染率(41.4±3.75)%和4代转染率(38.2±4.75)%],但传至4代GFP表达率的变化差异无统计学意义(P>0.05)。结论采用含有10%胎牛血清的DMEM/F12培养液可以从版纳微型猪骨髓中分离到MSCs,GFP慢病毒转染是标记MSCs的有效方法,连续传代4代不会显著影响GFP的表达。Objective This study was aimed to isolate and culture bone marrow mesenchymal stem cells（MSC） from inbred Banna minipig in vitro. Metheods Bone marrow was aspirated from posterior superior iliac spine of three male Banna minipigs under sterile conditions. Then, primary bone marrow mesenchymal stem cells were isolated and cultured. Adherent cells were purified and labeled at the fourth generation with fluorescent dyes GFP in vitro as a source of donor cells. Results Bone marrow mesenchymal stem cells were culRtred successfully in vitro using bone marrow adherent separation metheods. Flow cytometry rescults showed that CD13, CD29, CD90, CD105 were all highly expressed in the cells. However, CD34 were not exoressed. After passage, the GFP expression decreased slightly（42.3 % ±2.25 %, 41.6 % ±2.65 %, 41.4 % ±3.75 % and 38.2 %± 4.75 % for the 1-4 passages respectively, P 〉 0.05）. Conclusion Miniature pig bone marrow MSCs may proliferate in DMEM/F 12 medium with 10 % fetal bovine serum, and labeling of MSCs via GFP lentiviral transfection is effective and stable.

关 键 词：猪 骨髓 间质干细胞 细胞培养技术 生物学标记 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]