绿色荧光蛋白转基因大鼠骨髓间充质干细胞培养及鉴定  

Culture and Identification of Bone Marrow Mesenchymal Stem Cells from Enhanced Green Fluorescent Protein-transgenic Rats

作　　者：刘慧娟[1] 胡若愚[2] 戴王娟 刘元果[2] 闵波[2] 蒋犁[1] 

机构地区：[1]东南大学附属中大医院儿科,南京210009 [2]东南大学附属中大医院心胸外科,南京210009

出　　处：《中国医学科学院学报》2016年第1期9-15,共7页Acta Academiae Medicinae Sinicae

基　　金：国家自然科学基金(81370739);江苏省自然科学基金(BK20131303)~~

摘　　要：目的体外分离、培养和鉴定增强型绿色荧光蛋白(EGFP)转基因大鼠骨髓间充质干细胞(BMSCs)。方法取EGFP转基因大鼠胫、股骨骨髓,全骨髓贴壁法分离培养、纯化BMSCs;荧光显微镜观察细胞形态;流式细胞仪分析细胞表型;CCK-8法绘制细胞生长曲线并与野生型BMSCs增殖情况相比较;分别向成脂、成骨、成软骨诱导分化鉴定;将细胞经鼠尾静脉移植入大鼠体内,观察其在体内定植情况。结果获得稳定表达EGFP的BMSCs,以长梭形为主,融合后呈漩涡状排列;生长曲线示EGFP-BMSCs增殖能力旺盛,与野生型BMSCs相比差异无统计学意义(t=-0.023,P=0.982);细胞CD29、CD90、CD34、CD49d、CD45表达率分别为99.4%、96.4%、0.171%、0.049%、0.038%;成脂、成骨、成软骨诱导后,分别给予油红O、茜素红、甲苯胺蓝染色,结果均呈阳性;在肺组织内可检测到稳定的绿色荧光。结论成功获得高纯度、稳定表达EGFP的BMSCs,干细胞特性不受EGFP影响。细胞定植后,示踪效果良好,可用于后续实验研究。Objective To isolate,culture,and identify bone marrow mesenchymal stem cells（ BMSCs） from enhanced green fluorescent protein（ EGFP）-transgenic rats in vitro. Methods Bone marrows were isolated from tibia and femur of healthy EGFP-transgenic rats of specific pathogen free（ SPF） grade. Then,the whole bone marrow adherent method was used for isolation,culture,and purification of BMSCs. The morphological change was noted by continuous observation under inverted fluorescence microscope. The growth curve of cells was drawn through the method of CCK-8 and the proliferation compared with wild type BMSCs. The surface markers of BMSCs were detected by flow cytometry. The BMSCs were induced to differentiate into osteoblasts,adipocytes,and chondrocytes lineages. The EGFP-BMSCs were transplanted into the rats intravenously,and the expression of GFP was detected. Results BMSCs stably expressing EGFP gene were obtained successfully,with the fusiform-shaped appearance and the forming of circinate cell colonies. The growth curve of EGFP-MSCsshowed the characteristic of active proliferation,showing no significant difference compared with the wild-type BMSCs. The expression rates of the surface markers of BMSCs CD29,CD90,CD34,CD49 d,and CD45 were99. 4%,96. 4%,0. 171%,0. 049%,and 0. 038%. The GFP were detected in lung 3 days after transplantation. After osteogenic,adipogenic,and chondrogenic induction,oil red-O and alizarin red positive signals and toluidine blue positive cells were detected. Conclusions High-purity BMSCs stably expressing green fluorescent protein gene can be cultured using the whole bone marrow adherent method. EGFP does not affect the stem cell properties and expresses stably after transplantation. The cells can be used as seed cells for subsequent research.

关 键 词：骨髓间充质干细胞 绿色荧光蛋白 诱导分化 

分 类 号：R394.2[医药卫生—医学遗传学]