绿色荧光蛋白转基因大鼠脂肪间充质干细胞的培养及分化潜能鉴定  

Culture and Identification of Adipose-Derived Mesenchymal Stem in Green Fluorescent Protein-transgenic Rats

作　　者：范友芬[1] 潘艳艳 乐欣[1] 晋国营[1] 虞耀华 FAN You fen;PAN Yanyan;LE Xin(Ningbo No.2 Hospital,Ningbo 315000,Zhejiang,China)

机构地区：[1]宁波市第二医院,宁波315010

出　　处：《现代实用医学》2018年第2期150-152,187,共4页Modern Practical Medicine

基　　金：浙江省医药卫生科技计划(2013KYB234)

摘　　要：目的建立大鼠脂肪间充质干细胞(ADSCs)体外分离培养的方法,并鉴定其分化潜能。方法取SPF级绿色荧光蛋白(GFP)转基因大鼠腹股沟脂肪,用胶原酶法消化获取原代细胞并进行传代培养。获得的P3代ADSCs,观察其细胞形态及荧光强度衰减情况;用流式细胞仪分析其表面特异性标志物,进行细胞表型鉴定;分别用成脂诱导培养基和成骨诱导培养基诱导其向脂肪细胞与成骨细胞分化,组织化学染色检测细胞分化的情况;将细胞悬液多点注射于大鼠烧伤创面及创周皮下,观察其定植情况。结果从成体GFP大鼠脂肪组织中成功分离培养出ADSCs,该细胞呈长梭形、漩涡样生长,传代后细胞生长增殖速度加快。ADSCs对CD29、CD90及CD105呈现高表达;对CD34及CD45呈低表达;茜素红和油红O染色显示ADSCs具有成骨成脂多向分化能力;在创面可见散点状绿色荧光。结论成功获得高纯度、稳定表达的GFP转基因大鼠的ADSCs,且生长增殖能力强,具有多向分化潜能。采用GFP转基因大鼠来源的ADSCs简单易行且示踪效果良好,可用于后续实验研究。Objective To establish a method for isolation,culture and identify adipose-derived mesenchymal stem cells(ADSCs)from green fluorescent protein(GFP)transgenic Rats in vitro.Methods Adipose tissue was harvested from the inguinal fat pad of adult SPF grade GFP-transgenic rats under sterile conditions.Then,the whole Adipose tissue adherent method was used for isolation,cultrure,and purification of ADSCs.The morphology and fluorescence intensity attenuation of three passaged ADSCs was observed.The molecular phenotype and differentiation potentia-lity of three passaged ADSCs were identified.The GFP-ADSCs were transplanted into rats'bum wounds with multi-point injection,and the expression of GFP was detected.Results ADSCs stably expressing GFP gene were obtained successfully,with the fusiform-shaped appearance and the forming of circinate cell colonies.The ADSCs had the abil-ity to proliferate continuously and showed characteristics of multi-potent mesenchymal stem cells.The results of flow cytometry showed that ADSCs was highly expressed in CD 105,CD90 and CD29,while lowly expressed in hemat-opoietic cell surface marker CD34 and leukocyte common antigen CD45.Alizarin red and oil red O staining showed ADSCs could be differentiated to osteoblasts and adipocytes.The GFP were detected in wound 21 days after trans-plantation.ConclusionsHigh-purity ADSCs stably expressing GFP gene can be cultured using the adipose tissue adherent method.GFP does not affect the stem cell properties and expresses stably after transplantation.These can provide the basis for the following experiment in vivo.

关 键 词：脂肪间充质干细胞 绿色荧光蛋白类 大鼠 转基因 

分 类 号：R394.2[医药卫生—医学遗传学]