C型利钠利尿肽基因转染对骨髓间充质干细胞成软骨分化的影响  

Effect of C-type natriuretic peptide gene transfection on the chondrogenic differentiation of bone marrow mesenchymal stem cells

作　　者：杨烁[1] 时权[1] 钱智勇[2] 刘东华[2] 郭希民[2] 徐娟[1] 

机构地区：[1]解放军总医院口腔科,北京100853 [2]军事医学科学院基础医学研究所,北京100850

出　　处：《解放军医学院学报》2017年第6期531-535,544,共6页Academic Journal of Chinese PLA Medical School

基　　金：国家自然科学基金面上项目(31170926);国家自然科学基金应急项目(81541111);吴阶平医学基金会临床科研专项资助(320.6750.15029)~~

摘　　要：目的研究C型利钠利尿肽(C-type natriuretic peptide,CNP)基因转染对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成软骨分化能力的影响,为实现高质量基于间充质干细胞的组织工程软骨构建奠定基础。方法采用全骨髓培养法分离SD大鼠BMSCs并进行传代培养及多向分化能力鉴定。将已构建的含有人CNP基因和增强型绿色荧光蛋白(eGFP)基因的重组腺病毒(Ad-NPPC-eGFP)按照特定转染复数(multiplicity of infection,MOI)转染BMSCs,通过流式细胞仪检测转染效率,Real-time PCR检测转染后CNP mRNA表达情况,MTT实验检测转染后对细胞增殖的影响,阿尔新蓝染色检测转染后BMSCs体外诱导14 d及21 d时成软骨情况。结果成功获取了大鼠BMSCs,成脂、成骨分化诱导结果表明其具有较好的多向分化能力。腺病毒转染后细胞状态良好,转染效率较高,且未对细胞增殖产生显著影响(P>0.05),转染后BMSCs CNP mRNA高表达(P<0.05),体外成软骨能力强于未转染CNP基因组(P<0.05)。结论含有CNP基因的重组腺病毒可在SD大鼠BMSCs中稳定表达,并表现出较好的体外诱导成软骨能力。Objective To study the effect of C-type natriuretic peptide gene transfection on the chondrogenic differentiation of bone marrow mesenchymal stem cells （BMSCs） so as to lay a foundation for finding a new method of MSC-based cartilage tissue engineering construction with high quality. Methods Bone marrow mesenchymal stem cells were isolated from Sprague-Dawley rats by whole bone marrow adherence method and were subcultured. Multi-differentiation capacity of the cells was identified by traditional osteogenic and adipogenic differentiation in vitro. The BMSCs were then transfected by recombinant adenovirus vector containing C-type natriuretic peptide gene and enhanced green fluorescent protein （Ad-NPPC-eGFP） with the specific multiplicity of infection （MOI）. Flow cytometry was used to detect the transfection efficiency, real-time PCR was conducted to detect CNP mRNA expression, MTT assay was performed to examine the effect of transfection on cell proliferation, and alcian blue staining was performed to detect the extracellular matrix accumulation of the cells after chondrogenic differentiation in vitro for 14 days and 21 days. Results The rat BMSCs were successfully obtained, and osteogenic and adipogenic differentiation results revealed a good multi-diferentiafion capacity. Adenovirus transfected cells were in good condition with high transfection efficiency and no significant difference was shown on cell proliferation （P 〉 0.05）. The expression of CNP mRNA in BMSCs after transfection was significantly higher in Ad-NPPC- eGFP- transfected group （P 〈 0.05）. Alcian blue staining indicated significantly improved extracelluar matrix secretion （P 〈 0.05）. Conclusion Recombinant adenovirus containing CNP and eGFP gene can be stably expressed in BMSCs with better chondrogenic differentiation ability in vitro.

关 键 词：骨髓间充质干细胞 C型利钠利尿肽 重组腺病毒 软骨组织工程 

分 类 号：R318[医药卫生—生物医学工程]