调控脂肪间充质干细胞成软骨分化基因Sox-9和低氧诱导因子1α的表达  

Regulation of Sox-9 and hypoxia-inducible factor-1alpha in adipose-derived mesenchymal stem cells

作　　者：张传辉 李建军[2] 杨军[2] 

机构地区：[1]朝阳市中心医院骨外科,辽宁省朝阳市122000 [2]中国医科大学附属盛京医院创伤骨科,辽宁省沈阳市110004

出　　处：《中国组织工程研究》2016年第45期6766-6773,共8页Chinese Journal of Tissue Engineering Research

基　　金：辽宁省自然科学基金资助项目(20102264)~~

摘　　要：背景:有学者采用基因转染的方法诱导脂肪间充质干细胞向软骨细胞分化,使种子细胞能够稳定合成和分泌特定的细胞因子,从而达到长期稳定刺激种子细胞增殖、分化和软骨形成的目的。目的:观察胰岛素样生长因子1基因转染对兔脂肪间充质干细胞低氧诱导因子1α和Sox-9表达的影响。方法:(1)取成年新西兰大耳白兔颈后部皮下脂肪组织,分离、培养兔脂肪间充质干细胞,免疫荧光法鉴定后在脂质体介导下应用pc DNA3.1-IGF-1基因转染脂肪间充质干细胞,G418筛选,获得稳定转染的细胞株;(2)实验分为3组:正常传代培养的脂肪间充质干细胞;转染pc DNA3.1的脂肪间充质干细胞;转染pc DNA3.1-IGF-1的脂肪间充质干细胞。(3)RT-PCR及Western blot法检测转染后细胞中胰岛素样生长因子1的表达,MTT法绘制细胞增殖曲线,Dil荧光染料标记后计数细胞增殖效率,免疫荧光法分析转染后细胞低氧诱导因子1α和Sox-9的表达情况,DMMB定量测定总糖胺聚糖含量。结果与结论:(1)成功构建稳定表达pcD NA3.1-IGF-1的细胞株,RT-PCR及Western blot检测证实转染后的细胞株在m RNA水平上获得胰岛素样生长因子1的表达,并且成功翻译为蛋白;(2)MTT提示转染后细胞增殖活性增强;(3)胰岛素样生长因子1基因转染显著提高Dil标记脂肪间充质干细胞的增殖效率;(4)转染后细胞的低氧诱导因子1α和Sox-9表达阳性;(5)胰岛素样生长因子1基因转染组糖胺聚糖含量明显高于其他2组(P<0.01)。(6)结果说明,胰岛素样生长因子1基因转染能够能够有效促进脂肪间充质干细胞增殖,促使阳性表达低氧诱导因子1α和成软骨分化调控基因Sox-9,并有效促进细胞分泌软骨细胞外基质糖胺聚糖。BACKGROUND：Gene transfection of adipose-derived mesenchymal stem cel s （ADSCs） can stably synthesize seed cel s that secrete specific cytokines, thereby achieving long-term stable proliferation, differentiation and chondrogenic differentiation of seed cel s. OBJECTIVE：To observe the effect of insulin-like growth factor-1 （IGF-1） gene transfection on the expression of Sox-9 and hypoxia-inducible factor-1alpha （HIF-1α） in rabbit ADSCs. METHODS：ADSCs were harvested from the posterior subcutaneous adipose tissue from Adult New Zealand white rabbits, then the cel s were identified by immune fluorescent assay. After being transfected with pcDNA3.1-IGF-1, the cel s were selected with G418. RT-PCR and western blot were used to analyze the expression level of HIF-1αin normal ADSCs, pcDNA3.1-transfected ADSCs, and pcDNA3.1-IGF-1-transfected ADSCs. The growth curve of cel s was measured by MTT, and meanwhile, proliferation efficiency of cel s was measured by counting CM-DiL-labeled cel s. Immune fluorescent assay was used to analyze the expression of HIF-1αand Sox-9. And the content of glycosaminoglycan in each group was determined by using dimethylmethylene blue dye-binding assay. RESULTS AND CONCLUSION：The stably expressed pcDNA3.1-IGF-1 cel lines were successful y established and showed stable expression of IGF-1 at mRNA and protein levels. MTT and CM-Dil fluorescence results suggested that IGF-1-transfected cel s displayed stronger proliferation ability and efficiency, and these cel s showed a positive expression of HIF-1αand Sox-9 as wel as higher glycosaminoglycan content than the other two groups （P〈0.01）. Al these findings indicate that IGF-1 gene transfection can effectively promote the proliferation of ADSCs, and promote the positive expression of Sox-9 and HIF-1α. And the secretion of chondral extracel ular matrix such as glycosaminoglycan is also significantly improved.

关 键 词：干细胞 脂肪干细胞 胰岛素样生长因子1 脂肪间充质干细胞 低氧诱导因子1 SOX-9 辽宁省自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]