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

doi:10.3969/j.issn.2095-4344.2016.45.012

Abstract

Abstract:

BACKGROUND: Gene transfection of adipose-derived mesenchymal stem cells (ADSCs) can stably synthesize seed cells that secrete specific cytokines, thereby achieving long-term stable proliferation, differentiation and chondrogenic differentiation of seed cells.
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 cells were identified by immune fluorescent assay. After being transfected with pcDNA3.1-IGF-1, the cells were selected with G418. RT- PCR and western blot were used to analyze the expression level of HIF-1α in normal ADSCs, pcDNA3.1-transfected ADSCs, and pcDNA3.1-IGF-1- transfected ADSCs. The growth curve of cells was measured by MTT, and meanwhile, proliferation efficiency of cells was measured by counting CM-DiL-labeled cells. Immune fluorescent assay was used to analyze the expression of HIF-1α and 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 cell lines were successfully established and showed stable expression of IGF-1 at mRNA and protein levels. MTT and CM-Dil fluorescence results suggested that IGF-1-transfected cells displayed stronger proliferation ability and efficiency, and these cells showed a positive expression of HIF-1α and Sox-9 as well as higher glycosaminoglycan content than the other two groups (P < 0.01). All 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 extracellular matrix such as glycosaminoglycan is also significantly improved.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Mesenchymal Stem Cells, Insulin-Like Growth Factor I, Tissue Engineering

CLC Number:

R394.2

Zhang Chuan-hui, Li Jian-jun, Yang Jun. Regulation of Sox-9 and hypoxia-inducible factor-1alpha in adipose-derived mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(45): 6766-6773.

Figures/Tables 2

2.1 兔脂肪间充质干细胞的形态学观察及BD表型鉴定成功分离出的脂肪间充质干细胞接种后6 h就有少许贴壁，24 h贴壁增多，形态呈典型的长梭形或多角形细胞，呈团簇状生长，接种后5 d左右细胞融合超过80%时传代。细胞倍增时间约为2 d，传20代以上仍具有较高增殖速率，且形态未见显著改变，见图1。经荧光免疫法检测显示在脂肪间充质干细胞中CD44和CD109均呈阳性反应，见图2，进一步判定所获得细胞为脂肪间充质干细胞。 2.2 胰岛素样生长因子1质粒的鉴定以及细胞转染后的鉴定 XhoⅠ、EcoRⅠ双酶切pcDNA3.1-IGF-1经琼脂糖凝胶电泳鉴定、测序、G418筛选筛选后应用RT-PCR和Western blot方法对转染后细胞进行鉴定。 2.2.1 RT-PCR电泳结果转染组在电泳结果的300-500 bp的位置出现一条灰度较高的特异性条带，而未转染组和空载体组均未出现特异性条带，提示转染pcDNA3.1-IGF-1组的脂肪间充质干细胞在mRNA水平获得胰岛素样生长因子1的表达，见图3。 2.2.2 Western blot结果转染pcDNA3.1-IGF-1的脂肪间充质干细胞组有胰岛素样生长因子1蛋白的特异性表达，相对分子质量为7 600，未转染组和空载体组均未出现，提示质粒pcDNA3.1-IGF-1在转染组的脂肪间充质干细胞中获得良好的表达，胰岛素样生长因子1成功翻译为蛋白，见图4。 2.3 MTT法检测胰岛素样生长因子1基因转染对脂肪间充质干细胞增殖活性的影响 MTT比色法测定各实验组在490 nm波长处的A值，取均值列表绘制折线图，见图5。经统计学分析，转染PCDNA3.1的脂肪间充质干细胞组与正常传代培养的脂肪间充质干细胞组增殖速率基本一致，差异无显著性意义，转染PCDNA3.1- IGF-1的脂肪间充质干细胞组A值明显高于其他两组 (P < 0.01)。表明，负载pcDNA3.1-IGF-1的脂肪间充质干细胞增殖能力显著增强，有很好的生长活力。 2.4 Dil荧光染料标记后计数细胞增殖效率 Dil是一种较为理想的亲脂性碳花青染料，标记率可在95%以上[17]。荧光显微镜下观察细胞形态，可见胰岛素样生长因子1基因转染组细胞生长明显较对照组和空载体组旺盛，细胞形态饱满，见图6，细胞计数结果显示，转染PCDNA3.1-IGF-1的脂肪间充质干细胞组细胞增殖效率明显高于其他两组(P < 0.01)，正常传代培养的脂肪间充质干细胞、转染PCDNA3.1 的脂肪间充质干细胞组细胞计数平均值经组间比较，差异无显著性意义(P > 0.05)，见表2。表明经过胰岛素样生长因子1基因转染，显著提高脂肪间充质干细胞等增殖效率。 2.5 免疫荧光测定转染后细胞Sox-9和低氧诱导因子1α的表达免疫荧光法检测显示，转染pcDNA3.1- IGF-1的脂肪间充质干细胞组转染胰岛素样生长因子1后的脂肪间充质干细胞Sox-9和低氧诱导因子1α表达均为阳性，而正常传代培养的脂肪间充质干细胞和转染PCDNA3.1 的脂肪间充质干细胞组均为阴性，见图7。提示，胰岛素样生长因子1基因转染能够刺激脂肪间充质干细胞阳性表达低氧诱导因子1α和成软骨相关基因Sox-9，并有效促进其翻译成蛋白。 2.6 糖胺聚糖含量比较对各组总糖胺聚糖含量进行方差分析结果显示，空载体组与空白对照组之间糖胺聚糖含量差异无显著性意义(P > 0.05)，基因转染组总糖胺聚糖含量明显高于其他2组，差异有显著性意义(P < 0.01)，见表2。提示胰岛素样生长因子1基因转染能够促进细胞分泌软骨细胞外基质——糖胺聚糖。"

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