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目的:建立人胚骨髓间充质干细胞(MSCs)分离及培养的方法,探讨胰岛素样生长因子-I(IGF-I)对MSCs增殖的影响。方法:通过密度梯度离心,贴壁法分离培养人胚MSCs,取第4代MSCs,应用流式细胞术测定细胞周期和CD44、CD34、CD45表达。采用MTT法观察不同含量和不同作用时间的IGF-I对MSCs增殖的影响。结果:体外培养的MSCs呈现成纤维细胞形态,流式细胞仪检测结果显示,MSCs阳性表达CD44,阴性表达CD34,CD45。细胞周期分析显示IGF-I使S+G2+M期细胞数增加,占细胞总数(29.9±2.3)%(t=10.4,P<0.01)。1μg/LIGF-I促MSCs增殖作用不明显,10μg/LIGF-I即可促MSCs增殖,IGF-I含量为100μg/L时,其促增殖作用达最大值,吸光度为2.95。而当IGF-I含量大于100μg/L时,促细胞增殖作用反而下降;时效关系显示,IGF-I第1天发挥促增殖作用,第5天促增殖作用达高峰,维持时间可达7d。结论:外源性IGF-I能促进MSCs增殖,为实现MSCs体外快速扩增提供适宜的条件。
OBJECTIVE: To establish a method for the isolation and culture of human embryonic bone marrow mesenchymal stem cells (MSCs) and explore the effect of insulin-like growth factor-I (IGF-I) on the proliferation of MSCs. METHODS: Human embryonic MSCs were isolated and cultured by density gradient centrifugation and adherent method. The 4th generation MSCs were harvested, and the cell cycle and expression of CD44, CD34 and CD45 were detected by flow cytometry. MTT assay was used to observe the effect of IGF-I with different content and time on the proliferation of MSCs. Results: MSCs cultured in vitro showed fibroblast morphology. The results of flow cytometry showed that MSCs were positive for CD44 and negative for CD34 and CD45. Cell cycle analysis showed that IGF-I increased the number of cells in S + G2 + M phase, accounting for 29.9 ± 2.3% (t = 10.4, P <0.01). The effect of 1μg / L IGF-I on MSCs proliferation was not obvious. 10μg / L IGF-I could promote the proliferation of MSCs. When the IGF-I concentration was 100μg / L, its proliferation reached its maximum, with an absorbance of 2.95. However, when IGF-I concentration was more than 100μg / L, the proliferation of cells was decreased. The relationship of aging showed that IGF-I could promote the proliferation on the first day and reach the peak on the fifth day. The maintenance time could reach 7 days. Conclusion: Exogenous IGF-I can promote the proliferation of MSCs and provide suitable conditions for the rapid expansion of MSCs in vitro.