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目的探讨锌对骨髓间充质干细胞(bone marrow mesenchymal stem cells BMSCs)增殖及向神经样细胞诱导分化的作用。方法贴壁法体外分离培养BMSCs,MTT法检测不同浓度的锌处理BMSCs 24h、48h及72h后增殖活性。免疫细胞化学法检测不同浓度的锌处理BMSCs Ki67的表达。BMSCs分空白对照组、缺锌诱导组、正常诱导组、低浓度锌处理组和高浓度锌处理5组。除空白对照组外,其余4组在含有碱性成纤维细胞生长因子(bFGF)、表皮细胞生长因子(EGF)诱导分化培养基中培养5d,维甲酸继续培养14d,诱导BMSCs向神经样细胞分化。倒置显微镜观察细胞生长形态;免疫细胞化学法检测胶质纤维酸性蛋白(GFAP)和神经元特异性烯醇化酶(NSE)的表达。结果MTT结果显示,细胞暴露在锌浓度为200×10-7 mol/L时,不同时间点细胞活力均最强;免疫细胞化学法显示:缺锌组Ki67阳性表达显著低于空白对照组(P<0.05),低浓度锌处理组Ki67阳性表达显著高于空白对照组、缺锌组和高浓度锌处理组(P<0.05);缺锌组、正常诱导组和低浓度锌处理组GFAP和NSE阳性率显著高于空白对照组和高浓度锌处理组(P<0.05),正常诱导组和低浓度锌处理组的GFAP和NSE阳性率显著高于缺锌组(P<0.05),低浓度锌处理组的GFAP和NSE阳性率均显著高于正常诱导组(P<0.05)。结论锌在一定浓度范围内能够提高BMSCs增殖能力及促进BMSCs向神经样细胞分化。
Objective To investigate the effect of zinc on the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs) into neuron-like cells. Methods BMSCs were isolated and cultured in vitro. MTT assay was used to detect the proliferative activity of BMSCs treated with different concentrations of zinc for 24h, 48h and 72h. Immunocytochemistry was used to detect the Ki67 expression in different concentrations of zinc treated BMSCs. BMSCs were divided into blank control group, zinc-deficient induction group, normal induction group, low-concentration zinc treatment group and high-concentration zinc treatment group. Except for the blank control group, the other four groups were cultured in the medium containing basic fibroblast growth factor (bFGF) and epidermal cell growth factor (EGF) for 5 days, and retinoic acid was cultured for 14 days to induce the differentiation of BMSCs into neuron-like cells . The morphological changes of the cells were observed by inverted microscope. The expressions of glial fibrillary acidic protein (GFAP) and neuron specific enolase (NSE) were detected by immunocytochemistry. Results The results of MTT showed that cells exposed to zinc concentration of 200 × 10-7 mol / L had the strongest cell viability at different time points. Immunocytochemistry showed that Ki67 expression in zinc-deficient group was significantly lower than that in the blank control group (P <0.05). Ki67 expression in low concentration zinc treatment group was significantly higher than that in blank control group, zinc deficiency group and high concentration zinc treatment group (P <0.05); GFAP and NSE in zinc deficiency group, normal induction group and low concentration zinc treatment group (P <0.05). The positive rates of GFAP and NSE in normal induction group and low concentration zinc treatment group were significantly higher than those in zinc deficiency group (P <0.05). The positive rates of low concentrations of zinc The positive rates of GFAP and NSE in the treatment group were significantly higher than those in the normal induction group (P <0.05). Conclusion Zinc can increase the proliferation of BMSCs and promote the differentiation of BMSCs into neuron-like cells in a certain concentration range.