Objective To explore an effective and appropriate condition of inducing human bone marrow mesenchymal stem cells(hBMSCs) into insulin-producing cells.Methods hBMSCs were induced to insulin producing cells(IPCs) by glueagon like peptide-1 (GLP-l),extentin-4 and the additions of many cell stimulating growth factors according to a three-stage protocol.The expression of multiple genes related to Panereatic β-cell development and function was detected by RT-PCR.The identity of the IPCs was illustrated by the analysis of morphology,ditizone staining and immunocytochemistry and release of insulin.Results Typical islet-like cell clusters were observed at the end of Protocol (18 days).Ditizone staining and immunocytochemistry for insulin were both positive.These differentiated cells at the end of Protocol expressed PDX-l mRNA and insulin mRNA.Insulin was secreted by these cells in response to different concentration of glucose stimulation.In the situation of the incubation with high glucose solution(16.7 mmol/L),the insulin secretion in the induced group showed significantly increased as compared with that in the control group [(188.7 ± 1.5) mU/L vs(60.1 ± 0.9) mU/L,P ＜ 0.05].Conclusion With combined GLP-l and extentin-4,hBMSCs can improve the rate of insulin-producing cells differentiation,promote IPCs maturation and release of insulin.%目的 探讨并优化人骨髓间充质干细胞(hBMSCs)向分泌胰岛素细胞(IPCs)定向分化的条件,为糖尿病替代疗法寻找新出路.方法 hBMSCs复苏培养后,联合应用胰高血糖素样肽1(GLP-1)、Ex-tentin-4等细胞因子通过三步诱导方案进行体外向IPCs诱导分化.应用反转录-聚合酶链反应检测与β细胞发育和功能相关的基因表达;免疫细胞化学、双硫腙染色证实IPCs的生成;电化学发光法检测细胞胰岛素的分泌量.结果 诱导分化18d后,镜下观察到胰岛样细胞团的生成,双硫腙及免疫细胞化学染色胰岛素均呈现阳性反应;诱导分化的细胞在第18天则可以观察到胰十二指肠同源盒基因1、胰岛素的高表达;诱导分化的细胞接受葡萄糖刺激后能够分泌胰岛素,且胰岛素的分泌量随葡萄糖浓度的提高而增加,高糖胰岛素分泌量[(188.7±1.5)mU/L]与低糖胰岛素分泌量[(60.1±0.9)mU/L]相比,差异有统计学意义(P＜0.05).结论 体外联合应用GLP-l、Extentin-4、尼克酰胺等细胞因子采用三阶段诱导方案可以大大提高胰岛素细胞诱导分化率,并能促进IPCs的成熟和胰岛素的释放.
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