标题：BMP4促进诱导多能干细胞向感觉神经元分化

      作者：彭韬，苗刚勇，谭志强，刘斌    湖南省人民医院/湖南师范大学第一附属医院耳鼻咽喉头颈外科，湖南 长沙 410005

      卷次： 2019年30卷18期

      【摘要】 目的 探讨骨形成蛋白-4 (BMP4)在诱导多能干细胞(iPSCs)向感觉神经元分化中的作用。方法 采用碱性磷酸酶染色(AP)和拟胚体(EB)形成实验对 iPSCs进行鉴定。采用神经营养因子诱导法将 iPSCs向感觉神经元样细胞分化两周，对照组诱导方案为DMEM/12中加入2% B27、20 ng/mL碱性成纤维因子(bFGF)、200 ng/mL音猬因子(SHH)及10 μmol/L全反式维甲酸(ATRA)，BMP4组为对照组基础上添加10 ng/mL BMP4。通过细胞免疫荧光染色，检测诱导后细胞神经元活性，通过Realtime-PCR比较两组间细胞内感觉元相关基因mRNA相对表达量。结果 iPSCs呈现出典型的类似胚胎干细胞样克隆细胞团块，AP为阳性，EB随机分化实验中能表达三个胚层相关基因，符合 iPSCs生物学特性。BMP4组两周的神经元定向诱导后，iPSCs展现出双极或多极形态，神经元相关标志蛋白β-Tublin-Ⅲ表达阳性，表明诱导分化后的细胞具有神经元功能活性。Realtime-PCR结果显示在对照组 vsBMP4组中，BRN3A相对表达量为 (0.11±0.03) vs (0.25±0.04)、Neurog1为 (1.02±0.32) vs (1.93±0.52)、GFAP为(0.21±0.04) vs (0.65±0.07)，差异均具有显著统计学意义(P<0.01)，NeuroD为(0.45±0.05) vs (0.52±0.04)，差异无统计学意义(P>0.05)，表明BMP4能显著提升诱导后细胞BRN3A、Neurog1、GFAP相对表达量。结论 BMP4能显著提升iPSCs向感觉神经元样细胞诱导分化的效率，为后续实验研究提供了有力的保障。
      【关键词】 诱导多能干细胞；感觉神经元；骨形成蛋白-4；定向分化；细胞免疫荧光
      【中图分类号】 R338 【文献标识码】 A 【文章编号】 1003—6350（2019）18—2313—05

BMP4 promotes differentiation of induced pluripotent stem cells into sensory neurons.
PENG Tao, MIAOGang-yong, TANG Zhi-qiang, LIU Bin. Department of Otorhinolaryngology—Head and Neck Surgery, People's Hospital ofHunan Province/First Affiliated Hospital of Hunan Normal University, Changsha 410005, Hunan, CHINA
【Abstract】 Objective To investigate the role of bone morphogenetic protein-4 (BMP4) in the induction of plu-ripotent stem cells (iPSCs) into sensory neurons. Methods iPSCs were identified by alkaline phosphatase staining andembryoid body formation experiments. Using neurotrophic factor induction, iPSCs were differentiated into sensory neu-ron-like cells for 2 weeks. The control group was induced with DMEM/12 with 2% B27, 20 ng/mL basic fibroblast fac-tor (bFGF), 200 ng/mL sonic hedgehog homolog (SHH), and 10 μmol/L all-trans retinoic acid (ATRA). The BMP4group added 10 ng/mL BMP4 based on the treatment of the control group. The neuronal activity of the cells was detectedby immunofluorescence staining, and the relative expression levels of sensory neuron-related genes were compared by re-altime-PCR. Results iPSCs exhibited the morphological characteristics of embryonic stem cell, which were positivefor alkaline phosphatase staining. Three embryonic layer-related genes could be expressed in the embryonic body ran-dom differentiation experiment. After 2 weeks of neuronal induction in the BMP4 group, iPSCs exhibited bipolar ormultipolar morphology, and the sensory neuron-associated marker protein β-Tublin-Ⅲ was positive. Realtime-PCR re-sults showed that the relative expression of BRN3A, Neurog1, and GFAP in the control group vs BMP4 group were(0.11±0.03) vs (0.25±0.04), (1.02±0.32) vs (1.93±0.52), ( 0.21±0.04) vs (0.65±0.07), and the differences were statisticallysignificant (P<0.01). The relative expression of NeuroD was (0.45±0.05) vs (0.52±0.04), and the difference was not sig-nificant (P>0.05). Conclusion BMP4 can significantly improve the efficiency of iPSCs differentiation into sensoryneuron-like cells, which provides a powerful guarantee for subsequent experimental research.
      【Key words】 Induced pluripotent stem cell; Sensory neuron; Bone morphogenetic protein-4; Directional differen-tiation; Immunocytochemistry