含毛喉素的条件培养液诱导脂肪来源干细胞神经分化的体外实验研究  

Forskolin-containing conditioned medium induces neural differentiation of adipose-derived stem cells:An in vitro experimental study

作　　者：刘旭凌 胡颖涵 孙家悦 朱泽宇 陆家瑜 LIU Xuling;HU Yinghan;SUN Jiayue;ZHU Zeyu;LU Jiayu(Department of Stomatology,Sixth People's Hospital Affiliated Shanghai Jiao Tong University,School of Medicine,Shanghai 200233,China)

机构地区：[1]上海交通大学医学院附属上海第六人民医院口腔科,上海200233

出　　处：《口腔颌面外科杂志》2024年第3期170-179,共10页Journal of Oral and Maxillofacial Surgery

基　　金：国家自然科学基金(82071160、82370933);上海市高水平地方高校创新团队(SSMU-ZDCX20180900)。

摘　　要：目的:利用神经诱导液体外诱导大鼠脂肪来源干细胞(adipose-derived stem cells,ASCs)神经分化,探索对ASCs体外成神经分化的最佳毛喉素浓度,并研究该浓度的培养液对ASCs体外成神经分化的作用。方法:酶消化法获得原代ASCs,通过流式细胞术和细胞染色鉴定ASCs的表型和多向分化能力。体外用含不同浓度毛喉素的神经诱导条件培养液进行神经诱导分化,采用免疫荧光染色技术筛选最佳浓度。采用含最佳浓度毛喉素的神经诱导条件培养液诱导ASCs,镜下观察诱导前后细胞的形态学特征,采用实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)和免疫荧光检测诱导前后细胞中神经分化标志物的mRNA和蛋白表达变化情况。结果:神经诱导条件培养液中毛喉素的最佳浓度为10μmol/L,含该浓度毛喉素的神经诱导条件培养液诱导的细胞,能够迅速诱导ASCs向成神经方向分化。诱导后,细胞形态类似神经细胞,神经细胞标志物抗β-Ⅲ微管蛋白(classⅢβ-tubulin,Tuj1)、神经丝蛋白(neurofilament-M,NF-M)、巢蛋白(nestin)抗体和S100β的mRNA水平和蛋白水平均表达增加(P<0.05)。结论:含毛喉素浓度为10μmol/L的诱导培养液能诱导ASCs神经分化,诱导细胞有望成为周围神经种子细胞。Objective:To explore the neural differentiation of adipose-derived stem cells(ASCs) induced by neural-induced conditional medium in vitro,and to study the optimal concentration of forskolin and the effect of optimal concentration on neurogenic differentiation of ASCs in vitro.Methods:ASCs were isolated by enzyme digestion method.The phenotype and differentiation ability of stem cells are identified by flow cytometry and cell staining.Nerve induction differentiation was performed in vitro by neural-induced conditional medium containing different concentrations of forskolin,and the optimal concentration was screened by immunofluorescence staining.ASCs were induced by neural-induced conditional medium containing the optimal concentration of forskolin and the morphological changes of cells were observed by microscope before induction,6 h after induction and 24 h after induction.Effects of induction on rat ASCs were analyzed by real-time quantitative polymerase chain reaction(RT-qPCR) and immunofluorescence.Results:The optimal concentration of forskolin in neuralinduced conditional medium was 10 μmol/L,and the cells induced by neural-induced conditional medium containing 10 μmol/L forskolin could rapidly induce ASCs to differentiate in the direction of nerve formation.After induction,the cell morphology was similar to nerve cells.Results of RT-qPCR and immunofluorescence indicated that the expressions of anti-class Ⅲβ-tubulin(Tuj1),neurofilament-M(NF-M),nestin and S100 β at the mRNA level and protein level were increased after induction(P<0.05).Conclusion:Nerve differentiation of ASCs can be induced by conditional medium containing 10 μmol/L forskolin,and the induced cells are expected to become seed cells of facial nerve conduits.

关 键 词：脂肪来源干细胞 细胞分化 神经再生 毛喉素 

分 类 号：R782[医药卫生—口腔医学]