基于甲基丙烯酸酐化明胶悬滴培养小鼠毛乳头细胞制备仿生毛乳头球的生物活性及其在裸鼠中的毛发诱导功能  

Biological activity of biomimetic dermal papilla spheres prepared by culture of dermal papilla cells of mice based on hanging drops of gelatin methacrylate and its hair-inducing function in nude mice

作　　者：黄刚 陈亮 Huang Gang;Chen Liang(Department of Burns and Plastic Surgery,Deyang People's Hospital,Deyang 618000,China;Department of Plastic and Aesthetic Surgery,the First Affiliated Hospital of Army Medical University(the Third Military Medical University),Chongqing 400038,China)

机构地区：[1]德阳市人民医院烧伤整形科,618000 [2]陆军军医大学(第三军医大学)第一附属医院整形美容外科,重庆400038

出　　处：《中华烧伤杂志》2021年第8期770-780,共11页Chinese Journal of Burns

基　　金：重庆市自然科学基金(cstc2014yykfA110005);重庆市应用开发计划(2009BB5153)。

摘　　要：目的探讨基于甲基丙烯酸酐化明胶(GelMA)仿生微环境与悬滴法三维培养小鼠毛乳头细胞(DPC)制备仿生毛乳头球的生物活性及其在裸鼠中的毛发诱导功能。方法采用实验研究方法。采用酶消化法获取雄性5~6周龄C57BL/6J小鼠触须毛的DPC以及1 d龄C57BL/6J小鼠皮肤角质形成细胞(KC),经免疫荧光法鉴定前者第3代细胞稳定表达DPC标志物神经细胞黏附分子、碱性磷酸酶(ALP)、β连环蛋白及α平滑肌肌动蛋白,后者原代细胞稳定表达KC标志物角蛋白15。取第8代DPC,用GelMA重新悬浮并接种至Transwell孔板插件底面,光交联后倒置培养,于光学显微镜下观察培养0(即刻)、3 d GelMA悬滴中DPC聚集情况(聚集成球即仿生毛乳头球),采用活/死染色试剂盒检测培养3 d仿生毛乳头球中细胞活性。将传统二维培养的原代DPC、第8代DPC以及按前述方法制备的仿生毛乳头球分别设为原代DPC组、第8代DPC组、仿生毛乳头球组,利用高通量测序技术平台对每组培养3 d的3个样本中DPC进行转录组测序,利用基迪奥生物信息云工具平台(OmicShare Tools)对转录组数据进行主成分分析、Pearson相似性分析、差异表达基因筛选,利用时间序列趋势分析软件对差异表达基因进行表达模式聚类分析,利用基迪奥生物信息云工具平台对具有特定表达模式的差异表达基因进行京都基因与基因组百科全书(KEGG)和基因本体论(GO)富集分析。同前进行细胞分组,采用随机数字表法从差异表达基因中抽取性别决定区Y框蛋白8(SOX8)、基质金属蛋白酶9(MMP-9)、26型胶原纤维α1(COL26A1)、无翅型MMTV整合位点家族成员6(Wnt6),采用实时荧光定量反转录PCR(RT-PCR)法验证差异表达基因mRNA表达情况与测序结果的一致性(样本数为9);采用实时荧光定量RT-PCR法检测DPC生物功能标志物成纤维细胞生长因子7(FGF7)、Wnt10a、淋巴样增强因子1(LEF1)、ALP、β连环蛋白、多功�Objective To investigate the biological activity of biomimetic dermal papilla spheres(DPSs)prepared by three-dimensionally cultured dermal papilla cells(DPCs)of mice based on the biomimetic microenvironment of gelatin methacrylate(GelMA)and hanging drop method and its hair-inducing function in nude mice.Methods Experimental research methods were adopted.DPCs from the vibrissa of male C57BL/6J mice aged 5 to 6 weeks and keratinocytes(KCs)from the skin of 1 d old C57BL/6J mice were obtained by enzymatic digestion method.A stable expression of DPCs markers such as nerve cell adhesion molecules,alkaline phosphatase(ALP),β-catenin,andα-smooth muscle actin were identified by immunofluorescence method in the third passage of the former cells,while the latter primary cells stably expressed keratin 15,a marker of KCs.The 8th passage of DPCs were re-suspended with GelMA and inoculated on the bottom surface of the Transwell plate insert,and then the GelMA drops were photocrosslinked and cultured upside down later.The DPCs aggregation in GelMA drops after in cultures of 0(immediately)and 3 day(s)was observed under an optical microscope(the DPCs aggregates were the biomimetic DPSs).The cell viability of 3 day biomimetic DPSs culture was detected by live/dead staining kit.The primary DPCs and the 8th passage of DPCs derived from traditionally two-dimensional cultures,and the biomimetic DPSs prepared by the above-mentioned method were set as primary DPCs group,the 8th passage of DPCs group,and biomimetic DPSs group,respectively.Transcriptome sequencing was performed using the high-throughput sequencing technology platform,with 3 samples in each group analyzed after three days in culture.The principal component analysis,Pearson similarity analysis,and screening of differentially expressed genes were performed using OmicShare Tools based on the transcriptome data.Cluster analysis of expression patterns of differentially expressed genes was performed using time series trend analysis software.The Kyoto Encyclopedia of Genes and G

关 键 词：毛发 聚甲基丙烯酸类 仿生学 高通量核苷酸测序 毛乳头细胞 悬滴法 毛乳头球 

分 类 号：R63[医药卫生—外科学] R322.1+2[医药卫生—临床医学]