C6TSEDRVAJZ小分子化合物组合诱导人胎盘成纤维细胞分化为上皮样细胞  

C6TSEDRVAJZ,a combination of small-molecule compounds,induces differentiation of human placental fibroblasts into epithelioid cells in vitro

作　　者：代振佳 高群伟 应梦娇 王澳 洪娟 王春景 郭俣 刘长青 刘高峰 DAI Zhenjia;GAO Qunwei;YING Mengjiao;WANG Ao;HONG Juan;WANG Chunjing(,GUO Yu1,3,LIU Changqing1,2,LIU Gaofeng1,21School of Life Sciences,2Anhui Engineering Research Center for Neural Regeneration Technology and Medical New Materials,3School of Laboratory Medicine,Bengbu Medical University,Bengbu 233000,China)

机构地区：[1]蚌埠医科大学生命科学学院,安徽蚌埠233000 [2]蚌埠医科大学安徽省神经再生技术与医用新材料工程研究中心,安徽蚌埠233000 [3]蚌埠医科大学检验医学院,安徽蚌埠233000

出　　处：《南方医科大学学报》2025年第2期322-330,共9页Journal of Southern Medical University

基　　金：国家自然科学基金(82371382);安徽省高校自然科学基金项目(2024AH040193,2024AH051296);蚌埠医科大学大学生创新训练计划项目(bydc2024038)。

摘　　要：目的利用小分子化合物组合将人胎盘成纤维细胞(HPFs)重编程为化学诱导上皮样细胞(ciEP-Ls)。方法常氧条件下培养HPFs细胞,通过免疫荧光、PCR和染色体核型分析鉴定HPFs。生理性低氧条件下(37℃,5%O2),HPFs在含有小分子化合物C6TSEDRVAJZ(CHIR99021、616452、TTNPB、SAG、EPZ5676、DZNep、Ruxolitinib、VTP50469、Afuresertib、JNK-IN-8、EZM0414)的培养基中进行诱导,诱导期间每日定时观察细胞形态。通过免疫荧光法、Western blotting和PCR技术检测上皮细胞标志物的表达水平。对诱导细胞进行染色体核型分析,并计算诱导效率。结果HPFs免疫荧光结果显示,表达成纤维表面标记物CD34和Vimentin,不表达上皮表面标志物;PCR结果显示,成纤维特异性基因S100A4、COL1A1高表达,且具有人正常二倍体核型。在低氧条件下诱导1 d后,部分HPFs细胞形态发生改变,由多突的纺锤形变成圆形或多边形,具有ciEP-Ls形态特征。诱导处理完成第4天,免疫荧光和Western blotting结果显示,诱导细胞能够高表达上皮细胞特征性标志物E-Cadherin和Lin28A(P<0.05)。PCR结果表明,细胞显著表达上皮标志物Smad3,GLi3,PAX8,WT1,KRT19和KRT18,而成纤维细胞表面标记物表达均下调,且诱导细胞具有人正常二倍体核型,HPFs重编程效率为(64.53±2.80)%~(68.10±3.60)%。结论小分子组合C6TSEDRVAJZ在低氧条件下成功将HPFs诱导为ciEP-Ls,且具有较高的诱导效率。Objective To reprogram human placental fibroblasts(HPFs)into chemically induced epithelioid-like cells(ciEP-Ls)using a combination of small-molecule compounds.Methods HPFs cultured under normoxic conditions were identified using immunofluorescence assay,PCR and chromosomal karyotyping.Under hypoxic conditions(37℃,5%O_(2)),HPFs were cultured in a medium containing small-molecule compounds C6TSEDRVAJZ(CHIR99021,616452,TTNPB,SAG,EPZ5676,DZNep,Ruxolitinib,VTP50469,Afuresertib,JNK-IN-8,and EZM0414),and the cell morphology was observed daily.The expression levels of epithelial cell markers in the induced cells were detected by immunofluorescence,Western blotting and PCR.Chromosomal karyotyping of the induced cells was performed and the induction efficiency was calculated.Results Before induction,HPFs showed positive expressions of fibroblast surface markers CD34 and vimentin and were negative for epithelial surface markers.PCR results showed high expressions of fibroblast-specific genes S100A4 and COL1A1 in HPFs with a normal human diploid karyotype.After one day of induction,the HPFs underwent morphological changes from a multinodular spindle shape to a round or polygonal shape,which was morphologically characteristic of ciEP-Ls.On day 4 of induction,the cells exhibited high expressions of the epithelial cell markers E-cadherin and Lin28A.RT-qPCR results also showed that the cells expressed the epithelial markers Smad3,GLi3,PAX8,WT1,KRT19,and KRT18 with significantly down-regulated expressions of all the fibroblast surface markers and a normal human diploid karyotype.The reprogramming efficiency of HPFs into ciEP-Ls ranged from(64.53±2.8)%to(68.10±3.6)%.Conclusion The small-molecule compound combination C6TSEDRVAJZ is capable of inducing HPFs into ciEP-Ls under hypoxic conditions with a high induction efficiency.

关 键 词：化学重编程 小分子诱导 成纤维细胞 上皮样细胞 

分 类 号：R318[医药卫生—生物医学工程]