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

doi:10.12122/j.issn.1673-4254.2025.02.13

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (2): 322-330.doi: 10.12122/j.issn.1673-4254.2025.02.13

• • 上一篇

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

代振佳¹(), 高群伟¹, 应梦娇¹, 王澳¹, 洪娟¹, 王春景¹^,², 郭俣¹^,³, 刘长青¹^,², 刘高峰¹^,²()

^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

收稿日期:2024-08-20 出版日期:2025-02-20 发布日期:2025-03-03
通讯作者: 刘高峰 E-mail:zjdai000219@163.com;lgfmy@bbmu.edu.cn
作者简介:代振佳，在读硕士研究生，E-mail: zjdai000219@163.com
基金资助:
国家自然科学基金(82371382);安徽省高校自然科学基金项目(2024AH040193);蚌埠医科大学大学生创新训练计划项目(bydc2024038)

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

Zhenjia DAI¹(), Qunwei GAO¹, Mengjiao YING¹, Ao WANG¹, Juan HONG¹, Chunjing WANG¹^,², Yu GUO¹^,³, Changqing LIU¹^,², Gaofeng LIU¹^,²()

^1.School of Life Sciences, Bengbu Medical University, Bengbu 233000, China
^2.Anhui Engineering Research Center for Neural Regeneration Technology and Medical New Materials, Bengbu Medical University, Bengbu 233000, China
^3.School of Laboratory Medicine, Bengbu Medical University, Bengbu 233000, China

Received:2024-08-20 Online:2025-02-20 Published:2025-03-03
Contact: Gaofeng LIU E-mail:zjdai000219@163.com;lgfmy@bbmu.edu.cn
Supported by:
National Natural Science Foundation of China(82371382)

摘要/Abstract

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。

关键词: 化学重编程, 小分子诱导, 成纤维细胞, 上皮样细胞

Abstract:

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% O₂), 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.

Key words: chemical reprogramming, small molecule induction, fibroblasts, epithelioid-like cells

代振佳, 高群伟, 应梦娇, 王澳, 洪娟, 王春景, 郭俣, 刘长青, 刘高峰. 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[J]. 南方医科大学学报, 2025, 45(2): 322-330.

Zhenjia DAI, Qunwei GAO, Mengjiao YING, Ao WANG, Juan HONG, Chunjing WANG, Yu GUO, Changqing LIU, Gaofeng LIU. C6TSEDRVAJZ, a combination of small-molecule compounds, induces differentiation of human placental fibroblasts into epithelioid cells in vitro[J]. Journal of Southern Medical University, 2025, 45(2): 322-330.

图/表 5

表1 RT-PCR对差异表达miRNA的引物序列

Tab.1 Primer sequences for RT-PCR

Gene	Primersequence5'-3'
COL1A1	F: GCCAAGACGAAGACATCCCA R: GGCAGTTCTTGGTCTCGTCA
S100-A4	F: TTCTTCCCCTCTCTACAACCCTC R: ATGACAGCAGTCAGGATCAAACC
Smad3	F: TAATTTATTGCCGCCGCTCG R: GGCCATCCAGGGACTCAAAC
GLi3	F: CCGCCGCAGGGCATT R: AACGGCTTTCTCGCTCACAT
PAX8	F: CATTTGAGCGGCAGCACTAC R: AAGGGTGAGTGAGGATCTGC
WT1	F: CAGCGAAAGTTCTCCCGGT R: GCTGAAGGGCTTTTCACCTGTA
KRT19	F: AAATCAGTACGCTGAGGGGC R: GGTTCAATTCTTCAGTCCGGC
KRT18	F: CCTACAAGCCCAGATTGCCA R: TGGTGCTCTCCTCAATCTGC
Human GAPDH	F: AATGGGCAGCCGTTAGGAAA R: GCCCAATACGACCAAATCAGAG

图1 HPFs的生物学特征鉴定

Fig.1 Biological characterization of human placental fibroblasts (HPFs). A: Bright field observation of P3 generation HPFs in normal culture; B: Immunofluorescence detection of CD34, vimnetin, E-Cadherin and Lin28A in HPFs. C: RT-PCR showing the expressions of fibroblast specific genes S100A4 and COL1A1. D: HPFs chromosome G-banding showing chromosomes in metaphase (left) and karyotype (right).

图2 HPFs诱导分化为ciEP-Ls过程中细胞形态学变化

Fig.2 Morphological changes of HPFs during induced differentiation into ciEP-Ls. A: Flowchart of ciEP-Ls induction and differentiation. B: Summary of small molecules used for induced differentiation. C: Morphology of the cells induced for 4 days using afuresertib and AKT kinase inhibitor. D: Analysis of the reprogramming efficiency of the induced cells as a percentage of the total cells calculated from 3 independent experiments. Efficiency (%)=number of induced cells (ciEP-Ls)/number of seeded cells (HPFs)×100%. E: Morphological changes of the cells during induced differentiation.

图3 ciEP-Ls的免疫印迹与免疫荧光鉴定

Fig.3 Western blotting and immunofluorescence identification of ciEP-Ls. A: Immunofluorescence identification of E-Cadherin, Lin28A, CD34, and vimentin expression by ciEP-Ls. B: Western blotting of HPFs and ciEP-Ls proteins. *P<0.05.

图4 ciEP-Ls的PCR、qPCR与核型鉴定

Fig.4 PCR, qPCR and karyotype identification of ciEP-Ls. A: RT-PCR detection of ciEP-Ls markers Smad3, GLi3, PAX8, WT1, KRT19 and KRT18. B: Quantitative real-time PCR detection of ciEP-Ls markers Smad3, GLi3, PAX8, WT1, KRT19 and KRT18 (*P<0.05). C: Chromosome G-banding of ciEP-Ls showing chromosomes at the metaphase (left) and the karyotype (right).

参考文献 39

1	Willms RJ, Foley E. Mechanisms of epithelial growth and development in the zebrafish intestine[J]. Biochem Soc Trans, 2023, 51(3): 1213-24.
2	Luo LY, Zhang W, You SY, et al. The role of epithelial cells in fibrosis: Mechanisms and treatment[J]. Pharmacol Res, 2024, 202: 107144.
3	Kapsimali M. Epithelial cell behaviours during neurosensory organ formation[J]. Development, 2017, 144(11): 1926-36.
4	Lin H, Cheng Y, Zhang C. Research progress of pulmonary epithelioid hemangioendothelioma[J]. Zhongguo Fei Ai Za Zhi, 2019, 22(7): 470-6.
5	Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[J]. Cell, 2006, 126(4): 663-76.
6	González F, Boué S, Belmonte JCI. Methods for making induced pluripotent stem cells: reprogramming à la carte[J]. Nat Rev Genet, 2011, 12: 231-42.
7	Qin H, Zhao AD, Fu XB. Small molecules for reprogramming and transdifferentiation[J]. Cell Mol Life Sci, 2017, 74(19): 3553-75.
8	Ye J, Ge J, Zhang X, et al. Pluripotent stem cells induced from mouse neural stem cells and small intestinal epithelial cells by small molecule compounds[J]. Cell Res, 2016, 26(1): 34-45.
9	Liu D, Pavathuparambil Abdul Manaph N, Al-Hawwas M, et al. Small molecules for neural stem cell induction[J]. Stem Cells Dev, 2018, 27(5): 297-312.
10	Atkins H. Stem cell transplantation to treat multiple sclerosis[J]. JAMA, 2019, 321(2): 153-5.
11	Schmalzing M, Henes J, van Laar JM, et al. Editorial: Stem cell transplantation in autoimmune diseases (AID)[J]. Front Immunol, 2023, 14: 1150664.
12	Zhang DD, Wang GD, Qin LS, et al. Restoring mammary gland structures and functions with autogenous cell therapy[J]. Biomaterials, 2021, 277: 121075.
13	Xu W, Li H, Peng L, et al. Fish pluripotent stem-like cell line induced by small-molecule compounds from caudal fin and its developmental potentiality[J]. Front Cell Dev Biol, 2021, 9: 817779.
14	Nguyen KN, Bobba S, Richardson A, et al. Native and synthetic scaffolds for limbal epithelial stem cell transplantation[J]. Acta Biomater, 2018, 65: 21-35.
15	Rosa FF, Pires CF, Kurochkin I, et al. Direct reprogramming of fibroblasts into antigen-presenting dendritic cells[J]. Sci Immunol, 2018, 3(30): eaau4292.
16	Biddy BA, Kong WJ, Kamimoto K, et al. Single-cell mapping of lineage and identity in direct reprogramming[J]. Nature, 2018, 564: 219-24.
17	Liuyang SJ, Wang G, Wang YL, et al. Highly efficient and rapid generation of human pluripotent stem cells by chemical reprogramming[J]. Cell Stem Cell, 2023, 30(4): 450-9. e9.
18	Uko NE, Güner OF, Matesic DF, et al. Akt pathway inhibitors[J]. Curr Top Med Chem, 2020, 20(10): 883-900.
19	Wu JH, Limmer AL, Narayanan D, et al. The novel AKT inhibitor afuresertib suppresses human Merkel cell carcinoma MKL‐1 cell growth[J]. Clin Exp Dermatol, 2021, 46(8): 1551-4.
20	Xue WH, Yang L, Chen CX, et al. Wnt/β‑catenin-driven EMT regulation in human cancers[J]. Cell Mol Life Sci, 2024, 81(1): 79.
21	Lee S, Choi EJ, Cho EJ, et al. Inhibition of PI3K/Akt signaling suppresses epithelial-to-mesenchymal transition in hepatocellular carcinoma through the Snail/GSK-3/beta-catenin pathway[J]. Clin Mol Hepatol, 2020, 26(4): 529-39.
22	Spencer A, Yoon SS, Harrison SJ, et al. The novel AKT inhibitor afuresertib shows favorable safety, pharmacokinetics, and clinical activity in multiple myeloma[J]. Blood, 2014, 124(14): 2190-5.
23	Kim Y, Jeong J, Choi D. Small-molecule-mediated reprogramming: a silver lining for regenerative medicine[J]. Exp Mol Med, 2020, 52: 213-26.
24	Cao S, Yu S, Li D, et al. Chromatin accessibility dynamics during chemical induction of pluripotency[J]. Cell Stem Cell, 2018, 22(4): 529-42. e5.
25	Duan R, Du W, Guo W. EZH2: a novel target for cancer treatment[J]. J Hematol Oncol, 2020, 13(1): 104.
26	Ding R, Zheng J, Li N, et al. DZNep, an inhibitor of the histone methyltransferase EZH2, suppresses hepatic fibrosis through regulating miR-199a-5p/SOCS7 pathway[J]. PeerJ, 2021, 9: e11374.
27	Hou P, Li Y, Zhang X, et al. Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds[J]. Science, 2013, 341(6146): 651-4.
28	Chase A, Cross NCP. Aberrations of EZH2 in cancer[J]. Clin Cancer Res, 2011, 17(9): 2613-8.
29	Lin J, Wu Y, Tian G, et al. Menin “reads” H3K79me2 mark in a nucleosomal context[J]. Science, 2023, 379(6633): 717-23.
30	Alford JS, Lampe JW, Brach D, et al. Conformational-design-driven discovery of EZM0414: a selective, potent SETD2 inhibitor for clinical studies[J]. ACS Med Chem Lett, 2022, 13(7): 1137-43.
31	Guan JY, Wang G, Wang JL, et al. Chemical reprogramming of human somatic cells to pluripotent stem cells[J]. Nature, 2022, 605: 325-31.
32	Maherali N, Hochedlinger K. Tgfbeta signal inhibition cooperates in the induction of iPSCs and replaces Sox2 and cMyc[J]. Curr Biol, 2009, 19(20): 1718-23.
33	Zhao XX, An XL, Zhu XC, et al. Inhibiting transforming growth factor-β signaling regulates in vitro maintenance and differentiation of bovine bone marrow mesenchymal stem cells[J]. J Exp Zool B Mol Dev Evol, 2018, 330(8): 406-16.
34	Zhang YE. Non-smad pathways in TGF-beta signaling[J]. Cell Res, 2009, 19(1): 128-39.
35	Zheng J, Dai Q, Han K, et al. JNK-IN-8, a c-Jun N-terminal kinase inhibitor, improves functional recovery through suppressing neuroinflammation in ischemic stroke[J]. J Cell Physiol, 2020, 235(3): 2792-9.
36	Zhao M, Chen S, Yang ML, et al. Vitamin A regulates neural stem cell proliferation in rats after hypoxic-ischemic brain damage via RARɑ-mediated modulation of the β-catenin pathway[J]. Neurosci Lett, 2020, 727: 134922.
37	Janesick A, Wu SC, Blumberg B. Retinoic acid signaling and neuronal differentiation[J]. Cell Mol Life Sci, 2015, 72(8): 1559-76.
38	Jin Y, Teh SS, Lau HLN, et al. Retinoids as anti-cancer agents and their mechanisms of action[J]. Am J Cancer Res, 2022, 12(3): 938-60.
39	Das M, Pethe P. Differential expression of retinoic acid alpha and beta receptors in neuronal progenitors generated from human embryonic stem cells in response to TTNPB (a retinoic acid mimetic)[J]. Differentiation, 2021, 121: 13-24.

[1]	展俊平, 黄硕, 孟庆良, 范围, 谷慧敏, 崔家康, 王慧莲. 缺氧微环境下补阳还五汤通过抑制BNIP3-PI3K/Akt通路抑制类风湿关节炎滑膜成纤维细胞的线粒体自噬[J]. 南方医科大学学报, 2025, 45(1): 35-42.
[2]	骆金光, 陶怀祥, 闻志远, 陈龙, 胡昊, 关翰. 肿瘤相关成纤维细胞上调hsa-miR-18b-5p靶向FBXL3促进前列腺癌的增殖及转移[J]. 南方医科大学学报, 2024, 44(7): 1284-1296.
[3]	王梓凝, 杨明, 李双磊, 迟海涛, 王军惠, 肖苍松. 心肌梗死后心肌纤维化小鼠心肌线粒体功能和能量代谢重塑相关性的转录组学分析[J]. 南方医科大学学报, 2024, 44(4): 666-674.
[4]	邵珊, 白薇超, 邹鹏程, 罗敏娜, 赵新汉, 雷建军. 二甲双胍阻断乳腺癌细胞-间质细胞的交互作用：基于抑制肿瘤相关成纤维细胞缺氧诱导因子-1α的表达[J]. 南方医科大学学报, 2024, 44(3): 428-436.
[5]	郗雪艳, 邓婷, 杜伯雨. 结直肠成纤维细胞通过激活ERK信号通路促进结直肠癌细胞的恶性生物学行为[J]. 南方医科大学学报, 2024, 44(10): 1866-1873.
[6]	舒萍, 袁孟珂, 杨珂, 何伟志, 刘丽. 槲皮素通过抑制NLRP3/Caspase-1/GSDMD信号通路抑制小鼠成纤维细胞焦亡[J]. 南方医科大学学报, 2024, 44(10): 1874-1880.
[7]	邓婷, 杜伯雨, 郗雪艳. 结直肠癌细胞通过激活成纤维细胞的ERK通路诱导癌症相关成纤维细胞的形成[J]. 南方医科大学学报, 2023, 43(6): 943-951.
[8]	杨盼, 王圆圆, 高群伟, 刘阳, 王翼, 郭俣, 刘长青, 刘高峰. 小分子化合物组合将人胚胎成纤维细胞体外编程为神经前体细胞[J]. 南方医科大学学报, 2023, 43(3): 360-367.
[9]	肖姝喆, 成燕玲, 朱云, 唐芮, 古建标, 兰林, 何志华, 刘丹琼, 耿岚岚, 程旸, 龚四堂. WNT2b高表达的成纤维细胞破坏肠道黏膜屏障[J]. 南方医科大学学报, 2023, 43(2): 206-212.
[10]	李京娟, 李凡凡, 牛璐, 安斌斌, 成晓琼, 刘媛媛, 王金羊. 司美格鲁肽对高糖诱导的心肌成纤维细胞增殖的作用研究[J]. 南方医科大学学报, 2023, 43(11): 1935-1940.
[11]	黄永祺, 喻伟, 游月华. 槟榔碱通过促进巨噬细胞分泌含miR-155-5p外泌体诱导人口腔成纤维细胞的活化[J]. 南方医科大学学报, 2023, 43(1): 60-67.
[12]	江文豪, 钱垂文. 新型温敏性重组人釉原蛋白载体与传统丙二醇藻酸酯载体的体外测试效果比较[J]. 南方医科大学学报, 2022, 42(9): 1418-1425.
[13]	文君, 朱慧敏, 李雪梅, 黄家贵, 陈月, 杨琴. 抑制Sonic Hedgehog信号能抑制脑缺血性损伤后纤维瘢痕形成且不利于神经功能恢复[J]. 南方医科大学学报, 2022, 42(6): 840-848.
[14]	李舒雨, 杨启鑫, 左安娜, 田林华, 霍金海, 蒙艳丽, 汤庆发, 王伟明. 鲜广地龙纯化蛋白的制备及其体外抗肺纤维化活性评价[J]. 南方医科大学学报, 2022, 42(4): 618-624.
[15]	文君, 朱慧敏, 李雪梅, 黄家贵, 陈月, 杨琴. TGF-β1通过上调Shh 信号诱导大鼠脑膜成纤维细胞向肌成纤维细胞转化[J]. 南方医科大学学报, 2022, 42(2): 250-255.

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

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 5

参考文献 39

相关文章 15

编辑推荐

Metrics

本文评价