基于人诱导多能干细胞的组织工程化软骨再生

doi:10.12439/kqhm.1005-4979.2024.01.003

摘要/Abstract

摘要：

目的：构建一种稳定高效的人诱导多能干细胞（induced pluripotent stem cells，iPSCs）向软骨中胚层方向分化的培养方法，初步探究hiPSCs来源的软骨用于关节软骨再生的可能。方法：通过模拟体内发育过程和三维（three dimensions，3D）悬浮培养体系，逐步诱导hiPSCs向软骨中胚层和成熟的软骨组织分化；利用实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）、蛋白质印迹法（Western blotting）、免疫荧光及免疫组织化学技术检测整个培养过程中多能干性、中胚层及软骨相关基因和蛋白的动态变化；最后利用裸鼠皮下模型研究hiPSCs软骨球软骨表型的稳定性及成瘤性。结果：通过模拟体内发育过程，成功逐步诱导hiPSCs向软骨中胚层方向分化，最终分化为成熟的软骨组织。结论：本研究成功构建了一种稳定高效诱导hiPSCs向软骨中胚层方向分化的培养体系，为探究hiPSCs介导的软骨组织发育和再生提供了一个研究平台。

关键词: 人类诱导多能干细胞, 中胚层, 悬浮培养, 软骨球

Abstract:

Objective: To establish a stable and efficient culture approach for guiding human induced pluripotent stem cells (hiPSCs) differentiation towards chondrogenic mesodermal lineage, and preliminarily explore the possibility of hiPSCs-derived cartilaginous pellets for articular cartilage regeneration. Methods: By simulating the development process in vivo, hiPSCs were gradually induced to differentiate into chondrogenic mesodermal lineage and mature cartilage using three dimensions (3D) suspension culture system. Real-time quantitative polymerase chain reaction(RT-qPCR), Western blotting, immunofluorescence and immunohistochemistry were used to investigate the dynamic expression change of pluripotent, mesodermal and chondrongenic related genes and proteins during the whole culture procedure. Finally, subcutaneous model of nude mice was used to investigate the stability of chondrogenic phenotypes and tumorigenicity of hiPSCs derived-cartilaginous pellets.Results: By simulating the development process in vivo, hiPSCs were gradually induced to differentiate into chondrogenic mesodermal lineage, and finally differentiated into mature cartilage tissues. Conclusion: In this study, a stable and efficient culture approach for inducing hiPSCs differentiation towards chondrogenic mesoderm was successfully established, which provide a research platform for investigation of hiPSCs-mediated cartilage development and regeneration.

Key words: human induced pluripotent stem cells, mesoderm, suspension culture, cartilaginous pellets

中图分类号:

R782

李阳阳, 张茂林, 邹多宏, 张志愿. 基于人诱导多能干细胞的组织工程化软骨再生[J]. 《口腔颌面外科杂志》, 2024, 34(1): 22-28.

LI Yangyang, ZHANG Maolin, ZOU Duohong, ZHANG Zhiyuan. Tissue-engineered cartilage regeneration based on human induced pluripotent stem cells[J]. 《Journal of Oral and Maxillofacial Surgery》, 2024, 34(1): 22-28.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2024/V34/I1/22

图/表 5

表1 RT-qPCR 引物序列

Table 1 Primers sequence for RT-qPCR

基因	正向引物(5'-3')	反向引物(5'-3')
ACTB	TGGCACCACACCTTCTACAATGAGC	GCACAGCTTCTCCTTAATGTCACGC
OCT3/4	GACAACAATGAGAACCTTCA	TTCTGGCGCCGGTTACAGAA
Nang	CAAAGGCAAACAACCCACTT	CTGGATGTTCTGGGTCTGGT
Brachyury	TATGAGCCTCGAATCCACATAGT	CCTCGTTCTGATAAGCAGTCAC
Sox9	AGCGAACGCACATCAAGAC	CTGTAGGCGATCTGTTGGGG
Col2a1	TTTCCCAGGTCAAGATGGTC	CTTCAGCACCTGTCTCACCA

图1 hiPSCs体外培养与多能性维持（×100） A、B. 多能性标志物Nanog、SSEA4细胞免疫荧光染色；C. 碱性磷酸酶染色；D. Western blotting检测多能性标志物Sox2、SSEA4及Nanog的表达；E. RT-qPCR检测多能性标志物Nanog、OCT3/4的表达。

Figure 1 In vitro culture and pluripotency maintenance of hiPSCs（×100）

图2 hiPSCs软骨中胚层方向诱导分化培养体系 A. hiPSCs软骨中胚层方向诱导分化培养体系示意图；B. hiPSCs克隆（×100）；C. 中胚层诱导第3天时的细胞形态（×40）；D—F. 软骨诱导不同阶段的细胞形态（×40）；G. 培养至49 d，hiPSCs来源的软骨球大体观。

Figure 2 Culture system of hiPSCs cartilage mesodermal induced differentiation

图3 标志物Nanog、Brachyury、Sox9及Col2a1的表达分析及hiPSCs来源的软骨球组织学分析 A. RT-qPCR检测标志物Nanog、Brachyury、Sox9及Col2a1的mRNA表达，**表示P<0.01,与另外2组比较；B. hiPSCs来源软骨球组织形态学分析，分别为苏木精-伊红（hematoxylin and eosin，HE）、番红O、阿利新蓝及Ⅱ型胶原染色（×40，×100）。

Figure 3 Expression analysis of markers Nanog, Brachyury, Sox9 and Col2a1, and histological analysis of hiPSCs-derived cartilaginous pellets

图4 hiPSCs来源的软骨球皮下移植及组织化学分析(×100) A. hiPSCs来源的软骨球（箭头所示）皮下移植4周；B. 体内移植软骨球组织形态学分析，分别为HE染色、番红O染色、阿利新蓝染色及Ⅱ型胶原免疫组织化学。

Figure 4 Subcutaneous transplantation of hiPSCs-derived cartilaginous pellets and histological analysis (×100)

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