马脐带不同部位间充质干细胞的分离培养及成软骨诱导分化

doi:10.16431/j.cnki.1671-7236.2022.01.008

摘要/Abstract

摘要： [目的] 通过对比马雌性胎儿同一脐带3个不同部位分离的脐带间充质干细胞（umbilical cord mesenchymal stem cells，UC-MSCs）的增殖率、表面标记分子、多潜能性基因表达和软骨分化潜能的差异性，明确更适合作为分离培养间充质干细胞（mesenchymal stem cells，MSCs）的马脐带部位。[方法] 采用组织块分离法分离脐带靠近胎儿的部分（近端）、整段脐带的中心（中段）和靠近胎盘部分（远端）3个部分的MSCs，通过绘制细胞生长曲线对比UC-MSCs的增殖能力，通过实时荧光定量PCR检测多潜能性基因和表面标记分子的表达，经软骨诱导检测成软骨分化的潜能。[结果] 近端、中段和远端分离得到的MSCs均贴壁生长，呈成纤维状；细胞倍增时间分别为35.4、25.5和34.9 h，且都表达多潜能性基因NANOG（NANOG homeobox）、八聚体结合转录因子（POU class 5 homeobox 1，POU5F1）和Y染色体性别决定区基因盒2（SRY-box transcription factor 2，SOX2），高表达5'-核苷酸外切酶（5'-nucleotidase ecto，CD73）、Thy-1细胞表面抗原（Thy-1 cell surface antigen，CD90）、内皮糖蛋白（endoglin，CD105）和造血祖细胞抗原（CD34 molecule，CD34），低表达单核细胞分化抗原CD14（CD14 molecule，CD14）、B-淋巴细胞表面抗原B4（CD19 molecule，CD19）、蛋白酪氨酸磷酸酶受体C型（protein tyrosine phosphatase receptor type C，CD45）和B细胞抗原受体复合物相关蛋白α链（CD79a molecule，CD79a）。多潜能性基因NANOG相对表达量在P1代中段细胞显著高于近端细胞（P<0.05），POU5F1相对表达量在P5代近端细胞显著高于中段细胞（P<0.05），且极显著高于远端细胞（P<0.01）。CD14、CD19、CD73和CD90的相对表达量在P5代近端细胞均显著或极显著高于中段和远端细胞（P<0.05；P<0.01）；CD34、CD79a的相对表达量在P5代远端细胞均显著或极显著高于近端和中段细胞（P<0.05；P<0.01）。近端、中段和远端分离得到的UC-MSCs均可诱导分化为软骨细胞，在诱导分化第14天，近端细胞SOX9的相对表达量极显著高于远端细胞（P<0.01），诱导21 d时远端细胞SOX9的相对表达量极显著高于中段细胞（P<0.01）；在诱导第7和14天，中段细胞蛋白聚糖（aggrecan，ACAN）和Ⅱ型胶原蛋白1链（collagen type Ⅱ alpha 1 chain，COL2A1）相对表达量均极显著高于近端和远端细胞（P<0.01），第21天时显著高于近端和远端细胞（P<0.05）。[结论] 马脐带的近端、中段和远端均可用于制备MSCs，都表达多潜能性基因和MSCs表面标记分子，且均可诱导分化为软骨细胞，中段MSCs增殖能力和软骨分化潜能优于近端和远端，更适合用于分离MSCs。

关键词: 马; 脐带间充质干细胞(UC-MSCs); 多潜能性; 表面标记分子; 成软骨分化

Abstract: [Objective] By comparing the differences of proliferation rate, surface marker molecules, pluripotent gene expression and cartilage differentiation potential of umbilical cord mesenchymal stem cells (UC-MSCs) isolated from three different parts of the same umbilical cord of female equine fetus, the umbilical cord site which was suitable for isolation and culture of mesenchymal stem cells (MSCs) was identified. [Method] MSCs were isolated from the umbilical cord near the fetus (proximal end), the center of the umbilical cord (middle segment) and the placenta (distal end), and the proliferation ability of UC-MSCs was compared by plotting cell growth curve, the expression of multipotent genes and surface marker molecules were detected by Real-time quantitative PCR, and the potential of cartilage differentiation was detected by cartilage induction. [Result] The MSCs isolated from the proximal end, middle segment, and distal end attached to the wall and grew in a fibrous shape and the doubling time were 35.4, 25.5 and 34.9 h, respectively, and all of them expressed NANOG homeobox (NANOG), POU class 5 homebox 1 (POU5F1) and SRY-box transcription factor 2 (SOX2), overexpression of 5'-nucleotidase ecto (CD73), Thy-1 cell surface antigen (CD90), endoglin (CD105) and hematopoietic progenitor cell antigen (CD34), low expression of CD14 molecule (CD14), B-lymphocyte surface antigen B4 (CD19), protein tyrosine phosphatase receptor type C (CD45) and B cell antigen receptor complex associated protein α chain (CD79a). The relative expression of pluripotent gene NANOG in middle segment P1 cells was significantly higher than that in proximal end P1 cells (P<0.05), the relative expression of POU5F1 in P5 proximal end cells was significantly higher than that in middle segment cells (P<0.05), and it was extemely significantly higher than that of distal end cells (P<0.01). The relative expressions of CD14, CD19, CD73 and CD90 in P5 proximal end cells were significantly or extemely significantly higher than those in middle segment and distal end cells (P<0.05; P<0.01), the relative expressions of CD34 and CD79a in distal end cells of P5 generation was significantly or very significantly higher than that in proximal end and middle segment cells (P<0.05; P<0.01). The UC-MSCs derived from the proximal end, middle segment and distal end could be induced to differentiate into chondrocytes. On the 14th day of induced differentiation, the relative expression of SOX9 in proximal cells was extremely significantly higher than that in distal cells (P<0.05), and on the 21st day of induction, the relative expression of SOX9 in distal cells was extremely significantly higher than that in middle cells (P<0.01). The relative expressions of specific gene aggrecan (ACAN) and collagen type Ⅱ alpha 1 chain (COL2A1) in middle segment cells were extremely significantly higher than those in proximal and distal cells on the 7th and 14th day of induction (P<0.01), and significantly higher than those in proximal and distal cells on the 21st day (P<0.05). [Conclusion] The proximal end, middle segment and distal end of umbilical cord could be used to prepare MSCs, which could express pluripotent genes and MSCs surface marker molecules, and could be induced to differentiate into chondrocytes. The proliferation ability and cartilage differentiation potential of MSCs from the middle segment were better than those from the proximal and distal ends, so they were more suitable for isolating MSCs.

Key words: equine; umbilical cord mesenchymal stem cells (UC-MSCs); pluripotency; surface marker molecule; chondrogenic differentiation

中图分类号:

Q254

唐小云, 周桂珍, 周正娜, 邓雅迪, 张慧, 张欣如, 王旭光. 马脐带不同部位间充质干细胞的分离培养及成软骨诱导分化[J]. 中国畜牧兽医, 2022, 49(1): 70-80.

TANG Xiaoyun, ZHOU Guizhen, ZHOU Zhengna, DENG Yadi, ZHANG Hui, ZHANG Xinru, WANG Xuguang. Preparation and Chondrogenic Differentiation of Equine Umbilical Cord Mesenchymal Stem Cells from Different Parts of Umbilical Cord[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 70-80.

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