盘羊脐带间充质干细胞的分离培养及诱导分化

doi:10.16431/j.cnki.1671-7236.2021.05.009

摘要/Abstract

摘要： 试验旨在研究野生盘羊脐带间充质干细胞（umbilical cord mesenchymal stem cells，UC-MSCs）的分离、培养及体外多向分化潜能等生物学特性。取盘羊分娩后的新生雄性胎儿脐带，采用组织块培养法分离盘羊UC-MSCs后进行体外培养，并对UC-MSCs进行成骨、成软骨和成脂诱导分化，检测其多向分化潜能。结果显示，应用组织块培养法获得的盘羊UC-MSCs具有UC-MSCs特有的呈簇、成纤维状的特性，细胞呈"S"型曲线生长，细胞的倍增时间平均为33.50 h，且可以向成骨细胞、成软骨细胞和成脂细胞分化。诱导分化特异性染色结果表明，野生盘羊UC-MSCs经诱导后的成骨细胞经茜素红S染色呈现典型的橙红色钙沉积物聚集，成软骨细胞经阿利新蓝染色呈现蓝色的软骨基质，成脂细胞经油红O染色呈现深红色的脂滴。实时荧光定量PCR检测结果发现，随着诱导时间的延长，成骨分化的细胞中骨内γ-羧基谷氨酸蛋白（BGLAP）、骨桥蛋白基因（OPN）、Runt相关转录因子2（RUNX2）基因的相对表达量极显著升高（P<0.01）；成软骨分化的细胞中光蛋白聚糖（LUM）基因的表达量明显增加，而双糖链蛋白多糖（BGN）和Y染色体性别决定区基因盒9（SOX9）基因表达量极显著提高（P<0.01）；成脂分化的细胞中脂蛋白酶（LPL）和过氧化物酶体增殖因子活化受体γ（PPAR-γ）基因的相对表达量极显著上升（P<0.01）。试验结果表明，用盘羊胎盘附带的脐带组织可以分离到盘羊UC-MSCs，且分离到的UC-MSCs具有分化为成骨细胞、成软骨细胞及成脂细胞等多向分化潜能。

关键词: 盘羊; 脐带间充质干细胞; 分离培养; 体外诱导分化

Abstract: This experiment was aimed to study the isolation,culture,multi-lineage differentiation potential of umbilical cord mesenchymal stem cells (UC-MSCs) from argali in vitro.UC-MSCs were isolated from umbilical cord of newborn male fetus after delivery in argali by tissue block culture and cultured in vitro.UC-MSCs were induced to differentiate into osteoblasts,chondroblasts and adipoblasts to detect their multi-lineage differentiation potential.The results showed that the UC-MSCs of argali had the unique biological characteristics of being cluster and fibrous by tissue block culture,and the growth curve was "S" shape.The average doubling time of the cells was 33.50 h,and it could differentiate into osteoblasts,chondroblasts and adipoblasts.The osteoblasts were stained with Alizarin red S showing typical orange-red calcium deposits,chondroblasts were stained with Alcian blue showing a blue cartilage matrix,and adipoblasts were stained with oil red O showing dark red lipid droplets.Real-time quantitative PCR detection results revealed that with the extension of inducting time,the relative expression of bone gamma-carboxyglutamate protein (BGLAP),osteopontin (OPN) and Runt-related transcription factor 2 (RUNX2) genes in the osteogenic differentiation cells increased extremely significantly (P<0.01).The expression of lumican (LUM) gene in the chondrogenic differentiation cells increased significantly,while the expression of biglycan (BGN) and sex determining region Y-box 9 (SOX9) genes increased extremely significantly (P<0.01).The relative expression of lipoprotein lipase(LPL) and peroxisome proliferator-activated receptor γ(PPAR-γ) genes in the adipogenic differentiation cells increased extremely significantly (P<0.01).The results indicated that UC-MSCs could be isolated from umbilical cord tissue attached to placenta and differentiated into osteoblasts,chondroblasts and adipoblasts.

Key words: argali; umbilical cord mesenchymal stem cells; isolation and culture; in vitro differentiation induction

中图分类号:

Q254

周桂珍, 周正娜, 唐小云, 毕佳颖, 王旭光, 李娟. 盘羊脐带间充质干细胞的分离培养及诱导分化[J]. 中国畜牧兽医, 2021, 48(5): 1584-1592.

ZHOU Guizhen, ZHOU Zhengna, TANG Xiaoyun, BI Jiaying, WANG Xuguang, LI Juan. Isolation,Culture and Induced Differentiation of Umbilical Cord Mesenchymal Stem Cells from Argali[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(5): 1584-1592.

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