荷斯坦牛脐带间充质干细胞分离鉴定及分化研究

doi:10.13304/j.nykjdb.2023.0401

摘要/Abstract

摘要：

动物干细胞作为重要遗传种质资源和再生医学的可行性资源，拥有广阔研究和应用前景。对荷斯坦牛脐带细胞（umbilical cord mesenchymal stem cells，UCMSCs）进行分离、鉴定，并对其多向分化潜能等生物学特性进行研究。取3月龄健康荷斯坦牛胎牛脐带，采用Ⅳ型胶原酶分离细胞进行体外培养，获得的细胞贴壁后呈长梭形，生长曲线呈典型的“Ｓ”形，群体倍增时间随着代次的升高逐渐下降。免疫荧光和RT-PCR的检测结果都表明，分离的细胞中CD29、CD73、CD90、CD166均阳性表达，而CD45不表达。成脂肪诱导后，可见被油红染色液染色的脂滴，成脂肪关键基因LPL和PPAR-γ都阳性表达；成骨诱导分化后，可见被茜素红染色的钙结节，其表达成骨关键基因骨桥蛋白OPN和Ⅰ型胶原蛋白基因COL-Ⅰ；成软骨诱导分化后，可见被阿利新蓝染色的软骨组织，表达成软骨细胞关键基因转录因子基因SOX9和ACAN。成功从3月胚龄的荷斯坦牛脐带中分离出其具有良好的增殖活力及成脂肪、骨和成软骨能力的间充质干细胞，可为动物种质资源保存和再生医学提供依据和潜在可利用细胞资源。

关键词: 荷斯坦牛, 干细胞, 体外培养, 诱导分化

Abstract:

As important genetic germplasm resources and new potential cells for regenerative medicine， animal stem cells are broadly researched and utilized. In this study， Holstein bovine umbilical cord mesenchymal stem cells （UCMSCs） were isolated and investigated， and their multi-directional differentiation potential etc. biological characteristics were researched.The cells were isolated from 3-month-old fetal umbilical cords of Holstein cows using type Ⅳ collagenase， cultured in vitro. The obtained cells showed a typical “S” shape growth curve， and the population doubling time gradually decreased with the increase of generation.The expression of specific surface marker genes in isolated cells were identified by immunofluorescence and RT-PCR techniques，the results showed that CD29， CD73， CD90 and CD166 expressed， while CD45 didn’t express. After induction of adipogenesis， lipid droplets stained with oil red stain were seen， and adipogenic genes LPL and PPAR-γ both expressed. After induction of osteogenic differentiation， calcium nodules were stained with alizarin red， and osteogenic genes OPN and collagen type Ⅰ gene COL-?Ⅰ expressed； after induction of chondrogenic differentiation， the chondrocytes were stained with Alisin blue， and they expressed chondrogenic key gene transcription factor gene SOX9 and ACAN. In this study， mesenchymal stem cells with good proliferation viability and adipogenic， osteogenic and chondrogenic abilities were successfully isolated from the umbilical cord of Holstein cattle at 3-month-old embryonic， which provided basis for the conservation of animal germplasm resources and regenerative medicine as well as potential cell resources.

Key words: Holstein cattle, stem cells, in vitro culture, induced differentiation

中图分类号:

S823

宗俊霖, 王亚芹, 刘彦辰, 许龙, 关伟军. 荷斯坦牛脐带间充质干细胞分离鉴定及分化研究[J]. 中国农业科技导报, 2023, 25(9): 105-112.

Junlin ZONG, Yaqin WANG, Yanchen LIU, Long XU, Weijun GUAN. Identification and Differentiation of Holstein Cattle Umbilical Cord Mesenchymal Stem Cells[J]. Journal of Agricultural Science and Technology, 2023, 25(9): 105-112.

图/表 9

表1 引物信息

Table 1 Primers information

基因Gene	ID	引物序列Primer sequence（５’-３’）	产物长度Size/bp
GAPDH	NM_001034034.2	F：CGGCACAGTCAAGGCAGAGAAC R：GATGGCGTGGACAGTGGTCATAAG	473
CD29	NM_174368.3	F：GAAACTTGGTGGCATCGT R：CTCAGTGAAGCCCAGAGG	593
CD73	NM_174013.3	F：CAATGGCACGATTACCTG R：GATCTTCAACTGCTGGATA	479
CD90	NP_001029937.1	F：TCGCCCTGCTAACAGTCTTAC R：CCTCATCCTTGGTGGTGAAGTTGG	372
CD166	NM_001146392.1	F：TATCAGGATGCTGGAAAC R：TAGCCAATAGACGACACC	498
CD45	NM_001206523.1	F：CTACCCAACCTTCTACTCAA R：TTCACATCCAGGAGGTTC	221
LPL	NM_001206523.1	F：GGAGTGACCGAATCTGTGGCTAAC R：CCAAGGCTGTATCCCAAGAGATGC	308
PPAR-γ	NM_181024.2	F：GACGGGAAAGACGACAGACA R：GGTTCACAAAGCCGGGGATA	215
COL-I	NM_001034039.2	F：GGAATCACCCACGGACCAAGAAAC R：GGAGACCACGAGGACCAGAAGG	231
OPN	NM_174187.2	F：CATTCCGACGAGTCTCACCATTCTG R：AAGTCCTCCTCTGTGGCATCTGG	296
ACAN	NM_173981.2	F：GGAAGTCCCCTTCGATGGTC R：CAACCACAACAGACCAGAAGC	332
SOX9	XM_024981096.1	F：CGGAACAGACGCACATCTCG R：GACGTCGAAGGTCTCCATGTT	386

图1 荷斯坦牛脐带UCMSCs形态

Fig. 1 Cell morphology of Holstein cattle UCMSCs

图2 荷斯坦牛UCMSCs生长曲线和群体倍增时间

Fig. 2 Growth curves and population doubling time of Holstein cattle UCMSCs.

图3 荷斯坦牛UCMSCs克隆团形态

Fig. 3 Clone group morphology of Helstein cattle UCMSCs

图4 免疫荧光检测荷斯坦牛UCMSCs表面标志物

Fig. 4 UCMSCs markers detection by immunofluorescence staining in Holstein cattle

图5 RT-PCR检测标记基因的表达

Fig. 5 Marker genes expression by RT-PCR

图6 荷斯坦牛UCMSCs分化成骨染色和RT-PCR结果

Fig. 6 Osteoblast differentiation of Helstein UCMSCs and RT-PCR result

图7 荷斯坦牛UCMSCs分化成脂染色和RT-PCR结果

Fig. 7 Adipogentic differentiation of Helstein UCMSCs and RT-PCR result

图8 荷斯坦牛UCMSCs分化成软骨染色和RT-PCR结果

Fig. 8 Chondrocyte differentiation of Helstein UCMSCs and RT-PCR result

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