牛脂肪间充质干细胞的分离培养及分化潜能的研究

doi:10.16431/j.cnki.1671-7236.2023.09.009

摘要/Abstract

摘要： 【目的】对荷斯坦牛脂肪来源的间充质干细胞(adipose derived mesenchymal stem cells,AD-MSCs)进行体外分离与培养,研究其生物学特性,为干细胞临床研究提供一种新型可用的种子细胞。【方法】无菌收集2月龄荷斯坦牛胚胎的腹股沟脂肪组织,使用Ⅰ型胶原酶消化收集细胞进行培养,使用荧光抗体技术和RT-PCR检测细胞膜表面特异性标记物(CD29、CD44、CD73、CD166、CD34、CD45),利用纯化后的细胞绘制P4、P10、P16代生长曲线并计算群体倍增时间,利用细胞克隆形成试验计算克隆形成率,染色体核型分析判断细胞遗传是否稳定,通过向AD-MSCs培养皿中添加诱导液,验证其向成脂、成骨、成软骨的分化潜能。【结果】体外分离培养的细胞呈贴壁漩涡状生长,细胞形态为长梭形。荧光抗体技术和RT-PCR检测结果表明,CD29、CD44、CD73和CD166细胞膜表面抗原被表达,CD34和CD45不被表达,确定分离的细胞为AD-MSCs。绘制的细胞生长曲线为S型,P4、P10、P16代细胞群体倍增时间分别为33、35、44 h,克隆形成率分别为(51.67±1.53)%、(38.00±2.00)%、(25.00±1.00)%,2个指标在3个代次间差异均显著(P＜0.01;P＜0.05)。核型结果表明,AD-MSCs为正常二倍体(2n=60,XX),染色体形态均为端着丝粒,无畸变。诱导分化结果显示,在体外AD-MSCs具有成脂、成骨、成软骨的分化潜能,分别出现明显的脂滴、钙结节和软骨团,且分别表达成脂诱导基因脂蛋白脂肪酶(LPL)和过氧化物酶体增殖物激活受体(PPAR-γ)、成骨诱导基因Ⅰ型胶原蛋白(COL Ⅰ)和骨桥蛋白(OPN)、成软骨诱导基因SRY样HMG盒9(SOX-9)和软骨蛋白聚糖抗体(ACAN)。【结论】用荷斯坦牛胚胎能成功在体外分离原代AD-MSCs,细胞形态具有典型的间充质干细胞形态,具有成脂、成骨、成软骨细胞的分化潜能,且细胞具有增殖能力快、稳定等特点,可以为组织和器官修复提供种质资源。

关键词: 脂肪间充质干细胞; 分离培养; 诱导分化; 荷斯坦牛

Abstract: 【Objective】 To isolate and culture adipose derived mesenchymal stem cells (AD-MSCs) from Holstein cattle in vitro, study their biological properties, and provide a new type of usable seed cells for stem cell clinical research.【Method】 The inguinal adipose tissue of 2-month-old Holstein bovine embryos was collected aseptically,and the cells were digested with type Ⅰ collagenase and cultured.Fluorescent antibody technology and RT-PCR were used to detect the specific markers (CD29,CD44,CD73,CD166,CD34 and CD45) on the cell membrane surface.The growth curves of P4,P10 and P16 generations were calculated using the purified cells and calculate population doubling time.The clone formation rate was calculated using the clone formation assay,the karyotype analysis was performed to determine whether the cells were genetically stable,and the differentiation potential to lipogenesis,osteogenesis and chondrogenesis were verified by adding induction solution to AD-MSCs culture dishes.【Result】 Cells isolated and cultured in vitro grew in an appressed swirling pattern with a long shuttle-shaped cell morphology.The results of fluorescent antibody technique and RT-PCR showed that CD29,CD44,CD73 and CD166 cell membrane surface antigens were expressed,CD34 and CD45 were not expressed,and the isolated cells were identified as AD-MSCs.The cell growth curve was plotted in S-shape,and the cell population multiplication time was 33,35 and 44 h for P4,P10 and P16 generations,respectively,and the clone formation rates were (51.67±1.53)%,(38.00±2.00)%,and (25.00±1.00)%,respectively.The two indicators showed significant differences between the three generations (P＜0.01 or P＜0.05).The karyotype results showed that AD-MSCs were normal diploids (2n=60,XX),and all chromosome morphologies were telomeric with no aberrations.Induced differentiation results showed that in vitro AD-MSCs had lipogenic,osteogenic and chondrogenic differentiation potential,with distinct lipid droplets,calcium nodules and cartilage clusters,respectively,and expressed lipogenic inducible genes lipoprotein lipase (LPL) and peroxisome proliferator-activated receptor (PPAR-γ),osteogenic inducible genes type Ⅰ collagen (COL Ⅰ) and osteochondrogenic bridge protein (OPN),osteogenic chondrogenic gene SRY-like HMG cassette 9 (SOX-9) and chondroprotein glycan antibody (ACAN).【Conclusion】 Primary AD-MSCs could be successfully isolated in vitro using Holstein bovine embryos,and the cell morphology had typical mesenchymal stem cell morphology,with differentiation potential of lipogenic, osteogenic and chondrocytes,and the cells had fast proliferation ability and stability,which could provide germplasm resources for tissue and organ repair.

Key words: adipose mesenchymal stem cells; isolation and culture; induction of differentiation; Holstein cattle

中图分类号:

S823

王亚芹, 宗俊霖, 张涛, 关伟军. 牛脂肪间充质干细胞的分离培养及分化潜能的研究[J]. 中国畜牧兽医, 2023, 50(9): 3530-3540.

WANG Yaqin, ZONG Junlin, ZHANG Tao, GUAN Weijun. Isolation,Culture and Differentiation Potential of Bovine Adipose Mesenchymal Stem Cells[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(9): 3530-3540.

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