荷斯坦牛肺干细胞分离培养与生物学特性研究

doi:10.11843/j.issn.0366-6964.2024.02.012

摘要/Abstract

摘要： 本研究利用荷斯坦牛肺间质干细胞(LR-MSCs),通过体外试验观察LR-MSCs在不同条件下的成骨分化能力,以期为临床应用及组织工程提供新的种子细胞。本项目以荷斯坦牛(健康荷斯坦牛胎牛,0日龄,1只,雄性)为对象,通过无菌方法获得肺组织,通过组织块贴壁方法获得LR-MSCs,并将其原代培养。通过绘制3代、9代、15代细胞的生长曲线,测定其增殖时间及成体能力。利用RT-PCR技术对干细胞表面标志物(CD29、CD44、CD73、CD90、CD106、CD166、CD34和CD45)的表达进行检测。对该基因进行染色体核型分析,并对其进行遗传转化,观察该基因的多向性。结果显示,这种方法能获得LR-MSCs,它们在体外培养时表现为长梭形和螺旋形,并呈现典型的"S"曲线。第9世代的倍增时间比15世代的短,但比3世代的长(P<0.01);第9代LR-MSCs的成株率较3代明显降低,而15代较高,差异有统计学意义(P<0.05)。应用RT-PCR技术检测到在LR-MSCs中特异表达的CD29、CD44、CD73、CD90。前期研究发现,LR-MSCs可特异性地表达CD29、CD44、CD73、CD90、CD106、CD166,而CD34、CD45则不表达。对LR-MSCs进行染色体组测序,发现其为正常二倍体(2n=60,XY)。通过对荷斯坦牛LR-MSCs的免疫荧光染色及RT-PCR分析,证明LR-MSCs具有向脂肪、骨、软骨等多种细胞定向分化的能力。这可能为今后的组织工程研究提供了一种新的种子细胞。

关键词: 肺间充质干细胞组织, 原代培养, 诱导分化, 检测, 多向潜在能力研究

Abstract: This study utilized Holstein bovine lung resident mesenchymal stem cells (LR-MSCs) and observed their osteogenic differentiation ability in vitro under different conditions, with the hope of providing new seed cells for clinical application and tissue engineering. The Holstein calf (healthy Holstein fetal calf, 0 days old, male) was used to obtain lung tissue via sterile methods and to isolate LR-MSCs through techniques like tissue adherence. The cells were primarily cultured. The growth curves were drew, proliferation timings, and potency for the 3rd, 9th, and 15th generations were measured. The expression of stem cell surface markers (CD29, CD44, CD73, CD90, CD106, CD166, CD34 and CD45) was detected using RT-PCR. Chromosomal karyotype analysis was performed on these genes, and their pluripotency was observed after genetic transformation. The results indicate that LR-MSCs could be obtained through both techniques, exhibiting spindle and spiral shapes in vitro and showing a classic "S" growth curve. The 9th generation had a shorter doubling time than the 15th generation, but longer than the 3rd (P<0.01); colony-forming rates for the 9th generation LR-MSCs were significantly decreased compared to the 3rd generation, but higher for the 15th generation, showing statistical significance (P<0.05). RT-PCR revealed the specific expression of CD29, CD44, CD73, CD90 in LR-MSCs. The preliminary studies found that LR-MSCs could specifically express CD29, CD44, CD73, CD90, CD106, CD166, but not CD34, CD45. Chromosome sequencing showed LR-MSCs to be of a normal diploid karyotype (2n=60, XY). Immunofluorescence staining and RT-PCR analysis proved the multilineage differentiation potential of Holstein bovine LR-MSCs into adipocytes, osteocytes, and chondrocytes, which may offer a new seed cell type for upcoming tissue engineering research.

Key words: lung resident mesenchymal stem cells, primary culture, induced differentiation, assay, multidirectional potential study

中图分类号:

S823.1

刘晏辰, 周世莹, 张洋, 高扬, 关伟军. 荷斯坦牛肺干细胞分离培养与生物学特性研究[J]. 畜牧兽医学报, 2024, 55(2): 540-551.

LIU Yanchen, ZHOU Shiying, ZHANG Yang, GAO Yang, GUAN Weijun. Isolation, Culture and Biological Characteristics Study of Holstein Bovine Lung Stem Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 540-551.

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