西门塔尔牛胰腺间充质干细胞原代培养及其多向分化潜能研究

doi:10.11843/j.issn.0366-6964.2022.11.007

Abstract

Abstract: The purpose of the experiment was to primary culture and study the in vitro differentiation potential of Simmental bovine pancreatic mesenchymal stem cells (PMSCs), and to provide new seed cells for cell therapy and tissue engineering. Pancreatic tissue was aseptically isolated from 3-month-old Simmental cattle embryos, and PMSCs were isolated by collagenase digestion and tissue block adherence, respectively, and then primary cultured. The growth curves of 3rd, 9th and 15th passage cells were drawn and the population doubling time and clone formation ability were determined. Stem cell surface markers (CD29, CD44, CD73, CD90, CD34 and CD45) were detected by immunofluorescence, and stem cell surface markers (CD29, CD44, CD73, CD90, CD106, CD166, CD34 and CD45) were detected by RT-PCR. Its gene stability was detected by karyotype analysis, the multi-directional differentiation potential was detected by inducing adipogenic, osteogenic, chondrogenic and hepatocyte-like cells. The results showed that PMSCs could be successfully isolated by both methods. After the cells adhered, they were all long spindle-shaped, swirling growth, and the growth trend was typical S-shaped. The doubling time of the cell population in the 9th passage was significantly lower than that in the 15th passage and higher than that in the 3rd passage (P<0.05); The clone formation rate of the 9th passage of PMSCs was significantly lower than that of the 3rd passage and significantly higher than that of the 15th passage (P<0.05); Immunofluorescence results showed that CD29, CD44, CD73 and CD90 specifically expressed in PMSCs, and RT-PCR results showed that CD29, CD44, CD73, CD90, CD106 and CD166 specifically expressed in PMSCs, but the hematopoietic cell surface markers CD34 and CD45 did not express, which were consistent with international corresponding to the surface markers of MSCs designated by the Tissue Stem Cell Committee of the Society for Cell Therapy; Karyotype analysis showed that PMSCs were normal diploid (2n=60, XY), and there was no mutation in the chromosomal genome; Specific staining and RT-PCR results showed that PMSCs obtained from Simmental cattle could differentiate into adipocytes, osteocytes, chondrocytes and hepatocyte-like cells. This experiment confirmed that both methods could successfully establish the in vitro separation and culture system of Simmental cattle PMSCs. PMSCs showed good proliferation activity, similar biological properties and multiple differentiation potentials with MSCs. The results could provide new seed cells for tissue engineering.

Key words: pancreatic mesenchymal stem cells, Simmental cattle, primary culture, biological characteristics, differentiation potential

CLC Number:

S823.1

WANG Yunan, WU Yue, SONG Hanan, ZHANG Tao, GUAN Weijun. Primary Culture of Simmental Bovine Pancreatic Mesenchymal Stem Cells and Their Multi-directional Differentiation Potential[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 3786-3796.

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Primary Culture of Simmental Bovine Pancreatic Mesenchymal Stem Cells and Their Multi-directional Differentiation Potential

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