西门塔尔牛胰腺间充质干细胞分离培养及生物学特性研究

doi:10.16431/j.cnki.1671-7236.2025.06.006

摘要/Abstract

摘要： 【目的】分离培养西门塔尔牛胰腺间充质干细胞，建立西门塔尔牛胰腺间充质干细胞系，研究其生物学特性，为西门塔尔牛种质资源的保存提供参考。【方法】采集西门塔尔牛胰腺组织，利用酶消法和组织块贴壁法分离培养胰腺间充质干细胞，并对胰腺间充质干细胞的生物学特性进行鉴定；利用免疫荧光试验和半定量PCR测定细胞表面标记物(CD34、CD73、CD90、CD166)的表达情况；通过绘制P4、P10、P16代细胞生长曲线及计算群体倍增时间测定细胞增殖能力；通过细胞克隆试验测定细胞克隆形成率，通过划痕试验计算细胞迁移率；对纯化后的细胞分别进行成骨细胞、脂肪细胞、软骨细胞诱导分化，检测胰腺间充质干细胞多向分化能力，并通过染色体核型分析试验检测细胞的遗传稳定性。【结果】采用组织块贴壁法培养可得到胰腺间充质干细胞，且传代到第3代基本纯化。半定量PCR结果显示，表面标记物(CD73、CD90、CD166)呈阳性表达，而与造血细胞相关的表面抗原CD34呈阴性表达。西门塔尔牛胰腺间充质干细胞呈现典型的S型生长曲线，P4、P10、P16代细胞群体倍增时间分别为40.22、43.09和44.50 h，细胞克隆形成率分别为26.25%、19.00%和13.50%。划痕试验测定第3代细胞迁移率为44.44%。在体外诱导分化胰腺间充质干细胞后，结果呈现出清晰的脂滴、软骨团和钙结节。核型分析结果判定，西门塔尔牛胰腺间充质干细胞为正常二倍体核型(2n=60，XY)，染色体组未发生变异。【结论】从西门塔尔牛胰腺组织分离培养的胰腺间充质干细胞能够稳定遗传，增殖能力强，且具有向成骨细胞、脂肪细胞、软骨细胞分化的潜能。研究结果可为西门塔尔牛组织修复提供新方法，为西门塔尔牛种质资源的保护和保存提供种子细胞。

关键词: 西门塔尔牛; 胰腺间充质干细胞; 分离培养; 诱导分化

Abstract: 【Objective】 The purpose of this study was to isolate and culture pancreatic mesenchymal stem cells (PMSCs) in Simmental cattle,establish PMSCs line in Simmental cattle,study its biological characteristics,and provide reference for the preservation of germplasm resources in Simmental cattle.【Method】 PMSCs were isolated and cultured from pancreas tissue in Simmental cattle by enzyme digestion and tissue block adhesion method,and the biological characteristics of PMSCs were identified.The expressions of cell surface markers (CD34,CD73,CD90 and CD166) were measured by immunofluorescence and semi-quantitative PCR.The cell proliferation ability was determined by drawing cell growth curves of P4,P10 and P16 generations and calculating population doubling time.The cell cloning rate was determined by cell cloning test,and the cell migration rate was calculated through scratch test.The purified cells were induced into osteoblasts,adipocytes and chondrocytes respectively to detect the multipotent differentiation ability of PMSCs,and the cell genetic stability was detected by chromosome karyotype analysis test.【Result】 PMSCs could be cultured using tissue block adhesion method and passaged to the third generation for basic purification.Semi-quantitative PCR results showed that the surface markers (CD73,CD90 and CD166) were positive,but CD34,a surface antigen related to hematopoietic cells,was negative.The growth curve of PMSCs in Simmental cattle showed a typical S curve,the doubling time of P4,P10 and P16 generation cells were 40.22,43.09 and 44.50 h,and the colony formation rate were 26.25%,19.00% and 13.50%,respectively.The migration rate of the third passage cells was 44.44% by scratch test.The clear lipid droplets,cartilage masses and calcium nodules were observed after differentiation of PMSCs in vitro.Karyotype analysis results showed that PMSCs in Simmental cattle were normal diploid karyotype (2n=60,XY),and the chromosome set did not change.【Conclusion】 PMSCs isolated and cultured from pancreas tissue in Simmental cattle could stably inherit and strongly proliferate,and had the potential to differentiate into osteoblasts,adipocytes and chondrocytes.The results could provide new method for tissue repair and seed cells for the protection and preservation of germplasm resources in Simmental cattle.

Key words: Simmental cattle; pancreatic mesenchymal stem cells; isolation and culture; induced differentiation

中图分类号:

S852.2

王仲发, 刘晏辰, 闫炎, 李旻娟, 何玉楠, 关伟军, 刘文忠. 西门塔尔牛胰腺间充质干细胞分离培养及生物学特性研究[J]. 中国畜牧兽医, 2025, 52(6): 2519-2530.

WANG Zhongfa, LIU Yanchen, YAN Yan, LI Minjuan, HE Yunan, GUAN Weijun, LIU Wenzhong. Study on Isolation Culture and Biological Characteristics of Pancreatic Mesenchymal Stem Cells in Simmental Cattle[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(6): 2519-2530.

参考文献

[1] YUN F,ZHAORIGEN B,HAN X,et al.Islet like cells induced from umbilical cord mesenchymal stem cells with neonatal bovine pancreatic mesenchymal exosomes for treatment of diabetes mellitus[J].Hormone and Metabolic Research,2024,56(6):463-470.
[2] BEREBICHEZ-FRIDMAN R,MONTERO-OLVERA P R.Sources and clinical applications of mesenchymal stem cells:State-of-the-art review[J].Sultan Qaboos University Medical Journal,2018,18(3):264.
[3] BECKER A D,RIET I V.Homing and migration of mesenchymal stromal cells:How to improve the efficacy of cell therapy?[J]. World Journal of Stem Cells,2016,8(3):73.
[4] SHI X,MAO J,LIU Y.Pulp stem cells derived from human permanent and deciduous teeth:Biological characteristics and therapeutic applications[J].Stem Cells Translational Medicine, 2020,9(4):445-464.
[5] GU N,JEONG S Y,LEE S,et al.Comparison of characteristics of mesenchymal stem cells from bovine[J].Journal of the Preventive Veterinary Medicine,2021,45(2):63-69.
[6] MALDONADO V V,POKHAREL S,POWELL J G,et al.Phenotypic and functional characterization of bovine adipose-derived mesenchymal stromal cells[J].Research Square, 2024,14(9):1292.
[7] BUYL K,DE K J,NAJAR M,et al.Characterization of hepatic markers in human Wharton’s jelly-derived mesenchymal stem cells[J].Toxicology in Vitro, 2014,28(1):113-119.
[8] LEE J,BYEON J S,GU N Y,et al.Bovine tongue epithelium-derived cells:A new source of bovine mesenchymal stem cells[J]. Bioscience Reports, 2020,40(4):BSR20181829.
[9] MORAES C N D,MAIA L,DIAS M C,et al.Bovine endometrial cells:A source of mesenchymal stem/progenitor cells[J]. Cell Biology International,2016,40(12):1332-1339.
[10] FERRONI L,GARDIN C,BELLIN G,et al.Bovine pericardium membrane as new tool for mesenchymal stem cells commitment[J].Journal of Tissue Engineering and Regenerative Medicine,2019,13(10):1805-1814.
[11] ROSSI B,MERLO B,COLLEONI S,et al.Isolation and in vitro characterization of bovine amniotic fluid derived stem cells at different trimesters of pregnancy[J]. Stem Cell Reviews and Reports,2014,10(5):712-724.
[12] BOZSO S J,KANG J J H,EL-ANDARI R,et al.Recellularized bovine pericardium with autologous mesenchymal stem cells reduces immune activation[J].Xenotransplantation,2022,29(6):e12774.
[13] NOLTA J A.Mechanisms of modulation and differentiation in mesenchymal stem/stromal cells[J].Stem Cells,2021,39(1):1-2.
[14] SHI Y,WANG Y,LI Q,et al.Immunoregulatory mechanisms of mesenchymal stem and stromal cells in inflammatory diseases[J]. Nature Reviews Nephrology,2018,14(8):493-507.
[15] CHEN W,LV L,CHEN N,et al.Immunogenicity of mesenchymal stromal/stem cells[J].Scandinavian Journal of Immunology,2023,97(6):e13267.
[16] DREGALLA R C,HERRERA J A,DONNER E J.Red blood cells and their releasates compromise bone marrow-derived human mesenchymal stem/stromal cell survival in vitro[J].Stem Cell Research & Therapy,2021,12(1):547.
[17] ZHANG X,JIANG X,JIANG S,et al.Schwann cells promote prevascularization and osteogenesis of tissue-engineered bone via bone marrow mesenchymal stem cell-derived endothelial cells[J].Stem Cell Research & Therapy,2021,12(1):382.
[18] WU Y,GU S,COBB J M,et al.E2-loaded microcapsules and bone marrow-derived mesenchymal stem cells with injectable scaffolds for endometrial regeneration application[J].Tissue Engineering Part A,2024,30(3-4):115-130.
[19] LI Y,XIA W,MERWE L V D,et al.Efficacy of stem cell therapy for burn wounds:A systematic review and Meta-analysis of preclinical studies[J].Stem Cell Research & Therapy,2020,11(1):322.
[20] SUROWIECKA A,CHRAPUSTA A,KLIMECZEK-CHRAPUSTA M,et al.Mesenchymal stem cells in burn wound management[J].International Journal of Molecular Sciences,2022,23(23):15339.
[21] LI M,QIU L,HU W,et al.Genetically-modified bone mesenchymal stem cells with TGF-β3 improve wound healing and reduce scar tissue formation in a rabbit model[J].Experimental Cell Research,2018,367(1):24-29.
[22] MEDICI D,SHORE E M,LOUNEV V Y,et al.Conversion of vascular endothelial cells into multipotent stem-like cells[J].Nature Medicine,2010,16(12):1400-1406.
[23] BOURIN P,BUNNELL B A,CASTEILLA L,et al.Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells:A joint statement of the international federation for adipose therapeutics and science (IFATS) and the international society for cellular therapy(ISCT)[J].Cytotherapy,2013,15(6):641-648.
[24] LAN T,LUO M,WEI X.Mesenchymal stem/stromal cells in cancer therapy[J].Journal of Hematology & Oncology,2021,14(1):195.
[25] MOHAMAD Y F,HIGASHI Y.Mesenchymal stem/stromal cells for therapeutic angiogenesis[J]. Cells,2023,12(17):2162.
[26] WANG Y,FANG J,LIU B,et al.Reciprocal regulation of mesenchymal stem cells and immune responses[J].Cell Stem Cell,2022,29(11):1515-1530.
[27] CHEN G,TONG K,LI S,et al.Extracellular vesicles released by transforming growth factor-beta 1-preconditional mesenchymal stem cells promote recovery in mice with spinal cord injury[J].Bioactive Materials,2024,35(6):135-149.
[28] DADFAR S,YAZDANPANAH E,PAZOKI A,et al.The role of mesenchymal stem cells in modulating adaptive immune responses in multiple sclerosis[J].Cells,2024,13(18):1556.
[29] MEZEY É.Human mesenchymal stem/stromal cells in immune regulation and therapy[J].Stem Cells Translational Medicine,2022,11(2):114-134.
[30] NOMBELA-ARRIETA C,RITZ J,SILBERSTEIN L E.The elusive nature and function of mesenchymal stem cells[J].Nature Reviews Molecular Cell Biology,2011,12(2):126-131.
[31] ZHOU J,SHI Y.Mesenchymal stem/stromal cells (MSCS):Origin,immune regulation,and clinical applications[J].Cellular & Molecular Immunology,2023,20(6):555-557.
[32] INGRID U S.Mesenchymal stem cells and their microenvironment[J]. Stem Cell Research & Therapy,2011,16(1):2271.
[33] FRISBIE L,BUCKANOVICH R J,COFFMAN L.Carcinoma-associated mesenchymal stem/stromal cells:Architects of the pro-tumorigenic tumor microenvironment[J].Stem Cells,2022,40(8):705-715.