鸡软骨细胞体外分离培养鉴定以及过表达miR-15a对软骨细胞的影响

doi:10.11843/j.issn.0366-6964.2022.06.009

Abstract

Abstract: The study aimed to investigate the expression of miR-15a in different tissues of broilers and explore the effect of overexpression of miR-15a on chondrocytes. In this study, the chondrocytes were isolated and culture, and it was identified by inverted microscope observation, PCR, gel electrophoresis and toluidine blue staining. Real-time fluorescence quantitative PCR was used to detect the expression level of miR-15a in each tissue of broilers in the valgus-varus deformity (VVD) group and the healthy group (3 each). After that, the cell proliferation of chondrocytes in miR-15a overexpression group was detected by CCK8 and EDU assay. After chondrocytes were transfected with miR-15a mimics, the expression of marker genes of chondrocytes Collagen-2, Aggrecan, Collagen-10, mature differentiation genes Runx2, Sox9, VEGF, MMP9, inflammatory factors IL-1β, IL- 6, IL-8, IL-10, TNF-α, TGF-β3 and the apoptosis genes Fas, FasL and Bcl-2 were detected by qPCR. The wild-type vector and mutant vector of FKBP5 3'UTR were constructed, and the targeting relationship between miR-15a and FKBP5 was detected by dual-luciferase detection reporter. The results showed that the trypsin, collagenase II and hyaluronidase combined digestion method used in this study could successfully isolate chondrocytes. The qPCR results showed that miR-15a was expressed in all detected tissues. The expression of miR-15a in the liver (P < 0.01), spleen (P < 0.05) and thymus (P < 0.01) in the VVD group were significantly higher compared with the healthy group. And the expression level in the heart and pectoral muscle tissues was significantly lower in the VVD group (P < 0.01). The results of CCK8 and EDU showed that, compared with the NC group, the proliferation rate of chondrocytes in the miR-15a overexpression group was significantly decreased (P < 0.01), and the number of proliferating cells was significantly decreased (P < 0.01). The qPCR results showed that, the expression of Aggrecan, Sox9 and Runx2 genes in the miR-15a mimics group were significantly decreased (P < 0.05), and the expression of Fas gene was extremely significantly increased (P < 0.01), the expression of FasL gene and anti-apoptotic gene Bcl-2 were extremely significantly decreased (P < 0.01) compared with the NC group. The FKBP5 3'UTR wild-type and mutant vectors were successfully constructed, and the dual-luciferase detection reporter showed that the predicted target gene FKBP5 had no targeting relationship with miR-15a. In this study, chicken chondrocytes were successfully isolated and identified, and the results suggest that the overexpression of miR-15a can inhibit the proliferation, maturation, differentiation and promote apoptosis of the chicken chondrocytes.

Key words: chicken, chondrocyte isolation in vitro, miR-15a, tissue expression profile, inhibit cell proliferation, cell apoptosis

CLC Number:

S831.2

ZHANG Zhenzhen, LI Jianzeng, JIANG Ruirui, MA Yanchao, CAI Chunxia, ZHANG Lujie, GUO Yujie, JI Jinqing, HAN Lu, TIAN Yadong, KANG Xiangtao, HAN Ruili. Isolation, Culture and Identification of Chicken Chondrocytes and the Effect of Overexpression of miR-15a on Chondrocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(6): 1749-1758.

References 34

[1]	LETERRIER C, NYS Y. Clinical and anatomical differences in varus and valgus deformities of chick limbs suggest different aetio-pathogenesis[J]. Avian Pathol, 1992, 21(3): 429-442.
[2]	BRADSHAW R H, KIRKDEN R D, BROOM D M. A review of the aetiology and pathology of leg weakness in broilers in relation to welfare[J]. Avian Poult Biol Rev, 2002, 13(2): 45-103.
[3]	GUO Y P, TANG H H, WANG X N, et al. Clinical assessment of growth performance, bone morphometry, bone quality, and serum indicators in broilers affected by valgus-varus deformity[J]. Poultry Sci, 2019, 98(10): 4433-4440.
[4]	YANG Q. Animal histology and embryology[M]. Beijing: China Agricultural University Press, 2008. (in Chinese)杨倩. 动物组织学与胚胎学[M]. 北京: 中国农业大学出版社, 2008.
[5]	CHEN X D, LIN J H. The continuous observation of chondrocyte degeneration in vitro of rats[J]. Chinese Journal of Gerontology, 2008, 28(7): 652-655. (in Chinese)陈晓东, 林建华. 体外培养大鼠软骨细胞退变现象的连续观察[J]. 中国老年学杂志, 2008, 28(7): 652-655.
[6]	LIN Y Y. Advances in molecular mechanism of chondrocyte dedifferentiation[J]. Guangxi Medical Journal, 2020, 42(18): 2437-2443. (in Chinese)林阳洋. 软骨细胞去分化的分子机制的研究进展[J]. 广西医学, 2020, 42(18): 2437-2443.
[7]	CHEN F L, MAO T Q, FENG X, et al. Isolation, purification and identification of rabbit joint cartilage cell in vitro[J]. Journal of Modern Stomatology, 1998, 12(4): 263-265. (in Chinese)陈富林, 毛天球, 冯雪, 等. 兔关节软骨细胞的体外分离、纯化及鉴定[J]. 现代口腔医学杂志, 1998, 12(4): 263-265.
[8]	BARTEL D P. MicroRNAs: genomics, biogenesis, mechanism, and function[J]. Cell, 2004, 116(2): 281-297.
[9]	AMBROS V. The functions of animal microRNAs[J]. Nature, 2004, 431(7006): 350-355.
[10]	SEIDL C I, MARTINEZ-SANCHEZ A, MURPHY C L. Derepression of MicroRNA-138 contributes to loss of the human articular chondrocyte phenotype[J]. Arthritis Rheumatol, 2016, 68(2): 398-409.
[11]	MOULIN D, SALONE V, KOUFANY M, et al. MicroRNA-29b contributes to collagens imbalance in human osteoarthritic and dedifferentiated articular chondrocytes[J]. Biomed Res Int, 2017, 2017: 9792512.
[12]	ZHANG Z Z, TANG H H, MA Y C, et al. Identification of key miRNAs affecting broilers with Valgus-varus Deformity by RNA sequencing and analysis of miRNA-mRNA interactions[J]. Mol Omics, 2021, 17(5): 752-759.
[13]	HU E B, JIN Q, FANG F J, et al. Expression of miR-15a in exosomes of patients with multiple myeloma and the effect of exosomes on tumor cell proliferation and apoptosis[J]. Journal of Chinese Oncology, 2020, 26(6): 491-495. (in Chinese)胡恩赑, 金强, 方福瑾, 等. miR-15a在多发性骨髓瘤患者外泌体中的表达及外泌体对肿瘤生长的影响[J]. 肿瘤学杂志, 2020, 26(6): 491-495.
[14]	CHEN D C, NING F Y, ZHANG J Y, et al. NF-κB pathway took part in the development of apoptosis mediated by miR-15a and oxidative stress via mitochondrial pathway in ammonia-treated chicken splenic lymphocytes[J]. Sci Total Environ, 2020, 729: 139017.
[15]	SULLIVAN R P, LEONG J W, SCHNEIDER S E, et al. MicroRNA-15/16 antagonizes Myb to control NK cell maturation[J]. J Immunol, 2015, 195(6): 2806-2817.
[16]	MOON H G, YANG J C, ZHENG Y J, et al. miR-15a/16 regulates macrophage phagocytosis after bacterial infection[J]. J Immunol, 2014, 193(9): 4558-4567.
[17]	WEI H, WU Q, SHI Y M, et al. MicroRNA-15a/16/SOX5 axis promotes migration, invasion and inflammatory response in rheumatoid arthritis fibroblast-like synoviocytes[J]. Aging, 2020, 12(14): 14376-14390.
[18]	YAN S J. Expression of miR-15a in human knee articular chondrocytes and effects of miR-15a on proliferation and apoptosis of articular chondrocytes[D]. Xi'an: Air Force Medical University, 2014. (in Chinese)颜世举. miR-15a在人膝关节软骨细胞中的表达及其对软骨细胞增殖与凋亡作用的研究[D]. 西安: 第四军医大学, 2014.
[19]	ARRON J R, CHOI Y. Bone versus immune system[J]. Nature, 2000, 408(6812): 535-536.
[20]	AMARASEKARA D S, YUN H, KIM S, et al. Regulation of osteoclast differentiation by cytokine networks[J]. Immune Netw, 2018, 18(1): e8.
[21]	DE BENEDETTI F, RUCCI N, DEL FATTORE A, et al. Impaired skeletal development in interleukin-6-transgenic mice: a model for the impact of chronic inflammation on the growing skeletal system[J]. Arthritis Rheum, 2006, 54(11): 3551-3563.
[22]	KANESHIRO S, EBINA K, SHI K, et al. IL-6 negatively regulates osteoblast differentiation through the SHP2/MEK2 and SHP2/Akt2 pathways in vitro[J]. J Bone Miner Metab, 2014, 32(4): 378-392.
[23]	SCHRODER K, HERTZOG P J, RAVASI T, et al. Interferon-γ: an overview of signals, mechanisms and functions[J]. J Leukoc Biol, 2004, 75(2): 163-189.
[24]	YAO Z Q, LI P, ZHANG Q, et al. Tumor necrosis factor-α increases circulating osteoclast precursor numbers by promoting their proliferation and differentiation in the bone marrow through up-regulation of c-Fms expression[J]. J Biol Chem, 2006, 281(17): 11846-11855.
[25]	PONZETTI M, RUCCI N. Updates on osteoimmunology: what's new on the cross-talk between bone and immune system[J]. Front Endocrinol (Lausanne), 2019, 10: 236.
[26]	ZHU J, GARRETT R, JUNG Y, et al. Osteoblasts support B-lymphocyte commitment and differentiation from hematopoietic stem cells[J]. Blood, 2007, 109(9): 3706-3712.
[27]	FAN Z Y, LIU Y H, SHI Z L, et al. MiR-155 promotes interleukin-1β-induced chondrocyte apoptosis and catabolic activity by targeting PIK3R1-mediated PI3K/Akt pathway[J]. J Cell Mol Med, 2020, 24(15): 8441-8451.
[28]	GUO Y P. Identification and study of phenotypes and susceptibility genes in broilers affected by valgus-varus deformity[D]. Zhengzhou: Henan Agricultural University, 2020. (in Chinese)郭亚苹. 肉鸡VVD腿病表型及易感基因的鉴定与研究[D]. 郑州: 河南农业大学, 2020.
[29]	LIU W, SONG H, DONG P. Advances in osteoimmunology[J]. Journal of Clinical Orthopedics and Research, 2020, 5(2): 124-126. (in Chinese)刘伟, 宋慧, 董鹏. 骨免疫学研究进展[J]. 骨科临床与研究杂志, 2020, 5(2): 124-126.
[30]	OKAMOTO K, NAKASHIMA T, SHINOHARA M, et al. Osteoimmunology: the conceptual framework unifying the immune and skeletal systems[J]. Physiol Rev, 2017, 97(4): 1295-1349.
[31]	TSUKASAKI M, TAKAYANAGI H. Osteoimmunology: evolving concepts in bone-immune interactions in health and disease[J]. Nat Rev Immunol, 2019, 19(10): 626-642.
[32]	YOULE R J, STRASSER A. The BCL-2 protein family: opposing activities that mediate cell death[J]. Nat Rev Mol Cell Biol, 2008, 9(1): 47-59.
[33]	DENG Y P, FENG P. The role of Fas and Fasl in cell apoptosis[J]. International Journal of Immunology, 1997, 20(4): 213-216. (in Chinese)邓友平, 冯璞. Fas和Fasl在细胞凋亡中的作用[J]. 国外医学免疫学分册, 1997, 20(4): 213-216.
[34]	PANAYIOTIDIS P, GANESHAGURU K, FORONI L, et al. Expression and function of the FAS antigen in B chronic lymphocytic leukemia and hairy cell leukemia[J]. Leukemia, 1995, 9(7): 1227-1232.

Isolation, Culture and Identification of Chicken Chondrocytes and the Effect of Overexpression of miR-15a on Chondrocytes

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