The Effects of Bone Morphogenetic Protein 4 Overexpression on Proliferation and Chondrogenesis of Rabbit Bone Marrow Stem Cells

doi:10.3969/j.issn.1674-5817.2020.02.001

Abstract

Abstract: Objective To investigate the effects of bone morphogenetic protein 4 (BMP-4) overexpression on the proliferation and chondrogenesis of rabbit bone marrow stem cells (BMSCs) infected by adenovirus. Methods The BMSCs were isolated and cultured by whole bone marrow adherence method, the purity of BMSCs was improved by subculture, the cells were identified by cell morphology and surface molecular marker by flow cytometry, and the optimal multiplicity of infection (MOI) of adenovirus was detected by inverted phase contrast microscopy and flow cytometry. The effects of BMP-4 overexpression on the secretion of type Ⅱ collagen (COLL Ⅱ), sry-related high mobility group-box gene 9 (Sox9) and BMP-4 in chondrocytes were analyzed by enzyme linked immunosorbent assay (ELISA) and Western blotting. The effect of BMP-4 overexpression on the proliferation of BMSCs in vitro was detcted by cell counting kit-8 (CCK-8). Results Primary cells were round, elliptical and spindle-shaped. The second generation cells were typical fibroblast-like long spindle-shaped, vortex-like distribution, with low expression of hematopoietic stem cell surface molecular markers CD34(1.94%) and CD45(2.13%), and high expression of CD44(98.2%), CD29(99.7%) and CD90(98.8%). MOI for 100, with higher infection efficiency and smallest damage to cells, is the most appropriate MOI. Compared with the control group, the overexpression of BMP-4 could significantly promote the proliferation of BMSCs and secrete a large number of COLL Ⅱ、Sox9 and BMP-4 proteins (P<0.05). Conclusion Overexpression of BMP-4 can significantly promote the proliferation and differentiation of BMSCs into chondrocytes in rabbit.

Key words: Bone marrow mesenchymal stem cells, Bone morphogenetic protein 4, Proliferation, Chondrogensis

CLC Number:

R-33
Q95-33

ZHANG Fei, DANG Yuan, XUE Laien, WEN Fuli, ZHENG Heping, HU Deqing. The Effects of Bone Morphogenetic Protein 4 Overexpression on Proliferation and Chondrogenesis of Rabbit Bone Marrow Stem Cells[J]. Laboratory Animal and Comparative Medicine, 2020, 40(2): 87-94.

References

[1] Craft AM, Rockel JS, Nartiss Y, et al.Generation of articular chondrocytes from human pluripotent stem cells[J]. Nat Biotechnol, 2015, 33(6):638-645.
[2] Fahy N, Alini M, Stoddart MJ, et al.Mechanical stimulation of mesenchymal stem cells: Implications for cartilage tissue engineering[J]. J Orthop Res, 2018, 36(1):52-63.
[3] Kode JA, Mukherjee S, Joglekar MV, et al.Mesenchymal stem cells: immunobiology and role in immunomodulation and tissue regeneration[J]. Cytotherapy, 2009, 11(4):377-391.
[4] Wang JC, Kanim LE, Yoo S, et al.Effect of regional gene therapy with bone morphogenetic rotein-2-producing bone marrow cells on spinal fusion in rats[J]. J Bone Joint Surg Am, 2003, 85(5):905-911.
[5] 江华, 肖增明. 骨髓间充质干细胞在骨科疾病修复中的应用[J]. 中国临床康复, 2006, 10(45):118-120.
[6] 黄涛, 孟志斌, 贾丙申, 等. 全骨髓贴壁法分离培养rBMSCs及成骨诱导探讨[J]. 广东医学, 2010, 31(9):1089-1091.
[7] Abdallah BM, Alshammary A, Skagen P, et al.CD34 defines an osteoprogenitor cell population in mouse bone marrow stromal cells[J]. Stem Cell Res, 2015, 15(3):449-458.
[8] Zeng YL, Zheng H, Chen QR, et al.Bone marrow-derived mesenchymal stem cells overexpressing miR-21 efficiently repair myocardial damage in rats[J]. Oncotarget, 2017, 8(17):29161-29173.
[9] Wang X, Li Y, Han R, et al.Demineralized bone matrix combined bone marrow mesenchymal stem cells, bone morphogenetic protein-2 and transforming growth factor-β3 gene promoted pig cartilage defect repair[J]. PLoS One, 2014, 9(12):e0125948.
[10] Shang J, Liu H, Zhou Y.Roles of microRNAs in prenatal chondrogenesis, postnatal chondrogenesis and cartilage-related diseases[J]. J Cell Mol Med, 2013, 17(12):1515-1524.
[11] Thanunchai M, Hongeng S, Thitithanyanont A.Mesenchymal stromal cells and viral infection[J]. Stem Cells Int, 2015, 2015:860950.
[12] 李松南, 毛晓波, 冯义柏, 等. 大鼠Cx43基因慢病毒表达载体的构建及转染骨髓间充质干细胞表达的观察[J]. 临床心血管病杂志, 2010, 26(11):854-858.
[13] Laurencin CT, Attawia MA, Lu LQ, et al.Poly(lactide-co-glycolide)/hydroxyapatite delivery of BMP-2-producing cells: a regional gene therapy approach to bone regeneration[J]. Biomaterials, 2001, 22(11):1271-1277.
[14] 毛颖佳, 郑源强, 石艳春. 慢病毒载体及其应用的研究进展[J]. 中国生物制品学杂志, 2009, 22(2):196-200.
[15] Sándor GK, Tuovinen VJ, Wolff J, et al.Adipose stem cell tissue-engineered construct used to treat large anterior mandibular defect: a case report and review of the clinical application of good manufacturing practice-level adipose stem cells for bone regeneration.[J]. J Oral Maxillofac Surg, 2013, 71(5):938-950.
[16] 杨林, 徐军美, 王亚平, 等. 骨形态发生蛋白2/4在神经系统中的作用及其研究进展[J]. 中南大学学报: 医学版, 2018, 43(2):222-228.
[17] Lópiz Y, Abarrategi A, Ramos V, et al.In vivo comparison of the effects of RHBMP-2 and RHBMP-4 in osteochondral tissue regeneration[J]. Eur Cell Mater, 2010, 20(20):367-378.