Expression and significance of Wnt-5a gene in a model of intervertebral disc degeneration

doi:10.3969/j.issn.2095-4344.0092

Abstract

Abstract:

BACKGROUND: Important extracellular matrixes are reduced with the prolongation of duration of cyclic pressure in the endplate of the intervertebral disc. Meanwhile, the expression of Wnt-5a gene is significantly decreased. There is an important relationship between Wnt-5a gene and intervertebral disc degeneration (IDD).
OBJECTIVE: To investigate the expression of Wnt-5a gene under cyclic pressure in a rabbit model of IDD and to explore its role in IDD progress.
METHODS: Lumbar intervertebral discs were removed from the 6-month-old New Zealand white rabbits to prepare IDD models and were then randomly divided into experimental (cyclic pressure ) and control (no intervention) groups. The morphological changes of intervertebral discs were observed by hematoxylin-eosin staining and safranin O-fast green staining. The mRNA expression levels of proteoglycan, collagen type II, and Wnt-5a were detected by real-time PCR. The protein expression level of Wnt-5a was detected by western blot assay.
RESULTS AND CONCLUSION: The morphology of intervertebral discs cultured for 7 days in the experimental and control groups showed a certain change, but was still intact; expression levels of aggrecan, type II collagen, Wnt-5a showed differences from the intervertebral discs cultured for 0 day. On day 14, the damage to the histomorphology was severer in the experimental group than the 0-day control group. The mRNA expression levels of proteoglycan, collagen type II, and Wnt-5a were decreased in both groups, especially the experimental group, at 7 and14 days. The mRNA and protein expression levels of Wnt-5a revealed the same change trend with time. To conclude, regulation of Wnt-5a expression may alter the process of endplate cartilage degeneration, and thus providing new ideas for the prevention and treatment of IDD.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Wnt Proteins, Intervertebral Disk, Organ Culture Techniques, Collage Type II, Tissue Engineering

CLC Number:

R318

Yang Xiao-ming, Zhao Quan-lai, Gao Zhi, Xu Hong-guang, Wang Hong, Liu Ping. Expression and significance of Wnt-5a gene in a model of intervertebral disc degeneration[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(4): 570-575.

Figures/Tables 3

2.1 实验动物数量分析纳入35只大白兔，随机分为5组，每组7只。其中5只因感染发生脱失，未脱失中每组随机取3只进行结果分析。 2.2 苏木精-伊红染色观察椎间盘整体器官矢状面石蜡切片苏木精-伊红染色结果显示，与培养0 d相比较，加力组与对照组椎间盘整体培养器官，培养至7 d各组椎间盘大体形态结构完整；培养至14 d的椎间盘整体器官大体组织学结构发生明显的改变，表现为纤维环出现裂伤，结构紊乱，与髓核组织界限不清，髓核组织出现纤维化，体积缩小，这些表现以14 d加力组椎间盘整体培养器官更为明显，见图1。 2.3 番红O固绿染色观察椎间盘整体器官冠状面石蜡切片番红O固绿染色结果显示，与培养0 d相比较，培养7 d对照组椎间盘整体培养器官显示基质完全匀称着色，组织整体性保持完好；培养7 d的加力组、培养14 d的对照组和加力组椎间盘整体器官染色不均匀，染色较培养7 d对照组浅，组织整体性缺损、层次混乱，见图2。 2.4 椎间盘整体器官软骨细胞表型基因以及Wnt-5a基因的检测 Real time PCR检测蛋白多糖表达：对照组14 d与0 d两两比较，蛋白多糖表达下调，且差异有显著性意义(P=0.003)；培养7 d时2组比较，加力组蛋白多糖表达下调，且差异有显著性意义(P=0.021)；培养14 d时2组比较，加力组蛋白多糖表达明显下调，且差异有显著性意义(P=0.000)，见表2。检测Ⅱ型胶原表达：对照组14 d与0 d两两比较，Ⅱ型胶原表达明显下调，且差异有显著性意义(P=0.004)；培养7 d时2组比较，加力组Ⅱ型胶原表达下调，且差异有显著性意义(P=0.015)；培养14 d时2组比较，加力组Ⅱ型胶原表达明显下调，且差异有显著性意义(P=0.006)，见表3。检测Wnt-5a基因表达：对照组14 d与0 d两两比较，Wnt-5a基因表达明显下调，且差异有显著性意义(P=0.005)；培养7 d时2组比较，加力组Wnt-5a基因表达下"

References

[1] Natvig B, Ihlebæk C, Grotle M, et al. Neck pain is often a part of widespread pain and is associated with reduced functioning. Spine. 2010;35(23):E1285-1289.

[2] Yang H, Cao C, Wu C, et al. TGF-betal Suppresses Inflammation in Cell Therapy for Intervertebral Disc Degeneration. Sci Rep. 2015;5: 13254.

[3] Manek NJ, MacGregor AJ. Epidemiology of back disorders: prevalence, risk factors, and prognosis. Curr Opin Rheumatol. 2005;17(2):134-140.

[4] Xu HG, Zheng Q, Song JX, et al. Intermittent cyclic mechanical tension promotes endplate cartilage degeneration via canonical Wnt signaling pathway and E-cadherin/beta-catenin complex cross-talk. Osteoarthritis Cartilage. 2016;24(1):158-168.

[5] Wu B, Meng C, Wang H, et al. Changes of proteoglycan and collagen II of the adjacent intervertebral disc in the cervical instability models. Biomed Pharmacother. 2016;84: 754-758.

[6] Tomaszewski KA, Adamek D, Pasternak A, et al. Degeneration and calcification of the cervical endplate is connected with decreased expression of ANK, ENPP-1, OPN and TGF-beta1 in the intervertebral disc. Pol J Pathol. 2014; 65(3):210-217.

[7] Xu HG, Zhang XH, Wang H, et al. Intermittent Cyclic Mechanical Tension-Induced Calcification and downregulation of ankh gene expression of end plate chondrocytes. Spine. 2012;37(14):1192-1197.

[8] Grant MP, Epure LM, Bokhari R, et al. Human cartilaginous endplate degeneration is induced by calcium and the extracellular calcium-sensing receptor in the intervertebral disc. Eur Cell Mater. 2016;32:137-151.

[9] Mariani E, Pulsatelli L, Facchini A. Signaling pathways in cartilage repair. Int J Mol Sci. 2014;15(5): 8667-8698.

[10] Bradley EW, Drissi MH. WNT5A regulates chondrocyte differentiation through differential use of the CaN/NFAT and IKK/NF-kappaB pathways. Mol Endocrinol. 2010; 24(8): 1581-1593.

[11] 徐宏光,章平治,宋俊兴,等. 循环机械压力诱导下兔椎间盘退变器官模型的建立及意义[J].中国骨与关节外科, 2014, 7(1):45-51.

[12] Xu HG, Ma MM, Zheng Q, et al. P120-Catenin Protects Endplate Chondrocytes From Intermittent Cyclic Mechanical Tension Induced Degeneration by Inhibiting the Expression of RhoA/ROCK-1 Signaling Pathway. Spine (Phila Pa 1976). 2016;41(16): 1261-1271.

[13] Xiao L, Xu HG, Wang H, et al. Intermittent Cyclic Mechanical Tension Promotes Degeneration of Endplate Cartilage via the Nuclear Factor-kappaB Signaling Pathway: an in Vivo Study. Orthop Surg. 2016;8(3):393-399.

[14] Lindblom K. Intervertebral-disc degeneration considered as apressureatrophy. J Bone Joint Sur Br. 1957;39A: 933-945.

[15] Huang YC, Urban JP, Luk KD. Intervertebral disc regeneration: do nutrients lead the way? Nat Rev Rheumatol. 2014; 10(9): 561-566.

[16] Colombier P, Clouet J, Hamel O, et al. The lumbar intervertebral disc: from embryonic development to degeneration. Joint Bone Spine. 2014;81(2): 125-129.

[17] Louati K, Berenbaum F. Joint Biochemical Markers for Cartilage, Bone, Cartilage Degradation, Bone Remodeling, and Inflammation. 2016.

[18] Gantenbein B, Illien-Junger S, Chan SC, et al. Organ culture bioreactors--platforms to study human intervertebral disc degeneration and regenerative therapy. Curr Stem Cell Res Ther. 2015;10(4): 339-352.

[19] Thompson JP, Pearce RH, Schechter MT, et al. Preliminary evaluation of scheme for grading the gross morphology of the human intervertebral disc. Spine(Phila Pa 1976). 1990;15 (5): 411-415.

[20] Sobajima S, Kompel JF, Wallach CJ, et al. A slowly progresssive and reproducible animal model of intervertebral disc degeneration characterized by MRI, X-ray, and Histology. Spine(Phila Pa 1976). 2005;30(1): 15-24.

[21] Rieppo J, Toyras J, Nieminen MT, et al. Structure function relationships in enzymatically modified articular cartilage. Cells Tissues Organs. 2003;175(3): 121-132.

[22] Wodarz A, Nusse R. Mechanisms of Wnt signaling in development. Ann Rev Cell Dev Biol. 1998;14: 59-88.

[23] Eisenmann DM. Wnt signaling. Worm Book, 2005: 1-17.

[24] Bougault C, Briolay A, Boutet MA, et al. Wnt5a is expressed in spondyloarthritis and exerts opposite effects on enthesis and bone in murine organ and cell cultures. Transl Res. 2015; 166(6): 627-638.

[25] 邓刚,司维柯,潘静,等. Wnt-5a基因在淋巴细胞系恶性肿瘤中的表达分析[J].第三军医大学学报,2008,30(19):1832-1835.

[26] Miclea RL, Silbelt M, Finos L, et al. Inhiibion of Gsk3β in cartilage induces osteoarthritic features through activation of the canonical Wnt signaling pathway. Osteoarthritis Cartilage. 2011;19(11):1363.

[27] Yang Y, Topol L, Lee H, et al. Wnt5a and Wnt5b exhibit distinct activities in coordinating chondrocyte proliferation and differentiation. Development. 2003;130(5): 1003-1015.

[28] 贾瑞平,徐宏光,张小海. Wnt信号通路对软骨细胞影响研究进展[J]. 国际骨科学,2010,31(4):200-203.

[29] Kuss P, Kraft K, Stumm J, et al. Regulation of cell polarity in the cartilage growth plate and perichondrium of metacarpal elements by HOXD13 and WNT5A. Dev Biol. 2014;385(1): 83-93.

[30] Ge XP, Ma XC, Meng JH, et al. Role of Wnt-5a in Interleukin-1β-Induced Matrix Meta lloproteinase Expression in Rabbit Temporomandibular Joint Condylar Chondrocytes. Arthritis Rheum. 2009;60(9): 2714-2722.

[31] Yang Y, Topol L, Lee H, et al. Wnt5a and Wnt5b exhibit distinct activities in coordinating chondrocyte proliferation and differentiation. Development. 2003;130(5):1003-1015.

[32] Sassi N, Laadhar L, Allouche M, et al. Wnt signaling is involved in human articular chondrocyte de-differentiation in vitro. Biotech Histochem.2014;89(1):29-40.