Type <b>Ⅰ</b> and <b>Ⅱ</b> collagen in intervertebral discs of animal models with different injury-type changes in the content of tissues _West China Medical Journal

Authors：

ZHAO Xiaodan ,  LIU Hao , HUANG Fuguo , LIU Xi , DENG Yuxiao

Department of Orthopaedics, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;

Corresponding author：

LIU Hao, Email: liuhao6304@163.com

Keywords：

Injury; Cartilage endplate; Type Ⅰ/Ⅱ collagen; Reverse transcriptase polymerase chain reaction

DOI：

10.7507/1002-0179.201807150

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ObjectiveTo quantitatively determine the levels of type Ⅰ and type Ⅱ collagen mRNA in the intervertebral disc cartilage endplate of injured animal model, and to clarify the cytological function of intervertebral disc chondrocytes during fibrosis repair after intervertebral disc injury.MethodsForty healthy New Zealand rabbits were randomly divided into fibrosus puncture group, upper cartilage endplate single puncture group, upper and lower cartilage endplate multiple puncture group, and sham group. Two experimental animals were randomly selected from each group on the 2nd day, and the 2nd, 8th, 12th, and 24th week after the animal modeling operation to obtain intervertebral disc specimens. The levels of type Ⅰ collagen and type Ⅱ collagen in cartilage endplate cells of the intervertebral disc were determined by reverse transcriptase polymerase chain reaction (PCR). RNA was extracted from the endplate of the intervertebral disc, and the RNA concentration and the ratio of RNA concentration to protein concentration were determined by nucleic acid analyzer. Reverse transcription was performed by Revertaid M-Mulv reverse transcriptase, type Ⅰ and type Ⅱ collagen primers were designed to establish a PCR reaction system, 2% agarose gel electrophoresis (120 V, 40 min) was prepared by using 0.5×TBE electrophoresis buffer. The amplification results were observed under ultraviolet light, and the gray values of different electrophoresis bands were determined.ResultsThe level of type Ⅰ collagen mRNA in each operation group showed a progressive increase after 8 weeks, and the magnitude of the increase was related to the degree of injury. The level of type Ⅱ collagen mRNA showed a transient increase in the fibrosis puncture group and the upper endplate single-puncture group in the first two weeks after the endplate punctures were completed, and then began to decline progressively; in the multiple puncture group, it showed a downward trend from the beginning of the operation. ConclusionsThe synthesis of type Ⅰ collagen in chondrocytes of the injured nucleus pulposus tissue continues to increase with time, while the synthesis of type Ⅱ collagen begin to decrease progressively after a small increase. The formation and change of type Ⅰ and type Ⅱ collagen in injured intervertebral disc chondrocytes are different from natural degeneration.

Citation： ZHAO Xiaodan, LIU Hao, HUANG Fuguo, LIU Xi, DENG Yuxiao. Type Ⅰ and Ⅱ collagen in intervertebral discs of animal models with different injury-type changes in the content of tissues . West China Medical Journal, 2018, 33(9): 1146-1152. doi: 10.7507/1002-0179.201807150 Copy

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3.	Antoriou J, Goudsouzian NM, Heathfield TF, et al. The human lumbar endplate. Evidence of changes in biosynthesis and denaturation of the extracellular matrix with growth, maturation, aging, and degeneration. Spine (Phila Pa 1976), 1996, 21(10): 1153-1161.
4.	Hao H, Cohen DA, Jennings CD, et al. Bleomycin-induced pulmonary fibrosis is independent of eosinophils. J Leukoc Biol, 2000, 68(4): 515-521.
5.	Wynn TA. Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J Clin Invest, 2007, 117(3): 524-529.
6.	Masuda K, Aota Y, Muehleman C, et al. A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture: correlation between the degree of disc injury and radiological and histological appearances of disc degeneration. Spine (Phila Pa 1976), 2005, 30(1): 5-14.
7.	Cinotti G, Della Rocca C, Romeo S, et al. Degenerative changes of porcine intervertebral disc induced by vertebral endplate injuries. Spine (Phila Pa 1976), 2005, 30(2): 174-180.
8.	Singh K, Masuda K, An HS. Animal models for human disc degeneration. Spine J, 2005, 5(Suppl 6): 267S-279S.
9.	王一, 戴国钢, 黄雷, 等. 腰椎退行性疾病矢状面失衡及结构因素研究. 华西医学, 2017, 32(4): 529-533.
10.	Fichtner-Feigl S, Strober W, Kawakami K, et al. IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and fibrosis. Nat Med, 2006, 12(1): 99-106.
11.	Cinotti G, Della Rocca C, Romeo S, et al. Degenerative changes of porcine intervertebral disc induced by vertebral endplate injuries. Spine(Phila Pa 1976), 2005, 30(2): 197-180.
12.	Chiaramonte MG, Donaldson DD, Cheever AW, et al. An IL-13 inhibitor blocks the development of hepatic fibrosis during a T-helper type 2-dominated inflammatory response. J Clin Invest, 2009, 104(6): 777-785.

1. 胡有谷, 吕振华, 陈晓亮, 等. 腰椎间盘的细胞、胶原与弹性蛋白. 中华骨科杂志, 1997, 17(12): 8-10.
2. 李书忠, 胡有谷, 陈培勋. 椎腰间盘胶原分布的研究. 中华骨科杂志, 1992, 12(3): 213.
3. Antoriou J, Goudsouzian NM, Heathfield TF, et al. The human lumbar endplate. Evidence of changes in biosynthesis and denaturation of the extracellular matrix with growth, maturation, aging, and degeneration. Spine (Phila Pa 1976), 1996, 21(10): 1153-1161.
4. Hao H, Cohen DA, Jennings CD, et al. Bleomycin-induced pulmonary fibrosis is independent of eosinophils. J Leukoc Biol, 2000, 68(4): 515-521.
5. Wynn TA. Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J Clin Invest, 2007, 117(3): 524-529.
6. Masuda K, Aota Y, Muehleman C, et al. A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture: correlation between the degree of disc injury and radiological and histological appearances of disc degeneration. Spine (Phila Pa 1976), 2005, 30(1): 5-14.
7. Cinotti G, Della Rocca C, Romeo S, et al. Degenerative changes of porcine intervertebral disc induced by vertebral endplate injuries. Spine (Phila Pa 1976), 2005, 30(2): 174-180.
8. Singh K, Masuda K, An HS. Animal models for human disc degeneration. Spine J, 2005, 5(Suppl 6): 267S-279S.
9. 王一, 戴国钢, 黄雷, 等. 腰椎退行性疾病矢状面失衡及结构因素研究. 华西医学, 2017, 32(4): 529-533.
10. Fichtner-Feigl S, Strober W, Kawakami K, et al. IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and fibrosis. Nat Med, 2006, 12(1): 99-106.
11. Cinotti G, Della Rocca C, Romeo S, et al. Degenerative changes of porcine intervertebral disc induced by vertebral endplate injuries. Spine(Phila Pa 1976), 2005, 30(2): 197-180.
12. Chiaramonte MG, Donaldson DD, Cheever AW, et al. An IL-13 inhibitor blocks the development of hepatic fibrosis during a T-helper type 2-dominated inflammatory response. J Clin Invest, 2009, 104(6): 777-785.

West China Medical Journal

Type Ⅰ and Ⅱ collagen in intervertebral discs of animal models with different injury-type changes in the content of tissues

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