初级纤毛在长骨生长板分布规律的小鼠实验研究

doi:10.3969/j.issn.1005-4979.2021.05.001

摘要/Abstract

摘要：

目的： 探究长骨发育过程中初级纤毛在生长板的分布规律。方法： 对1、4周龄和8周龄的小鼠股骨切片进行免疫荧光染色，分析初级纤毛在生长板的分布规律；培养生长板软骨细胞并诱导其肥大化，研究该过程中初级纤毛的改变；通过无血清饥饿法提高有纤毛软骨细胞比例后诱导软骨细胞肥大化，通过实时荧光定量聚合酶链式反应（real-time quantitative polymerase chain reaction, RT-qPCR）和免疫荧光染色检测肥大软骨细胞标志物的表达。结果： 1、4周龄小鼠的有纤毛软骨细胞主要分布在静息层、增殖层和前肥大层，肥大层有初级纤毛细胞显著减少；在体外诱导软骨细胞肥大化后，有纤毛细胞比例降低；提高有纤毛细胞比例后诱导软骨细胞肥大化，肥大软骨细胞标志性基因COL10、Prg4表达降低。结论： 初级纤毛主要分布在生长板静息层、增殖层和前肥大层，肥大层软骨细胞初级纤毛减少；通过无血清饥饿法提高有纤毛细胞比例能够降低肥大软骨细胞标志性基因的表达。

关键词: 初级纤毛, 生长板, 软骨细胞

Abstract:

Objective: To investigate the regularity of primary cilia distribution in growth plate during long bone development. Methods: Immunofluorescence staining was applied to label primary cilia in femur sections of 1-, 4- week-old mice to study the distribution of primary cilia in cartilage growth plates. Growth plate chondrocytes were cultured, and changes of primary cilia were observed during the process of hypertrophic differentiation induction; the proportion of ciliated chondrocytes was increased through serum-free starvation, and then hypertrophic differentiation induction were performed; the expression of hypertrophic chondrocyte markers was detected by real-time quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence staining. Results: Ciliated chondrocytes of 1-, 4- week-old mice were mainly distributed in the resting layer, the proliferating layer and the anterior hypertrophy layer, and primary ciliated cells were significantly reduced in the hypertrophy layer. It was found that the proportion of ciliated cells decreased after hypertrophic differentiation induction in vitro. After raising the proportion of ciliated cells, chondrocyte fertilization was induced, and the expression of COL10 and Prg4, the marker gene of hypertrophic chondrocytes, was significantly reduced. Conclusion: The primary cilia mainly existed in the resting zone, proliferation zone and the prehypertrophy zone of the growth plate. The chondrocytes in hypertrophy zone have less primary cilia. Increasing the proportion of ciliated cells by serum-free starvation can reduce the expression of the marker genes of hypertrophic chondrocytes.

Key words: primary cilia, growth plate, chondrocyte

丁允鹏, 陶狄坷, 张帅, 孙瑶. 初级纤毛在长骨生长板分布规律的小鼠实验研究[J]. 《口腔颌面外科杂志》, 2021, 31(5): 265-271.

DING Yunpeng, TAO Dike, ZHANG Shuai, SUN Yao. The distribution of primary cilia in the growth plate of long bone: A study in the mice[J]. 《Journal of Oral and Maxillofacial Surgery》, 2021, 31(5): 265-271.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2021/V31/I5/265

图/表 5

表1 RT-qPCR引物序列

Table 1 Primer sequence in RT-qPCR

基因	正向引物（5′-3′）	反向引物（5′-3′）
GAPDH	AGGTCGGTGTGAACGGATTTG	TGTAGACCATGTAGTTGAGGTCA
COL10a1	TTCTGCTGCTAATGTTCTTGACC	GGGATGAAGTATTGTGTCTTGGG
Prg4	GAAAATACTTCCCGTCTGCTTGT	ACTCCATGTAGTGCTGACAGTTA

图1 长骨发育过程中生长板软骨细胞存在初级纤毛 A~C. 1、4、8周龄时的股骨甲苯胺蓝染色图（×40）；D~F. 1、4、8周时的股骨切片Arl13b免疫荧光染色图（×100）；G~I.1、4、8周龄时股骨切片Arl13b免疫荧光染色图（×400），白色箭头指示初级纤毛；J. 不同年龄小鼠生长板纤毛细胞百分比定量。

Figure 1 Primary cilia of chondrocyte in femur growth plate of developing mouse

图2 感染患者清创后置入VSD的术后观

Figure 2 Postoperative view of debridement and installation of VSD in infected patient

图3 生长板软骨细胞肥大化过程中，初级纤毛略减少 A、B、D、E. 肥大软骨细胞诱导第0、7天时的免疫荧光染色图（×200）；C、F. 肥大软骨细胞诱导第0、7天时的免疫荧光染色图，白色箭头指示初级纤毛（×400）；G~H. RT-qPCR检测肥大软骨细胞标志性基因，*表示P<0.05，与诱导0 d时相比；I. 肥大软骨细胞诱导第0、7天时有纤毛细胞百分比定量。

Figure 3 The primary cilia were slightly reduced during growth plate chondrocytes differentiated into hypertrophic chondrocytes

图4 提高有纤毛细胞比例伴随肥大软骨细胞标志性基因的表达降低 A、B. 饥饿48 h后，Arl13b免疫荧光染色图（×400）；C. 肥大软骨细胞诱导7 d后有纤毛细胞百分比定量，*表示P<0.05，与对照组相比；D、E. 肥大软骨细胞诱导7 d后COL10免疫荧光染色（×200）；F、G. 肥大软骨细胞诱导7 d后，RT-qPCR检测肥大软骨细胞标志性基因表达，*表示P<0.05，与诱导0天时相比。

Figure 4 The proportion of ciliated chondrocytes was increased accompanied by the expression of marker genes of hypertrophic chondrocytes depressed

参考文献 20

[1]	Lyu R, Zhou J. The multifaceted roles of primary cilia in the regulation of stem cell properties and functions[J]. J Cell Physiol, 2017, 232(5): 935-938. doi: 10.1002/jcp.25683 pmid: 27861880
[2]	Hampl M, Cela P, Szabo-Rogers HL, et al. Role of primary cilia in odontogenesis[J]. J Dent Res, 2017, 96(9): 965-974. doi: 10.1177/0022034517713688 pmid: 28605602
[3]	Novarino G, Akizu N, Gleeson JG. Modeling human disease in humans: The ciliopathies[J]. Cell, 2011, 147(1): 70-79. doi: 10.1016/j.cell.2011.09.014 pmid: 21962508
[4]	Huber C, Cormier-Daire V. Ciliary disorder of the skeleton[J]. Am J Med Genet C Semin Med Genet, 2012, 160C(3): 165-174. doi: 10.1002/ajmg.c.v160c.3 URL
[5]	Hoey DA, Tormey S, Ramcharan S, et al. Primary cilia-mediated mechanotransduction in human mesenchymal stem cells[J]. Stem Cells, 2012, 30(11): 2561-2570. doi: 10.1002/stem.1235 pmid: 22969057
[6]	Yuan X, Cao J, He XN, et al. Ciliary IFT80 balances canonical versus non-canonical hedgehog signalling for osteoblast differentiation[J]. Nat Commun, 2016, 7: 11024. doi: 10.1038/ncomms11024 pmid: 26996322
[7]	Yuan X, Yang SY. Deletion of IFT80 impairs epiphyseal and articular cartilage formation due to disruption of chondrocyte differentiation[J]. PLoS One, 2015, 10(6): e0130618.
[8]	Morris SA. Single-cell RNA-seq steps up to the growth plate[J]. Trends Biotechnol, 2016, 34(7): 525-527. doi: S0167-7799(16)30053-1 pmid: 27260936
[9]	Rais Y, Reich A, Simsa-Maziel S, et al. The growth plate's response to load is partially mediated by mechano-sensing via the chondrocytic primary cilium[J]. Cell Mol Life Sci, 2015, 72(3): 597-615. doi: 10.1007/s00018-014-1690-4 pmid: 25084815
[10]	Mizuhashi K, Nagata M, Matsushita Y, et al. Growth plate borderline chondrocytes behave as transient mesenchymal precursor cells[J]. J Bone Miner Res, 2019, 34(8): 1387-1392. doi: 10.1002/jbmr.3719 pmid: 30888720
[11]	Papaioannou G, Petit ET, Liu ES, et al. Raf kinases are essential for phosphate induction of ERK1/2 phosphorylation in hypertrophic chondrocytes and normal endochondral bone development[J]. J Biol Chem, 2017, 292(8): 3164-3171. doi: 10.1074/jbc.M116.763342 pmid: 28073913
[12]	Song BE, Haycraft CJ, Seo HS, et al. Development of the post-natal growth plate requires intraflagellar transport proteins[J]. Dev Biol, 2007, 305(1): 202-216. doi: 10.1016/j.ydbio.2007.02.003 pmid: 17359961
[13]	Kobayashi T, Soegiarto DW, Yang YZ, et al. Indian hedgehog stimulates periarticular chondrocyte differentiation to regulate growth plate length independently of PTHrP[J]. J Clin Invest, 2005, 115(7): 1734-1742. pmid: 15951842
[14]	Mizuhashi K, Ono W, Matsushita Y, et al. Resting zone of the growth plate houses a unique class of skeletal stem cells[J]. Nature, 2018, 563(7730): 254-258. doi: 10.1038/s41586-018-0662-5
[15]	St-Jacques B, Hammerschmidt M, McMahon AP. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation[J]. Genes Dev, 1999, 13(16): 2072-2086.
[16]	Horowitz MC, Tommasini SM. Fat and bone: PGC-1α regulates mesenchymal cell fate during aging and osteoporosis[J]. Cell Stem Cell, 2018, 23(2): 151-153. doi: S1934-5909(18)30346-1 pmid: 30075123
[17]	Sasai N, Briscoe J. Primary cilia and graded sonic hedgehog signaling[J]. Wiley Interdiscip Rev Dev Biol, 2012, 1(5): 753-772. doi: 10.1002/wdev.v1.5 URL
[18]	Jenkins D. Hedgehog signalling: Emerging evidence for non-canonical pathways[J]. Cell Signal, 2009, 21(7): 1023-1034. doi: 10.1016/j.cellsig.2009.01.033 pmid: 19399989
[19]	He M, Agbu S, Anderson KV. Microtubule motors drive hedgehog signaling in primary Cilia[J]. Trends Cell Biol, 2017, 27(2): 110-125. doi: S0962-8924(16)30152-0 pmid: 27765513
[20]	Kronenberg HM. Developmental regulation of the growth plate[J]. Nature, 2003, 423(6937): 332-336. doi: 10.1038/nature01657 URL

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