| 陶凤华,蒋婷,向威.初级纤毛参与成纤维细胞生长因子18介导的软骨细胞增殖和表型调控的机制研究.骨科,2020,11(1): 67-73. |
| 初级纤毛参与成纤维细胞生长因子18介导的软骨细胞增殖和表型调控的机制研究 |
| Involvement of primary cilia in fibroblast growth factor 18-mediated regulation of chondrocyte proliferation and phenotype maintenance |
| 投稿时间：2019-09-09 |
| DOI：10.3969/j.issn.1674-8573.2020.01.013 |
| 中文关键词: 成纤维细胞生长因子18 初级纤毛 软骨细胞 |
| 英文关键词: Fibroblast growth factor 18 Primary cilia Chondrocyte |
| 基金项目:国家自然科学基金（81902300），中央高校基础科研业务费专项资金（2042019kf0064） |
|
| 摘要点击次数: 4930 |
| 全文下载次数: 2479 |
| 中文摘要: |
| 目的 探究初级纤毛在成纤维细胞生长因子18（fibroblast growth factor 18, FGF 18）介导的软骨细胞增殖和表型调控中的作用及机制。方法 通过CCK8法和免疫荧光染色检测不同浓度FGF18对大鼠软骨细胞增殖和初级纤毛表达的影响；设置对照组、FGF18组、水合氯醛组和FGF18+水合氯醛组，通过免疫荧光染色检测初级纤毛表达，甲苯胺蓝染色检测细胞外基质分泌，Live/dead实验检测细胞活性，qPCR检测成软骨相关基因COL Ⅱ和SOX9的表达变化，Western blot检测细胞周期蛋白D1（CyclinD1）、表型蛋白COL Ⅱ和细胞外调节蛋白激酶（extracellular regulated protein kinases, ERK）通路蛋白表达。结果 不同浓度FGF18对软骨细胞增殖均有促进作用，20 ng/ml时效应最明显；FGF18能下调初级纤毛的发生率（0 ng/ml，77.91%±5.53%；5 ng/ml，52.91%±5.61%；10 ng/ml，42.12%±5.20%；20 ng/ml，36.53%±4.88%；40 ng/ml，33.44%±5.98%），但上调其平均长度[0 ng/ml，（1.63±0.67） μm；5 ng/ml，（2.67±0.90） μm；10 ng/ml，（2.71±0.97） μm；20 ng/ml，（2.76±1.37） μm；40 ng/ml，（2.79±1.13） μm]；FGF18能维持细胞活性并促进细胞外基质分泌，上调COLⅡ和SOX9基因表达；但水合氯醛破坏初级纤毛结构后，纤毛发生率为（9.10±2.44）%，活细胞占比为72.86%±2.95%，水合氯醛+FGF18组纤毛发生率为（10.01±2.23）%，活细胞占比为（76.94±5.62）%，相比对照组[纤毛发生率为（77.91±5.53）%，活细胞占比为（96.81±1.38）%]和FGF18组[纤毛发生率为（36.53%±4.88）%，活细胞占比为（96.29±2.17）%]均明显降低，同时，FGF18的促增殖和促基质分泌作用受到抑制，COL Ⅱ和SOX9基因表达下调；FGF18能促进CyclinD1、COL Ⅱ蛋白表达，并上调P-ERK/T-ERK的比值，破坏纤毛结构则抑制FGF18对相关蛋白的促表达效应。结论 FGF18能促进软骨细胞增殖和维持软骨表型，并且初级纤毛参与FGF18介导的软骨细胞生长发育调控。 |
| 英文摘要: |
| Objective To explore the role and mechanism of primary cilia in fibroblast growth factor 18 (FGF18)-mediated regulation of chondrocytes proliferation and differentiation development. Methods CCK-8 assay and immunefluorescence staining were used to detect the effects of different concentrations of FGF18 on chondrocytes proliferation, and the expression and length changes of primary cilia in vitro. Then the ciliary structure was destroyed by chloral hydrate, and the following groups were set up: the control group, FGF18 group, chloral hydrate group and FGF18+chloral hydrate group. The expression of primary cilia was detected by immunofluorescence staining, and the extracellular matrix secretion was examined by toluidine blue staining, as well as cell viability was measured by Live/dead assay. The expression of phenotype gene COL Ⅱ and SOX9 was detected by qPCR. The expression levels of proliferating protein CyclinD1, phenotype protein COL Ⅱ and ERK signaling pathways were detected by Western blotting. Results Different concentrations of FGF18 could promote chondrocytes proliferation, and the promoting effect was most obvious at 20 ng/ml. Different concentrations of FGF18 could down-regulate the expression of primary cilia (0 ng/ml: 77.91%±5.53%; 5 ng/ml: 52.91%±5.61%; 10 ng/ml: 42.12%±5.20%; 20 ng/ml: 36.53%±4.88%; 40 ng/ml: 33.44%±5.98%), but increase their average lengths [0 ng/ml: (1.63±0.67) μm; 5 ng/ml: (2.67±0.90) μm; 10 ng/ml: (2.71±0.97) μm; 20 ng/ml: (2.76±1.37) μm; 40 ng/ml: (2.79±1.13) μm]. FGF18 could facilitate the maintenance of cells viability, promote cartilage extracellular matrix secretion, and enhance the expression of COL Ⅱ and SOX 9 genes. After destroying the structure of primary cilia by chloral hydrate, the incidence of primary cilia in chloral hydrate group was (9.10±2.44)%, and the proportion of living cells was (72.86±2.95)%. Further, the incidence of cilia in the chloral hydrate+FGF18 group was (10.01±2.33)%, and the percentage of living cells was (76.94±5.62)%, which were obviously reduced as compared with the control group [for ciliary incidence: (77.91±5.53)%; for ration of living cells: (96.81±1.38)%] and FGF18 group [for ciliary incidence: (36.53±4.88)%; for ratio of living cells: (96.29±2.17)%]. The promoting effects of FGF18 on the proliferation of chondrocytes were inhibited after disturbing ciliary structure by chloral hydrate, and these results were statistically different (P＜0.05). Meanwhile, the promoting effect of FGF18 on cartilage matrix secretion was inhibited, and the expression of COL Ⅱ and SOX 9 genes was down-regulated. FGF18 could promote the expression of proliferative protein CyclinD1 and phenotype protein COL Ⅱ, as well as up-regulate the ratio of P-ERK/T-ERK. But the disruption of ciliary structure could inhibit the promoting effects of FGF18 on CyclinD1, COL Ⅱ and P-ERK. Conclusion FGF18 can promote the proliferation and maintain the phenotype of chondrocytes, and primary cilia are involved in FGF18-mediated chondrocytes growth and development regulation. |
|
查看全文
下载PDF阅读器 |
| 关闭 |
|
|
|
