lncRNA HOXA11-AS抑制骨关节炎大鼠软骨细胞凋亡与基质降解的作用研究

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摘要目的研究长链非编码RNA(long-nocoding RNA，lncRNA)HOXA11-AS在骨关节炎(osteoarthritis，OA)的大鼠软骨细胞中的表达情况，及其对软骨细胞凋亡、基质降解以及核因子κB(nuclear factor kappa B，NF-κB)信号通路的影响。方法采用弗氏完全佐剂注射大鼠构建OA大鼠模型，造模后分离培养软骨细胞；小干扰RNA(small interfering RNA，siRNA)技术抑制软骨细胞中HOXA11-AS表达；细胞计数试剂盒(cell counting kit-8，CCK-8)检测转染siRNA不同时间后软骨细胞的活力；实时荧光定量聚合酶链式反应(quantitative real-time PCR，qRT-PCR)检测软骨细胞中HOXA11-AS及基质金属蛋白酶13(matrix metallo protein-13，MMP-13)、血小板结合蛋白基序的解聚蛋白样金属蛋白酶5(a disintesrin and metalloprotease with thrombospondin type 5 motifs，ADAMTS-5)、Ⅱ型胶原(Collagen Ⅱ)、聚集蛋白聚糖(aggrecan)mRNA的表达情况；流式细胞术检测软骨细胞的凋亡情况；酶联免疫吸附测定法(enzyme-linked immune sorbent assay，ELISA)检测软骨细胞上清液中Bcl-2相关X蛋白(Bcl 2-associated X protein，Bax)、B淋巴细胞瘤-2(B-cell lymphoma-2，Bcl-2)、半胱氨酸蛋白酶3(caspase-3)的表达情况；蛋白质印迹法(western blot)检测MMP-13、ADAMTS-5、CollagenⅡ、aggrecan和抗核因子κB抑制蛋白(anti-nuclear factor kappa B inhibitory protein，IκBα)、抗磷酸化核因子κB抑制蛋白(anti-phosphorylated nuclear factor kappa B inhibitory protein，p-IκBα)、抗核因子κB 65(nuclear factor kappa B，NF-κB 65)的表达，免疫荧光染色检测NF-κB在软骨细胞细胞核中的表达情况。结果与正常组大鼠软骨细胞相比，HOXA11-AS在OA大鼠软骨细胞中高表达；与NC siRNA组比较，HOXA11-AS siRNA组细胞活力显著下降，细胞凋亡率及促凋亡因子Bax、Caspase-3的表达水平显著升高，而抑凋亡因子Bcl-2的表达水平显著下降；同时，与NC siRNA组比较，HOXA11-AS siRNA组细胞collagen Ⅱ和aggrecan的基因和蛋白表达水平明显降低，MMP-13和ADAMTS-5基因及蛋白表达水平明显升高，IκBα蛋白表达水平显著下降而p-IκBα和NF-κB p65的表达水平显著增高，此外，NF-κB在HOXA11-AS siRNA组细胞核中明显表达。结论 lncRNA HOXA11-AS可以抑制骨关节炎大鼠软骨细胞的凋亡以及基质降解，并阻止NF-κB信号通路的激活。

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	钱亮
	李宏军
	施源
	邓新超 *

关键词 ： HOXA11-AS, 软骨细胞, 细胞凋亡, 基质降解, NF-κB信号通路, 表达水平

Abstract：Objective To investigate the expression of long-nocoding RNA (lncRNA) HOXA11-AS in osteoarthritis (OA) rat chondrocytes and its effect on chondrocyte apoptosis，matrix degradation and nuclear factor κB (Nuclear factor kappa B，NF-κB) .Methods Rats were injected with Freund's complete adjuvant to construct OA rat model，and chondrocytes were isolated and cultured after modeling.Small interfering RNA (Small interfering RNA，siRNA) technology was used to inhibit the expression of HOXA11-AS in chondrocytes；Cell Counting Kit-8 (CCK-8) was used to to detect the viability of chondrocytes after transfection of siRNA at different times.Quantitative Real-time PCR (qRT-PCR) detected the expression of HOXA11-AS and matrix metallo protein-13(MMP-13)a disintesrin and metalloprotease with thrombospondin type 5 motifs(ADAMTS-5)，CollagenⅡ，aggrecan in chondrocytes.Flow cytometry was used to detect chondrocyte apoptosis.Enzyme-linked immuno sorbent assay (ELISA)was used to detect the expression of BCL2-Associated X Protein（Bax）、B-cell lymphoma-2（Bcl-2）、Caspase-3 in chondrocyte supernatant.And Western blot was used to detect the expression of extracellular matrix related proteins MMP-13，ADAMTS-5，CollagenⅡ，aggrecan and anti-nuclear factor kappa B inhibitory protein（IκBα），anti-phosphorylated nuclear factor kappa B inhibitory protein （p-IκBα）、nuclear factor kappa B（NF-κB 65）.Immunofluorescence staining was used to detect the expression of NF-κB in chondrocyte nuclei.Results Compared with the normal group of chondrocytes，HOXA11-AS was highly expressed in chondrocytes of OA rats.Compared with NC siRNA group，the cell viability of HOXA11-AS siRNA group was significantly decreased.Apoptosis rate，proapoptotic factor Baxandthe expression level of Caspase-3 significantly increased，while the expression level of anti-apoptotic factor Bcl-2 was significantly decreased.Meanwhile，compared with NC siRNA group，the protein and gene expression levels of collagen II and aggrecan in HOXA11-AS siRNA group were significantly higher.The expression levels of MMP-13 and ADAMTS-5 genes and proteins were significantly increased，the expression level of IκBα protein was significantly decreased，and the expression levels of p-IκBα and NF-κB p65 were significantly increased.In addition，NF-κB was significant expression in the nucleusin HOXA11-AS siRNA group.Conclusion lncRNA HOXA11-AS can inhibit the apoptosis and matrix degradation of chondrocytes in rats with osteoarthritis and prevent the activation of NF-κB signaling pathway.

Key words： HOXA11-AS chondrocytes；apoptosis matrix degradation NF-κB signaling pathway expression level

基金资助:湖北省中医药科研项目(ZY2019M042)

通讯作者: 邓新超

作者简介: 钱亮(1981－ )，男，主治医师，湖北省武汉市第八医院骨科，430010。

引用本文:

钱亮，李宏军，施源，邓新超 *. lncRNA HOXA11-AS抑制骨关节炎大鼠软骨细胞凋亡与基质降解的作用研究[J]. 实用骨科杂志, 2020, 26(7): 614-623.
Qian Liang，Li Hongjun，Shi Yuan，et al. Effect of lncRNA HOXA11-AS on the Inhibition of Chondrocyte Apoptosis and Matrix Degradation in Osteoarthritis Rats. sygkzz, 2020, 26(7): 614-623.

链接本文:

http://www.sygkzz.com/CN/ 或 http://www.sygkzz.com/CN/Y2020/V26/I7/614

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