敲低SOX5对骨关节炎软骨细胞生物学功能的影响

Biological functions of transcription factor SOX5 in osteoarthritis cartilage cells

目的探讨转录因子SOX5在骨关节炎软骨细胞中的生物学功能。方法分离培养人骨关节炎(osteoarthritis,OA)软骨细胞;运用脂质体2000转染法将si SOX5转染OA软骨细胞(OA-si SOX5),以转染NCsi RNA为阴性对照(OA-NCsi RNA);运用Real time PCR检测转染的OA软骨细胞中SOX5、白介素6(IL-6)、白介素1β(IL-1β)、II型胶原(COL2A1)及蛋白多糖(ACAN)m RNA的表达水平;酶联免疫法(ELISA)检测OA软骨细胞培养上清中IL-6和IL-1β浓度;Western blot检测SOX5、基质金属蛋白酶1(MMP-1)和MMP-13蛋白表达水平;运用Annexin V-FITC及流式细胞术检测细胞凋亡。结果 OA-si SOX5细胞中IL-6(0.72±0.05)和IL-1βm RNA(0.64±0.07)表达显著低于OA-NCsi RNA细胞(1.32±0.08、1.64±0.09)(P<0.05);COL2A1(1.27±0.08)和ACAN m RNA(2.38±0.17)显著高于OA-NCsi RNA细胞(0.58±0.04,1.25±0.10),(P<0.05);OA-si SOX5细胞中IL-6(175.2±14.5)pg/ml和IL-1β(102.6±20.3)pg/ml浓度显著低于OA-NCsi RNA细胞[(584.6±74.5)pg/ml和(268.4±25.3)pg/ml)](P<0.05);细胞凋亡率OA-si SOX5[(3.04±0.86)%]显著低于OA-NCsi RNA细胞[(18.35±2.74)%](P<0.05);OA-si SOX5细胞中MMP-1(0.53±0.05)和MMP-13蛋白水平(0.46±0.08)明显低于OA-NCsi RNA细胞(1.95±0.11、2.48±0.24)(P<0.05)。结论下调SOX5表达可能通过抑制基质金属蛋白酶的表达、促进细胞外基质的合成与分泌、抑制细胞凋亡和炎症反应进而缓减OA的发展。

Objective To investigate biological functions of transcription factor SOX5 in osteoarthritis cartilage cells. Methods Human osteoarthritic cartilage chondrocytes （ OA cartilage cells ） were isolated and cultured. OA cartilage cells were transfected with siSOX5 using lipidosome 2000 and named as OA-siSOX5 cells, while OA cartilage cells transfected with NCsiRNA served as negative control. Levels of SOX5, interleukin 6 （ IL-6 ）, IL-1β, Collagen II （ COL2A1 ）, protein polysaccharide （ ACAN ） mRNA expression in OA cartilage cells transfected with siSOX5 or NCsiRNA were detected using Real time PCR. Concentration of IL-6 and IL-1β in culture supernatants of OA cartilage cells transfected with siSOX5 or NCsiRNA were measured using ELISA. The levels of SOX5, matrix metalloproteinase 1 （ MMP-1 ） and MMP-13 protein expression were detected using Western blot and apoptosis was measured using AnnexinV-FITCI and flow cytometry. Results Levels of IL-6 （ 0.72 ± 0.05 ） and IL-1β mRNA （ 0.64 ± 0.07 ） in OA-siSOX5 cells were significantly higher than OA-NCsiRNA cells （ 1.32 ± 0.08, 1.64 ± 0.09 ） （ P 〈 0.05 ）, while the levels of COL2A1 （ 1.27 ± 0.08 ） and ACAN mRNA （ 2.38 ± 0.17 ）, and the concentration IL-6 （ 175.2 ± 14.5 ） pg / ml and IL-1β （ 102.6 ± 20.3 ） pg / ml in OA-siSOX5 cells were notable lower compared with OA-NCsiRNA cells [ （ 584.6 ± 74.5 ） pg / ml, （ 268.4 ± 25.3 ） pg / ml; P 〈 0.05 ]. The levels of MMP-1 （ 0.53 ± 0.05 ） and MMP-13 protein （ 0.46 ± 0.08 ） in OA-siSOX5 cells were significantly lower than OA-NCsiRNA cells （ 1.95 ± 0.11, 2.48 ± 0.24; P 〈 0.05 ）. Conclusions Down-expressed SOX5 could relieve the development of OA by inhibiting the expression of matrix metalloproteinase, apoptosis and inflammation, promoting synthesis and excretion of the extracellular matrix.