白细胞介素1β诱导大鼠骨性关节炎软骨细胞模型的效果评价

Evaluation of rat osteoarthritis chondrocyte models induced by interleukin-1beta

背景:建立骨性关节炎软骨细胞模型,对进一步解释骨性关节炎的病理过程,评估和筛选骨性关节炎治疗药物具有重要意义。目的:评价白细胞介素1β诱导大鼠骨性关节炎软骨细胞模型的效果,为进一步探索药物治疗骨性关节炎提供参考。方法:取3周龄SD大鼠髋关节软骨,机械剪切和酶消化分离软骨细胞并进行鉴定。软骨细胞随机分为3组:对照组、5 ng/mL和10 ng/mL白细胞介素1β组,分别诱导24 h和48 h。MTT法检测软骨细胞的增殖活力;Real-Time PCR检测Ⅱ型胶原蛋白、蛋白聚糖、性别决定区Y框蛋白9、基质金属蛋白酶13、解聚蛋白样金属蛋白酶5 mRNA表达;Western blot检测Ⅱ型胶原蛋白、性别决定区Y框蛋白9、基质金属蛋白酶13、解聚蛋白样金属蛋白酶5蛋白表达。结果与结论:①成功分离并培养原代大鼠软骨细胞;②10 ng/mL白细胞介素1β诱导软骨细胞24 h可使细胞增殖活力显著下降(P<0.05),5 ng/mL白细胞介素1β组则需诱导48 h;③Real-Time PCR结果显示,同对照组相比,5 ng/mL和10 ng/mL白细胞介素1β诱导软骨细胞24,48 h,Ⅱ型胶原蛋白、蛋白聚糖、性别决定区Y框蛋白9 mRNA表达水平均显著降低(P<0.05),基质金属蛋白酶13、解聚蛋白样金属蛋白酶5 mRNA表达水平均显著增加(P<0.05);相比10 ng/mL组,5 ng/mL白细胞介素1β诱导软骨细胞48 h可显著增加基质金属蛋白酶13、解聚蛋白样金属蛋白酶5 mRNA表达(P<0.05);④Western blot结果显示,相比对照组,10 ng/mL白细胞介素1β诱导软骨细胞24 h,Ⅱ型胶原蛋白、性别决定区Y框蛋白9蛋白水平显著下降(P<0.05),基质金属蛋白酶13、解聚蛋白样金属蛋白酶5蛋白水平显著增加(P<0.05);5 ng/mL白细胞介素1β诱导软骨细胞48 h,Ⅱ型胶原蛋白、性别决定区Y框蛋白9蛋白水平显著下降(P<0.05),基质金属蛋白酶13、解聚蛋白样金属蛋白酶5蛋白水平显著增加(P<0.05);⑤提示干预24 h后10 ng/mL白细胞介素1β对软骨细胞的诱导效果可能更明显;干预48 h后5 ng/mL和10 ng/mL白细胞介素1β的诱导效果相似。

BACKGROUND:Establishing a chondrocyte model of osteoarthritis is of great significance for further explaining the pathological process of osteoarthritis and evaluating and screening the therapeutic drugs of osteoarthritis.OBJECTIVE:To evaluate the effect of interleukin-1βto induce osteoarthritis models in rat chondrocyte models,thereby providing a reference for further exploration of drug treatment of osteoarthritis.METHODS:Chondrocytes were isolated from the hip cartilage of 3-week-old Sprague-Dawley rats by mechanical shearing and enzymatic digestion,and then identified.Chondrocytes were randomly divided into three groups:control group,5 ng/mL interleukin-1β-induced group,10 ng/mL interleukin-1β-induced group,with induction times of 24 and 48 hours.Chondrocyte proliferation activity was detected by MTT.Real-Time PCR was used to detect the expression of type II collagen,Aggrecan,sex-determining region Y-box protein 9(Sox9),matrix metalloproteinase 13,and a disintegrin and metaloproteinase with thrombospondin-like motifs-5(Adamts5)mRNA.Western blot was used to detect the expression of type II collagen,Sox9,matrix metalloproteinase 13 and Adamts5.RESULTS AND CONCLUSION:Primary rat chondrocytes were successfully isolated and cultured.Induction of chondrocytes by interleukin-1βat 10 ng/mL for 24 hours could significantly reduce cell proliferation and viability(P<0.05),while the 5 ng/mL interleukin-1β-induced group required 48 hours of induction to cause a significant decrease in cell proliferation and viability.Real-Time PCR results showed that compared with the control group,5 or 10 ng/mL interleukin-1βinduction for 24 and 48 hours significantly reduced the expression levels of type II collagen,Aggrecan,Sox9 mRNAs(P<0.05)and increased the expression levels of matrix metalloproteinase 13 and Adamts5 mRNAs(P<0.05).Compared with the 10 ng/mL interleukin-1β-induced group,5 ng/mL interleukin-1βinduction significantly increased the mRNA expression of matrix metalloproteinase 13 and Adamts5 in chondrocytes after 48 hours of induction(P<0.05).Western blot results showed that compared with the control group,10 ng/mL interleukin-1βinduction for 24 hours and 5 ng/mL interleukin-1βinduction for 48 hours significantly decreased the protein expression of type II collagen and Sox9 in chondrocytes(P<0.05),and significantly increased the protein expression of matrix metalloproteinase 13 and Adamts5(P<0.05).To conclude,compared with 5 ng/mL interleukin-1β,10 ng/mL interleukin-1βmay have more obvious effects on chondrocytes for 24 hours,while 5 and 10 ng/mL interleukin-1βhave similar effects after 48 hours of intervention.