鹿茸多肽对软骨终板细胞基质蛋白及其降解酶基因表达的影响

Effects of Pilose Antler Polypeptides on Matrix Protein and Matrix Degradation Enzyme mRNA Expression of Rat Cartilage Endplate Cell

目的:观察鹿茸多肽对IL-1β诱导的大鼠椎间盘软骨终板细胞的增殖及基质蛋白(Ⅱ,Ⅹ型胶原)、基质降解酶(金属蛋白酶-13)基因表达的影响。方法:选取1月龄的健康SD大鼠,对其椎间盘软骨终板细胞进行分离与培养,取第3代软骨终板细胞实验,空白对照组不做任何处置,诱导组单加入10μg/L的IL-1β,药物处理组分三组,分别在培养基中加入10μg/L的IL-1β和10μg/mL,30μg/mL,50μg/mL的鹿茸多肽。MTT比色法检测三个时间节点(24h,48h,72h)各组椎间盘软骨终板细胞的增殖情况;qPCR检测法对软骨终板细胞中基质Ⅱ,Ⅹ型胶原蛋白、基质金属蛋白酶-13基因的表达情况。结果:在24h,48h,72h时间点,MTT法检测10μg/mL鹿茸多肽组在各时间点软骨终板细胞增殖情况与IL-1β组比较,差异无统计学意义(P>0.05);而30μg/mL组和50μg/mL组均能减轻IL-1β对软骨终板细胞増殖的抑制作用,差异有统计学意义(P<0.05,P<0.01)。qPCR检测结果显示:IL-1β诱导组的Ⅱ型胶原蛋白基因的表达减弱,Ⅹ型胶原蛋白、基质金属蛋白酶-13基因的表达增强,与空白组对比差异有统计学意义(P<0.05);鹿茸多肽各浓度组均能上升Ⅱ型胶原蛋白基因的表达,下调Ⅹ型胶原蛋白、基质金属蛋白酶-13基因的表达水平,差异有统计学意义(P<0.05,P<0.01),以50μg/mL组效果显著,差异有统计学意义(P<0.01)。结论:一定浓度的鹿茸多肽(50μg/mL组)能有效地拮抗IL-1β对软骨终板细胞增殖的抑制作用,上升Ⅱ型胶原蛋白基因的表达,下调Ⅹ型胶原蛋白、基质金属蛋白酶-13基因的表达,改善了基质的代谢,起到延缓椎间盘软骨终板退变的作用。

Objective:To observe effects of pilose antler polypeptides(PAP)at different concentrations on matrix protein(collagen typeⅡand typeⅩ)and Matrix degradation enzyme(MMP-13)mRNA expression of rat cartilage endplate cell induced by the IL-1β.Methods:Chondrocytes from endplate of 4-weeks-old SD rat’s intervertebral discs were obtained and cultured.Chondrocytes at passage 3 were divided into five groups:normal group without any treatment,induced group with IL-1β(10μg/L)and PAP groups with IL-1β(10μg/L)divided into three groups adding 10μg/mL,30μg/mL and50μg/mL PAP respectively.MTT method was used to measure the proliferation at three time points(24 h,48 h,and72 h),while qPCR method was used to detect the expression of collagen typeⅡ,typeⅩand MMP-13 mRNA.Results:At 24 h,48 h,and 72 htime point,the proliferation of 10μg/mL PAP group and induced group were measured by MTT and no significant differences were found(P>0.05);while the proliferation were increased in 30 ug/ml and 50μg/mL PAP groups with significant differences(P<0.05,P<0.01).By qPCR,collagen typeⅡmRNA expression was decreased,while collagen typeⅩmRNA and MMP-13 mRNA expression were increased in induced group.Compared to the normal group,there was significant difference(P<0.05).Compared to the induced group,Collagen typeⅡmRNA expression was increased,while collagen typeⅩmRNA and MMP-13 mRNA expression were decreased in all the PAP groups with significant differences(P<0.05,P<0.01),especially the 30μg/mL PAP group and 50μg/mL PAP group(P<0.01).Conclusion:This study demonstrated PAP at certain concentrations can decrease the inhibition of endplate chondrocytes induced by IL-1β,increase the expression of collagen typeⅡmRNA and inhibit the expression of collagen typeⅩ mRNA and MMP-13 mRNA,improving matrix metabolism,resulting in relief the cartilage endplate of intervertebral disc degeneration.