大鼠颞下颌关节骨关节炎髁突软骨细胞自噬功能下降对细胞凋亡的影响

Loss of autophagy in condylar chondrocytes causes increased apoptosis rate in temporomandibular joint osteoarthritis of rats

目的观察大鼠颞下颌关节骨关节炎(temporomandibular joint osteoarthritis,TMJOA)髁突软骨细胞自噬功能改变对细胞凋亡的影响。方法选择雄性2个月龄无特定病原体(specific pathogen free,SPF)级SD大鼠40只,分为对照组(n=20)和实验组(n=20)。实验组所有大鼠均造单侧前牙反模型模拟TMJOA。8周后处死所有大鼠,并取颞下颌关节,分别提取两组大鼠髁突软骨细胞进行体外培养至第3代。应用免疫荧光技术检测软骨细胞中Ⅱ型胶原、基质金属蛋白酶-13(matrix metalloproteinase-13,MMP-13)及细胞自噬水平标志物轻链蛋白-3(light chain-3,LC-3)表达水平;应用免疫组化技术检测细胞糖原累积指标糖原蛋白-1和细胞凋亡指标胱天蛋白酶-3(caspase-3)表达水平。应用Tunel技术检测两组细胞72 h凋亡率。裂解细胞提取全蛋白,应用蛋白质印迹法检测Ⅱ型胶原、MMP-13、LC-3、糖原蛋白-1及胱天蛋白酶-3的表达水平并做灰度分析。结果免疫荧光结果显示,与对照组相比,实验组髁突软骨细胞内Ⅱ型胶原和LC-3表达减少,MMP-13、糖原蛋白-1及胱天蛋白酶-3表达均增多。实验组软骨细胞的72 h凋亡率[(17.3±4.4)%]显著高于对照组[(5.6±2.1)%](t=10.732,P<0.001);蛋白质印迹法条带灰度统计结果显示,在实验组软骨细胞内,Ⅱ型胶原表达量(0.43±0.21)显著低于对照组(0.71±0.26)(t=2.409,P=0.043);LC-3表达量(0.09±0.04)显著低于对照组(0.39±0.18)(t=3.638,P=0.007);MMP-13的表达量(0.73±0.31)显著高于对照组(0.24±0.10)(t=3.364,P=0.010);糖原蛋白-1表达量(0.68±0.30)显著高于对照组(0.29±0.17)(t=2.529,P=0.035);胱天蛋白酶-3表达量(0.19±0.08)显著高于对照组(0.05±0.02)(t=3.796,P=0.005)。结论大鼠发生TMJOA时,髁突软骨细胞自噬水平下降、糖原累积增多,软骨细胞凋亡率增高、数量减少,最终髁突软骨组织出现退变。

Objective To observe the effect of autophagy of condylar chondrocytes on apoptosis in temporomandibular joint osteoarthritis(TMJOA)of rats.Methods Fourty male 2-month-old SPF SD rats were equally divided into sham group(n=20)and experimental group(n=20).UAC metal prosthesis was cemented to the left incisors of maxilla and mandible of the rats in experimental group rats.After 8 weeks,all rats were sacrificed and the temporomandibular joint was taken.Two groups of rat condylar chondrocytes were extracted and cultured in vitro to the third generation.Immunofluorescence technique was used to detect the levels of collagenⅡand matrix metalloproteinase-13(MMP-13)in chondrocytes.The level of light chain-3(LC-3),an autophagy marker of chondrocytes,was detected.Immunohistochemical technique was used to detect the level glycogenin-1,a glycogen formation marker of chondrocyte,was detected.The level of caspase-3,an apoptosis marker of chondrocyte,was also detected.Tunel technique was used to detect the apoptosis rate of the two groups at 72 h.Cracking cell extraction of total protein,Western-blotting(WB)technology to detect the levels of collagenⅡ,MMP-13,LC-3,glycogenin-1,caspase-3 and make gray analysis.Results Compared with sham group,the level of collagenⅡdecreased,MMP-13 increased,LC-3 decreased,glycogenin-1 increased and caspase-3 increased in experimental group.The apoptosis rate of chondrocytes in experimentaal group[(17.3±4.4)%]at 72h was higher than that in control group[(5.6±2.1)%](t=10.732,P<0.001).WB bands gray statistical results show that the level of collagenⅡin chondrocytes of experimental group(0.43±0.21)was lower than that of control group(0.71±0.26)(t=2.409,P=0.043),the level of MMP-13 in chondrocytes of experimental group(0.73±0.31)was higher than that of control group(0.24±0.10)(t=3.364,P=0.010),the level of LC-3 in chondrocytes of experimental group(0.09±0.04)was lower than that of control group(0.39±0.18)(t=3.638,P=0.007),the level of glycogenin-1 in chondrocytes of experimental group(0.68±0.30)was higher than that of control group(0.29±0.17)(t=2.529,P=0.035),the level of caspase-3 in chondrocytes of experimental group(0.19±0.08)was higher than that of control group(0.05±0.02)(t=3.796,P=0.005).Conclusions The level of autophagy of condylar chondrocytes in temporomandibular joint of rats decreased,glycogen accumulation increased,the rate of chondrocyte apoptosis increased,and the number of chondrocytes decreased,resulting in degeneration of condylar cartilage tissue.