STAT-1对白介素1β诱导的关节软骨细胞衰老的影响及其可能机制研究

Influence of STAT-1 on Articular Cartilage Cells Senescence Induced by IL-1β and the Possible Mechanism

目的探讨STAT-1对白介素1β(IL-1β)诱导的关节软骨细胞衰老的影响及可能机制。方法软骨细胞同步化后分为:正常对照组(正常培养的软骨细胞);IL-1β组(加入IL-1β,浓度为10 ng/ml);阴性对照组(加入IL-1β和空白质粒);STAT-1转染组(加入IL-1β和siRNA-STAT-1重组质粒)。检测各组细胞的β-半乳糖苷酶染色阳性率;采用MTT法检测4组细胞培养24、48、72 h时的增殖情况;Western blotting法检测4组细胞STAT-1蛋白的表达水平;采用端粒酶检测试剂盒进行端粒酶活性测定。结果细胞培养24 h时,各组细胞的增殖率比较,差异无统计学意义(F=3.037,P=0.087)。细胞培养48 h和72 h时,各组细胞的增殖率比较,差异均有统计学意义(F=3.754,P<0.05;F=3.871,P<0.05);其中IL-1β组、阴性对照组细胞增殖率均分别低于正常对照组、STAT-1转染组,差异均有统计学意义(P<0.05)。正常对照组β-半乳糖苷酶染色阳性率为(12.11±1.34)%,IL-1β组为(65.62±2.56)%,阴性对照组为(60.73±3.21)%,STAT-1转染组为(21.32±2.21)%,4组细胞β-半乳糖苷酶染色阳性率比较,差异有统计学意义(F=2.877,P<0.05);其中IL-1β组和阴性对照组均高于正常对照组和STAT-1转染组,差异均有统计学意义(P<0.05)。4组STAT-1表达水平比较,差异有统计学意义(F=236.150,P<0.05);其中IL-1β组和阴性对照组STAT-1表达水平均高于正常对照组和STAT-1转染组,差异均有统计学意义(P<0.05)。正常对照组端粒酶活性为(81.65±5.28)%,IL-1β组为(60.32±5.32)%,STAT-1转染组为(76.76±3.45)%。3组端粒酶活性比较,差异有统计学意义(F=26.040,P<0.05);其中IL-1β组端粒酶活性低于正常对照组和STAT-1转染组,差异均有统计学意义(P<0.05)。结论 STAT-1在IL-1β诱导的衰老关节软骨细胞中表达水平升高,可能通过影响端粒酶活性而参与软骨细胞的衰老。

Objective To study the influence of STAT-1 on articular cartilage cellular senescence induced by IL-1βand the possible mechanism. Methods After the synchronization of articular cartilage cells,they were divided into four groups：normal control group（cartilage cells normally cultured）,IL-1β group（IL-1β was added with an concentration of 10 ng/ ml）, negative control group（ IL-1β and empty plasmid were added）and STAT-1 transfection group（ IL-1β and siRNA-STAT-1 recombinant plasmid were added）. The positive rate of β - galactosidase staining of each group was detected;MTT method was adopted to observe the cell proliferation of the four groups at 24,48 and 72 h of cell culture;Western blotting method was used to detect the STAT-1 protein expression of the four groups;telomerase detection kit was used to determine telomerase activity. Results At 24 h of cell culture,the four groups were not significantly different in proliferation level（F = 3. 037,P = 0. 087）. The four groups were significantly different in the proliferation among each group at 48 h and 72 h of cell culture（F = 3. 754,P〈 0. 05;F = 3. 871,P 〈 0. 05）;IL-1β group and negative control group were lower than normal control group and STAT-1 transfection group in cell proliferation level（P 〈 0. 05）. The positive rate of β - galactosidase staining was（12. 11 ± 1. 34）%for normal control group, （65. 62 ± 2. 56）% for IL-1β group, （60. 73 ± 3. 21）% for negative control group and（21. 32 ± 2. 21）% for STAT-1 transfection group. The four groups were significantly different in the positive rate of β - galactosidase staining（F = 2. 877,P 〈 0. 05）;IL-1β group and negative control group were higher than normal control group and STAT-1 transfection group in the positive rate（P 〈 0. 05）. The four groups were significantly different in STAT-1 protein expression level （F = 236. 150,P 〈 0. 05）;IL-1β group and negative control group were higher than normal control group and STAT-1 transfection group in the expression level（P 〈 0. 05）. The telomerase activity was（81. 65 ± 5. 28）% for normal control group, （60. 32 ± 5. 32）% for IL-1β group and（76. 76 ± 3. 45）% for STAT-1 transfection group. The three groups were significantly different in telomerase activity（ F = 26. 040,P 〈 0. 05 ）;IL-1β group was lower than normal control group and STAT-1 transfection group in telomerase activity（P 〈 0. 05）. Conclusion There is higher STAT-1 protein level in the articular cartilage cellular senescence induced by IL-1β. STAT-1 may participate in the articular cartilage cells senescence by influencing telomerase activity.