老年血液循环因素对年轻小鼠椎间盘衰老表型的影响

Influences of old circulatory factors on young mouse’s intervertebral disc aging phenotype

摘要:

目的：本研究通过异时联体共生（Heterochronic Parabiosis）模型评估年老小鼠血液循环因素对年轻小鼠脊柱椎间盘衰老表型的影响。方法：本研究通过手术建立年轻和年老小鼠同时（Isochronic Parabiosis，IP）、异时联体共生模型。随机选择3个月龄的年轻及18个月龄年老雌性小鼠。联体8周后分成同时联体年轻小鼠组(Young isochronic，Y-Y )，异时联体年轻小鼠组(Young heterochronic，Y-O), 同时联体年老小鼠(Old isochronic，O-O)。通过组织切片H & E染色观察椎间盘组织学改变，提取椎间盘组织蛋白行蛋白印痕（western blot）和RT-qPCR检测衰老基因P16、 P21及金属蛋白酶MMP13、ADAMTS4蛋白和基因表达的差异性。取脊柱椎间盘组织进行组织免疫荧光（Immunofluorescence, IF）检测aggrecan基因表达差异、组织化学染色（immunohistochemistry, IHC）检测自噬因子LC3的表达差异性。结果：H & E染色结果显示Y-Y组椎间盘纤维环结构完整，纤维环及髓核组织没有裂痕，纤维环及髓核分界清楚，髓核组织含大量髓核细胞，基质完整；而Y-O组可见纤维环结构破损并出现裂痕，纤维环及髓核分界不清，髓核细胞数相比Y-Y组明显减少；O-O组表现为老年椎间盘形态特征：髓核细胞减少、髓核基质及纤维环基质出现裂痕。IF检测aggrecan基因表达量Y-Y组明显高于Y-O及O-O组，各组间表达有差异性（P<0.05）。Western Blot显示P16蛋白表达Y-O组比Y-Y组升高77.1±20.5%（P<0.05）；P21蛋白表达Y-O组比Y-Y组有升高，但无明显统计学差异（P>0.05）；金属蛋白酶MMP13及ADAMTS4蛋白在Y-Y组表达低于Y-O及O-O组，组间统计学有差异性（P<0.05）。RT-qPCR检测显示P16基因异时联体后Y-O组有升高的趋势，但组间无统计学意义（P>0.05）；P21基因Y-O组比Y-Y组升高40.5%±6.7%、O-O组比Y-Y组升高95.5%±24.2%，组间有统计学意义（P<0.05）；MMP13基因在Y-O组中较Y-Y组升高接近4倍，组间统计有差异P<0.05），ADAMTS4基因表达Y-O组比同时联体Y-Y组升高33.5±22.6%，但组间统计无明显意义（P>0.05）。结论：血液循环因素可以影响椎间盘衰老退变，老年血液循环因素可以加速年轻小鼠椎间盘衰老退变。

Abstract:

Objective: The purpose of this study was to investigate the parabiosis model effect on mouse intervertebral disc aging. Methods: With the means of surgical pairing on wildtype C57B6 female mice; Young (Y, age 3 month) and Old (O, age 18 month) using the Heterochronic Parabiosis and Isochronic Prarbiosis model. Mice were sacrificed eight weeks after surgical pairing. After separation three groups, Young Isochronic (Y-Y), Young Heterochronic (Y-O), Old Isochronic (O-O). After the treatment, matrix gene expression, western blot and immunofluorescence analysis were measured to determine the effects of Heterochronic parabiosis model. Results: From the histological analysis, we found decreased nucleus pulposus (NP) cells and increased exhibit clefts in annulus fibrosus (AF)compartment, after exposure young disc in old blood circulation environment. Disc aggrecan immunofluorescence decreased in young mice paired with old mice（Y-O） compared to young mice paired with young mice(Y-Y), there was a statistical difference between groups (P < 0.05).We found P16 increased 77.1±20.5% in Y-O group than Y-Y group by Western blot analysis (P < 0.05). We also found P16 increased in Y-O group than Y-Y group by RT-qPCR analysis, but there was no statistical difference between groups (P > 0.05). Q-PCR analyzed p21 gene increased 40.5%±6.7% in Y-O group compared to Y-Y group and increased 95.5%±24.2% in O-O group compared to Y-Y group (P < 0.05). Western blot analyzed p21 protein analyzed increased in Y-O group compared to Y-Y group, but there was no statistical difference between groups (P > 0.05). We also found that MMP13、ADAMTS4 protein and gene expression increased in young mice paired with old mice compared to young mice paired with young mice. The difference had statistically significance (P < 0.05). Conclusion: The results of this study suggest the existence of the global blood circulating factors controlling the disc aging process.