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 circulatory factors of aged mice on intervertebral disc aging phenotype of young mice

目的: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),同时联体年老小鼠组(old isochronic,O-O)。通过组织切片H&E染色观察椎间盘组织学改变,提取椎间盘组织行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检测aggrecan基因表达差异。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。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检测P16蛋白表达Y-O组比Y-Y组升高77.1±20.5%(P<0.05),RT-qPCR检测P16基因Y-O组有升高的趋势,但组间无统计学差异(P>0.05);Western Blot检测P21蛋白Y-O组比Y-Y组有升高,但无明显统计学差异(P>0.05),但RT-qPCR检测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%(P<0.05);金属蛋白酶MMP13及ADAMTS4蛋白在Y-Y组表达明显低于Y-O及O-O组(P<0.05),RT-qPCR检测MMP13及ADAMTS4基因表达差异性与蛋白表达相符合。结论:老年小鼠血液循环因素可以加速年轻小鼠椎间盘衰老退变。

Objective:The purpose of this study was to investigate the effect of blood circulation factors in aged mice parabiosis model on intervertebral disc aging.Methods:The Young(Y,age 3 month)and Old(O,age 18 month)wildtype C57B6 female mice were randomly selected to build Heterochronic Parabiosis and Isochronic Prarbiosis model.Mice were separated into three groups,Young Isochronic(Y-Y),Young Heterochronic(Y-O),old Isochronic(O-O).The H-E staining was used to observe changes in disc histology.The differences in protein and gene expression of aging genes P16,P21 and metalloproteinase MMP13,ADAMTS4 were detected by Western blot and RT-qPCR.Differences in aggrecan gene expression were detected by immunofluorescence.Results:The results suggested decreased nucleus pulposus(NP)cells and increased clefts in annulus fibrosus(AF)compartment,after exposing young disc to old blood circulation environment.The expression of aggrecan gene decreased in(Y-O)and O-O groups compared to Y-Y group with significant statistical difference among groups(P<0.05).We found P16 protein increased in Y-O group by 77.1±20.5% than that in Y-Y group(P<0.05).The P16 gene also increased,but there was no statistical difference between them(P>0.05).The p21 gene increased 40.5%±6.7%in Y-O group and 95.5%±24.2% in O-O group compared to Y-Y group(P<0.05).The P21 protein increased in Y-O group compared to Y-Y group without statistical difference(P>0.05).We also found the expression of MMP13,ADAMTS4 protein increased in Y-O and O-O group compared to Y-Y group with significant statistical difference(P<0.05).RT-qPCR detected differences in gene expression between MMP13 and ADAMTS4 that are consistent with protein expression.Conclusion:The results of this study suggest that the old global blood circulating factors may accelerate the disc aging process.