异时连体共生对小鼠椎旁肌肉衰老退变的影响

Effect of heterochronic parabiosis model on old mouse spine muscle aging

中文关键词:  连体共生  脊柱退变  年轻血液  衰老

英文关键词:Parabiosis  Spine degeneration  Young blood  Aging

中文摘要:

  【摘要】 目的：通过异时连体共生（heterochronic parabiosis，HP）模型检测年轻小鼠血液循环环境对年老小鼠脊柱椎旁肌肉的影响。方法：选择4月龄的年轻小鼠及18月龄年老雌性小鼠，通过手术建立年轻和年老小鼠同时（isochronic parabiosis，IP）、异时连体共生模型。连体8周后将小鼠分成同时连体年轻小鼠组（young isochronic，YI），异时连体年轻小鼠组（young heterochronic，YH）；同时连体年老小鼠（old isochronic，OI）和异时连体年老小鼠组（old heterochronic，OH）4组，解剖并提取椎旁肌肉。通过流式细胞仪吸光度检测各组椎旁肌肉糖胺聚糖（glycosaminoglycans，GAG）及各组DNA的含量，提取肌肉组织蛋白行蛋白印痕（Western blot）检测衰老基因P53、P21、P16及自噬基因LC3、金属蛋白酶MMP13基因的表达。取脊柱椎旁肌肉组织进行组织免疫荧光（immunofluorescence，IF）检测Aggrecan、P16及炎症因子IL8的表达差异性。结果：GAG定量分析显示YI、YH、OH、OI各组分别为2.91±0.17、2.01±0.21、2.23±0.36、1.18±0.09，其中YI与YH、OH与OI组之间有统计学差异（P<0.05）；通过IF检测Aggrecan表达呈下降趋势，YI、YH、OH、OI各组分别为717.25±64.44、611.59±28.45、683.04±17.95、570.34±31.81，其中OH与OI组间有统计学差异（P<0.05），YI与YH组间无统计学意义（P＞0.05）；LI8基因表达YI、YH、OH、OI各组分别为494.20±28.35、561.62±44.72、602.35±45.57、726.36±58.40；P16基因表达YI、YH、OH、OI各组分别为701.30±27.21、695.92±31.06、754.82±25.78、815.54±24.70，有上升趋势但YI与YH、OH与OI组间无明显差异（P>0.05）。Western blot检测自噬基因LC3Ⅱ/Ⅰ蛋白表达呈下降趋势YI、YH、OH、OI各组分别为0.39±0.09、0.29±0.05、0.26±0.04、0.16±0.01；衰老基因P21蛋白表达YI、YH、OH、OI各组分别为1.09±0.23、1.32±0.12、1.54±0.03、1.86±0.06；金属蛋白酶MMP13蛋白表达YI、YH、OH、OI组分别为0.59±0.11、0.68±0.13、0.88±0.15、1.11±0.18。在LC3 Ⅱ/Ⅰ、P21、MMP13表达中，YI与YH组间无明显差异性（P>0.05），OH与OI组间有统计学差异（P<0.05）。结论：全身血液循环可以影响椎旁肌肉退变，年轻血液环境可以延缓年老小鼠脊柱椎旁肌肉衰老退变。

英文摘要:

  【Abstract】 Objectives: Heterochronic parabiosis model was used to achieve the chimerism of young and elderly individuals through blood circulation, so as to reverse paravertebral muscle aging. Methods: With the means of surgical pairing on wild type C57B6 female mouse, young(Y, age 4 mth) and old(O, age 18 mth) by using the heterochronic parabiosis and isochronic prarbiosis model. Mouse were sacrificed eight weeks after surgical pairing. After separating four main groups, young isochronic(YI), young heterochronic(YH), old isochronic(OI) and old heterochronic(OH) were obtained. After the treatment, matrix gene expression, total glycosaminoglycans, Western blot and immunofluorescence analysis were used to determine the effects of heterochronic parabiosis model. Results: GAG assay showed that the YI, YH, OH, OI data presented a decreasing trend in each group, YI 2.91±0.17, YH 2.01±0.21, OH 2.23±0.36, OI 1.18±0.09, respectively. There was significant difference between the two groups(P＜0.05). The immunofluorescence demonstrated increased Aggrecan protein, YI 717.25±64.44, YH 611.59±28.45, OH 683.04±17.95, OI 570.34±31.81, respectively. There was a statistical difference between the OH and OI group(P<0.05). And the LI8 expression showed YI 494.20±28.35, YH 561.62±44.72, OH 602.35±45.57, OI 726.36±58.40; the P16 expression showed YI 701.30±27.21, YH 695.92±31.06, OH 754.82±25.78, OI 815.54±24.70, with no statistical significance among the groups. The expression of autophagy gene LC3 Ⅱ/Ⅰ showed a decreasing trend by using Western blot, which was YI 0.39±0.09, YH 0.29±0.05, OH 0.26±0.04, OI 0.16±0.01; the senescence gene P21 protein expression was YI 1.09±0.23, YH 1.32±0.12, OH 1.54±0.03, OI 1.86±0.06. The expression of MMP13 protein was YI 0.59±0.11, YH 0.68±0.13, OH 0.88±0.15, OI 1.11±0.18. Among the LC3 Ⅱ/Ⅰ, P21 and MMP13 expressions, there was no significant difference between YI and YH group(P>0.05), but significant difference in OH compared with OI group(P<0.05). Conclusions: Our findings provide evidence supporting the hypothesis that systemic blood circulation can affect paravertebral muscle degeneration. The heterochronic parabiosis model through the young blood circulation can variably promote tissue proliferation, reduce tissue damage, and restore organ function.