山东医药

目的 基于HE染色法、改良的Masson三色法、Verh?eff弹力纤维染色法、苦味酸-天狼猩红染色法观察不同月龄小鼠窦房结组织起搏细胞（P细胞）、移行细胞（T细胞）数量及间质组织成分变化。方法 选择C57野生型雄性小鼠20只，其中10日龄小鼠5只作为新生组，3～6月龄小鼠5只作为青年组，10～15月龄小鼠5只作为中年组，18～24月龄小鼠5只作为老年组。各组小鼠处死后取心脏，制作窦房结石蜡切片，采用HE染色法、改良的Masson三色法、Verh?eff弹力纤维染色法、苦味酸-天狼猩红染色法进行窦房结组织细胞学观察，记录其P细胞计数、T细胞计数、P/T细胞比例及总细胞密度、胶原纤维面积占比，Verh?eff弹力纤维染色法观察弹力纤维面积占比。结果 HE染色法观察结果：新生组、青年组、中年组、老年组P细胞计数依次降低，胶原纤维面积占比依次升高（P均<0.05）；新生组、青年组、中年组T细胞计数依次升高，而老年组低于青年组、中年组（P均<0.05）；新生组、青年组P/T细胞比例高于中年组、老年组，老年组总细胞密度低于其余各组（P均<0.05）。改良的Masson三色法观察结果：新生组、青年组、中年组、老年组P细胞计数及P/T细胞比例依次降低，胶原纤维面积占比依次升高（P均<0.05）；新生组、青年组、中年组T细胞计数依次升高，而老年组低于中年组（P均<0.05）；老年组总细胞密度低于其余各组（P均<0.05）。Verh?eff弹力纤维染色法观察结果：新生组、青年组、中年组、老年组P细胞计数依次降低，弹力纤维及胶原纤维面积占比均依次升高（P均<0.05）；新生组、青年组、中年组T细胞计数依次升高而P/T细胞比例依次降低，老年组T细胞计数高于青年组而P/T细胞比例低于青年组（P均<0.05）；老年组总细胞密度低于其余各组（P均<0.05）。苦味酸-天狼猩红染色法观察结果：与新生组比较，中年组、老年组P细胞计数均降低而胶原纤维面积占比均升高，且老年组变化更明显（P均<0.05）；青年组、中年组、老年组T细胞计数均高于新生组，中年组高于青年组（P均<0.05）；新生组P/T细胞比例高于其余各组，老年组总细胞密度低于其余各组（P均<0.05）。结论 随着月龄增长，小鼠窦房结组织P细胞计数呈下降趋势，T细胞计数呈先升高后降低趋势，间质组织中的胶原纤维、弹力纤维逐渐增多，出现间质重构现象。

Objective To observe the quantitative changes of pacemaker cells(P cells) and transitional cells(T cells) as well as interstitial tissue components in sinoatrial nodes of mice at different ages using HE staining, modified Masson's trichrome staining, Verh?eff's elastic fiber staining, and picrosirius red staining. Methods Twenty male C57 wildtype mice were divided into four groups(n=5 each): neonatal group(10-day-old), young group(3-6 months), middleaged group(10-15 months), and old group(18-24 months). After euthanasia, the hearts were harvested to prepare paraffin sections of the sinoatrial node. HE staining, modified Masson's trichrome staining, Verh?eff's elastic fiber staining, and picrosirius red staining were performed to evaluate P cell count, T cell count, P/T cell ratio, total cell density, and proportion of collagen fiber area; Verh?eff's elastic fiber staining was used to observe the the proportion of elastic fiber area. Results HE staining results: The P cell count decreased sequentially in the neonatal, young, middle-aged, and old groups, while the proportions of collagen fiber area increased(all P<0. 05). The T cell count increased in the neonatal, young, and middle-aged groups, and the T cell count was lower in the old group than in the young and middle-aged groups(all P<0. 05). The P/T cell ratio was higher in the neonatal and young groups than in the middle-aged and old groups, and the total cell density was lower in the old group than in the other groups(all P<0. 05). Modified Masson's trichrome staining results: The P cell count and P/T cell ratio decreased sequentially, while the proportions of collagen fiber area increased in the neonatal, young, middle-aged, and old groups(all P<0. 05). The T cell count increased in the neonatal, young, and middle-aged groups but decreased in the old group in comparison with that of the middle-aged group(all P<0. 05). The total cell density was lower in the old group than in the other groups(P<0. 05). Verh?eff's elastic fiber staining results: The P cell count decreased, while the elastic fiber and the proportions of collagen fiber area increased in the neonatal, young, middle-aged, and old groups(all P<0. 05). The T cell count increased, whereas the P/T cell ratio decreased in the neonatal, young, and middle-aged groups; the old group had a higher T cell count but a lower P/T cell ratio than the young group(both P<0. 05). The total cell density was lower in the old group than in the other groups(all P<0. 05). Picrosirius red staining results: Compared with the neonatal group, the middle-aged and old groups showed reduced P cell counts and increased proportions of collagen fiber area, with more pronounced changes in the old group(all P<0. 05). The young, middle-aged, and old groups had higher T cell counts than the neonatal group, and the middleaged group had a higher count than the young group(all P<0. 05). The P/T cell ratio was higher in the neonatal group than in the other groups, and the total cell density was lower in the old group than in the other groups(all P<0. 05).Conclusion With aging, the P cell count in the sinoatrial node decreases, the T cell count first increases and then decreases, and the collagen and elastic fibers in the interstitial tissues gradually increase, indicating interstitial remodeling.