不同年龄大鼠胸椎椎体生长板的组织学观察及增殖能力测定

摘要

Objective To compare the histological features of the thoracic vertebral body growth plates (VBGPs) of rats at different ages and assess their proliferative capability. Methods The thoracic VBGPs obtained from rats aged 1 day and 1, 4, 8, 16 and 28 weeks were identified using safranin O-fast green staining, and the height of the hypertrophic zone, proliferative zone, and resting zone were measured. The chondrocytes were isolated from these VBGPs with a modified trypsin-collagenase type Ⅱ digestion method for primary culture in vitro. The expressions of proliferating cell nuclear antigen (PCNA) mRNA and protein was detected by real time-PCR and Western blotting, respectively. Results The 1-day- and 1-week-old rats showed significantly greater hypertrophic zone and proliferative zone in the VBGPs than older rats (P<0.01); the proliferative zone was significantly greater in rats aged 4 weeks than in those aged 28 weeks (P＜0.05). The resting zone was obviously greater in rats aged 1 day and 1 week than in older rats (P＜0.05), and also greater in rats aged 4 weeks than in those aged 16 and 28 weeks (P<0.05). Obvious ossification in the resting zone occurred at 16 weeks, and most of the resting zone became ossified at 28 weeks. The expression of PCNA decreased at both the mRNA and protein levels as the rats grew. Conclusion The 3 zones of VBGPs are greater in rats aged 1 day and 1 week than in older ones. Ossification in the resting zone begins at 16 weeks, and till 28 weeks, most of the resting zone is ossified. The proliferation ability of VBGP chondrocytes decreases with the increase of age of the rats.%目的 比较不同年龄大鼠胸椎椎体生长板的组织学差异并对不同年龄大鼠椎体生长板软骨细胞的增殖能力进行测定.方法 采用番红O-固绿染色法对1 d及1、4、8、16、28周龄大鼠胸椎椎体生长板进行染色鉴定,测定生长板软骨肥大区、增殖区、静止区的高度.采用改良的胰酶和Ⅱ型胶原酶序贯消化法对以上年龄阶段大鼠胸椎椎体生长板软骨细胞进行原代培养.采用实时定量PCR检测不同年龄大鼠胸椎椎体生长板体外培养软骨细胞增殖细胞核抗原(PCNA)的基因表达水平,采用Western blot法测定不同年龄大鼠胸椎椎体生长板体外培养软骨细胞PCNA蛋白的表达.结果 1 d及1周大鼠肥大区和增殖区明显高于其他年龄组(P＜0.01);4周大鼠增殖区高于28周大鼠(P＜0.05);1 d及1周大鼠静止区高于其他年龄组(P＜0.05),4周大鼠静止区高于16周、28周大鼠(P＜0.05).16周大鼠椎体生长板静止区内开始出现骨化现象,28周龄大鼠椎体生长板静止区大部分出现骨化现象.PCNA在基因和蛋白水平的表达随年龄增加而降低.结论 1 d及1周大鼠椎体生长板各区的高度明显高于年龄较大的大鼠.16周大鼠椎体生长板静止区内开始有骨长入,28周龄大鼠椎体生长板静止区大部分出现骨化现象.随年龄增加椎体生长板软骨细胞的增殖能力逐渐下降.