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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。
Objective To compare the histological differences of thoracic vertebral growth plate in different age rats and determine the proliferative capacity of chondrocytes in vertebral growth plate of rats of different ages. Methods The thoracic vertebral body growth plate was stained with Safranin O-solid green staining method on day 1 and 1, 4, 8, 16, and 28 weeks old to determine the height of cartilage hypertrophy zone, proliferation zone and quiescent zone . Chondrocytes of the thoracic vertebral growth plate in the above age group were primarily cultured with the modified trypsin and collagenase Ⅱ sequential digestion method. Real-time quantitative PCR was used to detect the gene expression of PCNA in thoracic vertebral growth plate of rats of different ages. The expression of PCNA protein in chondrocytes of thoracic vertebral growth plate in different ages was detected by Western blot expression. Results Compared with other age groups (P <0.01), the area of proliferation and proliferation of rats in 1 d and 1 week were significantly higher than that in 28 weeks (P <0.05) The resting area was higher than other age groups (P <0.05), and the resting area of rats in 4 weeks was higher than those in 16 weeks and 28 weeks (P <0.05). At 16th week, ossification began to appear in the quiescent zone of the vertebral body growth plate. Most of the quiescent zone of 28-week-old rat’s vertebral body growth plate appeared ossification. The expression of PCNA at gene and protein level decreases with age. Conclusions The height of each area of rat ’s vertebral body growth plate in 1 d and 1 week is obviously higher than that in the older rats. At 16th week, the inguinal growth plate began to grow in the quiescent zone of the vertebral body, and the ossification occurred in most of the 28-week-old rats. With age increased proliferation of chondrocytes of vertebral growth plate gradually decreased.