Chrysis Dionisios, Nilsson Ola, Ritzen E Martin, Sävendahl Lars
Department of Woman and Child Health, Pediatric Endocrine Unit, Astrid Lindgren Children's Hospital, Karolinska Institute, Stockholm, Sweden.
Endocrine. 2002 Aug;18(3):271-8. doi: 10.1385/ENDO:18:3:271.
Apoptosis occurs in the growth plate during normal and abnormal longitudinal growth. To investigate the role of apoptosis during growth plate maturation, apoptosis and apoptosis-related proteins were studied in rat tibial growth plates at 2, 4, 8, and 11 wk of age. Apoptosis was studied by the terminal deoxynucleotidyl transferase (TdT)-mediated deoxy-UTP nick end-labeling (TUNEL) method, and immunohistochemistry was used to detect p53, caspase-3 and -6, the antiapoptotic proteins Bcl-2 and Bcl-x, and the proapoptotic proteins Bax and Bad. In all age groups studied, most apoptotic chondrocytes were terminal hypertrophic chondrocytes (THPCs) with a significant increase during development. At 2 wk, 0.108 +/- 0.026 THPCs were found to be apoptotic per millimeter of growth plate width; at 4 wk, 0.355 +/- 0.048; at 8 wk, 0.394 +/- 0.043; and at 11 wk, 1.084 +/- 0.069 (p < 0.001; 11 wk vs 2, 4, and 8 wk). THPCs were negative for p53 immunoreactivity at 2 and 4 wk, whereas some THPCs were positive at 8 and 11 wk. Caspase-3 and -6 were found in proliferative and early hypertrophic cells at 2 wk, whereas mature hypertrophic cells and THPCs were negative. At later stages of development, mature hypertrophic cells and THPCs were stained for both caspase-3 and -6. Bcl-2 and Bcl-x were present in proliferative and early hypertrophic cells at 2 wk, whereas at older ages a decrease in staining was observed. At 2 wk of age, Bax and Bad immunoreactivities were localized in proliferative and early hypertrophic cells, whereas at 8 and 11 wk many mature hypertrophic cells and THPCs were immunoreactive for Bax and Bad. Our results show that apoptosis is developmentally regulated in the rat growth plate. In older animals, with decreased growth rate and growth plate height, apoptosis is significantly increased, especially in THPCs.
细胞凋亡发生于正常及异常纵向生长过程中的生长板。为研究细胞凋亡在生长板成熟过程中的作用，我们对2周龄、4周龄、8周龄和11周龄大鼠胫骨生长板中的细胞凋亡及凋亡相关蛋白进行了研究。采用末端脱氧核苷酸转移酶（TdT）介导的脱氧UTP缺口末端标记（TUNEL）法研究细胞凋亡，免疫组织化学法检测p53、半胱天冬酶-3和-6、抗凋亡蛋白Bcl-2和Bcl-x以及促凋亡蛋白Bax和Bad。在所有研究的年龄组中，大多数凋亡软骨细胞为终末肥大软骨细胞（THPCs），且在发育过程中显著增加。2周龄时，每毫米生长板宽度发现0.108±0.026个凋亡的THPCs；4周龄时为0.355±0.048；8周龄时为0.394±0.043；11周龄时为1.084±0.069（p<0.001；11周龄与2周龄、4周龄和8周龄相比）。2周龄和4周龄时THPCs的p53免疫反应性为阴性，而8周龄和11周龄时部分THPCs为阳性。2周龄时在增殖细胞和早期肥大细胞中发现半胱天冬酶-3和-6，而成熟肥大细胞和THPCs为阴性。在发育后期，成熟肥大细胞和THPCs均被半胱天冬酶-3和-6染色。2周龄时Bcl-2和Bcl-x存在于增殖细胞和早期肥大细胞中，而在较大年龄时染色减少。2周龄时，Bax和Bad免疫反应性定位于增殖细胞和早期肥大细胞，而8周龄和11周龄时许多成熟肥大细胞和THPCs对Bax和Bad呈免疫反应性。我们的结果表明，大鼠生长板中的细胞凋亡受发育调控。在年龄较大的动物中，随着生长速率和生长板高度降低，细胞凋亡显著增加，尤其是在THPCs中。
