晚发型脊柱骨骺发育不良伴进行性骨关节病患者的成骨细胞增殖分化及基因表达谱改变  

Proliferation, differentiation, and gene expression profile of osteoblast of patient with spondyloepiphyseal dysplasia tarda with progressive arthropathy

作　　者：周后德[1] 黄秋霞[1] 谢辉[1] 刘敏[1] 郭丽娟[1] 袁凌青[1] 隋国良[1] 翟木绪[1] 彭依群[1] 谷卫[2] 倪江东[3] 张湘生[3] 廖二元[1] 

机构地区：[1]中南大学湘雅二医院代谢内分泌研究所,长沙410011 [2]浙江大学医学院附属二医院内分泌科 [3]中南大学湘雅二医院骨科,长沙410011

出　　处：《中华医学杂志》2005年第19期1310-1314,共5页National Medical Journal of China

基　　金：国家自然科学基金资助项目(30400218);国家"九五"攻关基金资助项目(969060505)

摘　　要：目的探讨晚发型脊柱骨骺发育不良伴进行性骨关节病(SEDT PA)患者WISP3基因突变所造成的软骨及周围骨组织病变和可能的发病机制。方法从SEDT PA患者及正常同龄个体的股骨头松质骨分离成骨细胞进行体外培养,对培养的成骨细胞进行细胞增殖、骨特异性碱性磷酸酶(BAP)、骨钙素、护骨素测定,利用cDNA表达芯片进行基因表达谱分析,并用免疫组织化学方法对基因芯片进行验证。比较SEDT PA患者及正常对照的成骨细胞在细胞增殖、分化及基因表达谱等方面的改变。同时采用Northern杂交检测成骨细胞的WISP3表达情况。结果与正常人成骨细胞相比,SEDT PA患者成骨细胞的增殖能力较强(0.86±0.04vs0.71±0.10),氚胸腺嘧啶(3H TDR)的掺入量明显较高(1363cpm±350cpmvs867cpm±128cpm)。SEDT PA患者的成骨细胞骨钙素的表达水平明显低于正常同龄人(3.62ng/μg蛋白±0.3ng/μg蛋白vs0.85ng/μg蛋白±0.06ng/μg蛋白),护骨素的表达明显低于正常同龄人(9.43pg/μg蛋白±0.41pg/μg蛋白vs1.98pg/μg蛋白±0.03pg/μg蛋白),而BAP的表达未发生明显改变;WISP3基因的表达极低。cDNA表达芯片分析结果发现,与正常对照比较,SEDT PA患者的成骨细胞有22个基因表达明显上调,16个基因表达明显下调,这些基因包括有成骨细胞的标记分子。Objective To explore the proliferation, differentiation, and gene expression profile of the osteoblasts (OBs) of patient with spondyloepiphyseal dysplasia tarda with progressive arthropathy (SEDT-PA). Methods OBs from the head of femur of a SEDT-PA patient resected during operation were cultured. ELISA was used to examine the bone-specific alkaline phosphatase (BAP), osteocalcin (OC), and osteoprotegrin (OPC) in the lysate of OBs. The proliferation and survival of the OBs were evaluated with MTT method and 3H-TDR incorporation. Flow cytometry was used to observe the cell cycle. Northern blotting was used to evaluate the mRNA expression of WISP3, a member of the CCN family, mRNA in the OBs. Gene differential expression microarray was used to determine the cDNA expression. Osteoblasts from the head of femur of a patient about the same age with fracture of femur neck was used as control. Results^The cultured OBs from the SEDT-PA patient showed a higher survival capacity (0.86±0.04 vs 0.71±0.10) and more 3H-TDR incorporation (1363±350 vs 867±128). The expressions of OC and OPG were down-regulated to the 1/4.3 and 1/4.69 of the control respectively. There was no significant difference in the expression of BAP. WISP3 was very lowly expressed in the OBs of the SEDT-PA patient. In comparison with the normal control, there were up-regulation of 22 genes, including those coding chemotactic factors and inflammatory factors, and down-regulation of 16 genes in the OBs of the SEDT-PA patient with a synthetic effect of more rapid proliferation of OBs and reduction of synthesis of extracellular matrix, resulting in osteopenia. Conclusion Not significantly different between SEDT-PA patient and normal person, the WISP3 expression may not be a direct factor of SEDT-PA.

关 键 词：晚发型脊柱骨骺发育不良 进行性骨关节病 成骨细胞 增殖分化 基因表达 免疫组织化学 

分 类 号：R684[医药卫生—骨科学]