人骨髓基质细胞向成骨细胞分化过程中差异表达基因的筛选

生理学报 Acta Physiologica Sinica , August 25, 2006, 58 (4): 370-376

370

Received 2006-03-30 Accepted 2006-06-02

This work was supported by the National Natural Science Foundation of China (No. 30400251).

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Corresponding author. Tel: +86-10-62179164; Fax: +86-10-62173457; E-mail: jmchun@

人骨髓基质细胞向成骨细胞分化过程中差异表达基因的筛选

刘美玲，石心泉，周万灏，刘洪文，李 东，贾孟春*

国家人口计划生育委员会科学技术研究所，北京 100081

摘 要：为了探讨人骨髓基质细胞(bone marrow stromal cells, BMSCs)向成骨细胞分化过程中差异表达的基因，本实验采用体外培养人BMSCs ，诱导向成骨细胞分化。分别选取培养12和21 d 的细胞作为驱动方(driver)和实验方(tester)，进行抑制消减杂交，构建cDNA 消减文库，将挑选出的阳性克隆与GenBank 人基因库中已公布的核酸序列进行同源性比较分析。结果表明，从培养21 d 的BMSCs 中，筛查出5个差异基因，与人基因库中已知基因的同源性分别达到90%以上。有兴趣的是，核心蛋白聚糖和Bax inhibitor 1在培养21 d 的BMSCs 中差异表达。RT-PCR 检测显示，核心蛋白聚糖基因在培养21d 的细胞中高表达，而在12 d 的细胞中未检测到表达；Bax inhibitor 1基因在培养21 d 细胞中的表达明显高于12 d 的细胞。关键词：骨髓基质细胞；成骨细胞；核心蛋白聚糖；基因中图分类号：R 329

Screening differentially expressed genes in human bone marrow stromal cells at defined stage of differentiation

LIU Mei-Ling, SHI Xin-Quan, ZHOU Wan-Hao, LIU Hong-Wen, LI Dong, JIA Meng-Chun *

National Population Research Institute for Family Planning, Beijing 100081, China.

Abstract: To screen differentially expressed genes involved in osteogenic differentiation of human bone marrow stromal cells (BMSCs)at defined stages, subtractive cDNA library was established by means of suppression subtractive hybridization. The BMSCs cultured for 12 and 21 d were used as driver and tester, respectively. A subtract library was successfully constructed and five positive clones were selected from the library. Sequencing analysis and homology comparison showed that the five clones differentially expressed in BMSCs cultured for 21 d were at least 90% homologous with the known genes in human GenBank. It was interestingly found that the osteogenic BMSCs cultured for 21 d differentially expressed decorin and Bax inhibitor 1. RT-PCR was performed to confirm the differentially expressed genes. The results showed that the expression of Bax inhibitor 1 was significantly higher in the cells of 21-day than that of 12-day, while the expression of decorin was only detected in the cells of 21-day.Key words: bone marrow stromal cells; osteoblast; decorin; gene

正常骨量的维持需要骨祖细胞不断提供足够的成骨细胞，而正常骨转换中所需要的成骨细胞来源于骨髓基质细胞(bone marrow stromal cells, BMSCs)。BMSCs 是骨髓基质的一种间充质细胞，它能产生骨祖细胞系。BMSCs 具有多向分化潜能，在一定的诱导条件下可向脂肪细胞、肌细胞、软骨细胞，甚至神经细胞等分化[1,2]。定向分化的骨祖细胞首先增

殖并分化为前成骨细胞，同时表达I 型胶原和碱性磷酸酶(alkaline phosphatase, ALP)。I 型胶原的合成在细胞增殖期占主导地位，并与细胞的进一步分化有很大的关系。随后前成骨细胞分化为早期的成骨细胞并表达骨桥蛋白(osteopontin, OPN)，同时具有了骨基质形成的能力。最终，分化成熟的成骨细胞开始骨钙素(osteocalcin, OC)的表达，并进一步分化