结缔组织生长因子（CTGF)在再生障碍性贫血发病机制中的作用研究

It's known that, in aplastic anemia, hematopoietic stem/progenitor cells are ordinarily injured by a viriety of adverse factors. Yet with the increasing research of pathological mechanisms of apalstic anemia (AA), abnormality of bone marrow hematopoietic microenvironment was also found to be involved in the development of this disease. Studies indicate that bone marrow-dirived mesenchymal stem cells (BM-MSC) manifest all kinds of defects including biological characteristics as well as cellular functions. BM-MSC are identified to secret abundant connective tissue growth factor (CTGF) into the hematopoietic microenvironment. In recent years, it was reported that in the BM-MSC of mouse origin, when expression of CTGF was silenced by infection of shRNA ,the cellular proliferation was dramatically inhibited and the cells underwent adipogenic differentiation easily. A series of research revealed that CTGF not only influenced cellular proliferation and adipogenic differentiation, but also inhibited cellular apoptosis and promoted angiogenesis in vitro as well as in vivo. Our examination of the mRNA microarry showed that mRNA expression level of CTGF in BM-MSC from AA patiants was largely reduced compared with the normal control. According to the literature and mRNA microarray results, in the current applicaion, we aim to measure the the concentration of CTGF in bone marrow microenvironment and the amount of CTGF that BM-MSC produce in vitro, then further explore its relationship with the functional defects of BM-MSC in AA and its possible participation in the pathogenesis of AA.

随着对再生障碍性贫血（AA）发病机制研究的不断深入，发现该病不仅存在造血干/祖细胞损伤，还涉及骨髓造血微环境的异常。再生障碍性贫血患者骨髓间充质干细胞(BM-MSC)在生物学特性和功能上几乎存在全方位异常。BM-MSC可以分泌产生丰富的结缔组织生长因子CTGF至骨髓微环境中。近年研究发现，小鼠BM-MSC转染shRNA沉默CTGF的表达后，细胞增殖受抑，向脂肪细胞分化显著增强。一系列的研究发现，CTGF不仅影响细胞的增殖，抑制细胞的脂肪分化，而且还可以抑制多种细胞凋亡，在体内和体外均促进血管新生。我们比较AA患者和正常人BM-MSC表达谱芯片，发现AA的BM-MSC中CTGF的mRNA表达水平比正常人显著减少。本项目，我们拟分析AA骨髓微环境中的CTGF水平和BM-MSC分泌表达CTGF的量和AA临床疾病参数的相关性，并且研究其与BM-MSC功能缺陷的关系，进一步探索AA病理机制。

再生障碍性贫血（AA)不仅存在造血干/祖细胞损伤，还涉及造血微环境异常，其骨髓间充质干细胞在生物学特性和功能上几乎存在全方位异常。骨髓间充质干细胞可以分泌产生丰富的结缔组织生长因子（CTGF)至骨髓微环境。一系列研究发现，CTGF不仅影响细胞增殖，抑制细胞脂肪分化，还可以抑制多种细胞凋亡，在体内和体外均能促进血管新生。我们比较了再障患者和正常人骨髓间充质干细胞的表达谱芯片，发现AA的骨髓间充质干细胞的CTGF的mRNA表达水平比正常人明显减少。本研究通过一系列实验，旨在阐明CTGF在AA的骨髓间充质干细胞的功能异常和AA发病中的作用，并探索其相关作用机制，从造血微环境的角度研究再障的病理机制。我们收集正常人和AA患者骨髓标本，培养获得骨髓间充质干细胞并进行鉴定。通过诱导成脂分化比较正常人和AA患者间充质干细胞向脂肪分化的能力；使用RT-PCR， ELISA和Western Blot方法检测比较间充质干细胞CTGF的表达水平；流式细胞术分析间充质干细胞的凋亡水平；使用Brdu Elisa的方法检测骨髓间充质干细胞的增殖情况；在骨髓间充质干细胞培养体系中加入外源性CTGF，比较其成脂、增殖、凋亡等方面的变化；使用慢病毒转染方法过表达再障患者骨髓间充质干细胞的CTGF后，比较细胞在成脂、增殖、凋亡等反面的变化。研究结果显示，与正常人相比，AA患者骨髓间充质干细胞成脂能力增强，增殖能力下降，凋亡水平增加；成脂诱导分化后PPAR-g和CEBP-d表达增强；细胞内CTGF mRNA表达水平下降，培养上清中CTGF因子分泌水平下降；培养体系红加入外源性CTGF后细胞凋亡减少，增殖能力增强，成脂能力下降；使用慢病毒转染过表达CTGF后，细胞增殖能力增强，成脂能力下降，凋亡有下降趋势。再生障碍性贫血是血液科的常见病、多发病和难治病，但其确切发病机制却仍不明确。本研究阐明了CTGF在AA的骨髓间充质干细胞功能异常和AA发病中的作用，并探索其相关机制，从一个新的角度研究AA的病理机制，具有一定的理论意义，并且有潜在性临床指导意义。