成骨细胞抑制因子dickkopf 1促进多发性骨髓瘤溶骨病变发生机制研究

Multiple myeloma (MM) is unique in its propensity to cause bone destruction in eighty percent of patients. This complication is a significant clinical problem for which there is no effective cure and not reversible. The mechanism of MM bone disease is due to stimulation of osteoclasts (OC) and inhibition of osteoblasts (OC). However, the molecular mechanism is not clear yet. Dickkopf1(DKK1) is a strong factor secreted by myeloma cells to inhibit OB fuction through blocking Wnt signaling pathway. Based on preliminary data, we have found serum level of stromal cell derived factor 1α (SDF-1α), which mediates hematopoietic stem cell homing together with its surface ligand CXCR4, positively correlated with serum DKK1 level in newly diagnosed MM patients. SDF-1α enhanced DKK1 mRNA expression in RPMI 8226 myeloma cell line. To further study the interreaction between SDF-1α and DKK1, we plan to recruit 100 cases of newly diagnosed MM. Serum SDF-1α and DKK1 levels will be detected by ELISA. The relationship between these two factors and clinical prognostic parameters and severity of bone disease will be analyzed, including stage, cytogenetic abnormalities, alkaline phosphatase (ALP), renal dysfunction, numbers of osteolytic lesions, plasma cell leukemia etc. In addition, DKK1 mRNA and protein level will be tested by real time PCR and ELISA in primary myeloma cells treated with SDF-1α or CXCR4 antagonist AMD3100. Primary bone marrow stromal cells (BMSC) of MM patients are induced to differentiate into OB or adipocyts when treated with SDF-1α or DKK1. Once OB differentiation is inhibited, adipocytes may have the predominance to grow. The capability of differentiation into OB will be tested by cytoplasma ALP and mineralization, while adipocyts by oil red O staining. When BMSC are treated with DKK1, Wnt3a, which is a stimulator of Wnt signaling pathway, will be added to salvage βcatenin synthesis. SDF-1α mRNA and protein in BMSC will be tested by real time PCR and ELISA after treatment of DKK1 and Wnt3a. This study is to further explain the molecular mechanism of myeloma bone disease, expecially emphasizing the liaison between OB dysfunction and OC activation. To block the interreaction between SDF-1α and DKK1 potentially leads to the improvement of myeloma bone disease.

80%多发性骨髓瘤(MM)患者并发骨病，严重程度与预后相关，为破骨细胞(OC)激活和成骨细胞(OB)受抑导致，但分子机制不明。Dickkopf1(DKK1)是瘤细胞分泌的强有力OB抑制剂。基质细胞衍生因子(SDF)1α介导瘤细胞归巢粘附并激活OC。前期结果显示MM患者血清DKK1与SDF1α水平存在正相关，且SDF1α体外显著促进瘤细胞株DKK1 mRNA表达。本研究拟收集100例初治MM患者临床资料，测定血清DKK1和SDF1α并分析和骨病、预后的关系。免疫磁珠分选原代瘤细胞，实时PCR和ELISA检测SDF1α对瘤细胞DKK1基因和蛋白表达的影响。培养MM患者骨髓基质细胞(BMSC)，评价DKK1对BMSC分泌SDF1α及向OB和脂肪细胞分化的影响。本课题旨在深入探讨MM骨病的分子机制，强调破骨激活和成骨抑制之间的密切联系，并初步研究阻断DKK1和SDF1α对MM骨病的改善作用。

多发性骨髓瘤（MM）患者80%并发骨质疏松和溶骨病变，骨病严重程度和预后相关，为破骨细胞（OC）异常激活和成骨细胞(OB)功能抑制造成，但其分子机制不明。Dickkopf1(DKK1)是瘤细胞分泌的强有力OB抑制剂。基质细胞衍生因子(SDF)1α介导瘤细胞归巢粘附并激活OC。为进一步揭示骨病发生机制，本研究主要完成以下内容（1）评价血清DKK1和SDF1α水平和临床骨病、预后指标之间的相关性。（2）评价SDF-1α对骨髓细胞株和纯化的原代肿瘤细胞DKK1基因和蛋白表达的影响。（3）评价DKK1对MM患者骨髓基质细胞（BMSC）分泌SDF1α及向OB和脂肪细胞分化的影响。.主要研究结果如下：.1..临床部分：共收集46例新诊断多发性骨髓瘤患者的临床资料，血清测定SDF-1α和DKK-1, CT三维骨重建判断骨病严重程度。结果显示高分辨胸部CT骨重建用于评估MM骨病比X线更敏感，5-10mm和10mm以上的病灶CT检出率均显著高于X线。另外高危细胞遗传学阳性的MM患者更多出现CT溶骨病变而不是骨折。溶骨病变对染色体13q缺失的意义更大。.2..实验室部分.1).MM患者血清SDF-1α和DKK-1之间存在正相关，而正常对照者中未见相关性。.2).SDF-1α促进骨髓瘤细胞株DKK1 mRNA表达。原代骨髓瘤细胞经SDF-1α体外作用后DKK-1mRNA表达呈现异质性。在完成的9例患者中，有4例患者基线表达极低水平DKK-1，而其他5例表达DKK-1mRNA的患者经SDF-1α作用后均有不同程度升高，且升高具有显著性。.3).DKK1对MM患者骨髓基质细胞（BMSC） SDF-1α转录水平的影响：直接作用下骨髓瘤患者BMSC中SDF-1αmRNA的表达并无明显变化，当加入Wnt通路促进剂Wnt-3a后SDF-1αmRNA表达下降，而同时加入DKK-1后表达有恢复，证明DKK-1对BMSC表达SDF-1α的影响通过Wnt通路实现。但对BMSC的分化无阳性发现。. 本研究旨在深入探讨骨髓瘤骨病的分子机制，确立DKK1和SDF1α对MM骨病乃至疾病预后的重要意义；并初步研究阻断DKK1和SDF1α对MM骨病的改善作用。后续工作包括收集骨髓瘤患者血清原代细胞，继续研究DKK和SDF相互促进的分子通路机制。