慢性淋巴细胞性白血病骨髓基质细胞和正常骨髓基质细胞的比较性分析

Comparison and Analysis between CLL-hBMSC and N-hBMSC

本研究旨在对慢性淋巴细胞性白血病原代骨髓基质细胞(CLL-human bone marrow stromal cells,CLL-hBMSC)和正常原代骨髓基质细胞(normal hBMSC,N-hBMSC)进行比较性分析,为建立CLL-CLL-hBMSC相互作用模型,从而更好地模拟CLL体内环境提供理论依据。从CLL患者和正常供者骨髓中分离单个核细胞,进行原代骨髓基质细胞(hBMSC)培养;采用实时荧光定量PCR(real-time PCR)检测CLL-hBMSC和N-hBMSC表面粘附分子如血管细胞粘附分子1(vascular cell adhesion molecule 1,VCAM-1)和细胞间粘附分子1(intercellular adhesion molecule 1,ICAM-1)的mRNA水平;采用real-time PCR和Western blot法分别检测两类不同来源的hBMSC中淋巴毒素β受体(lymphotoxin beta receptor,LTβR)的mRNA和蛋白质表达水平;采用Western blot法检测NF-κB家族成员的表达并分析细胞因子LTα1β2对NF-κB家族成员表达的影响;建立hBMSC和CLL细胞共培养体系,利用碘化丙啶(PI)染色检测细胞死亡情况。结果表明:从CLL患者骨髓中能成功地培养出贴壁的成纤维样hBMSC。与N-hBMSC相似,表面粘附分子VCAM-1和ICAM-1在两类hBMSC中的mRNA表达一致;两类hBMSC中LTβR的mRNA和蛋白表达水平相似;各NF-κB家族成员在两类hBMSC中都有表达,且蛋白水平无显著性差异;细胞因子LTα1β2可诱导hBMSC中经典性和非经典性NF-κB信号通路的活化;体外实验表明,两类hBMSC都能够有效地支持CLL细胞的体外存活。结论:两类hBMSC培养效率以及细胞形态上无明显差异。LTβR-NF-κB信号分子在两类hBMSC的表达以及活化情况相似。

This study was purposed to compare and analyze the chronic lymphocytic leukemia human bone marrow stromal cells（CLL-hBMSC） and normal hBMSC（N-hBMSC） so as to provide theoretical evidence for establishment of CLL-hBMSC interaction model to imitate CLL microenvironment. Mononuclear cells （MNC） were isolated from bone marrow of CLL patients and healthy donors and then were cultured, hBMSC were established by expanding for at least five passages. The mRNA expression of adhesion molecules, such as vascular cell adhesion molecule 1 （ VCAM-1 ） and intercellular adhesion molecule 1（ICAM-1）, was analyzed by real-time PCR. The mRNA and protein expression of lymphotoxin beta receptor （LTβR） were determined by real-time PCR and Western blot, respectively. The individual NF-KB members at protein level of CLL-hBMSC and N-hBMSC were examined by Western blot. The effect of LTα1β2 on individual NF-KB family members at protein level in CLL-hBMSC and N-hBMSC was also examined by Western blot. The death of CLL cells was determined by flow cytometry with PI staining when cultured with or without CLL- hBMSC and N-hBMSC at different time points. The results showed that the hBMSC could be established successfully from bone marrow of CLL patients, which were similar to N-hBMSC. Adhesion molecules, such as VCAM-1 and ICAM- 1, were found to be expressed at similar mRNA levels in CLL-hBMSC and N-hBMSC. LTβR expressions at mRNA and protein levels were comparable between CLL-hBMSC and N-hBMSC. The protein expression of the individual NF-KB family members could be detected in CLL-hBMSC and N-hBMSC with similar expression levels. LTα1β2 sitimulation activated both the classical （ RelA/p50 ） and alternative （ RelB/p52 ） NF-KB complexes in CLL-hBMSC and N-hBM- SC. The capacities of CLL-hBMSC and N-hBMSC to protect CLL cell survival were similar. It is concluded that there is no statistical difference between bone marrow from healthy donors and CLL patients in the efficiency of generating of hBMSC. LTI3R-NF-KB signaling molecules are expressed and activated on hBMSC with a similar pattern.