骨髓内皮细胞产生造血抑制因子

Hematopoietic Inhibitors Elaborated by Bone Marrow Endothelial Cells

本研究探讨骨髓内皮细胞分泌的造血抑制因子在扩增造血祖细胞中的作用并初步探讨其作用机制。收集无血清骨髓内皮细胞条件培养液 (BMEC CM) ,离心超滤 ,获得 >1 0kD和 <1 0kD的组分 ,以观察BMEC CM原液及其 >1 0kD和 <1 0kD组分对CFU GM及HPP CFC的增殖作用的影响 ;检测骨髓内皮细胞及BMEC CM中抑制因子的表达 ,用抗体中和实验检测BMEC CM中的抑制因子对造血集落的抑制活性 ;用膜杂交法检测抑制因子联合造血刺激因子扩增造血细胞时增殖与分化相关基因的改变。结果表明 :①BMEC CM ,>1 0kD及 <1 0kD组分分别加入集落培养体系 ,BMEC CM对CFU GM及HPP CFC的生成无明显影响 ,>1 0kD组分增加CFU GM及HPP CFC的生成 ,<1 0kD组分抑制CFU GM及HPP CFC的生成。②BMEC CM ,>1 0kD及 <1 0kD组分分别加入骨髓细胞液体培养体系培养 2 4小时后 ,BMEC CM组CFU GM明显减少 ,HPP CFC显著增加 ;>1 0kD组分组CFU GM明显增加 ,HPP CFC无显著改变 ;<1 0kD组分组CFU GM及HPP CFC均明显减少。③小鼠骨髓内皮细胞表达MIP 2 ,MIP 1α ,MSP ,TGF β,TNF α,IFN γ及Tβ4。BMEC CM中存在有MIP 2 ,MIP 1α ,MSP ,TGF β ,TNF α和Tβ4。④抗体中和实验结果表明在BMEC CM中的TGF β ,MSP ,MIP 1α,IFN γ和Tβ4有明显抑制活性。

In this study, the roles of hematopoietic inhibitors elaborated by bone marrow endothelial cells in the proliferation and differentiation of hematopoietic progenitors were investigated. Murine bone marrow endothelial cell conditioned medium (BMEC CM) was collected and the components with >10 kD and <10 kD were obtained by centrifugal ultrafiltration. The effect of BMEC CM and its components on proliferation of hematopoietic progenitors was evaluated by CFU GM and HPP CFC assay and antibody neutralization test. The expression of the inhibitors in BMEC and BMEC CM was detected by RT PCR and Western blot, and change of proliferation and differentiation related genes during expansion of hematopoietic progenitors was examined by membrane hybridization technique. The results: (1)When BMEC CM and its components directly were added to CFU GM and HPP CFC culture system, BMEC CM had no effect on colony formation, >10 kD component enhanced and <10 kD component inhibited the formation of CFU GM and HPP CFC. (2)When BMEC CM and its components were added to liquid culture system of marrow cells, after 24 hours incubation, CFU GM decreased and HPP CFC increased significantly in BMEC CM group, CFU GM increased and HPP CFC had no significant change in >10 kD component group; and both CFU GM and HPP CFC reduced in <10 kD group. (3)MIP 2, MIP 1α, MSP, TGF β, TNF α, IFN γ and Tβ4 were expressed in murine marrow endothelial cells, and MIP 2, MIP 1α, MSP, TGF β, TNF α and Tβ4 were existed in BMEC CM. (4)Antibody neutralization test results demonstrated that TGF β, MSP, MIP 1α, IFN γ and Tβ4 exsited in BMEC CM had significant suppressive effects on CFU GM and HPP CFC. (5)Tβ4 combined with 5 hematopoietic cytokines (SCF, IL 3, IL 6, GM CSF and EPO) added to CD34 + cells expansion culture system, HPP CFC significantly increased compared with 5 cytokines group. Tβ4 could downregulated the expression of proliferation and differentiation related genes and signal transduction related genes. It is concluded that BMEC CM promotes the proliferation of early hematopoietic progenitor cells, and this effect is related with the inhibitors existed in BMEC CM and it could be executed via influencing cell proliferation and differentiation related genes and signal related genes.