李书坛 黄纯兰

[摘要] 目的 研究BMP-2联合人骨髓间充质干细胞（MSC）造血干细胞（HSC）体外增殖的影响。 方法 培养MSC至第三代，磁珠分选仪分选HSC，并用流式细胞术鉴定MSC与HSC。将获得的MSC与HSC利用Transwell非接触共培养，100 ng/mL的骨形态发生蛋白（BMP-2）干预，即实验分四组：HSC组、HSC+BMP2组、HSC+MSC组、HSC+MSC+BMP2组。培养3 d后比较不同培养条件下HSC计数、RNA浓度的不同，并通过实时荧光定量PCR法及免疫荧光方法检测HSC的Ki67的mRNA及蛋白的表达。 结果 HSC的计数、总RNA含量、Ki67的表达在HSC+BMP2组高于HSC组、HSC+MSC组高于HSC组、HSC+MSC+BMP2组高于HSC+MSC组（P < 0.05）。 结论 MSC协同BMP-2蛋白可促进HSC的增殖。

[关键词] 造血干细胞；间充质干细胞；BMP-2；增殖

[中图分类号] R331.2 [文献标识码] A [文章编号] 1673-7210（2018）04（c）-0004-05

The effect of bone marrow mesenchymal stem cell synergistic BMP-2 in the proliferation of hematopoietic stem cell

LI Shutan HUANG Chunlan

Department of Hematology， the Affiliated Hospital of Southwestern Medical University， Sichuan Province， Luzhou 646000， China

[Abstract] Objective To explore the proliferation effect of bone morphogenetic protein （BMP）-2 on hematopoietic stem cell （HSC） cocultured with mesenchymal stem cell （MSC）. Methods MSCs were expanded to the third passage （P3） in the plastic dish； CD34+ cells were sorted by MACS Miltenyi Biotec. MSCs（P3） and HSCs were identified by FCM separately， and then MSC （P3） and HSC were co-cultured in the Transwell interfered with BMP-2 protein， four groups in the study： HSC group， HSC+BMP2 group， HSC+MSC group， HSC+MSC+BMP2 group. Cells were collected after 3 d to test HSC proliferation using the following methods：counting of the number， detecting of the total RNA， the mRNA and protein expression of Ki67 were detected by fluorescence qRT-PCR and immunofluorescence methods. Results The number of HSC， totel RNA， Ki67 expression of the HSC+BMP2 group was higher than HSC group， HSC+MSC group was higher than HSC group， HSC+MSC+BMP2 group was higher than HSC+MSC group （P < 0.05）. Conclusion MSCs synergistic with BMP-2 can enhance MSC proliferation effect on HSC.

[Key words] Hematopoietic stem cell； Mesenchymal stem cell； BMP-2； Proliferation

造血干细胞（hematopoietie stem cells，HSC）自我更新及多向分化等生理过程都离不开其生存的微环境增殖。骨髓基质细胞及其前体间充质干细胞（mesenchymal stem cell，MSC）以及它分泌的黏附分子、細胞因子和细胞外基质形成复杂的信号网络，它们共同构成的微环境即微龛“niche”调控着HSC的增殖等。从微环境的角度研究MSC对HSC的增殖作用及其机制是当前的研究热点[1-3]。BMP-2蛋白是多功能蛋白，可直接参与造血的调控，对造血表现出抑制、促进[4-5]等作用，此外BMP-2能促进新骨的形成，同时新生出血管。已有大量研究证实MSC对HSC具有增殖作用[6]，其依赖于细胞直接接触间的相互作用、分泌的细胞外基质和细胞因子[7-8]，且接触比非接触共培养具有更强的扩增作用[9]。本研究利用Transwel的上室底部网板微孔直径仅0.4 μm，使得上室的HSC不能进入下室，排除了细胞间直接接触对HSC增殖的影响，但细胞分泌的细胞因子可以通过该微孔，利于探讨这种作用可能是通过MSC分泌的细胞因子促进了HSC的增殖。……