白血病骨髓基质细胞促进SHI－1细胞体外侵袭及其作用机制探讨

Bone marrow stromal cells isolated from leukemia patients promotes the invasion capacity of SHI-1 cells in vitro and its underlying mechanism

目的:研究白血病患者骨髓基质细胞(bone marrow stromal cells,BMSCs)与白血病SHI-1细胞互相接触对后者侵袭能力的影响及其作用机制。方法:收集白血病患者BMSC及其条件培养液,采用RT-PCR法检测细胞外基质金属蛋白酶诱导因子(extracellular matrix metalloproteinase inducer,EMMPRIN)在细胞SHI-1和BMSC中的表达。按1?10比例将BMSC与SHI-1细胞进行混合,接种于铺有matrigel基质胶的Transwell小室内,并加入终浓度为2μg/mL的CXC趋化因子受体4(CXCchemokine receptor 4,CXCR4)或EMMPRIN功能阻断抗体,或以BMSC条件培养液进行侵袭实验。采用实时定量PCR法检测共同培养前后SHI-1细胞的基质金属蛋白酶(matrix metalloproteinase,MMP)-2、MMP-9、基质金属蛋白酶组织抑制因子2(tis-sue inhibitor of metalloproteinase 2,TIMP-2)和CXCR4 mRNA表达的改变。应用ELISA法测定无血清培养上清液中基质细胞衍生因子1(stromal cell-derived factor1,SDF-1)的含量。结果:SHI-1和BMSC细胞均表达EMMPRIN。SHI-1细胞与BMSC共培养后,其侵袭能力有显著提高,但能被CXCR4和EMMPRIN抗体阻断,条件培养液未明显提高SHI-1细胞的侵袭能力。共同培养后,SHI-1细胞的MMP-2、MMP-9、TIMP-2和CXCR4 mRNA以及无血清上清液中SDF-1的含量均有显著提高。结论:白血病患者的BMSC与白血病SHI-1细胞接触时,前者可通过其细胞表面的多种分子途径来诱导SHI-1细胞跨matrigel侵袭能力的提高,这可能是导致白血病细胞向髓外浸润的一种重要机制。

Objective：To investigate the effects of bone marrow stromal cells （BMSCs） isolated from leukemia patients on the invasion capacity of leukemia SHI-1 cells in vitro and the underlying mechanism. Methods： BMSCs were isolated from leukemia patients and their conditional culture medium were collected. The expressions of extracellular matrix metalloproteinase inducer （EMMPRIN） in SHI-1 cells and BMSC were detected by RT-PCR. The BMSC and SHI-1 cells were mixed at 1： 10 and inoculated in Matrigel-coated transwells. Then CXC chemokine receptor 4 （ CXCR4, final concentration of 2μg/mL） or functional antibody of EMMPRIN were added. The BMSC cultured medium was used as control on cell invasion test. The alteration of matrix metalloproteinase （MMP） -2, MMP- 9, tissue inhibitor of metalloproteinase 2 （ TIMP-2 ） , and CXCR4 mRNA were determined by real-time PCR before and after co-culture of SHI-1 cells and BMSC. The content of stromal cell-derived factor 1 （ SDF-1 ） in serum-free supernatent was measured by ELISA. Results：Both SHI-1 and BMSC expressed EMMPRIN. The invasion capacity of SHI-1 cells increased significantly after co-culture with BMSC, which could be blocked by CXCR4 and functional antibody of EMMPRIN. The cultured medium of BMSC did not increase the invasion capacity of SHI-1 ceils. The mRNA expression levels of MMP-2, MMP-9, TIMP-2, and CXCR4 as well as SDF-1 contents in serum-free supernatent increased significantly after the SHI-1 cells were co-cultured with BMSC. Conclusion：When BMSC islated from leukemia patients contacted with leukemia SHI-1 cells, they increases the invasion capacity of SHI-1 cells through multiple molecule pathways on the surface of them on cell invasion test. It may be an important mechanism responsible for the invasion of leukemia cells to the outer space of bone marrow.