骨髓间充质干细胞与多发性骨髓瘤细胞共培养后CX43表达及SDF-1α分泌水平的变化及其意义  

Alterations of connexin 43 expression and SDF-la secretion of bone marrow mesenchymal stem cells co-cultured with myeloma cells

作　　者：张晓慧[1] 孙谕[1] 王子妍[1] 黄湛平[1] 傅晋翔[1] 

机构地区：[1]苏州大学附属第二医院血液科,215004

出　　处：《中华血液学杂志》2013年第9期788-793,共6页Chinese Journal of Hematology

基　　金：基金项目：国家自然科学基金（81071934、81272631）

摘　　要：目的构建多发性骨髓瘤（MM）细胞与骨髓间充质干细胞（Msc）共培养体系，探讨共培养后MSC连接蛋白43（CX43）表达及基质细胞衍生因子（SDF）-1a分泌水平变化及其意义。方法用Westernblot、免疫荧光法检测MM细胞系及MM原代细胞CX43的表达及SDF-1a分泌水平。建立间接及直接共培养体系共培养MM细胞和MSC，然后用CD138磁珠法分离RPMI8226细胞及MSC。用实时定量PCR、Westernblot法检测共培养前后MSCCX43表达，免疫荧光法检测CX43分布；划痕实验检测共培养后MSC间间隙连接通讯（GJIC）变化；微孔隔离实验检测18a-甘草次酸（18a—GA）对MSC诱导的RPMI8226细胞迁移的影响。ELISA法检测共培养后的MSCSDF—1a分泌水平。结果MM细胞系RPMI8226、U266、1／3MM细胞及MM原代细胞CX43mRNA呈中、低度表达，XG-4、XG-7细胞不表达CX43。骨髓MSC高表达CX43。直接或间接共培养后骨髓MSC的CX43mRNA相对表达量明显提高，分别是单独培养时的1．36倍和2．10倍，Westernblot检测显示共培养后MSCCX43蛋白表达水平也上调，免疫荧光染色显示增高的CX43主要分布在胞质。划痕实验显示在MSC与RPMI8226直接共培养后荧光染料在细胞间扩散距离增加。MSC与RPMI8226细胞直接和间接共培养体系中，MSC培养上清SDF．1et水平分别为（373．02±10．11）和（309．714-10．71）pg／ml，高于共培养前[（237．84±9．23）pg／m1]（P〈0．01），该作用可被18a-GA抑制降为（126．01±4．80）和（106．99±3．39）pg／ml。18et—GA可抑制MSC诱导的RPMI8226细胞迁移，其作用前后RPMI8226细胞迁移率分别为（8．00--0．67）％及（4．82：50．19）％。结论MM细胞与MSC直接和间接共培养均可上调MSCCX43表达水平，并促进SDF—let的分泌。间隙连接阻断剂18et—GA可降低共培养体系中SDF—1a的分泌并抑制MSC诱导的MM细胞迁移。Objective To construct a co-culture system of mesenchymal stem cells （MSC） and multiple myeloma （MM） cells and investigate the alterations of connexin 43 （CX43） expression and stromal derived growth factor （SDF）-let secretion of MSC. Methods CX43 expression and SDF-la secretion of MM cell lines （RPMI8226） and human primary MM cells were analyzed by western blot and immunofluorescence. Western blot, RT-PCR and immunofluorescence were employed to detect the alterations of CX43 expression and distribution in MSC directly and indirectly co-cultured with myeloma cells. Lucifer yellow dye spread was utilized to evaluate gap junctional intercellular communication （GJIC） between co-cultured MSC. Transwell was applied to study the transmigration of RPMI8266 induced by MSC under the condition of 18a-glycyrrhetinic acid （18a-GA）. The level of SDF-1a was detected by EILSA. Results RPMI8266, U266 and one-third primary MM cells expressed CX43 at low or moderate levels. CX43 wasn＇ t expressed in XG-4 and XG-7 cells but highly expressed in MSC. The expressions of CX43 mRNA of MSC were up-regulated after directly and indirectly co-cultured with RPMI8226, 1.36 and 2.10 times that of MSC cultured alone respectively. Western blot analysis showed that CX43 protein expression of MSC was also up-regulated, mainly distributed in cytoplasm. Lucifer yellow dye spread showed that GJIC was up-regulated in MSC. SDF-let concentration in supernatant of MSC directly and indirectly co-cultured with RPMI8226 were （373.02±10.11） pg/ml and （309.71±10.71） pg/ml respectively, which were higher than that of MSC cultured alone （237.8±9.23） pg/ml （P〈0.01）, and could be inhibited by 18et-GA[（237.84±9.23） pg/ml and （94.31±6.44） pg/ml] respectively （P〈0.01）. 18a-GA could inhibit the transmigration of RPMI8226 induced by MSC, decrease from （8.00±0.67）% to （4.82±0.19）%. Conclusion CX43 expression of MSC was up-regulated after directly and indirectly co-cultured with MM cells, which

关 键 词：多发性骨髓瘤 间质干细胞 连接蛋白43 基质细胞衍生因子-1a 细胞运动 

分 类 号：R733.3[医药卫生—肿瘤]