[Effect of the Connexin 43 Coupling to the Biobehavior of Multiple Myeloma Cells]

To investigate the effect of gap junction intercellular communication (GJIC) combined by connexin43 (Cx43) and its signal to the biobehavior of multiple myeloma (MM) cells, and its possible mechanism.The mesenchymal stem cell (MSC) cells were isolated and cultured from patients with MM and normal donors. The expression of connexin43 (Cx43) in MSC cells from different sources was detected by RT-PCR and Western blot. The side population (SP) cells were sorted by flow cytometry (FCM). The effect of MSC cells from different sources to the cell cycle, Cx43 expression, colony formation in vitro, stem cell related genes expression, cytokines secretion and chemoresistance in MM SP cells as well as with or without Cx43 inhibitor 18α-glycyrrhetinic acid (18α-GA) was observed.There was no significantly difference between the MSC isolated from normal donor and MM patients. Western blot showed that Cx43 expression in SP cells was up-regulated when the cells were incubated with MSC, and medium containing 18α-GA could partially inhibit it, moreover, it was more significant in MSC cells of MM patients. The ability of colony formation of SP cells in vitro was higher than those of MM cells and MM-MSC could promote the colony formation in a co-culture manner. The effect of MM-MSC to SP cells was down-regulated after 18α-GA was added. RT-PCR showed that there was several important stem cell-related genes including c-myc, Oct-4 Klf-4, and Sox-2 were found in RPMI 8226 cells, but those cells were up-regulated in SP cells (P0.001). Meanwhile, MM-MSC could up-regulate the expression of c-myc, Klf-4 and Sox-2 (P0.001), but down-regulate Oct-4 gene in the SP cells. The expression of those genes decreased after 18α-GA was added, but showed no significant difference (P0.05). Cytometry bead array assays showed that MM-MSCs could secrete high level of IL-6, but the levels of IL-6, IL-10 and TGF-β increased significantly when the MM-MSCs were co-cultured with SP cells (P0.05), especially the levels of IL-6 and IL-10 were significantly higher than cultured alone. There was no significant change in the levels of bFGF and IL-17 before and after co-cultured. The levels of IL-6, IL-10 and TGF-β in supernatant decreased significantly after GJ inhibitor 18α-GA was added. PI/Annexin V assay showed that MM cells were sensitive to bortezomib (BTZ)-induced apoptosis, but the sensitivity for SP cells was weaker. The ratio of cell apoptosis was 75.2%±0.77% and 8.12%±0.86% (P0.001), respectively. MM-MSC could down-regulate the cell apoptosis induced by BTZ, while the sensitivity of MM cells to BTZ could be partially recovered after GJ inhibitor was added.MSC derived from MM patients can enhance GJIC to maintain its "hematopoiesis" by up-regulating the expression of Cx43 in MM cells, and at the same time promote cell proliferation and drug recistance by secreting multiple cytokines, which finally contributes to the relapse of MM.连接蛋白43偶合对多发性骨髓瘤细胞生物学行为的影响.观察由连接蛋白43（Cx43）组成的细胞间隙连接通讯（GJIC）及其介导的信号对多发性骨髓瘤（MM）细胞生物学行为的影响，并探讨其可能机制。.分离、培养MM患者、正常人骨髓间充质干细胞（MSC）；采用RT-PCR及蛋白印迹检测不同来源MSC中Cx43的表达，流式细胞术分选MM侧群细胞（SP），直接共培养后观察不同来源MSC对SP细胞周期、Cx43表达、体外集落形成、干细胞相关基因表达、细胞因子分泌和耐药的变化以及加入GJIC抑制剂18α甘草次酸（18α-GA）的影响。.MM患者及正常人骨髓所获的MSC形态及表型无明显区别，蛋白印迹证实，这两种MSC均表达较高水平的Cx43，与SP细胞共培养后可上调其Cx43表达，18α-GA可部分抑制该作用，对来源于MM患者的MSC作用更为显著（P0.001）；SP细胞具较强的体外集落形成能力，MM-MSCs具促进作用，加入18α-GA后SP细胞体外集落形成能力下降；RT-PCR检测证实RPMI 8266细胞存在少量c-myc、Klf-4、Sox-2和Oct-4基因表达，但SP细胞亚群中该类基因明显上调（P0.001），MM-MSC可显著上调SP细胞c-myc、Klf-4和Sox-2基因的表达（P0.001），而下调Oct-4基因表达，加入GJ阻断剂后，上调的基因均有不同程度下调，但无明显差异（P0.05）；CBA分析结果显示，MM-MSC分泌的高水平白介素（IL），与SP细胞共培养后，其上清中IL-6、IL-10及转化生长因子-β（TGF-β）表达上调（P0.05），尤其是IL-6和IL-10较单独培养时显著上调（P0.01），碱性成纤维细胞生长因子和IL-17共培养前后则无明显变化，加入18α-GA后，上清中IL-6、IL-10和TGF-β水平均明显降低（P0.05）；PI/Annexin V检测证实，MM细胞对硼替佐米诱导的凋亡敏感，但SP细胞敏感性较差，凋亡率分别为75.2%±0.77%和8.12%±0.86%（P0.001），MM-MSC可显著减少硼替佐米介导的细胞凋亡 （P0.05），加入18α-GA可部分恢复MM细胞对硼替佐米的敏感性。.MM患者来源的MSC通过上调MM细胞Cx43表达，强化GJIC维持其“干”性，同时通过分泌多种细胞因子促进细胞增殖和耐药，是最终导致MM复发的原因之一。.