细胞间隙连接在多发性骨髓瘤SP细胞生物学行为中的作用  

Role of intercellular junctions in the biological behavior of SP cells of multiple myeloma

作　　者：王子妍 张晓慧 芮明忠 周敏 傅晋翔 WANG Ziyan;ZHANG Xiaohui;RUI Mingzhong;ZHOU Min;FU Jinxiang(Department of Hematology,Soochow Hopes hematonosis hospital,Suzhou 215000,China)

机构地区：[1]苏州弘慈血液病医院血液科,江苏苏州215000

出　　处：《药学实践杂志》2022年第4期326-334,共9页Journal of Pharmaceutical Practice

基　　金：国家自然科学基金(81272631)资助。

摘　　要：目的观察不同来源间充质干细胞(MSCs)中由连接蛋白43(Cx43)组成的细胞间隙连接(GJIC)及其介导的信号对多发性骨髓瘤(MM)侧群细胞(SP细胞)生物学行为的影响,并探讨其可能机制。方法分离培养不同来源的间充质干细胞(MSCs);应用流式细胞术分选MM细胞株RPMI 8266的SP细胞;采用RT-PCR技术及蛋白印迹(Western blot)法检测不同来源MSCs、RPMI 8266、SP细胞中Cx43基因及蛋白水平表达;直接共培养观察不同来源MSCs对SP细胞周期、Cx43蛋白表达、体外集落形成能力、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连接通道抑制剂18α甘草次酸(α-GA)后的影响。结果MM-MSCs与ND-MSCs形态及表型无明显区别,与RPMI 8266细胞均表达较高水平的Cx43;与MM-MSCs共培养可使更多SP细胞进入G0期(P<0.001),SP细胞的c-myc、KIF4和SOX2基因表达显著上调,而Oct-4基因表达下调,加入α-GA后,c-myc、KIF4和SOX2均有不同程度下调,但无显著性差别;使Cx43表达上调,分别为(31.00±2)%和(39.00±2)%;使体外集落形成能力上调,加入α-GA可部分抑制该作用;RPMI 8266存在少量c-myc、KIF4、SOX2和Oct-4基因表达,SP细胞亚群中该类基因明显上调,MM-MSCs分泌高水平的白介素(IL)-6,与SP细胞共培养后,其上清液中IL-6、IL-10及TGF-β表达上调(P=0.0072,P=0.037);bFGF和IL-17则无明显变化。加入α-GA后,上清液中IL-6、IL-10和TGF-β水平降低;MM细胞对硼替佐米诱导的凋亡敏感,但SP细胞敏感性较差,与MM-MSCs共培养显著减少硼替佐米介导的细胞凋亡,加入α-GA可部分恢复MM细胞对硼替佐米的敏感性。结论MM-MSCs与多发性骨髓瘤SP细胞上调Cx43蛋白表达,形成更多GJIC,并通过改变MSCs细胞因子分泌谱,促进SP细胞增殖和耐药,可能是最终导致MM复发的原因之一。Objective To observe the effects of the intercellular gap junction(GJIC)composed of connexin 43(Cx43)in mesenchymal stem cells(MSCs)from different sources and their signals on the biological behavior of multiple myeloma(MM)lateral population cells(SP cells),and to explore its possible mechanism.Methods Mesenchymal stem cells(MSCs)from different sources were isolated and cultured.SP cells of MM cell line RPMI 8266 were sorted by flow cytometry.RT-PCR and Western blot were used to detect the expression of Cx43 gene and protein in MSCs,RPMI 8266 and SP cells from different sources.The effects of MSCs from different sources on SP cell cycle,Cx43 protein expression,colony formation ability in vitro,stem cell related gene expression,cytokine secretion and drug resistance were observed.Results There was no significant difference in morphology and phenotype between MM-MSCs and ND-MSCs.Both MM-MSCs and RPMI 8266 cells expressed a higher level of Cx43.Co-culture with MM-MSCs induced more SP cells to enter G0 phase(P<0.001).The expressions of c-myc,Kif4 and Sox2 genes in SP cells were significantly up-regulated,while the expression of Oct-4 gene was down-regulated.After addingα-GA,c-myc,Kif4 and Sox2 were down-regulated in varying degrees,but there was no significant difference.The expression of Cx43 was up-regulated by(31.00±2)%and(39.00±2)%,respectively.The colony formation ability in vitro was up-regulated,and the addition ofα-GA could partially inhibit this effect.A small amount of c-myc,Kif4,Sox2 and Oct-4 genes were expressed in RPMI 8266.These genes were significantly up-regulated in SP cell subpopulation.MM-MSCs secreted high levels of interleukin(IL)-6.After co-culture with SP cells,the expressions of IL-6,IL-10 and TGF-βin the supernatant of MM-MSCs were up-regulated(P=0.0072,P=0.037).bFGF and IL-17 had no significant change.After addingα-GA,the levels of IL-6,IL-10 and TGF-βin the supernatant decreased.MM cells were sensitive to bortezomib(BTZ)induced apoptosis,but SP cells were less sensitive.Co-culture w

关 键 词：多发性骨髓瘤 细胞间隙连接 肿瘤微环境 

分 类 号：R96[医药卫生—药理学]