【作者】 张晓慧；

【导师】 傅晋翔；

【作者基本信息】 苏州大学 ， 内科学血液病， 2014， 博士

【摘要】 多发性骨髓瘤（MM）是浆细胞在骨髓中异常增生的恶性肿瘤，这些异常增生的浆细胞与骨髓基质细胞密切相关。MM细胞与骨髓微环境可通过直接接触及分泌细胞因子的方式间接相互作用。在MM的发展过程中，骨髓微环境逐渐表现出支持MM细胞生存和增殖的特征。骨髓间充质干细胞（BMSCs）参与构建骨髓微环境，参与促进MM细胞的生长及骨破坏的发生；同时，MM细胞对骨髓微环境重塑使其成为适合MM细胞生存和增殖场所。目前，此过程中涉及的具体机制尚未完全阐明。间隙连接是普遍存在的一种细胞间连接方式，相邻细胞间通过间隙连接介导的细胞间隙连接通讯(GJIC)进行着信息和能量物质的交换，对细胞增殖、分化等生理过程起着重要的调控作用。间隙连接蛋白43(Cx43)是在人体表达最高的一种间隙连接物质，可在包括造血干细胞、基质细胞等多种细胞表达，参与造血调控。Cx43表达及功能异常在多种肿瘤的发生、发展中发挥重要作用。那么在MM的发生、发展中，MM细胞及BMSCs如何相互作用，是否发生间隙连接的表达或功能改变，其改变又有怎样的生物学效应？我们通过建立MM细胞与BMSCs的共培养体系，研究MM细胞对BMSCs Cx43表达及功能的影响以及此过程中MM细胞及BMSCs的生物学行为的变化，从而进一步探讨MM的发病机制。本课题为国家自然科学基金项目（NO81071934/H1616）“接头蛋白Cx43在多发性骨髓瘤成骨细胞巢重塑中的作用”的重要研究内容，该实验完成为此项目的结题、文章发表及延伸课题的申报奠定基础。第一部分Cx43在骨髓瘤细胞和骨髓间充质干细胞的表达及生物学功能目的：检测人MM细胞及BMSCs的Cx43表达，探讨间隙连接在BMSCs诱导的MM细胞的迁移、粘附中的作用及其对BMSCs的基质细胞衍生因子-1α（SDF-1α）分泌的影响。方法：贴壁培养法分离培养人BMSCs，CD138磁珠及midi MACs分选原代MM细胞，流式细胞仪测定BMSCs及原代MM细胞的免疫表型。Westernblot及免疫荧光检测Cx43在BMSCs的表达。CCK-8法检测间隙连接阻断剂18α-甘草次酸（18α-GA）对MM细胞及BMSCs增殖的作用；微孔隔离实验检测18α-GA对BMSCs诱导的MM细胞迁移、粘附能力的影响。ELISA检测BMSCs SDF-1α分泌水平。结果：1）MM细胞系RPMI8226、U266及1例原代MM细胞中、低度表达Cx43，XG-4、XG-7细胞不表达Cx43。BMSCs高表达Cx43。初诊MM患者的BMSCs（MM-MSC）Cx43表达高于正常供者的BMSCs（ND-MSC）。2）18α-GA对RPMI8226细胞和BMSCs的增殖无明显影响。3）18α-GA抑制BMSCs的SDF-1α分泌，其作用前后SDF-1α浓度分别为（237.84±9.23）pg/ml、（94.31±6.44）pg/ml（P<0.01）。4）18α-GA可抑制BMSCs诱导的MM细胞迁移，BMSCs经其作用前后RPMI8226细胞迁移率分别为（8.0±0.673）%及（4.82±0.186）%(P<0.01)，XG-7的细胞迁移率分别为（0.88±0.036）%、（0.58±0.020）%（P<0.05）。5）18α-GA可抑制MM细胞在BMSCs的粘附，其作用前后RPMI8226细胞粘附率分别为（16.967±1.55）%、（11.1±0.819）%（P<0.05），XG-7的细胞粘附率分别为（9.5±1.323）%、（6.63±0.551）%（P<0.05）。结论：BMSCs及部分MM细胞表达Cx43, MM-MSC Cx43表达高于ND-MSC。阻断间隙连接可抑制BMSCs诱导的MM细胞的迁移和粘附，并可抑制BMSCs的SDF-1α分泌。第二部分三维BMSCs细胞球的建立及其特点观察目的：建立BMSCs的三维培养方法以模拟骨髓微环境，并观察MM细胞在三维培养的BMSCs细胞球中的迁移。方法：在琼脂糖包被的圆底培养板中进行BMSCs的三维培养，倒置显微镜、HE染色及电镜下观察三维培养的BMSCs（3D-BMSCs）的形态特点，RT-PCR检测3D-BMSCs和普通二维培养的BMSCs（2D-BMSCs）的Cx43mRNA、SDF-1α mRNA的表达。免疫荧光检测18α-GA对RPMI8226细胞在3D-BMSCs细胞球中迁移的影响。结果：BMSCs在琼脂糖包被的圆底培养板中可呈球形生长，4天后细胞球直径约450μm。细胞球石蜡切片HE染色示，细胞球外层多为长梭形细胞，内部为多角形、不规则细胞。扫描电镜示，BMSCs细胞及其胞外丝状突起、基质样物质形成细胞球的外层结构，外层BMSCs仍为长梭形。与2D-BMSCs相比，3D-BMSCs的SDF-1αmRNA表达明显升高，Cx43mRNA表达无明显差别；18α-GA可抑制RPMI8226细胞在3D-BMSCs细胞球中的迁移。结论：3D-BMSCs细胞球在一定程度上可模拟骨髓微环境，其SDF-1α mRNA表达较2D-BMSCs明显升高，并且其Cx43表达对诱导MM细胞的迁移有重要作用。第三部分MM细胞与BMSCs共培养体系中Cx43表达变化及其生物学效应目的：观察MM细胞与BMSCs共培养体系中Cx43表达水平的变化及其对MM细胞及BMSCs生物学行为的影响。方法：建立RPMI8226细胞和BMSCs的间接及直接共培养体系，用CD138磁珠分离直接共培养的RPMI8226细胞及BMSCs。实时定量PCR、Western blot检测共培养前后BMSCs的Cx43表达，免疫荧光法检测Cx43分布；划痕实验检测共培养后BMSCs间隙连接通讯（GJIC）的变化；ELISA检测共培养前后BMSCsSDF-1α分泌水平变化。Western blot检测共培养前后BMSCs的beclin、LAMP、LC3表达及自噬抑制剂3-甲基腺嘌呤（3-MA）对共培养体系中BMSCs LC3及Cx43表达的影响。Vonkossa染色检测RPMI8226细胞和3-MA对BMSCs成骨分化的影响。结果：1）直接或间接共培养后BMSCs的Cx43mRNA相对表达量明显提高，分别是单独培养时的1.36倍和2.1倍。2）Western blot检测示共培养后BMSCs Cx43蛋白表达水平也上调，免疫荧光示增高的Cx43主要分布在胞质。3）划痕实验显示在BMSCs与RPMI8226直接共培养后荧光染料在细胞间扩散距离增加。4）与BMSCs直接共培养后，RPMI8226细胞Cx43表达降低，间接共培养后Cx43表达无明显变化。5）在BMSCs与RPMI8226细胞直接和间接共培养体系中，BMSCs培养上清SDF-1α水平分别为（373.02±10.11）和（309.71±10.71）pg/ml，均高于共培养前（237.84±9.23）pg/ml（P<0.01，P<0.05）。经18α-GA作用后，直接和间接共培养体系中SDF-1α分别降为（126.01±4.80）（P<0.001）和（106.99±3.39）pg/ml（P<0.01）。6）RPMI8226与BMSCs共培养48h后， BMSCs的自噬相关蛋白LC3-Ⅱ、beclin均较对照组升高，溶酶体相关蛋白LAMP1也升高。3-MA抑制共培养体系中BMSCs的自噬水平，同时BMSCs的Cx43水平较前升高。RPMI8226细胞可抑制BMSCs的成骨分化，3-MA可减轻该抑制作用。结论：MM细胞可通过上调BMSCs Cx43的表达并增强BMSCs的GJIC，从而促进BMSCs SDF-1α的分泌。MM细胞可通过上调BMSCs的自噬水平抑制BMSCs的成骨分化。更多还原

【Abstract】 Multiple myeloma (MM) is a neoplasm characterized by the clonal expansion ofmalignant plasma cells that accumulate mainly in the bone marrow (BM) and are closelyrelated to the surrounding stromal microenvironment. The interaction of MM cells with theBM microenvironment, either directly via adhesion molecules or indirectly via thestimulation of autocrine/paracrine production of cytokines, activates a broad range ofproliferative and anti-apoptotic signaling pathways.It has been found that alterations in thelocal microenvironment are not only supportive of tumor growth but also required fortumorigenesis. During the process of MM development, BM stromal cells graduallydevelop special characteristics that support MM cells. For example, BM mesenchymalstem cells(BMSCs) can increase MM cell adhesion to BM, protecting the cells fromchemotherapy and aiding their accumulation within the BM. Meanwhile, during theprocess of MM cell migration and homing to the BM, MM cells remodel the BMmicroenvironment and make it a place that is suitable for the survival and proliferation ofMM cells. At present, the detailed mechanisms involved in this process have not beencompletely elucidated.The gap junction (GJ) is a common type of cell-cell junction and is involved in theregulation of many physiological process, such as cell proliferation and differentiation. Theexchange of information and energy between adjacent cells occurs via GJ-mediatedintercellular communication(GJIC). Connexin-43(Cx43) is the major component of GJs inhuman tissue, and it is expressed in many cell types, including hematopoietic cells andstromal cells. Dysregulation of Cx43expression and dysfunction of GJIC are related touncontrolled proliferation and malignant phenotypes and may be two of the genetic eventsinvolved in tumorigenesis. GJs and connexins may be new therapeutic targets in cancer. The aim of this study was to assess the alteration of Cx43expression in BMSCs,evaluate the interactions between BMSCs and MM cells in coculture systems. We alsoaimed to determine the effects of altered Cx43expression on the migration and adhesion ofMM cells.Part1Cx43Expression In Myeloma Cells And Bone MarrowMesenchymal Stem Cells And Its Biological FunctionObjective To analyse Cx43expression in multiple myeloma (MM) cells and bonemarrow mesenchymal stem cells (BMSCs) and investigate the role of gap junction playedin the process of BMSCs induced MM cells migration and adhesion and SDF-1α secretionof BMSCs.Methods BMSCs were isolated and cultured with adherent culture method. CD138magnetic beads and midi MACs system were employed to isolate primary MM cells. Theimmunophenotye of BMSCs and primary MM cells were detected by Flowcytometry.Cx43expression in MM cells and BMSCs were analysed by westernblot andimmunofluorescence. The influence of GJ inhibitor18α-GA on MM cells and BMSCsproliferation was determined by CCK-8. Transwell was applied to study the effect of18α-glycyrrhetinic acid（18α-GA）on MM cells transmigrion induced by BMSCs. SDF-1αsecretion was detected by ELISA.Results1) MM cell lines RPMI8266、U266and one of three primary MM cellsexpressed Cx43at moderate and low levels. Cx43was not expressed in XG-4、XG-7celllines but highly expressed in BMSCs. Cx43expression in BMSCs from patients newlydiagnosed with MM was stronger than that in BMSCs from healthy donors.2)18α-GA hadlittle effects on the proliferation of RPMI8226cells or BMSCs.3) SDF-1α concentrationin the supernatant of BMSCs cultured alone was237.84±9.23pg/ml, which decreased to94.31±6.44pg/mL（P<0.01）when the cells were incubated with18α-GA.4)18α-GAinhibited the migration of MM cells induced by BMSCs. The migration rate of ofRPMI8226and XG-7cells were （8.0±0.673）%、（0.88±0.036）%, respectively, whichdecreased to （4.82±0.186）%（P<0.01）、（0.58±0.020）%（P<0.05）after BMSCs were incubated with18α-GA.5)18α-GA inhibited MM cells adhesion to BMSCs. The adhesionrates of RPMI8226and XG-7cells were16.967±1.55%and9.5±1.323%, respectively,which decreased to11.1±0.819%（P<0.05）and6.63±0.551%（P<0.05）after incubationwith18α-GA.Conclusions BMSCs and proportion of MM cells express CX43. GJ inhibitor candownregulate SDF-1α secretion of BMSCs and inhibit the migration and adhesion of MMcells induced by BMSCs. Part2Construction Of three Dimentional Cultured Bone MarrowMesenchymal Stem Cells Spheroids And Investigation Of TheirCharacteristicsObjective To establish the method of three dimentional culture of BMSCs，andinvestigate the characteristics of BMSCs spheroids，and observe MM cells migration in theBMSCs spheroids.Methods Three dimentional cultured BMSCs spheroids （3D-BMSCs） werecultured in the agarose coated96well round bottom plates. The apperance of BMSCsspheroids were examined under an inverted microscope and HE staining and ScanningElectronic Microscopy. Cx43mRNA、SDF-1α mRNA expression of3D-BMSCs andcommon2dimentional cultured BMSCs （2D-BMSCs） were analysed with reversetranscription polymerase chain reaction （RT-PCR）. The effects of18α-GA on themigration of MM cells in3D-BMSCs spheroids were detected by immunofluorescence.Results After4days, BMSC formed spheroid structures of approximately450mmdiameters in the agarose coated96well round bottom plates. Scanning electronicmicroscopy showed that cells with numerous filopodia-like projections contactedneighboring cells and formed a complex three-dimensional network.3D-BMSCs expressedhigher SDF-1α mRNA compared to2D-BMSCs. Cx43mRNA in3D-BMSCs and 2D-BMSCs had no difference.18α-GA inhibited MM cells migrated into BMSCsspheroid.Conclusion3D-BMSCs spheroid can mimic BM microenvionment, and its Cx43expression plays an important role in MM cells migration. Part3Alteration Of Cx43Expression And Its Biological Effects in TheCoculture Systems Of MM cells And BMSCsObjective To investigate the interplay of MM cells and BMSCs and alteration ofCx43expression in coculture systems.Methods Westernblot、qRT-PCR and immunofluorescence were employed todetect the alteration of Cx43expression and distribution in BMSCs directly and indirectlycocultured with myeloma cells. Lucifer yellow dye spread was utilised to evaluate GJICbetween BMSCs. SDF-1α secretion was detected by ELISA. Westernblot were employedto detect beclin、LAMP、LC3expression in BMSCs after cocultured with RPMI8226withor without autophgy inhibitor3-MA. Vonkossa staining were used to determine osteogenicdifferentiation of BMSCs.Results1) The expression of Cx43mRNA in BMSCs were both upregulated aftercoculture directly and indirectly with RPMI8226，1.36and2.1times that of BMSCscultured alone respectively.2) Westernblot analysis showed that Cx43protein expressionin BMSCs was also upregulated after coculture with RPMI8226, and the increased Cx43was mainly distributed in cytoplasm.3) Lucifer yellow dye spread showed that GJIC wasupregulated in BMSCs cocultured with RPMI8226.4) Cx43expression in RPMI8226cellswas downregulated after directly coculture with BMSCs and had no marked alteration afterindirectly coculture with BMSCs.5) SDF-1α concentration in supernant of BMSCsdirectly and indirectly cocultured with RPMI8226were（373.02±10.11）pg/ml and（309.71±10.71）pg/ml respectively, which were both higher than that of BMSCs cultured alone （237.84±9.23）pg/ml（P<0.01, P<0.05）, and could be inhibited by18α-GA to(126.01±4.80）and（106.99±3.39）pg/ml, respectively（P<0.001, P<0.01）.6) Autophagyassociated protein beclin、LAMP、LC3expression in BMSCs were upregulated aftercoculture with RPMI8226, which could be inhibited by3-MA.7) Vonkossa stainingshowed that RPMI8226inhibited osteogenic differentiation of BMSCs, which could beimproved by3-MA.Conclusion The Cx43expression in BMSCs is upregulated after directly andindirectly coculture with MM cells, which can improve SDF-1α secretion of BMSCs. MMcells can inhibit osteogenic differentiation of BMSCs via upregulating the autophgy ofBMSCs.更多还原