中华血液学杂志
中華血液學雜誌
중화혈액학잡지
Chinese Journal of Hematology
2013年
9期
788-793
,共6页
张晓慧%孙谕%王子妍%黄湛平%傅晋翔
張曉慧%孫諭%王子妍%黃湛平%傅晉翔
장효혜%손유%왕자연%황담평%부진상
多发性骨髓瘤%间质干细胞%连接蛋白43%基质细胞衍生因子-1α%细胞运动
多髮性骨髓瘤%間質榦細胞%連接蛋白43%基質細胞衍生因子-1α%細胞運動
다발성골수류%간질간세포%련접단백43%기질세포연생인자-1α%세포운동
Multiple myeloma%Mesenchymal stem cells%Connexin 43%Stromal derived factor-1α%Cell movement
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)-1α分泌水平变化及其意义.方法 用Western blot、免疫荧光法检测MM细胞系及MM原代细胞CX43的表达及SDF-1α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RPMI 8226细胞及MSC.用实时定量PCR、Western blot法检测共培养前后MSC CX43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；微孑隔离实验检测18α-甘草次酸(18α-GA)对MSC诱导的RPMI 8226细胞迁移的影响.ELISA法检测共培养后的MSC SDF-1α分泌水平.结果 MM细胞系RPMI 8226、U266、1/3MM细胞及MM原代细胞CX43 mRNA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CX43 mRNA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,Western blot检测显示共培养后MSC CX43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RPMI 8226直接共培养后荧光染料在细胞间扩散距离增加.MSC与RPMI 8226细胞直接和间接共培养体系中,MSC培养上清SDF-1α水平分别为(373.02±10.11)和(309.71±10.71)pg/ml,高于共培养前[(237.84±9.23)pg/ml](P＜0.01),该作用可被18α-GA抑制降为(126.01±4.80)和(106.99±3.39)pg/ml.18α-GA可抑制MSC诱导的RPMI 8226细胞迁移,其作用前后RPMI 8226细胞迁移率分别为(8.00±0.67)％及(4.82±0.19)％.结论 MM细胞与MSC直接和间接共培养均可上调MSC CX43表达水平,并促进SDF-1α的分泌.间隙连接阻断剂18α-GA可降低共培养体系中SDF-1α的分泌并抑制MSC诱导的MM细胞迁移.
目的 構建多髮性骨髓瘤(MM)細胞與骨髓間充質榦細胞(MSC)共培養體繫,探討共培養後MSC連接蛋白43 (CX43)錶達及基質細胞衍生因子(SDF)-1α分泌水平變化及其意義.方法 用Western blot、免疫熒光法檢測MM細胞繫及MM原代細胞CX43的錶達及SDF-1α分泌水平.建立間接及直接共培養體繫共培養MM細胞和MSC,然後用CD138磁珠法分離RPMI 8226細胞及MSC.用實時定量PCR、Western blot法檢測共培養前後MSC CX43錶達,免疫熒光法檢測CX43分佈；劃痕實驗檢測共培養後MSC間間隙連接通訊(GJIC)變化；微孑隔離實驗檢測18α-甘草次痠(18α-GA)對MSC誘導的RPMI 8226細胞遷移的影響.ELISA法檢測共培養後的MSC SDF-1α分泌水平.結果 MM細胞繫RPMI 8226、U266、1/3MM細胞及MM原代細胞CX43 mRNA呈中、低度錶達,XG-4、XG-7細胞不錶達CX43.骨髓MSC高錶達CX43.直接或間接共培養後骨髓MSC的CX43 mRNA相對錶達量明顯提高,分彆是單獨培養時的1.36倍和2.10倍,Western blot檢測顯示共培養後MSC CX43蛋白錶達水平也上調,免疫熒光染色顯示增高的CX43主要分佈在胞質.劃痕實驗顯示在MSC與RPMI 8226直接共培養後熒光染料在細胞間擴散距離增加.MSC與RPMI 8226細胞直接和間接共培養體繫中,MSC培養上清SDF-1α水平分彆為(373.02±10.11)和(309.71±10.71)pg/ml,高于共培養前[(237.84±9.23)pg/ml](P＜0.01),該作用可被18α-GA抑製降為(126.01±4.80)和(106.99±3.39)pg/ml.18α-GA可抑製MSC誘導的RPMI 8226細胞遷移,其作用前後RPMI 8226細胞遷移率分彆為(8.00±0.67)％及(4.82±0.19)％.結論 MM細胞與MSC直接和間接共培養均可上調MSC CX43錶達水平,併促進SDF-1α的分泌.間隙連接阻斷劑18α-GA可降低共培養體繫中SDF-1α的分泌併抑製MSC誘導的MM細胞遷移.
목적 구건다발성골수류(MM)세포여골수간충질간세포(MSC)공배양체계,탐토공배양후MSC련접단백43 (CX43)표체급기질세포연생인자(SDF)-1α분비수평변화급기의의.방법 용Western blot、면역형광법검측MM세포계급MM원대세포CX43적표체급SDF-1α분비수평.건립간접급직접공배양체계공배양MM세포화MSC,연후용CD138자주법분리RPMI 8226세포급MSC.용실시정량PCR、Western blot법검측공배양전후MSC CX43표체,면역형광법검측CX43분포；화흔실험검측공배양후MSC간간극련접통신(GJIC)변화；미혈격리실험검측18α-감초차산(18α-GA)대MSC유도적RPMI 8226세포천이적영향.ELISA법검측공배양후적MSC SDF-1α분비수평.결과 MM세포계RPMI 8226、U266、1/3MM세포급MM원대세포CX43 mRNA정중、저도표체,XG-4、XG-7세포불표체CX43.골수MSC고표체CX43.직접혹간접공배양후골수MSC적CX43 mRNA상대표체량명현제고,분별시단독배양시적1.36배화2.10배,Western blot검측현시공배양후MSC CX43단백표체수평야상조,면역형광염색현시증고적CX43주요분포재포질.화흔실험현시재MSC여RPMI 8226직접공배양후형광염료재세포간확산거리증가.MSC여RPMI 8226세포직접화간접공배양체계중,MSC배양상청SDF-1α수평분별위(373.02±10.11)화(309.71±10.71)pg/ml,고우공배양전[(237.84±9.23)pg/ml](P＜0.01),해작용가피18α-GA억제강위(126.01±4.80)화(106.99±3.39)pg/ml.18α-GA가억제MSC유도적RPMI 8226세포천이,기작용전후RPMI 8226세포천이솔분별위(8.00±0.67)％급(4.82±0.19)％.결론 MM세포여MSC직접화간접공배양균가상조MSC CX43표체수평,병촉진SDF-1α적분비.간극련접조단제18α-GA가강저공배양체계중SDF-1α적분비병억제MSC유도적MM세포천이.
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)-1α secretion of MSC.Methods CX43 expression and SDF-1α 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 18α-glycyrrhetinic acid (18a-GA).The level of SDF-1α 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-1α 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 (237.84±9.23) pg/ml (P＜0.01),and could be inhibited by 18α-GA [(237.84±9.23) pg/ml and (94.31±6.44) pg/ml] respectively (P＜0.01).18α-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 could improve the level of SDF-1α secretion of MSC.GJ inhibitor could downregulate SDF-1 α secretion of MSC and inhibit the transmigration of MM cells induced by MSC.
