CAJ | 학술논문

目的探讨骨髓基质细胞在骨髓瘤耐药中的作用及其分子机制.方法收集2011年5-7月中国医学科学院血液病医院淋巴瘤骨髓瘤诊疗中心初诊的多发性骨髓瘤(MM)患者骨髓标本5份,健康供者骨髓标本5份,分离培养其中骨髓基质细胞(BMSC),收集细胞培养上清液,酶联免疫吸附试验(ELISA)法检测细胞因子分泌水平.将MM患者BMSC与人骨髓瘤细胞系共同培养,分别加入美法仑、硼替佐米；采用四甲基偶氮唑盐(MTT)法观察骨髓瘤细胞增殖情况；采用实时定量PCR(qRT-PCR)方法检测骨髓瘤细胞微小RNA( miRNA) 15a/-16的表达.人骨髓瘤细胞系中加入细胞因子,qRT-PCR检测miRNA-15a/-16的表达.转染上调骨髓瘤细胞miRNA-15a表达,采用膜联蛋白(Annexin) V/碘化丙啶(PI)染色及流式细胞术观察miRNA-15a上调表达对细胞周期、细胞凋亡的影响.结果成功分离得到BMSC细胞,MM患者BMSC中白细胞介素6(IL-6)和血管内皮生长因子(VEGF)的表达水平均明显高于健康对照[(189±9)比(115±15) pg/ml,( 1497±40)比(1239±21)pg/ml,均P＜0.05].美法仑、硼替佐米均上调骨髓瘤细胞miRNA-15a和miRNA-16的表达(均P＜0.05)；而MM患者BMSC与骨髓瘤细胞共培养后,可抑制美法仑和硼替佐米对miRNA-15a和miRNA-16的上调作用[美法仑:4.690±0.050比34.440±4.100,0.760±0.070比12.030±1.020；硼替佐米:1.440±0.230比11.480±1.488,0.880 ±0.040比3.680±0.420；均P＜0.05].IL-6可抑制骨髓瘤细胞miRNA-15a/-16表达,并呈时间和剂量依赖性(P＜0.05).转染上调骨髓瘤细胞miRNA-15a表达,导致细胞周期G1/S阻滞,抑制细胞增殖(P＜0.05).结论骨髓瘤微环境中基质细胞可能通过分泌高水平IL-6抑制骨髓瘤细胞miRNA-15a/-16表达,抑制细胞增殖,降低骨髓瘤细胞对化疗药物的敏感性,参与MM耐药的发生.
목적 탐토골수기질세포재골수류내약중적작용급기분자궤제.방법 수집2011년5-7월중국의학과학원혈액병의원림파류골수류진료중심초진적다발성골수류(MM)환자골수표본5빈,건강공자골수표본5빈,분리배양기중골수기질세포(BMSC),수집세포배양상청액,매련면역흡부시험(ELISA)법검측세포인자분비수평.장MM환자BMSC여인골수류세포계공동배양,분별가입미법륜、붕체좌미；채용사갑기우담서염(MTT)법관찰골수류세포증식정황；채용실시정량PCR(qRT-PCR)방법검측골수류세포미소RNA( miRNA) 15a/-16적표체.인골수류세포계중가입세포인자,qRT-PCR검측miRNA-15a/-16적표체.전염상조골수류세포miRNA-15a표체,채용막련단백(Annexin) V/전화병정(PI)염색급류식세포술관찰miRNA-15a상조표체대세포주기、세포조망적영향.결과 성공분리득도BMSC세포,MM환자BMSC중백세포개소6(IL-6)화혈관내피생장인자(VEGF)적표체수평균명현고우건강대조[(189±9)비(115±15) pg/ml,( 1497±40)비(1239±21)pg/ml,균P＜0.05].미법륜、붕체좌미균상조골수류세포miRNA-15a화miRNA-16적표체(균P＜0.05)；이MM환자BMSC여골수류세포공배양후,가억제미법륜화붕체좌미대miRNA-15a화miRNA-16적상조작용[미법륜:4.690±0.050비34.440±4.100,0.760±0.070비12.030±1.020；붕체좌미:1.440±0.230비11.480±1.488,0.880 ±0.040비3.680±0.420；균P＜0.05].IL-6가억제골수류세포miRNA-15a/-16표체,병정시간화제량의뢰성(P＜0.05).전염상조골수류세포miRNA-15a표체,도치세포주기G1/S조체,억제세포증식(P＜0.05).결론 골수류미배경중기질세포가능통과분비고수평IL-6억제골수류세포miRNA-15a/-16표체,억제세포증식,강저골수류세포대화료약물적민감성,삼여MM내약적발생.
Objective To explore the effects and mechanism of bone marrow stromal cells (BMSCs) on the drug resistance of multiple mycloma (MM).Methods Fresh untreated MM patient (5 samples) and health donor (5 samples) bone marrow samples were collected from May 2011 to July 2011 al our hospital.Their BMSCs were separated respectively.The supernatant expression of cytokines in BMSCs was detected by enzyme-linked immunosorbent assay (ELISA).Myeloma cells were co-cultured with BMSCs and treated with melphalan or bortezomib.Cell proliferation and miRNA-15a/-16 expression of myeloma cells were measured in different culture conditions with thiazoyl blue tetrazolium bromide (MTT) assay and quantitative real-time PCR (qRT-PCR). miRNA-15a was transfected into myeloma cells. And the miRNA-15a functions on cell cycle and cell apoptosis were detected by flow cytometry.Results ELISA assay showed that cytokine IL-6 and VEGF were at higher levels in MM-BMSCs than healthy BMSCs ( (189 ±9) vs( 115 ± 15) pg/ml,( 1497 ±40) vs(1239 ±21) pg/ml,both P ＜0.05).The miRNA-15a/-16 expressions of myeloma cells were up-regulated after the treatment of melphalan and bortezomib ( all P ＜0.05 ).However,the miRNA-15a/-16 up-regulation by melphalan or bortezomib became inhibited when MM cells were co-cultured with MM-BMSCs ( melphalan:4.690 ± 0.050 vs 34.440 ± 4.100,0.760 ± 0.070 vs 12.030 ± 1.020,bortezomib:1.440 ± 0.230 vs 11.480 ± 1.488,0.880 ± 0.040 vs 3.680 ± 0.420,all P ＜0.05). Furthermore,IL-6 suppressed the expression of miRNA-15a/-16 in a dose and time-dependent pattern.The transfection of miRNA-15a led to the arrest of MM cell cycle iu G1/S phase. Conelusions BMSCs suppress the proliferation of myeloma cells and regulate the drug sensitivity of myeloma cells through the inhibited expression of miRNA-15a/-16.IL-6 plays a pivotal role in the occurrence of drug resistance.