抵抗素通过抑制细胞死亡和上调ABC转运蛋白的表达诱导多发性骨髓瘤多药耐药

发布时间：2018-09-18 13:02

【摘要】：实验目的:近年来,由于新药的研发,多发性骨髓瘤的治疗取得了很大的进展,但是由于复发、耐药的存在,多发性骨髓瘤仍是一种不可治愈的血液肿瘤。多发性骨髓瘤的耐药机制现在仍不甚清楚,最近有研究表明,骨髓微环境中的脂肪细胞通过其分泌的脂肪细胞因子参与骨髓瘤的多药耐药,但是哪些脂肪细胞因子参与这一过程及具体的机制尚不清楚。本实验探讨新型脂肪细胞因子抵抗素在多发性骨髓瘤多药耐药中的作用及相关分子学机制。实验方法:1.用马法兰、硼替佐米或卡非佐米诱导多发性骨髓瘤细胞系和骨髓瘤患者骨髓CD138+浆细胞凋亡,同时加入不同浓度的抵抗素,用Annexin V-FITC/PI双标记流式细胞术检测凋亡。2.用Western blot检测凋亡相关蛋白和重要的骨髓瘤细胞存活信号通路的相关蛋白表达情况,同时应用特异性的抑制剂阻断信号通路,用Annexin V-FITC/PI双标记流式细胞术对比细胞凋亡率的变化。3.首先用流式细胞术检测抵抗素对ABC转运蛋白功能的影响,其次用实时定量PCR检测ABCB1、ABCB3、ABCC1、ABCC3、ABCC4、ABCC5、ABCG2的表达变化,最后Western blot验证ABCC5和ABCG2蛋白表达量的变化。同时用小干扰RNA(si RNA)技术抑制ABCG2和ABCC5的表达,并观察ABCG2和ABCC5 si RNA在抵抗素诱导多发性骨髓瘤细胞系多药耐药中的影响。4.用甲基化特异性PCR(methylation-specific PCR,MS-PCR)检测抵抗素对ABCG2和ABCC5基因甲基化水平的影响,同时用实时定量PCR和Western blot检测抵抗素对甲基化转移酶(DNA methyltransferase,DNMT)的作用。5.选取多发性骨髓瘤细胞系ARP-1建立重症联合免疫缺陷(severe combined immune deficiency,SCID)小鼠成瘤实验模型,将20只小鼠随机分为四组,待3周骨髓瘤形成后,用马法兰和/或抵抗素处理小鼠。之后,用ELISA检测小鼠血浆中的M蛋白含量,用流式细胞术检测小鼠骨髓中CD138+细胞,用Annexin V-FITC/PI双标记流式细胞术和DNA断裂的原位末端标记法(TUNEL)法检测骨髓瘤细胞的凋亡。实验结果:抵抗素能显著抑制马法兰诱导的多发性骨髓瘤细胞系ARP-1、MM.1S和U266的细胞凋亡,并具有浓度依赖性。同时,抵抗素也能抑制硼替佐米和卡非佐米诱导的ARP-1、MM.1S和RPMI8226诱导的细胞凋亡。并且,抵抗素也可显著减少马法兰诱导的多发性骨髓瘤患者骨髓浆细胞CD138+的凋亡。在ARP-1和MM.1S细胞中,与马法兰处理组相比,抵抗素和马法兰处理组细胞凋亡相关蛋白cleaved caspase-9、cleaved caspase-3、cleaved PARP和Bax显著减少,而凋亡抑制蛋白Bcl-2和Bcl-Xl显著增多。进一步,我们发现抵抗素能显著增加IκBa,Akt,and ERK1/2的磷酸化水平,并且NF-κB和PI3K抑制剂能明显减弱抵抗素对骨髓瘤细胞系的抗凋亡保护作用。同时,我们发现抵抗素能显著提高ABC转运蛋白的外排功能和m RNA的表达,其中ABCG2和ABCC5转运蛋白在多个骨髓瘤细胞系中均明显增多,干扰ABCG2和ABCC5基因表达后,抵抗素对马法兰、硼替佐米和卡非佐米诱导的骨髓瘤细胞凋亡无保护作用。进一步研究发现,抵抗素能使ABCG2和ABCC5基因启动子甲基化水平明显降低,非甲基化水平明显提高,同时,抵抗素能显著减少DNMT1和DNMT3a的m RNA和蛋白表达量。体内试验中,我们用血浆中M蛋白含量来反映小鼠的肿瘤负荷,发现与马法兰处理组相比,抵抗素和马法兰处理组小鼠血浆M蛋白含量明显增高,骨髓中CD138+细胞明显增多,并且CD138+细胞凋亡率明显降低,同时小鼠骨髓切片TUNEL染色也进一步验证了此结果。结论:1.抵抗素能抑制多种化疗药物诱导的骨髓瘤细胞凋亡。2.抵抗素通过激活抗凋亡信号通路NF-κB and PI3K/Akt发挥其对骨髓瘤细胞的保护作用。3.抵抗素能减少甲基化转移酶DNMT1和DNMT3a的表达,增加ABC转运蛋白的表达量和药物外排功能,从而发挥其抗凋亡作用。4.体内实验进一步验证了抵抗素在多发性骨髓瘤多药耐药中的促进作用。
[Abstract]:EXPERIMENTAL OBJECTIVES: In recent years, great progress has been made in the treatment of multiple myeloma due to the development of new drugs, but multiple myeloma is still an incurable hematological tumor due to recurrence and drug resistance. Cells participate in multidrug resistance of myeloma by secreting adipocytokines, but it is not clear which adipocytokines are involved in this process and the specific mechanism. This study explores the role of novel adipocytokine resistin in multidrug resistance of multiple myeloma and its related molecular mechanisms. Experimental methods: 1. Marfan, boron Tezomib or Carfezomib induced apoptosis of bone marrow CD138 + plasma cells from multiple myeloma cell lines and patients with myeloma. At the same time, different concentrations of resistin were added. Apoptosis was detected by Annexin V-FITC/PI double-labeled flow cytometry. 2. Apoptosis-related proteins and important myeloma cell survival signal pathway-related proteins were detected by Western blot. Specific inhibitors were used to block the signaling pathway and Annexin V-FITC/PI double-labeled flow cytometry was used to compare the changes of apoptosis rate. 3. Firstly, the effect of resistin on the function of ABC transporter protein was detected by flow cytometry. Secondly, the expression of ABCB1, ABCB3, ABCC1, ABCC3, ABCC4, ABCC5, ABCG2 was detected by real-time quantitative PCR. The expression of ABCG2 and ABC5 was inhibited by small interfering RNA (si RNA) technique, and the effect of ABCG2 and ABC5 Si RNA on resistance-induced multidrug resistance in multiple myeloma cell lines was observed. 4. Methylation-specific PCR (MS-PCR) was used to detect the expression of ABCG2 and ABC5 Si RNA. The effects of antibiotics on methylation of ABCG2 and ABC5 genes were detected by real-time quantitative PCR and Western blot. 5. Severe combined immune deficiency (SCID) mice were established by multiple myeloma cell line ARP-1. Twenty mice were randomly divided into four groups and treated with melphalan and/or resistin after 3 weeks of myeloma formation. The M protein content in plasma was detected by ELISA, CD138 + cells in bone marrow were detected by flow cytometry, and Annexin V-FITC/PI flow cytometry and DNA fragmentation in situ end labeling (TUNEL) were used. The results showed that resistin significantly inhibited the apoptosis of MAF-induced multiple myeloma cell lines ARP-1, MM.1S and U266 in a concentration-dependent manner. At the same time, resistin also inhibited the apoptosis of ARP-1, MM.1S and RPMI8226 cells induced by bortezomib and carfezomib. The apoptosis-related proteins cleaved caspase-9, cleaved caspase-3, cleaved PARP and Bax were significantly decreased in ARP-1 and MM.1S cells compared with those in melphalan treatment group, while the inhibitor of apoptosis proteins Bcl-2 and Bcl-Xl were significantly decreased in resistin and melphalan treatment groups. Furthermore, we found that resistin significantly increased the phosphorylation levels of I kappa Ba, Akt, and ERK1/2, and that NF-kappa B and PI3K inhibitors significantly decreased the anti-apoptotic protective effect of resistin on myeloma cell lines. ABC5 transporters were significantly increased in multiple myeloma cell lines. After interfering with ABCG2 and ABC5 gene expression, resistin had no protective effect on myeloma cell apoptosis induced by melphalan, bortezomib and carfezomib. In vivo, we used the M protein content in plasma to reflect the tumor load in mice. We found that the M protein content in plasma of the mice treated with resistin and melphalan was significantly higher than that of the mice treated with melphalan, and the CD138 + cells in bone marrow were significantly increased. Conclusion: 1. Resistin can inhibit the apoptosis of myeloma cells induced by various chemotherapeutics. 2. Resistin exerts its protective effect on myeloma cells by activating the anti-apoptosis signaling pathway NF-kappa B and PI3K/Akt. Resistin can reduce the expression of methyltransferase DNMT1 and DNMT3a, increase the expression of ABC transporter protein and drug efflux function, so as to play its anti-apoptosis role. 4. In vivo experiments further confirmed the role of resistin in promoting multidrug resistance in multiple myeloma.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R733.3

【相似文献】