基于原代细胞移植建立硼替佐米耐药多发性骨髓瘤小鼠模型  

Establishment of a Bortezomib-Resistant Multiple Myeloma Xenotransplantation Mouse Model by Transplanting Primary Cells from Patients

作　　者：岳延华 周怡芳 缪颖洁 曹阳[1] 王飞 刘月 李枫 沈洋灵 郭嫣婷 黄玉辉 顾伟英[1] YUE Yan-Hua;ZHOU Yi-Fang;MIAO Ying-Jie;CAO Yang;WANG Fei;LIU Yue;LI Feng;SHEN Yang-Ling;GUO Yan-Ting;HUANG Yu-Hui;GU Wei-Ying(Department of Hematology,The Third Affliated Hospital of Soochow University/The First People's Hospital of Changzhou,Changzhou 213000,Jiangsu Province,China;Cyrus Tang Hematology Center,Soochow University,Suzhou 215123,China)

机构地区：[1]苏州大学附属第三医院/常州市第一人民医院血液科,江苏常州213000 [2]苏州大学唐仲英血液学研究中心,江苏苏州215123

出　　处：《中国实验血液学杂志》2025年第1期133-141,共9页Journal of Experimental Hematology

基　　金：江苏省卫生健康委重点项目(ZD2021043);常州市十四五卫生领军人才工程资助项目(KY20221336)。

摘　　要：目的:探索耐药性多发性骨髓瘤(MM)患者来源的异种移植物(PDX)小鼠模型的构建方法。方法:分别将MM患者来源的1.0×10^(7)个单个核细胞(MNC)、MM细胞株MM.1S和NCI-H929各2.0×10^(6)个细胞经皮下植入NOD.CB17-PrkdcscidIl2rgtm1/Bcgen(B-NDG)小鼠,接种体积为每只100μl,构建动物模型,应用苏木精-尹红、免疫组织化学染色、细胞周期、流式细胞术和荧光原位杂交研究PDX肿瘤的形态、表型、增殖及遗传学特征,通过细胞增殖、凋亡以及体内外实验研究PDX肿瘤对硼替佐米、安罗替尼单药或联合治疗的敏感性,利用免疫组织化学染色、TUNEL染色和荧光共聚焦显微镜分析安罗替尼治疗对肿瘤血管及细胞凋亡的影响。结果:通过皮下接种原代MNC的方法成功建立了MM PDX模型,该种PDX肿瘤细胞形态上类似成熟的浆细胞,阳性表达CD138和CD38,存在1q21扩增、Rb1缺失和IgH重排,细胞增殖活力低于MM细胞系。体外细胞增殖实验中,硼替佐米和安罗替尼分别处理PDX、MM.1S和NCI-H929细胞24 h,其中硼替佐米的IC50值分别为5716.486、1.025和2.775 nmol/L,安罗替尼的IC50值分别为55107.337、0.706和5.13μmol/L;安罗替尼处理增加了MM.1S细胞的凋亡(P<0.01),但不影响PDX肿瘤细胞的凋亡(P>0.05)。体内实验结果显示,硼替佐米单药组与对照组比较两组间肿瘤生长无统计学差异(P>0.05),而安罗替尼单药组和安罗替尼与硼替佐米联合组均有效抑制了PDX肿瘤的生长(均P<0.05);安罗替尼治疗组PDX肿瘤的血管灌注和血管密度均减少(均P<0.01);安罗替尼治疗组较对照组凋亡细胞数增加(P<0.05)。结论:通过在B-NDG小鼠皮下接种MM患者来源的MNC能够成功建立硼替佐米耐药的MM PDX模型,该种PDX模型保留了MM细胞的基本生物学特征,能够用于MM治疗新方案的研究。Objective:To explore the construction method of a resistant multiple myeloma(MM)patient-derived xenotransplantation(PDX)model.Methods:1.0×10^(7) MM patient-derived mononuclear cells(MNCs),2.0×10^(6)MM.1S cells and 2.0×10^(6) NCI-H929 cells were respectively subcutaneously inoculated into NOD.CB17-Prkdc~(scid)Il2rg~(tm1)/Bcgen(B-NDG)mice with a volume of 100μl per mouse to establish mouse model.The morphologic,phenotypic,proliferative and genetic characteristics of PDX tumor were studied by hematoxylin-eosin staining,immunohistochemical staining(IHC),cell cycle analysis,flow cytometry and fluorescence in situ hybridization(FISH).The sensitivity of PDX tumor to bortezomib and anlotinib monotherapy or in combination was investigated through cell proliferation,apoptosis and in vitro and in vivo experiments.The effects of anlotinib therapy on tumor blood vessel and cell apoptosis were analyzed by IHC,TUNEL staining and confocal fluorescence microscope.Results:MM PDX model was successfully established by subcutaneously inoculating primary MNCs.The morphologic features of tumor cells from MM PDX model were similar to those of mature plasma cells.MM PDX tumor cells positively expressed CD138 and CD38,which presented 1q21 amplification,deletion of Rbl and IgH rearrangement,and had a lower proliferative activity than MM cell lines.In vitro,PDX,MM.1S and NCI-H929 cells were treated by bortezomib and anlotinib for 24 hours,respectively.Cell viability assay showed that the IC_(50)value of bortezomib were 5716.486,1.025 and 2.775 nmol/L,and IC_(50)value of anlotinib were 55107.337,0.706 and 5.13μmol/L,respectively.Anlotinib treatment increased the apoptosis of MM.1S cells(P<0.01),but did not affect PDX tumor cells(P>0.05).In vivo,there was no significant difference in PDX tumor growth between bortezomib monotherapy group and control group(P>0.05),while both anlotinib monotherapy and anlotinib combined with bortezomib effectively inhibited PDX tumor growth(both P<0.05).The vascular perfusion and vascular density of PDX tum

关 键 词：多发性骨髓瘤 硼替佐米耐药 PDX模型 安罗替尼 

分 类 号：R733.3[医药卫生—肿瘤]