沙利度胺对裸鼠移植骨髓瘤达雷妥尤单抗耐药的逆转作用及其机制

Reversal effect of thalidomide on drug resistance of daratumumab in transplanted myeloma in nude mice

目的观察沙利度胺对裸鼠移植骨髓瘤达雷妥尤单抗耐药的逆转作用,并探讨相关机制。方法将Balb/c裸鼠分为空白组、模型组、实验组,每组10只,模型组与实验组取对数生长期的裸鼠浆细胞瘤细胞MPC-11接种到裸鼠的右腋窝,建立裸鼠皮下骨髓瘤模型。实验组分别于成瘤后第1天、第8天皮下注射达雷妥尤单抗144 mg/kg,在第1次皮下注射达雷妥尤单抗注射后48 h给予沙利度胺15 mg/kg每天1次灌胃,空白组与模型组每日给予蒸馏水灌胃。实验组检测在注射达雷妥尤单抗不同时间点的NK细胞耗竭情况;实验组在裸鼠首次给予达雷妥尤单抗前、注射达雷妥尤单抗后NK细胞耗竭点、给予沙利度胺第3天及第二次给予达雷妥尤单抗前检测NK细胞变化情况;实验结束后统一采用流式细胞术检测各组NK细胞数量及CD38表达情况,采用ELISA法检测IL-15、IFN-γ、TNF-α。结果注射达雷妥尤单抗后,实验组NK细胞数量先呈上升趋势,在24 h达最高,之后呈下降趋势。模型组NK细胞数低于空白组,实验组NK细胞数高于模型组(P均<0.01)。模型组CD38表达高于空白组,实验组CD38表达低于模型组(P均<0.01)。首次给予达雷妥尤单抗前、注射达雷妥尤单抗后NK细胞耗竭点、给予沙利度胺第3天及第二次给予达雷妥尤单抗前,实验组裸鼠NK细胞数量分别为3.25±0.23、12.65±1.27、26.48±0.66、37.67±1.45,呈上升趋势。模型组血清IL-15、IFN-γ、TNF-α水平高于空白组,实验组血清IL-15、IFN-γ水平高于空白组(P均<0.05)。实验组血清IFN-γ、TNF-α水平低于模型组(P均<0.01)。结论NK细胞耗竭是骨髓瘤细胞达雷妥尤单抗耐药的重要原因之一,沙利度胺可能通过缓解NK细胞耗竭、调节炎症因子表达从而逆转小鼠移植骨髓瘤对达雷妥尤单抗的耐药。

Objective To observe the reversal effect of thalidomide on drug resistance of daratumumab in transplanted myeloma in nude mice and to explore the related mechanism.Methods Balb/c nude mice were divided into the blank group(n=10),model group(n=10),and experimental group(n=10),respectively.The MPC-11 cells of plasmacytoma in the logarithmic growth phase were inoculated into the right axilla of nude mice in the model group and experimental group to establish the models of subcutaneous myeloma in nude mice.In the experimental group,144 mg/kg was injected subcutaneously on the 1st and 8th days after tumor formation,and 15 mg/kg thalidomide was given to the stomach at 48 h after the first subcutaneous injection of daratumumab,once a day;while in the blank group and model group,distilled water was given daily.In the experimental group,NK cell depletion was detected at different time points after daratumumab injection;in the experimental group,we detected the NK cell changes before the first dose of daratumumab,and NK cell depletion point after daratumumab injection on day 3 of thalidomide and at the second dose of daratumumab.After the experiment,flow cytometry was used to detect NK cells and CD38 expression in each group.ELISA was used to detect interleukin(IL)-15,IFN-γ,and tumor necrosis factor(TNF)-αin nude mice serum.Results After injection of daratumum‐ab,the number of NK cells in the experimental group increased at first,reached the highest at 24 h,and then decreased.The number of NK cells in the model group was lower than that in the blank group,while the number of NK cells in the ex‐perimental group was higher than that in the model group(all P<0.01).The expression of CD38 in the model group was higher than that in the blank group,while the expression of CD38 in the experimental group was lower than that in the mod‐el group(both P<0.01).The numbers of NK cells in nude mice of the experimental group were 3.25±0.23,12.65±1.27,26.48±0.66,and 37.67±1.45,respectively,before the first administration of daratumumab,at the time point of depletion of NK cells after injection of thalidomide,on the third day after administration of thalidomide and before the second administration of daratumumab,showing an increasing trend.The serum levels of IL-15,IFN-γ,and TNF-αin the model group were higher than those in the blank group(all P<0.05),and the levels of IL-15 and IFN-γin the experimen‐tal group were higher than those in the blank group(all P<0.01).Conclusions The depletion of NK cells is one of the important reasons for the drug resistance of myeloma cells to daratumumab.Thalidomide may reverse the drug resistance of transplanted myeloma cells to daratumumab by alleviating the depletion of NK cells and regulating the expression of inflam‐matory factors.