【作者】 王莹；

【导师】 董晓荣；

【作者基本信息】 华中科技大学 ， 肿瘤学， 2021， 硕士

【摘要】 目的:我们前期研究显示抑制MIF/CD74信号通路可以促使脑转移瘤内放疗激活后的巨噬细胞由M2向M1型转化,发挥放射增敏的效应。有文献报道M2型巨噬细胞是肿瘤免疫抑制微环境中的主要组成部分,其表面标志物精氨酸酶1可以大量分解T细胞生长分化中所必须的精氨酸。因此本文重点将探究MIF通过诱导巨噬细胞表型转化来调控肿瘤浸润淋巴细胞(TILs)的相关机制。方法:慢病毒sh RNA干扰技术构建稳定沉默MIF的Lewis肺癌细胞,并建立肺癌脑转移模型;ELISA试剂盒检测细胞上清中MIF的分泌情况;WB实验检测Lewis细胞MIF,BV2细胞M1型标记物i NOS、M2型标记物Arg-1蛋白表达水平;qPCR技术检测i NOS和Arg-1的RNA表达水平;免疫荧光染色检测肺癌脑转移模型小鼠脑转移瘤组织中i NOS、Arg-1、i BA-1的表达;精氨酸试剂盒检测细胞上清和肿瘤组织中精氨酸的含量;流式细胞术检测肿瘤相关淋巴细胞的比例;CFSE检测原代T细胞的增殖;小动物活体成像技术检测肺癌脑转移模型小鼠肿瘤生长情况。结果:体内实验中,WB、PCR、免疫荧光染色结果显示敲低MIF联合全脑照射后,脑转移瘤内巨噬细胞i NOS表达增加,Arg-1表达减少,提示抑制MIF可以促使巨噬细胞的M2向M1表型转化。流式细胞术和活体成像结果显示,敲低MIF联合全脑照射可以提高脑转移瘤CD8+T/CD4+T和CD8+IFN-γ/CD4+FOXP3细胞比例,增强脑转移瘤对全脑照射的敏感性。而巨噬细胞脂质体清除剂使用后的结果显示敲低MIF联合全脑照射组的T细胞比例基本与对照组无差别;动物活体成像的结果也表明敲低MIF对肿瘤生长的抑制作用较巨噬细胞清除前减弱。提示敲低MIF联合全脑照射所产生的促进T细胞浸润、抑制脑转移瘤的效果可能是由巨噬细胞介导的。精氨酸试剂盒检测结果显示全脑照射和敲低MIF后精氨酸含量均有所上升,而巨噬细胞清除后各个分组间精氨酸含量总体升高,但各分组间差距不明显,提示全脑照射或敲低MIF是通过减少M2型巨噬细胞来增加组织中的精氨酸含量。外源性精氨酸加入后的流式和活体成像的结果显示CD8+/CD4+TILs比值升高,肿瘤生长受抑。综上所诉,抑制MIF可以减少放疗后脑转移瘤内M2型巨噬细胞的数量,增加精氨酸含量,提高TILs中CD8+杀伤性T细胞比例和IFN-γ的分泌,发挥免疫调节功能,抑制肿瘤生长。细胞实验中,将沉默MIF的Lewis细胞与照射激活的BV2细胞共培养24h,WB、PCR结果与对照组相比,下调Lewis细胞MIF表达可使BV2细胞i NOS表达显著增加,Arg-1表达显著降低。收集以上分组的细胞上清液,精氨酸试剂盒结果示精氨酸的含量变化与Arg-1蛋白的表达变化相反。提示抑制MIF可以减少Arg-1表达并升高细胞外的精氨酸水平。将上述两者的共培养上清作用于从肺癌脑转移瘤中提取的原代T细胞,流式结果表明全脑照射和敲低MIF都可以提高CD8+T/CD4+T细胞比例,联合使用效果更显著,而外源性补充精氨酸的效果最好。CFSE结果显示,全脑照射和敲低MIF都可以促进T细胞增殖,联合使用效果更显著,而外源性补充精氨酸的促增殖作用最明显。结论:抑制Lewis肺癌细胞的MIF表达促使脑转移瘤内被辐照激活的巨噬细胞由M2向M1型转换,减少微环境中M2型巨噬细胞表面标志物Arg-1的表达,从而提高精氨酸含量,促进肿瘤内淋巴细胞的浸润,改善免疫微环境,发挥放疗增敏的效应。更多还原

【Abstract】 ObjectiveOur previous studies showed that inhibition of MIF/CD74 signaling pathway can promote the transformation of intracranial macrophages after radiotherapy activation from m2 to M1 type,and play a role in radiosensitization.It is reported that M2 macrophages are the main components of tumor immunosuppression microenvironment,and the surface marker of the macrophage,arg-1,can decompose the essential arginine in the growth and development of T cells.Therefore,this paper will focus on the mechanism of inhibiting MIF through regulating macrophages to influence tumor infiltrating lymphocytes in radiotherapy of brain metastasis.MethodsMIF-Knockdown in Lewis cells were constructed by lentiviral sh RNA interference technique.The secretion of MIF in Lewis cell supernatant was detected by ELISA kit.The expression levels of MIF in Lewis and i NOS(M1 type marker),Arg-1(M2 type marker)in BV2 cells were detected by Western Blotting.The RNA expression levels of M1 and M2 tags were detected by qPCR.The expression of i NOS,Arg-1 and Iba-1in lung cancer BMs model mice was detected by immunofluorescence staining.The content of arginine in tumor tissue and cell supernatant was detected with Arginine kit.The proportion of TILs in vivo and in vitro was measured by flow cytometry.The proliferation of primary T cells was tested by CFSE.BMs tumor growth in mice was observed by Small Animals Imaging Technology.Result1.In animal experiments,WB、PCR and IF results showed that MIF knockdown(sh-MIF)combined with whole brain irradiation(IR)can raise the expression of i NOS,while the Arg-1 lessend,suggesting that downregulation of MIF can promote M1 phenotype and reduce M2 phenotype.Flow cytometry results showed that sh-MIF combined with IR increased the proportion of CD8+T/CD4+T and CD8+IFN-r+T/CD4+Foxp3+T cells.Small Animals Imaging results showed that inhibition of MIF enhanced the shrinkage of tumor induced by whole brain irradiation.After clearance of macrophage,Flow cytometry showed no difference between the sh-MIF group and the control group(NC).Small Animals Imaging results also showed that the inhibition of tumor growth by sh-MIF was less significant than macrophages presence.It is indicated that down-regulation of MIF expression combined with irradiation promote T cell infiltration and inhibit tumor may be mediated by macrophages.Arginine kit results showed that the content of arginine increased after IR or sh-MIF,while it increased overall after macrophage clearance,but the difference between each groups was not significant,suggesting that IR or sh-MIF could increase the content of arginine by reducing M2-type macrophages.Flow cytometry and animals imaging results showed that the CD8+/CD4+TILs ratio was increased and tumor growth was inhibited after the addition of exogenous arginine.In conclusion,inhibition of MIF can reduce M2-type macrophages,then increase the content of arginine to improve the proportion of TILs,and enhance the killing effect of IR on BMs.2.In cellular experiments,sh-MIF Lewis cell co-culture with BV2 cells activated by radiation of 24 h.The results of WB,PCR demonstrate that compared with the NC,inhibition of MIF can increased the i NOS expression significantly,decreased Arg-1expression markedly.Arginine kit is used to measure the above groups of cells supernatant,the results showed the change of arginine content is opposite to the Arg-1protein expression.It is suggested that inhibition of MIF can reduce the expression of Arg-1+M2 macrophages and increase the extracellular arginine level.The results of flow cytometry showed that,after treating primary T cells extracted from BMs with co-cultured supernatants,both sh-MIF and irradiation could increase the ratio of CD8+T/CD4+T cells,and the effect of combination was more significant,while the addition of arginine was the best.CFSE results showed that both sh-MIF and irradiation could promote the proliferation of T cells,the combination of the two is even better,while the exogenous arginine supplementation has the most obvious effect.ConclusionInhibitory MIF expression can reduce the expression of Arg-1 from M2 type macrophages in TME,thus raise the content of arginine,promote the infiltration of lymphocytes in tumors,improve the immune microenvironment,achieve radiotherapy sensitization finally.更多还原