CN116808034A - Cd47抑制剂联合idh1-r132h靶向抑制剂在治疗胶质瘤中的应用 - Google Patents
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Abstract
本发明公开了CD47抑制剂联合IDH1‑R132H靶向抑制剂在治疗胶质瘤中的应用。本发明通过体外细胞实验，对于RRx‑001和Ivosidenib联合用药应用后检测不同胶质瘤细胞系的细胞活力，进一步在C57BL/6小鼠颅内原位成瘤模型中验证抗肿瘤效果，实验结果表明RRx‑001和Ivosidenib联合用药在体内外均比单给药具有更强的抑制胶质瘤细胞生长的能力，二者联用对于胶质瘤的治疗作用具有协同增效的效果，因此，RRx‑001和Ivosidenib联合用药能为临床上治疗胶质瘤提供新思路。
Description
技术领域
本发明涉及CD47抑制剂联合IDH1-R132H靶向抑制剂在治疗胶质瘤中的应用，属于生物医药技术领域。
背景技术
脑胶质瘤是世界上最常见的原发性颅内肿瘤。我国脑胶质瘤年发病率为5～8/10万，5年病死率在全身肿瘤中仅次于胰腺癌和肺癌。IDH1-R132H是脑胶质瘤的主要突变之一，常见于低级别胶质瘤(Low-Grade Glioma，LGG)。LGG约占脑胶质瘤的30％，患者的发病年龄比高级别脑胶质瘤年轻，常位于或靠近重要功能区，如运动、语言、视空间和记忆等。其治疗方式仍旧以手术为主，药物治疗仍然是研究人员面临的挑战。
RNA甲基化修饰是近年来新发现的一种重要的参与肿瘤发生发展的修饰方式。其中m5C甲基化是常见修饰方式之一。NSUN5是RNA m5C甲基转移酶，可以在体外抑制胶质瘤的生长。有研究发现，NSUN5在大部分胶质瘤细胞系中低表达，通过调节核糖体的翻译来帮助肿瘤细胞抵抗较高的氧化应激负荷，这种低表达与DNA甲基化可能有关。TET家族是一类去甲基化酶，α-酮戊二酸是其去甲基化反应不可缺少的辅因子。IDH1-R132H可以通过减少α-酮戊二酸的产生来抑制去甲基化酶的作用，从而使得DNA的甲基化水平升高。Ivosidenib(艾伏尼布)是IDH1-R132H的靶向抑制剂，目前已经用于急性髓系白血病和原发性胆管癌的临床治疗，胶质瘤的临床试验仅处于I期阶段。Ivosidenib可能通过降低DNA甲基化水平提升NSUN5的表达从而发挥抗胶质瘤作用。目前NSUN5对于胶质瘤免疫微环境的作用未被报道，但是巨噬细胞是胶质瘤免疫微环境中最多见的免疫细胞。CD47是巨噬细胞表面的免疫检查点，可以抑制巨噬细胞对肿瘤细胞的吞噬。RRx-001是CD47的靶向抑制剂，目前也有临床试验开展。但是目前还没有关于RRx-001和Ivosidenib联用治疗胶质瘤的相关报道。
发明内容
本发明的目的是：为了解决如何提高胶质瘤的疗效的技术问题，本发明提供了CD47抑制剂联合IDH1-R132H靶向抑制剂在治疗胶质瘤中的应用，本发明的结果表明，CD47抑制剂与IDH1-R132H靶向抑制剂联用具有协同抗肿瘤的作用。
为达到解决上述问题的目的，本发明所采取的技术方案是提供CD47抑制剂联合IDH1-R132H靶向抑制剂在制备治疗IDH突变型胶质瘤中的应用，所述胶质瘤为IDH突变型胶质瘤。
优选地，所述CD47抑制剂为RRx-001，所述IDH1-R132H靶向抑制剂为Ivosidenib。
优选地，所述药物的剂型包括注射剂、片剂、粉剂、混悬剂、胶囊剂、丸剂或糖浆。
与现有技术相比，本发明的有益效果在于：
本发明通过体外细胞实验，对于RRx-001和Ivosidenib联合用药应用后检测不同胶质瘤细胞系的细胞活力，进一步在C57BL/6小鼠颅内原位成瘤模型中验证抗肿瘤效果，实验结果表明RRx-001和Ivosidenib联合用药在体内外均比单给药具有更强的抑制胶质瘤细胞生长的能力，二者联用对于IDH突变型胶质瘤的治疗作用具有协同增效的效果，因此，RRx-001和Ivosidenib联合用药能为临床上治疗IDH突变型胶质瘤提供新思路。
附图说明
图1展示了IDH1-R132H可以降低基因组甲基化水平并提高NSUN5蛋白水平；a：在人胶质瘤细胞系T98G中通过Westernblot检测IDH1-R132H过表达前后NSUN5蛋白水平；b：在小鼠胶质瘤细胞系GL261中通过Westernblot检测IDH1-R132H过表达前后NSUN5蛋白水平；ns：not significant，*p<0.05，**p<0.01，***p<0.001；
图2展示了体外实验中RRx-001和Ivosidenib联合给药比单给药更能抑制胶质瘤生长；a：通过Westernblot检测RRx-001和Ivosidenib不同给药方案下野生型和过表达IDH1-R132H的T98G中NSUN5、CD47的蛋白水平；b：CCK8实验结果，实验主要分为四组，第一组为对照组肝癌细胞系T98G；第二组培养基中含有2μM RRx-001的T98G；第三组为培养基中含有5μM Ivosidenib的T98G；第四组为培养基中同时含有2μM RRx-001和5μM Ivosidenib的T98G；ns：not significant，*p<0.05，**p<0.01，***p<0.001；
图3展示了体内实验中10mg/kg RRx-001(腹腔注射)和10mg/mg Ivosidenib(灌胃)联合给药比单给药更能抑制胶质瘤生长(从种瘤后第三天开始给药，每三天给一次药)；其中，a：分别使用稳定表达Vector和Idh1-R132H的小鼠胶质瘤细胞系GL261构建的原位胶质瘤C57BL/6小鼠，单独用药或联合给药的实验方案及分组示意图；b：各组C57BL/6小鼠原位瘤取出后进行HE染色的示意图；c～d：各组荧光定量分析结果；e～f：各组C57BL/6小鼠生存曲线对比；ns：not significant，*p<0.05，**p<0.01，***p<0.001。
具体实施方式
为使本发明更明显易懂，兹以优选实施例，并配合附图作详细说明如下。
下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例
本实施例提供了CD47抑制剂联合IDH1-R132H靶向抑制剂在IDH突变型胶质瘤中的协同抗肿瘤作用：
(一)实验方法：
1、细胞培养及处理
人胶质瘤细胞系T98G、小鼠胶质瘤细胞系GL261，来源于中国科学院细胞库(上海)。T98G细胞在添加10％胎牛血清(Yeasen)、100单位·mL-1青霉素和100μg·mL-1链霉素的DMEM培养基中维持在37℃、5％ CO2进行培养。
2、质粒构建
通过过表达IDH1-R132H来构建IDH1-R132H突变型胶质瘤细胞系及相应的小鼠模型，使用质粒pLVX来过表达IDH1-R132H和idh1-R132H。IDH1-R132H和idh1-R132H全长序列分别从人T98G细胞系和小鼠GL261细胞系cDNA中通过PCR扩增并使用同源重组试剂盒(Vazyme)接入pLVX载体中。
3、慢病毒包装
293T细胞在10cm细胞培养皿中培养，当细胞覆盖满60％时转染慢病毒包装质粒pMD2g和psPAX2质粒，经过48小时孵育后，收集含有包装好的慢病毒的上清液感染目的细胞。48小时后使用2μg/ml puromycin(invitrogen)进行筛选，得到稳定细胞株。
4、CCK8实验
细胞以2×103/孔的密度接种于96孔板中，RRx-001给药浓度为5μM，Ivosidenib给药浓度为5μM，将CCK8试剂(Yeason)以1:10的比例用培养基稀释并在每个孔中加入100μl稀释后的CCK8溶液，37℃孵育1.5小时。使用酶标仪在450nm波长处测量吸光值。
5、蛋白质印迹实验(Western blot)
为了检测目标蛋白在细胞中的表达含量，Western blot实验被用于蛋白的验证。采用RIPA(强)细胞裂解液被用来裂解细胞并收集蛋白质，每组取20μg总蛋白进行蛋白电泳。在转膜后，将膜与5％脱脂牛奶在22℃下孵育1小时，然后与一抗在4℃下孵育过夜。主要抗体及其稀释度如下：
β-actin(Proteintech)Rabbit mAb#81115-1-RR:按照1:5000比例稀释；
NSUN5(Proteintech)Rabbit mAb#15449-1-AP:按照1:1000比例稀释；
CD47(Proteintech)Rabbit mAb#20305-1-AP:按照1:1000比例稀释；
IDH1-R132H(Abcam)Rabbit mAb#ab264055:按照1:1000比例稀释；
随后，抗兔二抗(HRP连接抗体(#7074，CST，美国)：1:2000)在22℃(约室温)下使用1小时。使用化学发光ECL试剂盒(Tanon，上海，中国)可视化蛋白质条带。
6、C57BL/6小鼠原位胶质瘤实验
8周龄C57BL/6小鼠使用戊巴比妥腹腔注射麻醉，固定于立体定位仪，使用微量注射器将5μl含1×106个带有稳定转染luciferase肿瘤细胞的PBS溶液注射入颅内，从第3天开始按照图3a所示的给药方案每三天给一次药。地西他滨采用腹腔注射的方法，浓度为5mg/g体重，对照为等体积PBS；RRx-001采用灌胃法，浓度为5mg/g体重，对照为等体积10％DMSO+40％PEG400+5％Tween-20组成的混合液。于第7，14天向小鼠腹腔注射荧光素钾盐溶液，置于荧光活体成像仪中检测荧光强度间接反映肿瘤的大小。
7、HE染色
HE染色按照之前的研究进行。简而言之，C57BL/6原位肿瘤样品在实验终点取下后存放于4％多聚甲醛(Sigma-Aldrich，DK-2860，丹麦)中固定，由recordbio公司完成后续步骤。
(二)实验结果：
1、分别在人胶质瘤细胞系T98G和小鼠胶质瘤细胞系GL261中通过Westernblot检测IDH1-R132H过表达前后NSUN5蛋白水平，结果如图1a～b所示，表明IDH1-R132H可以提高NSUN5蛋白水平。
2、通过Westernblot检测RRx-001和Ivosidenib不同给药方案下野生型和过表达IDH1-R132H的T98G中NSUN5、CD47的蛋白水平，如图2a所示，在过表达的细胞系中，RRx-001和Ivosidenib联用相比单独用RRx-001或Ivosidenib处理组，具有协同提高NSUN5蛋白表达水平的作用，以及协同降低CD47蛋白表达水平的租用；CCK8实验结果如图2b所示，在过表达的细胞系中，RRx-001和Ivosidenib联用相比单独用RRx-001或Ivosidenib处理组，具有协同抑制胶质瘤细胞活力的作用，以上结果表明，体外实验中RRx-001和Ivosidenib联合给药比单给药更能抑制IDH突变型胶质瘤生长。
3、C57BL/6小鼠原位胶质瘤实验中，通过荧光活体成像仪中检测荧光强度间接反映肿瘤的大小，结果如图3c～d所示，各组小鼠生存曲线如图3e～f所示，以上结果表明，体内实验中RRx-001和Ivosidenib联合给药比单给药更能抑制IDH突变型胶质瘤肿瘤的生长。
上述实施例仅为本发明的优选实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
Claims (3)
1.CD47抑制剂联合IDH1-R132H靶向抑制剂在制备治疗IDH突变型胶质瘤中的应用。
2.如权利要求1所述的应用，其特征在于，所述CD47抑制剂为RRx-001，所述IDH1-R132H靶向抑制剂为Ivosidenib。
3.如权利要求1所述的应用，其特征在于，所述药物的剂型包括注射剂、片剂、粉剂、混悬剂、胶囊剂、丸剂或糖浆。
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