CN116212024A - Ube2t抑制剂的用途及抗恶性胶质瘤的药物组合物 - Google Patents
Ube2t抑制剂的用途及抗恶性胶质瘤的药物组合物 Download PDFInfo
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Abstract
本发明公开了UBE2T抑制剂在制备恶性胶质瘤治疗药物中的用途，所述UBE2T抑制剂包括化学抑制剂、通过基因工程手段抑制UBE2T表达的试剂、UBE2T上游靶点调节剂。同时还公开了UBE2T检测试剂在恶性胶质瘤替莫唑胺耐药状况检测中的用途，以及一种恶性胶质瘤替莫唑胺耐药性的检测试剂盒和一种抗恶性胶质瘤的药物组合物。UBE2T抑制剂单独作用即可发挥良好的抗恶性胶质瘤增殖效果，提供了UBE2T抑制剂作为恶性胶质瘤治疗药物的应用，为恶性胶质瘤治疗药物的开发提供了有价值的研究方向。
Description
技术领域
本发明属于生物技术领域，具体涉及UBE2T抑制剂在制备恶性胶质瘤治疗药物及替莫唑胺耐药状况检测中的用途治疗靶点，以及抗恶性胶质瘤的药物组合物。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
恶性胶质瘤(Glioblastoma,GBM)是最常见的恶性原发脑肿瘤，约占颅脑肿瘤的45％，其复发率高、易转移、生存率低。手术切除、放疗以及替莫唑胺(Temozolomide,TMZ)为基础的化疗是恶性胶质瘤的标准治疗方案。然而，由于分子异质性、促生存信号通路的过度激活、高生长速度、免疫抑制等原因，恶性胶质瘤患者往往对替莫唑胺治疗产生耐药性，预后较差。虽然人们为提高恶性胶质瘤的治疗效率做了许多尝试，但不幸的是，没有任何一种方案能达到预期的治疗效果。因此，阐明替莫唑胺耐药机制，开发新的靶向治疗方法具有重大临床意义。
泛素结合酶E2T(UBE2T，又称HSPC150)属于泛素E2家族，参与从E1激活酶到底物的泛素激活。UBE2T最初被报道为负调控范可尼贫血(FA)通路，其缺失导致DNA修复能力受损和异常染色体的形成。除与FA相关外，UBE2T也被证实在各种肿瘤的发生、进展和复发中发挥重要作用。最近的研究发现UBE2T在多种肿瘤的治疗耐药中起着关键作用，例如非小细胞肺癌、肝癌等。先前的研究发现UBE2T参与恶性胶质瘤的增殖、凋亡、侵袭和迁移。但是UBE2T在恶性胶质瘤替莫唑胺化疗耐药中的作用尚不清楚，UBE2T抑制剂能否治疗恶性胶质瘤尚不明确，靶向UBE2T能够克服恶性胶质瘤替莫唑胺耐药也尚未搞清。
发明内容
本发明针对上述问题展开了研究，发现UBE2T与恶性胶质瘤化疗耐药相关；采用UBE2T抑制剂应用于恶性胶质瘤治疗可以有效抑制恶性胶质瘤细胞增殖；采用UBE2T抑制剂与替莫唑胺共同应用于恶性胶质瘤治疗可以有效降低恶性胶质瘤对于替莫唑胺的耐药性，为恶性胶质瘤的治疗提供了新的用药依据和治疗方案。
本发明的技术方案如下：
本发明的第一个发明目的是提供UBE2T抑制剂作为恶性胶质瘤治疗药物的应用。
本发明发现使用siRNA敲低UBE2T能够显著增强替莫唑胺对恶性胶质瘤的抑制作用；转染质粒过表达UBE2T则明显较低替莫唑胺对恶性胶质瘤的抑制作用。进一步，通过CCK-8方法检测UBE2T抑制剂M435-1279对恶性胶质瘤细胞增殖的影响，结果表明UBE2T抑制剂可以以浓度梯度和时间梯度依赖的方式抑制恶性胶质瘤细胞增殖。体内实验中，相较于对照组，向裸鼠腹腔注射UBE2T抑制剂可以明显抑制细胞生长，减小荷瘤小鼠的肿瘤体积。这些结果证实UBE2T抑制剂可以作为恶性胶质瘤治疗药物加以开发的应用。
优选的，所述UBE2T抑制剂包括化学抑制剂、通过基因工程手段抑制UBE2T表达的试剂、UBE2T上游靶点调节剂等。
进一步优选的，所述化学抑制剂包括M435-1279。
本发明第二方面，提供UBE2T检测试剂作为恶性胶质瘤替莫唑胺耐药状况检测试剂的应用。
优选的，所述UBE2T检测试剂包括通过RT-qPCR检测方法、免疫印迹方法、免疫组化方法、免疫荧光检测等检测恶性胶质瘤中UBE2T的表达水平。
本发明第三方面，提供一种恶性胶质瘤替莫唑胺耐药检测试剂盒，所述试剂盒中包括上述UBE2T检测试剂。
本发明第四方面，提供UBE2T抑制剂与替莫唑胺的组合作为恶性胶质瘤治疗药物的应用。即一种抗恶性胶质瘤的药物组合物，所述药物组合物的活性成分为UBE2T抑制剂和替莫唑胺。
优选的，所述UBE2T抑制剂为M435-1279。
作为恶性胶质瘤替莫唑胺耐药治疗靶点的应用，恶性胶质瘤替莫唑胺的耐药是患者预后较差的主要原因之一，而替莫唑胺耐药机制不清，缺乏有效的逆转耐药方法。本发明针对UBE2T对替莫唑胺耐药的作用进行了系列研究。结果表明UBE2T在体内外促进恶性胶质瘤对替莫唑胺耐药。进一步发现UBE2T特异性抑制剂M435-1279在细胞和裸鼠水平明显抑制恶性胶质瘤细胞增殖，采用UBE2T抑制剂M435-1279与替莫唑胺联合应用于恶性胶质瘤治疗，治疗效果优于替莫唑胺单独治疗效果。
与现有技术相比，本发明的有益效果是：
1.相较于正常组织，UBE2T在多种肿瘤组织中表达水平升高，并且促进恶性肿瘤的增殖、转移和耐药。先前的研究同样表明UBE2T促进恶性胶质瘤的恶性进展。本发明证实UBE2T抑制剂单独作用即可发挥良好的抗恶性胶质瘤增殖效果，提供了UBE2T抑制剂作为恶性胶质瘤治疗药物的应用，为恶性胶质瘤治疗药物的开发提供了有价值的研究方向。
2.恶性胶质瘤患者对替莫唑胺耐药是恶性胶质瘤治疗失败的主要原因之一。本发明对恶性胶质瘤耐药机制进行了探究。证实UBE2T促进恶性胶质瘤对替莫唑胺耐药，提供了UBE2T抑制剂与替莫唑胺联合应用作为恶性胶质瘤治疗药物的应用。推动了恶性胶质瘤耐药机制及联合用药的研究。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。
图1为敲低UBE2T增强替莫唑胺作用效果：
其中，图中A为Western Blot方法检测UBE2T蛋白水平条带图，
B为敲低UBE2T后，使用替莫唑胺处理细胞增殖曲线图，
C为替莫唑胺处理条件下，流式细胞术检测细胞凋亡水平，
D为1C中细胞凋亡比率量化统计直方图，
E为替莫唑胺处理条件下，克隆形成实验检测细胞成克隆能力，
F为1E中细胞克隆数目量化统计直方图；
图2为过表达UBE2T促进替莫唑胺耐药：
其中，图中A为Western Blot方法检测UBE2T蛋白水平条带图，
B为过表达UBE2T后，使用替莫唑胺处理细胞增殖曲线图，
C为替莫唑胺处理条件下，流式细胞术检测细胞凋亡水平，
D为C中细胞凋亡比率量化统计直方图，
E为替莫唑胺处理条件下，克隆形成实验检测细胞成克隆能力，
F为E中细胞克隆数目量化统计直方图；
图3为UBE2T特异性抑制剂M435-1279显著抑制恶性胶质瘤细胞增殖：
其中，图中A为不同浓度抑制剂处理U251和U87细胞48h细胞存活率曲线图，
B为10μM抑制剂处理U251和U87细胞24h、48h和72h细胞存活率曲线图；
图4为M435-1279与替莫唑胺联合应用抑制恶性胶质瘤细胞增殖：
其中，图中A为CCK-8检测U251细胞存活率曲线图，
B为CCK-8检测U87细胞存活率曲线图，
C为克隆形成实验检测细胞成克隆能力，
D为C中细胞克隆数目量化统计直方图；
图5为在体内荷瘤裸鼠模型中，腹腔注射M435-1279显著减少肿瘤体积：
其中，图中A为荷瘤模型中小鼠肿瘤照片图，
B为肿瘤体积统计散点图；
图6为在体内荷瘤裸鼠模型中，M435-1279联合替莫唑胺抑制肿瘤体积增长：
其中，图中A为荷瘤模型中小鼠肿瘤照片图，
B为肿瘤体积统计散点图。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本发明提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本发明的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
正如背景技术所介绍的，针对现有技术中存在的不足，本发明提出了UBE2T作为恶性胶质瘤替莫唑胺耐药治疗靶点的应用。
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案，以下将结合具体的实施例详细说明本发明的技术方案。本发明涉及的动物实验研究均获得中国科学技术大学附属第一医院动物护理机构和使用委员会的批准。
以下各实施例中所使用的抗体和试剂均是已知可市购的产品，具体如下：
UBE2T(10105-2-AP,Proteintech)；
β-actin(66009-1-Ig,Proteintech)；
替莫唑胺(S1237，Selleck)；
M435-1279(S1180,Selleck)；
RIPA裂解(P0013B，碧云天)。
实施例1
实施例1.检测敲低UBE2T增强恶性胶质瘤对替莫唑胺敏感性。
使用慢病毒转染方法构建稳定敲低UBE2T的U251和U87细胞系(Ctr，对照组；sh-1，敲低组1；sh-2，敲低组2)，RIPA裂解提取细胞蛋白质，利用Western Blot方法检测UBE2T蛋白质水平(如图1中A)。将敲低UBE2T的细胞接种于96孔板中，每孔3000个细胞，每组6个复孔，使用100μM替莫唑胺(TMZ)处理细胞48h、72h和96h后，利用CCK-8方法检测细胞存活率(图1中B)。
将敲低UBE2T的细胞接种于6cm小皿中，使用100μM替莫唑胺处理细胞72h后，利用流式细胞术方法检测细胞凋亡水平(图1中C和D)。
将敲低UBE2T的细胞接种于6孔板中，每孔800个细胞，每组3个复孔，使用20μM替莫唑胺处理细胞3–7天，利用克隆形成实验检测细胞成克隆能力(图1中E和F)。
如图1所示，敲低UBE2T增强恶性胶质瘤细胞对替莫唑胺敏感性，敲低UBE2T促进替莫唑胺诱导的恶性胶质瘤细胞凋亡。UBE2T在细胞水平上促进恶性胶质瘤细胞对替莫唑胺耐药性。
实施例2.检测过表达UBE2T促进恶性胶质瘤对替莫唑胺耐药。
使用慢病毒转染方法构建稳定过表达UBE2T的U251和U87细胞系(Ctr，对照组；UBE2T，过表达组)，RIPA裂解提取细胞蛋白质，利用Western Blot方法检测UBE2T蛋白质水平(图2中A)。将过表达UBE2T的细胞接种于96孔板中，每孔3000个细胞，每组6个复孔，使用100μM替莫唑胺处理细胞48h、72h和96h后，利用CCK-8方法检测细胞存活率(图2中B)。
将过表达UBE2T的细胞接种于6cm小皿中，使用100μM替莫唑胺处理细胞72h后，利用流式细胞术方法检测细胞凋亡水平(图2中C和D)。
将过表达UBE2T的细胞接种于6孔板中，每孔800个细胞，每组3个复孔，使用20μM替莫唑胺处理细胞3–7天，利用克隆形成实验检测细胞成克隆能力(图2中E和F)。
如图2所示，过表达UBE2T减弱恶性胶质瘤细胞对替莫唑胺敏感性，过表达UBE2T降低替莫唑胺诱导的恶性胶质瘤细胞凋亡。UBE2T促进恶性胶质瘤细胞对替莫唑胺耐药。
实施例3.检测UBE2T抑制剂M435-1279对恶性胶质瘤细胞增殖水平的影响。
将3000个U251和U87恶性胶质瘤细胞接种于96孔板中，每组6个复孔，分别使用0、1、2、5、10、20、50、100μM M435-1279处理细胞48h，使用CCK-8方法检测细胞增殖水平(图3中A)。使用10μM M435-1279处理恶性胶质瘤细胞24、48、72和96h，使用CCK-8方法检测细胞增殖水平(图3中B)。
如图3所示，M435-1279能够明显抑制恶性胶质瘤细胞增殖。
实施例4.检测UBE2T抑制剂M435-1279与替莫唑胺联合应用于恶性胶质瘤治疗的效果。
将恶性胶质瘤细胞U251和U87接种于96孔板中，每孔3000个细胞，每组6个复孔。分别使用0、10、25、50、100、200、400、800μM替莫唑胺或/和5μMM435-1279(1279)处理细胞72h，使用CCK-8方法检测细胞存活率(图4中A和B)。将U251和U87细胞接种于6孔板中，每孔800个细胞，每组3个复孔，使用20μM替莫唑胺或/和2μM M435-1279处理细胞3–7天，利用克隆形成实验检测细胞成克隆能力(图4中C和D)。
如图4所示，M435-1279联合替莫唑胺处理杀伤效果明显优于替莫唑胺单独处理。
实施例5.检测体内荷瘤裸鼠模型中UBE2T促进恶性胶质瘤对替莫唑胺耐药
从北京唯尚立德公司订购4–6周大小裸鼠(20–25g)，将5×106个稳定过表达UBE2T(UBE2T组)或对照组U251(Ctr)细胞注射到裸鼠左侧腋窝皮下，待肿瘤体积增长至100mm3后腹腔注射50mg/kg替莫唑胺，15天后安乐法处死小鼠，取出肿瘤组织，拍照(图5中A)，使用公式：长×宽2×0.5计算肿瘤体积(图5中B)。
如图5所示，过表达UBE2T组小鼠肿瘤体积明显大于对照组，证明在体内，过表达UBE2T促进恶性胶质瘤对替莫唑胺耐药。
实施例6.检测体内荷瘤裸鼠模型中，M435-1279联合替莫唑胺对肿瘤的治疗作用
将裸鼠分成四组，每组6只。将5×106个U251细胞注射到裸鼠左侧腋窝皮下，待肿瘤体积增长至100mm3后，第一组作为对照组(Ctr)注射PBS、第二组腹腔注射50mg/kg替莫唑胺(TMZ)、第三组裸鼠的腹腔注射10mg/kg M435-1279(1279)、第四组裸鼠的腹腔注射50mg/kg TMZ+10mg/kg 1279(TMZ+1279)。15天后安乐法处死小鼠，取出肿瘤组织，拍照(图6中A)，使用公式：长×宽2×0.5计算肿瘤体积(图6中B)。
如图6所示，在体内荷瘤裸鼠模型中M435-1279联合替莫唑胺作用效果明显优于替莫唑胺单独作用效果。证明在体内，M435-1279联合替莫唑胺可以提高恶性胶质瘤治疗效果。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (7)
1.UBE2T抑制剂在制备恶性胶质瘤治疗药物中的用途，其特征在于：所述UBE2T抑制剂包括化学抑制剂、通过基因工程手段抑制UBE2T表达的试剂、UBE2T上游靶点调节剂。
2.根据权利要求2所述的用途，其特征在于：所述化学抑制剂为M435-1279。
3.UBE2T检测试剂在恶性胶质瘤替莫唑胺耐药状况检测中的用途。
4.根据权利要求3所述的用途，其特征在于：所述UBE2T检测试剂包括使用免疫印迹方法、免疫组化方法、RT-qPCR方法或免疫荧光方法检测恶性胶质瘤中UBE2T的表达水平。
5.一种恶性胶质瘤替莫唑胺耐药性的检测试剂盒，其特征在于：所述试剂盒中包括如权利要求3或4所述的UBE2T检测试剂。
6.一种抗恶性胶质瘤的药物组合物，其特征在于：所述药物组合物的活性成分为UBE2T抑制剂和替莫唑胺。
7.根据权利要求6所述的药物组合物，其特征在于：所述UBE2T抑制剂为M435-1279。
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