CN110308284B - 放射超敏蛋白Beclin1作为机体核辐射保护的靶点 - Google Patents
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Abstract
本发明公开了放射超敏蛋白Beclin1作为机体核辐射保护的靶点。本发明通过研究发现,Beclin1双等位基因敲除小鼠与致死剂量放射所致小鼠在死亡时间及体态特征及小鼠脏器系数、血量、血常规的变化上都呈现一致的表现,且致死剂量放射后1天小鼠骨髓细胞中Beclin1蛋白表达即消失,这和Beclin1敲除小鼠表型一致。因此证明可将Beclin1蛋白作为机体内放射超敏蛋白进行后续的研发,Beclin1蛋白有望作为机体内放射性损伤救治的靶标,筛选能提高机体内Beclin1蛋白表达或增强其稳定或筛选能抑制放射导致Beclin1降解的制剂可作为放射损伤救治的手段,为放射性救治提供全新理论依据和高效救治方案。
Description
技术领域
本发明属于生物技术领域,具体涉及放射超敏蛋白Beclin1作为机体核辐射保护的靶点。
背景技术
现代社会随着核技术和平使用范围的不断扩大,人类接触核辐射的机会也大大增加。除了核泄漏、核事故以及核战争等极端事件外,近年来随着航空航天项目的发展所带来的太空辐射,日常生活中的医疗辐射,消费品中应用的辐射源等各种人工辐射,都具有不同程度的放射性。核辐射对人体的损伤直接体现于对机体组织器官的损伤,组织器官的功能衰竭是核辐射后死亡的主要原因。减轻辐射对组织器官的损伤,提高机体组织器官的修复能力,是核辐射救治的根本途径。骨髓造血干细胞移植是救治放射性损伤致死的重要方法之一,可以恢复机体造血、增强组织器官的损伤修复。
但是对于大剂量核辐射所致损伤,造血干细胞移植虽可恢复造血,但大部分受照射者在后期仍会死于器官衰竭或感染等。如1990 年6月25日上海发生的核事故,其中2例吸收剂量分别为12Gy和 11Gy,为极重度骨髓型放射病,1999年9月30日发生在日本东海村的辐射事故,2例分别为4.5Gy中子和8.5Gyγ射线混合,2.9Gy 中子和4.5GyY线混合照射,2004年10月21日发生于山东济宁的 60Co源辐射事故,2例受照者受照剂量达20-25Gy(肠型急性放射病)和9-15Gy(极重度骨髓型急性放射病)。这些受照的患者在受照早期均进行了半相合或全相合的造血干细胞移植并显示移植成功,但是后期均相继死亡(分别为受照后90天、25天、81天、210 天、33天、75天),多死于脏器功能衰竭或严重的感染。
目前,治疗急性放射损伤主要有抗辐射药物、细胞因子、骨髓移植等方法。抗辐射药物包括氨磷汀等化学类、甾体激素类如尼尔雌醇、雌三醇等,但化学类、激素类等传统抗辐射药物治疗作用有限,仅对于轻中度放射病有效,且不良反应不易克服。细胞因子包括干细胞因子、促血小板生成因子、粒细胞集落刺激因子等,细胞因子的应用对于轻中度骨髓型急性放射病作用显著,但对于重度患者,由于体内造血干细胞残存数量过少,不能恢复自身造血,细胞因子作用也有限。骨髓移植或造血干细胞移植主要用于重度骨髓型放射病患者的救治,尤其是出现多器官损伤的患者,可以重建造血功能,减轻辐射所致的组织器官损伤,是救治放射性损伤致死的重要方法之一。不过对于极重度受照患者,造血干细胞移植虽可恢复造血,但大部分受照射者在后期仍会死于器官衰竭或严重感染等。因此寻找新的减轻器官衰竭,重塑组织器官的功能的辐射救治方法迫在眉睫。
Beclin 1基因最初是以Bcl-2结合蛋白的身份被发现的,随后其在自噬以及凋亡这两个重要的生物学途径中的作用被逐渐认识。近年来,Beclin1作为肿瘤抑制基因也得到了广泛的研究,在脑肿瘤、宫颈癌、卵巢癌等多种肿瘤中均出现Beclin1蛋白表达的下降。最近Beclin1基因的新功能也逐渐被发现,有研究者发现beclin1 敲除(beclin1-/-)的小鼠在胚胎时期全部死亡,beclin1杂合子 (beclin1+/-)的小鼠可以存活,而Atg5-/-和Atg7-/-等自噬基因缺失的小鼠能够正常出生,且未出现异常细胞增殖或者肿瘤形成。这些研究说明,beclin1可能存在自噬功能以外的更重要的功能。
我们实验室的早期研究显示,Beclin1在体外细胞系的钴源照射损伤修复中起作用。在极端情况下,Beclin1可通过非依赖自噬途径促进DNA钴源照射损伤修复,即通过与核内DNA解旋酶TopIIb 结合,促进DNA损伤修复。也有学者发现Beclin1通过与UVRAG相互作用,保护结肠直肠癌细胞免受辐射诱导的DNA损伤,以维持其中心体的稳定性。
但是我们以往的研究,在体外细胞细胞系中,Beclin1并不对放射敏感,放射并不能诱发其降解,相反,通过核移位促进DNA损伤修复。而我们最近的研究,更新、纠正了对Beclin1原来的认知:即在机体内情况恰恰相反,体内Beclin1对放射极其敏感,说明我们以往体外细胞系的研究结果没有真实反映哺乳类机体内Beclin1 的确切作用。
对人类放射损伤救治有真正指导意义的是体内研究结果,即我们最近发现的机体中Beclin1对放射的超级敏感(简称超敏)反应。对于机体,特别是对于哺乳类动物(包括啮齿类、灵长类),辐射后尤其是模拟致死核辐射剂量后Beclin1的变化及其作用尚无研究报道。因针对辐射超敏蛋白Beclin1的研发,对人类放射损伤救治中的作用意义重大。
发明内容
为了克服上述现有技术中存在的不足,本发明旨在提供一种放射超敏蛋白Beclin1作为机体核辐射保护的靶点,为放射损伤救治提供全新的、关键性研发基础和技术方案。
为实现上述技术目的,达到上述技术效果,本发明通过以下技术方案实现:
本发明提出了一种将Beclin1作为机体内放射超敏蛋白的应用,可作为放射性损伤治疗中的靶标,用于放射性损伤救治的后续研发。
进一步的,提高机体内Beclin1蛋白的表达或增强其稳定性,可用于放射性损伤的救治。
进一步的,筛选或制备诱导机体内Beclin1蛋白升高或抑制降解的制剂,可用于放射性损伤的救治。
为此本发明的发明人通过以下实验进行了验证,具体步骤如下:
1.利用实验室建立Beclin1f/f;Ubc-iCre小鼠,在用药物他莫西芬(TMXF)诱导后,得到Beclin1敲除小鼠,建立了Beclin1双等位基因条件性敲除小鼠模型。
2.将Beclin1敲除小鼠作为Beclin1-KO组;将同时进行他莫西芬(TMXF)腹腔注射的对照小鼠作为Ctrl-TMXF对照组;将经过致死剂量9Gy照射的野生型小鼠作为Irradiation组,将未经过照射的野生型小鼠作为Ctrl对照组。
3.通过Beclin1双等位基因敲除来模拟致死剂量的照射,分别观察Beclin1-KO组小鼠和Irradiation组小鼠的死亡情况。
4.通过Beclin1双等位基因敲除来模拟致死剂量的照射,分别观察Beclin1-KO组小鼠和Irradiation组小鼠的体态特征。
5.通过Beclin1双等位基因敲除来模拟致死剂量的照射,分别观察Beclin1-KO组小鼠和Irradiation组小鼠的心、脾、肝、胸腺、结肠等脏器重量和脏器系数的变化情况。
6.通过Beclin1双等位基因敲除来模拟致死剂量的照射,分别观察Beclin1-KO组小鼠和Irradiation组小鼠的血量变化。
7.通过Beclin1双等位基因敲除来模拟致死剂量的照射,分别观察Beclin1-KO组小鼠和Irradiation组小鼠的胫骨、腓骨颜色的变化情况。
8.通过Beclin1双等位基因敲除来模拟致死剂量的照射,分别观察Beclin1-KO组小鼠和Irradiation组小鼠的血常规变化。
通过上述实验后我们发现,Beclin1双等位基因敲除和致死剂量照射所致小鼠死亡时间和体态特征、小鼠脏器系数变化及血量变化、血常规变化都呈现一致的表现,并且致死辐射后1天小鼠骨髓细胞中Beclin1蛋白表达即消失,和Beclin1敲除小鼠表型一致。
因此以上研究提示,Beclin1可作为机体内放射超敏蛋白进行后续研发,在放射性损伤中,Beclin1蛋白有望作为放射性损伤救治的靶标,筛选能提高机体内Beclin1蛋白表达的制剂(如小分子化合物)或筛选能抑制放射导致Beclin1降解的制剂(如小分子化合物)可作为放射损伤救治的手段,从而为放射性救治提供全新的理论依据和高效的救治方案。
本发明的有益效果是:
本发明通过实验研究发现,Beclin1双等位基因敲除小鼠与致死剂量放射所致小鼠在死亡时间及体态特征、小鼠脏器系数变化及血量变化、血常规变化上都呈现一致的表现,并且致死剂量照射后 1天小鼠骨髓细胞中Beclin1蛋白表达即消失,这和Beclin1敲除小鼠表型一致。因此证明可以将Beclin1蛋白作为机体内放射超敏蛋白进行后续的研发。在放射性损伤中,Beclin1蛋白有望作为机体内放射性损伤救治的靶标,筛选能提高机体内Beclin1蛋白表达的制剂(如小分子化合物)或筛选能抑制放射导致Beclin1降解的制剂(如小分子化合物)可作为放射损伤救治的手段,从而为放射性救治提供全新的理论依据和高效的救治方案。
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例并配合附图详细说明如后。本发明的具体实施方式由以下实施例及其附图详细给出。
附图说明
此处所说明的附图用来提供对本发明的进一步理解,构成本申请的一部分,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。在附图中:
图1为本发明Beclin1f/f;Ubc-iCre小鼠模型的建立过程示意图;
图2A为本发明的Beclin1的敲除效果鉴定图;
图2B为本发明各组实验小鼠的骨髓细胞中Beclin1表达情况的对比图;
图2C为本发明各组实验小鼠的死亡数与实验天数的关系示意图;
图2D为本发明各组实验小鼠体重情况的对比图;
图2E为本发明各组实验小鼠体态特征的对照图;
图2F为本发明各组实验小鼠嘴唇及脚趾皮肤颜色情况的对照图;
图3A为本发明各组实验小鼠心脏重量及脏器系数的对照图;
图3B为本发明各组实验小鼠脾脏重量及脏器系数的对照图;
图3C为本发明各组实验小鼠肝脏重量及脏器系数的对照图;
图3D为本发明各组实验小鼠胸腺重量及脏器系数的对照图;
图3E为本发明各组实验小鼠胃重量及脏器系数的对照图;
图3F为本发明各组实验小鼠结肠重量及脏器系数的对照图;
图3G为本发明各组实验小鼠外周血量的对照图;
图3H为本发明各组实验小鼠胫骨、腓骨颜色的对照图;
图4A为本发明各组实验小鼠白细胞数量的对照图;
图4B为本发明各组实验小鼠淋巴细胞数量的对照图;
图4C为本发明各组实验小鼠单核粒细胞数量的对照图;
图4D为本发明各组实验小鼠中性粒细胞数量的对照图;
图4E为本发明各组实验小鼠嗜酸性细胞数量的对照图;
图4F为本发明各组实验小鼠CD41巨核细胞数量的对照图。
具体实施方式
下面将参考附图并结合实施例,来详细说明本发明。
本发明提出了一种将Beclin1作为机体内放射超敏蛋白的应用,可作为放射性损伤治疗中的靶标,用于放射性损伤救治的后续研发。
进一步的,提高机体内Beclin1蛋白的表达或增强其稳定性,可用于放射性损伤的救治。
进一步的,筛选或制备诱导机体内Beclin1蛋白升高或抑制降解的制剂,可用于放射性损伤的救治。
为此本发明的发明人通过以下实验进行了验证,具体步骤如下:
1.Beclin1双等位基因条件性敲除小鼠模型的建立。
本发明人利用实验室建立了Beclin1f/f;Ubc-iCre小鼠,具体过程为,在小鼠的Beclin1基因的3号外显子和9号外显子之间分别插入一个floxp位点,建立Beclin1f/f小鼠,然后与Ubc-iCre小鼠杂交,得到Beclin1f/f;Ubc-iCre小鼠,如图1所示,此小鼠可正常生存繁殖。在用药物他莫西芬(TMXF)诱导后,floxp位点之间的片段 被切除,Beclin1基因的功能区域即被切除,得到Beclin1敲除小鼠。
2.Beclin1双等位基因敲除模拟致死剂量辐射所致小鼠死亡情况和体态特征的观察。
Beclin1-KO组:七周龄Beclin1f/f;Ubc-iCre小鼠,进行他莫西芬(TMXF)腹腔注射诱导Beclin1的敲除,得到Beclin1敲除小鼠,敲除效果鉴定如图2A所示;
Ctrl-TMXF组:对照组小鼠同时进行他莫西芬(TMXF)腹腔注射;
Irradiation组:致死剂量9Gy照射野生型小鼠;
Ctrl组:未经过照射的野生型小鼠;
参见图2C所示,Beclin1f/f;Ubc-iCre纯合子小鼠(Beclin1-KO 组)在注射他莫西芬(TMXF)一周后开始出现死亡;致死剂量9Gy 照射野生型小鼠(Irradiation组),死亡时间为第8-第11天。
参见图2B所示,在照射后1天的致死剂量9Gy照射野生型小鼠 (Irradiation组)的骨髓细胞中Beclin1蛋白表达即消失,并且致死剂量9Gy照射野生型小鼠(Irradiation组)在濒死时出现和 Beclin1敲除小鼠(Beclin1-KO组)相似的体态特征,包括图2D 所示的弓背,图2E所述的体重减轻,图2F所示的嘴唇以及脚趾皮肤发白。
3.Beclin1双等位基因敲除模拟致死剂量辐射所致小鼠脏器系数变化及血量变化的观察。
参见图3A-3F所示,Beclin1f/f;Ubc-iCre小鼠在敲除Beclin1 基因后,小鼠的心、脾、肝、胸腺、结肠重量均减轻,脏器系数降低,胃重量增加,脏器系数变大。其中,参见图3A-3E所示,Beclin1 敲除小鼠(Beclin1-KO组)在心、脾、肝、胸腺、胃的变化上,和致死剂量9Gy照射野生型小鼠(Irradiation组)的变化一致;参见图3F所示,致死剂量9Gy照射野生型小鼠(Irradiation组)的结肠变化不明显。
参见图3G所示,Beclin1敲除小鼠(Beclin1-KO组)在外周血量的变化上,和致死剂量9Gy照射野生型小鼠(Irradiation组) 的变化也一致,均表现为血量减少;
参见图3H所示,Beclin1敲除小鼠(Beclin1-KO组)在胫骨、腓骨颜色的变化上,和致死剂量9Gy照射野生型小鼠(Irradiation 组)及对照组小鼠(Ctrl-TMXF组)的变化一致,都表现为颜色变红,可能与白细胞减少红细胞相对增多有关。
4.Beclin1双等位基因敲除模拟致死剂量辐射所致小鼠血常规变化的观察。
为了进一步检测外周血中各类细胞数量比例变化,采用五分类血细胞计数仪检测了各组小鼠外周血中的细胞数量。参见图4A-4F 所示,与对照组小鼠(Ctrl-TMXF组)相比,Beclin1敲除小鼠 (Beclin1-KO组)和致死剂量9Gy照射野生型小鼠(Irradiation 组)在白细胞、淋巴细胞、单核细胞、中性粒细胞、嗜酸性细胞、 CD41巨核细胞的数量上均出现了减少,变化一致。
终上所述,Beclin1双等位基因敲除和致死剂量放射所致小鼠死亡时间和体态特征、小鼠脏器系数变化及血量变化、血常规变化都呈现一致的表现,并且致死放射后1天小鼠骨髓细胞中Beclin1 蛋白表达即消失,和Beclin1敲除小鼠表型一致。
以上研究提示,Beclin1可作为机体内放射超敏蛋白进行后续的研发。在放射性损伤中,Beclin1蛋白有望作为机体内放射性损伤救治的靶标,筛选能提高机体内Beclin1蛋白表达的制剂(如小分子化合物)或筛选能抑制放射导致Beclin1降解的制剂(如小分子化合物)可作为放射损伤救治的手段,从而为放射性救治提供全新的理论依据和高效的救治方案。
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 (2)
1.放射超敏蛋白Beclin1作为机体内核辐射保护的靶点在制备或筛选核辐射治疗药物中的应用,其特征在于:放射超敏蛋白Beclin1在机体内受到核辐射后短时间内会降解。
2.根据权利要求1所述的放射超敏蛋白Beclin1作为机体内核辐射保护的靶点在制备或筛选核辐射治疗药物中的应用,其特征在于:可开发核辐射条件下基于维持Beclin1稳定表达的小分子化合物。
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