CN110124011A - 一种染色质重构复合物核心催化亚基在制备治疗或预防乙型肝炎病毒感染药物中的应用 - Google Patents
一种染色质重构复合物核心催化亚基在制备治疗或预防乙型肝炎病毒感染药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种染色质重构复合物核心催化亚基蛋白在制备治疗或预防乙型肝炎病毒感染药物中的应用。属于抗病毒药物领域。本发明将人BRG1的过表达质粒或小干扰RNA与能表达HBV的质粒pHBV1.3共转染到肝细胞系HepG2或Huh7中，随后检测细胞培养上清中HBV分泌抗原HBeAg和HBsAg的表达量和细胞内HBV复制中间体核衣壳相关DNA的水平，证明BRG1抑制HBV复制，具有抗乙型肝炎病毒的活性。因其抗病毒的机制多样化，不易使病毒产生耐药性，从而为临床上乙型肝炎的治疗提供一类安全高效且毒副作用小的多肽。
Description
技术领域
本发明涉及抗病毒药物领域，涉及一种染色质重构复合物核心催化亚基蛋白BRG1的新用途，更具体涉及一种染色质重构复合物核心催化亚基蛋白BRG1 在制备治疗或预防乙型肝炎病毒(Hepatitis B virus，HBV)感染药物中的应用。
背景技术
乙型肝炎病毒(Hepatitis B virus，HBV)感染是引起肝癌的主要因素之一，严重威胁着人类的生命健康。我国是乙型肝炎病毒感染的高发区，约有1.2亿慢性乙肝患者，每年约有30万人死于慢性乙肝引起的肝细胞癌。HBV属于嗜肝 DNA病毒科，基因组为部分双链环状DNA。HBV的核心颗粒直径为28nm，为20面体，病毒衣壳由核心蛋白HBc组成。核衣壳的表面有包膜，包膜上含有三种大小的表面抗原preS1，preS2和HBsAg。有感染性的病毒颗粒又称作 Dane颗粒，直径为42nm。HBV感染会导致一系列的肝脏疾病，从急性肝炎(包括暴发性肝功能衰竭)到慢性肝炎，肝硬化和肝细胞癌。HBV复制本身不直接导致细胞病变，多数的HBV携带者是无症状的，肝损害也很小，宿主的抗病毒免疫是导致肝损伤等临床症状的主要原因。HBV感染人体后的症状与被感染者的年龄和免疫力相关。新生儿中肝炎99％以上是无症状的，1–5岁儿童中，只有 10％是有症状的。三分之一的成人急性乙型肝炎患者有临床症状，其中约1％为爆发性肝炎，致死率达到70％。爆发性肝炎伴随着强烈的抗病毒免疫，病毒很快被清除。95％的成年人感染后会自发地清除病毒，而90％的幼儿和30％的1–5 岁的儿童则会导致慢性感染。HBV感染和相关疾病的诊断是根据一系列的临床的生化、组织和血清学的指标来进行的。检测病毒感染的指标主要有病毒的几种抗原，和相对应的特异性抗体以及HBV DNA；检测病人肝脏病变情况主要有血液丙氨酸和谷丙转氨酶(Alaninetransaminase，ALT)，以及肝组织检测炎症和纤维化程度。
目前临床上乙型肝炎病毒感染的治疗主要有两类药物：干扰素和核苷类似物。临床应用的干扰素包括IFNα2a，IFNα2b和IFNα1b，这些是传统的干扰素，还包括PEG-IFNα2a和PEG-IFNα2b。核苷类似物拉米夫定(Lamivudine，LAM) 和替比夫定(Telbivudine，LDT)；脱氧鸟苷类似物恩替卡韦(Entecavir，ETV)；核苷膦酸酯，阿德福韦酯(Adefovir，ADV)和富马酸替诺福韦酯(Tenofovir disoproxil fumarate，TDF)。其中ETV，TDF和PEG-IFN最常用。IFN用于治疗慢性HBV感染已经有超过30年的历史。临床上IFNα对于HBeAg阳性的病人，在6个月内，每周注射三次，有25％的病人HBeAg转阴。12个月的IFNα治疗结果显示10–17％的病人HBsAg转阴。对于HBeAg阴性的病人，12个月的IFNα治疗后60％病人血清中检验不到HBVDNA。经PEG修饰的IFN即 PEG-IFN，其抗病毒机理与IFNα相同，但明显提高了IFNα的药物代谢动力学，注射次数为每周一次，血清中IFNα浓度能维持较高的水平。2003年的一项研究表明，PEG-IFN的治疗效果优于IFN。但另一方面，PEG-IFN也有其副作用和局限，若治疗初期的效果不佳，则应停止用药，考虑其他治疗方案。核苷类似物通过抑制HBV RNA的逆转录来抑制HBV的复制。与IFN相比，核苷类似物有一些优势，如副作用小，方便等。但是也有一些缺点，如高复发率，低HBsAg转阴率，单一用药时容易产生耐药性。
染色质重构复合物核心催化亚基BRG1是表观遗传学SWI/SNF家族的重要 ATP酶亚基,其主要作用是利用ATP水解产生能量实现染色体重组。BRG1定位于19p13染色体,是一种磷酸化核蛋白,主要通过ATP依赖的方式破坏组氨酸 -DNA的接触、改变染色体结构从而调节基因的表达。人类BRG1基因在约90％的高钙血症型的卵巢小细胞癌中频繁突变,但在其他癌症类型中频率要低得多。 Shanahan等在结直肠癌细胞系DLD-1中敲除BRG1,结果发现BRG1通过抑制 PTEN的表达从而抑制了PI3K-Akt信号通路,导致cyclin D1的表达下调,所以在结直肠癌中BRG1和cyclinD1表达的相关性表明BRG1可能通过激活PI3K-Akt 信号通路从而促进了肿瘤的发展。Jose A等研究显示BRG1表达上调与乳腺癌、卵巢癌、肺腺癌、脂肪肉瘤和以及皮肤黑色素瘤,膀胱尿路上皮癌等预后相关, 表明BRG1的高表达可以用作这些肿瘤的预后标记。此外,也有一些研究表明,BRG1是肿瘤细胞增殖所必需的。Imbalzano等人也提出了BRG1在癌症中的双重作用。表明BRG1基因在不同的组织,不同类型的肿瘤中同时具有肿瘤抑制或致癌作用。目前未见BRG1的表达和HBV复制之间的关系研究报道。
发明内容
本发明的目的是提供一种染色质重构复合物核心催化亚基蛋白BRG1在制备治疗或预防乙型肝炎病毒感染药物中的应用，从而为临床上乙型肝炎的治疗提供一类安全高效且毒副作用小的多肽。通过单独使用或者与其他抗HBV药物联合使用，染色质重构复合物核心催化亚基蛋白BRG1能够在预防或者治疗乙型肝炎病毒感染过程中发挥重要作用。
为了实现上述的目的，本发明采用的技术方案是：
第一方面，本发明提供一种染色质重构复合物核心催化亚基蛋白BRG1在制备治疗或预防乙型肝炎病毒感染药物中的应用。
优选地，上述应用中所述的治疗或预防乙型肝炎病毒感染药物是以染色质重构复合物核心催化亚基蛋白BRG1作为药物活性成分，利用常规技术可制成片剂、胶囊、颗粒剂、口服液、缓释制剂、控释制剂、纳米制剂，其中活性成分含量5％(质量比，以下相同)，其余为相应辅料(90％淀粉和5％PVP)或注射剂(规格为1mg/mL，溶于生理盐水)。
在本发明的具体实施例中，将人BRG1的过表达质粒(本实验室构建)或小干扰RNA(广州锐博公司合成)与能表达HBV的质粒pHBV1.3(D型，构建方法见下文)共转染到肝细胞系HepG2(来自ATCC)或Huh7(来自ATCC)中，随后检测细胞培养上清中HBV分泌抗原HBeAg和HBsAg(上海科华)的表达量和细胞内HBV复制中间体核衣壳相关DNA的水平。
上述实验结果如图1所示。过表达和干扰实验都证明，BRG1能下调细胞培养上清中HBV分泌抗原HBeAg和HBs的表达量和细胞内HBV复制中间体核衣壳相关DNA的水平，说明BRG1抑制HBV复制，具有抗乙型肝炎病毒的活性。
所述的BRG1的过表达质粒，其构建过程见实施例1。
所述的质粒pHBV1.3，其构建过程是，提取HepG2.2.15细胞(来自ATCC) 的DNA，扩增HBV DNA并插入载体pBluescript II(购自Invitrogen)。
本发明与现有技术相比，具有以下优点和效果：
1.染色质重构复合物核心催化亚基蛋白BRG1是在人体内组成性表达的一种细胞因子，生理浓度下对人体没有毒副作用。
2.染色质重构复合物核心催化亚基蛋白BRG1的抗病毒作用机制是通过直接作用于病毒包膜，或影响病毒吸附，或改变细胞内的信号通路，从而抗病毒，因其抗病毒的机制多样化，不易使病毒产生耐药性。
附图说明
图1A为一种证明过表达BRG1抑制HBV的e抗原和s抗原分泌的示意图；
其中e抗原抑制约55％，s抗原抑制约35％。
图1B为一种证明过表达BRG1下调HBV的核衣壳相关DNA的量的示意图；
抑制约45％。
图1C为一种证明si-BRG1有较好的干扰效果的图示意；
干扰掉80％。
图1D为一种证明干扰BRG1促进HBV的e抗原和s抗原分泌的示意图；
e抗原和s抗原均上调2倍以上。
图1E为一种证明干扰BRG1上调HBV的核衣壳相关DNA的量的示意图；
上调约2倍。
图2为转染对应量的pCAGGS-BRG1质粒到HepG2细胞中检测细胞存活率。
具体实施方式
通过以下详细说明结合附图可以进一步理解本发明的特点和优点。所提供的实施例仅是对本发明方法的说明，而不以任何方式限制本发明揭示的其余内容。【实施例1】通过过表达实验评价染色质重构复合物核心催化亚基蛋白BRG1 的抗HBV活性。
一种染色质重构复合物核心催化亚基蛋白BRG1在制备治疗或预防乙型肝炎病毒感染药物中的应用，其步骤是：
1.实验材料：
1.1细胞、质粒：
HepG2细胞(来自ATCC)和pHBV1.3质粒。以从Huh7细胞中提取的总 mRNA为模板，先通过反转录PCR扩增总cDNA，再以该cDNA为模板，用特异引物扩增出完整BRG1基因片段，引物序列如下：
BRG1CDS Forward：5′-GACCAAGACCCGATGAACACCATCATGCAGCT-3′；
BRG1CDS Reverse：5′-GACTAGTCGCGTCGACTTATCCTTCTTCGCTGCC-3′。
通过特异限制性内切酶(EcoRI和XhoI)酶切、连接将BRG1基因克隆到载体pCAGGS(购自addgene)上，得到pCAGGS-BRG1的过表达质粒，并测序鉴定。
1.2试剂：
DMEM培养基购自GIBCO公司；限制性内切酶和T4连接酶购自NEB公司。
1.3实验仪器：
Roche LightCycler 480。
2.实验方法：
将人肝细胞系HepG2接种到24孔板，约12小时后，待细胞密度长到约70％后，进行转染。用Lipofectamine-2000将pCAGGS/pCAGGS-BRG1与pHBV1.3 共转染，转染后4小时换新鲜的培养液。转染后48小时后，用ELISA试剂盒检测细胞培养上清中的e抗原和s抗原的表达量；并提取和检测HBV核衣壳相关 DNA，方法如下：a.弃上清，每孔加入100微升的细胞裂解液(50mM Tris， 0.5％NP-40，1mM EDTA和100mM NaCl)，4℃摇床上裂解1小时，转移到1.5毫升EP管中。b.每管加入1微升1M氯化镁，和1微升DNaseI，37℃孵育2 小时。c.每管加入3.5微升0.5M的EDTA和22.5微升35％PEG，4℃孵育1 小时；4℃，12000rpm离心1分钟。d.弃上清，每管加入200微升重悬液(10mM Tris，100mM Nacl，1mM EDTA和1％SDS)，和5微升蛋白酶K(25mg/mL)， 55℃孵育过夜。e.每管加入100微升Tris饱和酚和100微升氯仿，振荡混匀，室温(20–25℃以下相同)12000rpm离心10分钟。f.取上清，加入等体积的异丙醇沉淀，室温，12000rpm离心10分钟，去上清，收沉淀。g.加入1毫升体积比为75％的乙醇，12000rpm离心5分钟，重复一次，吸干残余乙醇，干燥。 h.溶于20微升dH2O中。i.荧光定量PCR检测HBV，用梯度稀释的pHBV1.3 质粒做标准曲线，计算样品中的HBV DNA拷贝数。所用引物和探针序列为HBV DNA Forward：5’-ACCCAAGGCACAGCTTGGAGG-3’；HBV DNA Reverse： 5’-AGATGATTAGGCAGAGGTGAAAAA-3’；HBV探针序列： 5’-tggctagtttactagtgcaattttg-3’。
3.实验结果：
如图1A、1B所示，过表达BRG1下调e抗原和s抗原的表达量和细胞内 HBV复制中间体核衣壳相关DNA的水平，说明BRG1抑制HBV复制，BRG1 具有抗HBV活性。
【实施例2】通过干扰实验评价染色质重构复合物核心催化亚基蛋白BRG1的抗HBV活性。
一种染色质重构复合物核心催化亚基蛋白BRG1在制备治疗或预防乙型肝炎病毒感染药物中的应用，其步骤是：
1.实验材料：
干扰RNA套餐购自广州锐博公司。
2.实验方法
与实施例1相同。
3.实验结果：
首先检测了干扰RNA的干扰效果，如图1C所示，抑制效率约为80％。如图1D、1E所示干扰BRG1上调HBeAg和HBsAg的表达量和细胞内HBV复制中间体核衣壳相关DNA的水平，说明BRG1抑制HBV复制，BRG1具有抗HBV 活性。
其它步骤与【实施例1】相同。
以上实施例1和实施例2的实验结果说明，BRG1具有较好的抑制HBV的复制的作用，又由于BRG1是人体内组成性表达的分子量较小的分泌蛋白，毒副作用小，至今也未有BRG1或其衍生物开发出的抗病毒药物，具有很好的创新性。【实施例3】MTS检测转染pCAGGS-BRG1对细胞存活率的影响
如图2所示转染对应量的pCAGGS-BRG1质粒到HepG2细胞中，并用 pCAGGS空载作对照，转染后48小时用MTS(购自Promega)检测细胞存活率，实验结果表明，转染pCAGGS-BRG1对HepG2细胞存活率没有影响。
序列表
<110> 上海市浦东新区公利医院（第二军医大学附属公利医院）
<120> 一种染色质重构复合物核心催化亚基在制备治疗或预防乙型肝炎病毒感染药物中的应用
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gaccaagacc cgatgaacac catcatgcag ct 32
<210> 2
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gactagtcgc gtcgacttat ccttcttcgc tgcc 34
<210> 3
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<212> DNA
<213> 人工序列(Artificial Sequence)
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acccaaggca cagcttggag g 21
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<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
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agatgattag gcagaggtga aaaa 24
<210> 5
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tggctagttt actagtgcaa ttttg 25
Claims (2)
1.一种染色质重构复合物核心催化亚基蛋白BRG1 在制备治疗或预防乙型肝炎病毒感染药物中的应用。
2.根据权利要求1所述的应用，其特征在于，所述的治疗或预防乙型肝炎病毒感染药物是以染色质重构复合物核心催化亚基蛋白 BRG1 作为药物活性成分，利用常规技术可制成片剂、胶囊、颗粒剂、口服液、缓释制剂、控释制剂、纳米制剂 ，其中活性成分质量比含量5%，其余为相应辅料为质量比90%淀粉和5% 聚乙烯吡咯烷酮或BRG1含量为1 mg/mL生理盐水注射剂。
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