CN104480196B - 抗ang在hbv相关肝癌治疗中的应用 - Google Patents

抗ang在hbv相关肝癌治疗中的应用 Download PDF

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CN104480196B
CN104480196B CN201410630878.3A CN201410630878A CN104480196B CN 104480196 B CN104480196 B CN 104480196B CN 201410630878 A CN201410630878 A CN 201410630878A CN 104480196 B CN104480196 B CN 104480196B
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hbv
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liver cancer
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CN104480196A (zh
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周晓棠
陈智
楼国华
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Zhejiang University ZJU
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Abstract

本发明公开了抗ANG在HBV相关肝癌治疗中的应用，HBV促进HepG2细胞中血管生成素的表达及核转移。并公开了HBV促进ANG上调的分子机制，该发明将为HBV相关肝细胞肝癌的治疗提供新思路。

Description

抗ANG在HBV相关肝癌治疗中的应用
技术领域
本发明属于医药技术领域，具体涉及一种抗ANG在HBV相关肝癌治疗中的应用。
背景技术
HBV感染是威胁人类健康的重要公共卫生问题，全世界每年约有一百万人死于HBV感染引起的肝衰竭、肝硬化和肝癌。全球范围内，约有75％的肝细胞肝癌(hepatocelluarcarcinomas,HCC)是由HBV感染发展而来的。我国是HBV感染的高发区，随之HBV相关HCC的也相应高发。1970年Lacent首次报导了HBV与HCC的关系，之后不断有研究证实了这一结论。HBV的致癌性虽已明确，但HBV究竟通过何种机制致癌仍未有定论，基本上可以归纳为病毒自身、宿主与环境相互作用的结果。病毒的因素可包括激活原癌基因，病毒基因整合入宿主基因以及病毒自身蛋白等，这其中较为明确的是HBx蛋白。HBx蛋白可通过调节细胞信号通路、转录、细胞周期、DNA修复以及细胞凋亡等多种机制促进HCC的发生。
RNase5最初在人结肠癌细胞株HT-29中被发现，由于其具有促进血管生成的作用被命名为血管生成素(angiogenin，ANG)。之后随着对ANG研究的不断深入，发现其为RNaseA超家族成员，并命名为RNase5，随之，它的生物学功能也被逐渐认识，发现它在前列腺癌，乳腺癌，肺癌及肝癌等多种肿瘤中高表达，其机制涉及影响细胞凋亡信号通路、刺激rRNA转录以及参与tiRNA的合成等方面。有报导在卡波氏肉瘤病毒(KSHV)感染的内皮细胞(HMVEC-d)中，会促使ANG表达上调，上调的ANG与表面肌动蛋白结合，通过微管途径进入细胞并进入核内，进而通过促进45S RNA转录、抑制细胞凋亡、促使感染细胞迁徙扩散、血管生成等机制介导KSHV引起的宿主病理损伤，这一发现证实了病毒对ANG存在一定的调控作用。
发明内容
为克服上述缺点，本发明的提供一种抗ANG在HBV相关肝癌治疗中的应用。
本发明所采用的技术方案是，抗ANG在HBV相关肝癌治疗中的应用，HBV促进HepG2细胞中血管生成素的表达及核转移。
本发明的特点还在于，
上述的表达在于HBV促进IL-6及HIF-1α的表达，具体为HBV通过IL-6及HIF-1α上调ANG。
核转移被抑制后，细胞增殖被抑制。
本发明的有益效果是：本发明是通过细胞水平检测病毒感染或转染X蛋白细胞中的ANG表达情况，进而得出HBV可促进ANG高表达同时可促进其核转移，通过检测病毒感染细胞中IL-6及HIF-1α的表达情况及阻断其表达，得出病毒通过促进IL-6及HIF-1α的表达进而促进ANG的高表达；最后，通过抑制ANG表达或其核转移，得出抑制ANG活性后细胞增殖情况被抑制，这将为HBV相关肝癌的治疗提供新的思路。
附图说明
图1是HBV病毒复制对ANG的调节作用；
图2是X蛋白对ANG转录及翻译水平的调节；
图3是HBV病毒复制对IL-6及HIF-1α表达的影响；
图4是X蛋白对IL-6及HIF-1α表达的影响；
图5是IL-6对病毒表达细胞中ANG mRNA的影响；
图6是IL-6对病毒表达细胞中ANG蛋白分泌的影响；
图7是抗体封闭IL-6活性后对病毒复制细胞中ANG表达的影响；
图8是HIF-1α基因沉默后ANG表达变化；
图9是HBV对ANG核转移的影响；
图10是ANG抑制后对细胞增殖的影响。
具体实施方式
下面结合附图和具体实施方式对本发明进行详细说明。
实施例1 HBV病毒复制及HBx均可从转录及翻译水平上调ANG的表达水平
以Real time-PCR、Western Blot及Elisa分别对含有HBV完整颗粒并可进行病毒复制的HepG2.2.15及HepG2.2.15来源细胞--HepG2细胞中ANG的表达进行了测定。采用pCDNA3.1-HBx转染HepG2细胞，同时以pcDNA3.1转染HepG2细胞作为对照，以Western Blot及Elisa分别检测转染HBx基因的HepG2-HBx细胞及对照细胞内及细胞培养上清中ANG蛋白表达的变化。
实验结果：如图1所示，2215细胞内ANG mRNA及蛋白水平显著高于对照，如图2所示，转染X蛋白的HepG2细胞中ANG蛋白的表达及分泌高于对照。
以上结果表明，HBV病毒复制上调ANG的表达，这可能是通过HBx可从转录及翻译水平上调节ANG的表达实现的。
实施例2 HBV/Hbx上调ANG的机制
实验方法：以Western Blot检测负荷HBV/HBx蛋白的细胞内IL-6及HIF-1α蛋白；以外源性IL-6分别刺激HepG2及HepG2215细胞，以Real time-PCR及Elisa分别检测ANG mRNA及蛋白；以HIF-1αsiRNA沉默HIF-1α基因，采用Western Blot及Elisa检测ANG蛋白表达来探索HIF-1a通路和ANG上调的关系。
实验结果：HBV或其X蛋白表达细胞中IL-6及HIF-1α蛋白表达水平高于对照，如图3、4；如图5、6所示，IL-6促进2215细胞中ANG蛋白的表打及分泌，同时IL-6被阻断后ANG表达降低，如图7；如图8示，HIF-1α表达抑制后，ANG蛋白表达及分泌减少。
以上结果表明，HBV上调ANG的依赖于IL-6和HIF-1a的介导，抑制IL-6或HIF-1a后，ANG表达下调。
实施例3 HBV对ANG核转移的影响
以ANG特异性抗体26-2F(美国哈佛医学院和Tufts医学中心的胡国富教授赠予)进行免疫荧光染色，TO-PRO3染核，分别对HepG2及HepG2.2.15细胞中的ANG进行定位。通过ANGsiRNA(购至SANTA CRUZ公司，sc-39291)沉默ANG或以neomycine抑制ANG核转移，paromomycine作为对照，MTT检测细胞增殖情况。
实验结果：HepG2.2.15细胞核中ANG显著高于HepG2细胞，如图9；ANG表达或核转移抑制后，HBV感染的肝癌细胞HepG2.2.15细胞增殖明显被抑制，抑制肿瘤的发生，如图10。
以上结果表明，HBV促进ANG核转移，当其被抑制后可抑制2215细胞增殖。

Claims (3)

1.抗RNase5抗体在制备HBV感染引起的肝癌治疗药物中的应用，其特征在于，HBV感染促进HepG2.2.15细胞中RNase5的表达及核转移。
2.根据权利要求1所述的应用，其特征在于，所述的表达在于HBV感染通过促进IL-6及HIF-1α的表达上调RNase5。
3.根据权利要求1所述的应用，其特征在于，所述的核转移被抑制后，细胞增殖被抑制。
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Citations (3)

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CN1325907A (zh) * 2001-05-25 2001-12-12 陈志南 肝癌转移相关因子HAb18G及其反义RNA表达质粒载体PCI－asHAb18G
CN1884295A (zh) * 2006-07-04 2006-12-27 浙江大学 缺氧诱导因子1反义寡核苷酸及其用途
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