水动力转染技术在IFNβ-Luc小鼠模型与HBV树鼩模型研究中的应用
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摘要
水动力转染方法是一种简单、有效的将外源物质转染至活体动物细胞中的非病毒转染方法,这种方法是利用快速大量将溶液注射进血管,迅速产生压力增加血管内皮和细胞膜的通透性,将携带的物质转入细胞内。水动力转染方法越来越多的用在活体动物的研究上,包括动物模型建立、疾病的治疗、基因免疫等、药物评价等。该方法可用来将质粒DNA、基因组DNA、RNA、寡核苷酸、蛋白质等目的分子转染到活体动物的组织器官中。利用水动力转染的平台,本研究探索了两种实验动物模型,一种是用于与病毒感染密切相关的天然免疫分子IFN-β活性监测的小鼠模型,另一种是乙型肝炎病毒质粒转染的树鼩模型。以期在这两种模型的基础上进一步的研究肝炎病毒感染及免疫致病机制。
第一部分水动力转染方法建立IFN-β启动子调控荧光素酶表达小鼠模型
I型干扰素(Interferon ,IFN)在天然免疫反应中有着重要作用,其主要成员是IFN-α和IFN-β,干扰素最先被人们发现是因为它的抗病毒的作用,后来的研究发现它还参与器官发生、肿瘤的发展等生理病理学的机制。病毒的识别及蛋白信号级联反应的启动是引发抗病毒天然免疫的必要条件,病毒感染或是双链RNA、双链DNA都能够诱导I型干扰素的产生,随后I型干扰素通过自分泌或者旁分泌结合到细胞表面引发干扰素级联反应,通过正反馈诱发干扰素刺激基因ISGs的大量表达,这些基因编码一系列的抗病毒蛋白和炎性因子来阻止病毒的扩散避免宿主进一步的被感染。但是许多病毒在进化过程中逐渐产生一些逃避免疫的方式。一些细胞水平上有关干扰素的调节机制的研究丰富了天然免疫网络。但是在有着完整免疫力的活体动物内进行的研究相对较少。肝脏是蛋白合成和许多天然免疫成分活化的一个重要场所,许多免疫相关分子能够被肝细胞来源的信号活化,肝脏作为一个重要的免疫器官的说法在逐渐被人们接受。肝脏的天然免疫系统在肝炎病毒感染及其免疫致病中发挥何种效应,已经成为学者们研究的热点。
因此本研究构建了一种含有噬菌体整合酶识别位点attB的、由IFN-β启动子调控萤火虫荧光素酶表达的报告基因载体;先细胞水平通过荧光素酶检测和荧光成像来验证了该载体能够正常表达。然后通过水动力注射法将质粒转染至小鼠肝脏,并通过活体成像和Western blot证实了该质粒在小鼠肝脏的表达。进一步通过巢式PCR和活体荧光成像证实了表达载体在小鼠肝脏发生位点特异性整合。上述结果提示已成功建立IFN-β启动子调控荧光素酶表达小鼠模型。随后我们用经典的IFN-β的激活剂poly(I:C)来诱导小鼠模型肝脏中的荧光素酶表达,荧光素酶在6-8小时达到峰值。同时用ELISA法监测血清中的IFN-β,血清中的IFN-β的表达在时效上与荧光素酶表达一致,结果显示报告基因的表达能够指示IFN-β的活化。用不同剂量的poly(I:C)来激活小鼠肝脏的IFN-β,结果显示激活小鼠肝脏IFN-β最佳剂量为250μg/kg。已知HCV NS3/4A蛋白酶能够在人的细胞中阻断IFN-β信号通路,我们将HCV NS3/4A质粒水动力转染到小鼠肝脏,通过活体成像、血清IFN-β监测及Western blot检测,结果提示HCV NS3/4A蛋白酶能够在小鼠体内阻断IFN-β信号活化通路。
以上结果显示,我们建立了一种灵敏、稳定的在活体肝脏内评价IFN-β活性的模型体系,用此模型动物可以研究不同因素在体内对IFN-β启动子活性的调节作用,也可作为天然免疫中干扰素调节药物的评价模型。同时也有助于体内研究肝脏的天然免疫系统和肝炎病毒之间的相互作用。
第二部分水动力转染方法初步建立HBV转染树鼩模型
乙型肝炎病毒(HBV)是一种严重危害人类健康的嗜肝DNA病毒,全球约有1/4的人曾经感染过HBV,有约3.6亿慢性携带者。虽然大部分成年人在感染后病毒很快被清除,但婴幼儿感染HBV后却有很高的慢性化率。HBV慢性感染的机制至今未能明确,长期以来缺乏合适的动物模型阻碍了HBV的研究进展。HBV有严格的宿主特异性,已知的宿主有人,黑猩猩、和树鼩。黑猩猩等灵长类动物价格昂贵,实验还受到伦理学的限制。树鼩是一种生活在热带和亚热带地区的哺乳纲攀鼩类的小型动物,在生物进化上比啮齿类、猫、犬、兔等动物更接近灵长类。新陈代谢与解剖结构比啮齿类更接近人类,且易于驯养。因此,树鼩可能是应用于建立人类疾病动物模型的最佳动物之一。近年来在建立嗜肝病毒感染模型上有着较多的探索并取得了一定的进步。常规感染方法是用阳性血清或纯化的病毒颗粒接种树鼩,但是阳性率低,多为一过性感染。因此我们尝试用水动力转染HBV质粒的方法建立树鼩肝炎模型。
为了建立一种水动力注射将外源基因导入树鼩肝脏的方法,本研究摸索了水动力转染树鼩的适宜条件,确定了树鼩水动力注射以0.8ml/s速度注射100ml/kg的生理盐水为最佳条件。在确定了水动力转染的条件后用萤火虫荧光素酶和β-半乳糖苷酶(β-galactosidase,β-Gal)作为报告基因,通过树鼩大隐静脉将报告基因导入树鼩肝脏,通过活体成像和β-Gal染色观察到报告基因在肝脏特异性的表达,活体成像检测到肝脏中荧光光子数达到106～107。然后将pAAV/HBV1.2质粒用水动力转染的方法转入树鼩肝脏,检测到树鼩血清中ALT在注射后第1天达到峰值,3天后恢复到原来水平,实时荧光定量检测血清中HBV DNA在一周内一过性的升高,检测值在103～105,到第二周大部分树鼩血清HBV DNA降至检测线下。ELISA检测血清中HBsAg一过性升高,一周后大部分树鼩血清HBsAg转阴,部分树鼩在一周后出现anti-HBsAg。用环磷酰胺抑制树鼩免疫力后,水动力转染pAAV/HBV1.2质粒,血清标志物检测结果对照组和实验组未见有明显差别。水动力转染后树鼩血清标志物阳性率达到90%。由于树鼩不是模式化动物,树鼩之间个体差异大,所得数据差异也较大。
因此本研究结果显示大隐静脉水动力注射法能够作为外源基因导入树鼩肝脏的一种方法,用这种方法初步探索了树鼩的HBV转染模型。本研究可为建立树鼩感染肝炎病毒模型提供一种新的方法。
本研究以水动力转染技术作为平台,成功建立了IFN-β启动子调控荧光素酶表达小鼠模型,并用此技术初步探索了树鼩的HBV模型的建立。
Hydrodynamic delivery is a simple and effective non-viral method for intracellular delivery of exogenous substances in whole animals. This procedure employs a pressure generated by a rapid injection of large volume of solution into the vessel to expand the sinusoids and enhance the permeability of membrane for entrance of substance into cell. The gene delivery strategy is gradually popular for study in whole animal, including establishment of animal models, therapy of diseases, DNA immunization, evaluation of drug etc. The substance hydrodynamically delivered is not only DNA, but also protein, oligo nucleotides, genomic DNA and RNA. Based on the technique, we generated two kinds of animal model.
Part I Establishment of a mouse model with IFN-βpromoter-driven firefly luciferase reporter in the liver by hydrodynamic gene delivery
Type I IFNs, which is represented by IFN-αand IFN-β, play a critical role in the innate immune response. They were initially identified for their antiviral activity and effects on cells of the innate responses. Recognition of the invading virus by immune system and initiation of protein signaling cascades are prerequisites for triggering an effective anti-viral innate immune response. Type I IFNs can be induced by virus infection or by exposure to double-stranded RNA or DNA (dsRNA or dsDNA) in every cell type, and then binds to the cell surface receptors via autocrine or paracrine to trigger the IFN response signaling cascade which results in the expression of interferon-stimulated genes (ISGs). These genes encode a variety of antiviral proteins and inflammatory cytokines, leading to limit the virus spread and protect the host from further viral infection. But even so, most viruses can evade these host immune responses at various degrees. Researches targeting to the mechanism, regulation and application of type I IFN signaling pathways contribute to innate immune network. But few works have been carried out in animals, under a complex immune environment, to evaluate the type I IFN activation. The liver is a crucial site for proteins synthesis as well as metabolism. Many of the immunological components are triggered by signals from hepatocytes. Its function as a major immune organ is gradually appreciated. Many researches are gradually focusing on the effect producted by the innate immunological components in the liver during the infection of hepatitis virus.
So, we report a simple, sensitive and visualized method to measure activation of IFN-βin vivo. Mice received only a simple hydrodynamic injection of expression vectors of phage PhiC31o integrase and IFN-βpromoter-driven firefly luciferase reporter. The luciferase expression in mouse liver induced with synthesized dsRNA poly(I:C) can be monitored by bioluminescence imaging, which was consistent with the IFN-βin the sera. The optimal dose of poly(I:C) to induce luciferase expression is 250μg/kg. HCV NS3/4A protease not only inhibited IFN-βproduction, but also blocked the luciferase expression in mouse liver. We clearly demonstrated that our mouse model carried an IFN-βpromoter luciferase reporter gene is a sensitive and stable tool that can be applied to the evaluation of IFN-βpromoter activation under various conditions. The mouse model may not only help further understand the complex biological feature of the type I IFN system in liver, but also screen drug candidates for stimulator or inhibitor of IFN-βgene expression in vivo. It also contributed to the interaction mechanism between the innate immune system and hepatitis virus.
Part II Establishment of tree shrews models for HBV study by hydrodynamic gene delivery
Hepatitis B virus (HBV) has infected 25% of the world population. 360 million people were chronic infected. Although, the virus is cleared soon for most adults infected, there is a high rate of chronic infection in infants and young children. The mechanism of chronicity of HBV infection is still unclear because of the lack of suitable animal models. HBV has strict host specificity, It only infects some kinds of primates such as human, chimpanzees and tree shrews. However, some primates such as chimpanzee are an endangered species and expensive. Tree shrew lives in tropical and subtropical regions. It is closer to primates than rodents, cats, dogs, rabbits and other animals in biological evolution. Metabolism and anatomical structure are closer to humans than rodents, and easy to be domesticated. Therefore, Tree shrews can be used in many research fields. In recent years, tree shrew model of hepatotropic virus infection have made some progresses. HBsAg positive serum,or purified virus particles were commomly used to inoculate tree shrews for animal model generation. But the rate of infection is low. So we try to generate a tree shrews model of HBV transfection by hydrodynamic with plasmid pAAV/HBV1.2.
To establish a method for hydrodynamic-based gene delivery to tree shrews, we explored the conditions of the hydrodynamic transfection. The optimal injection speed is 0.8ml/s and volume is 100ml/kg. And the vectors containing firefly luciferase andβ-galactosidase reporter gene were hydrodynamically injected into tree shrew via the great saphenous vein. The reporter gene was only detected in the liver of tree shrew by bioluminescence imaging andβ-gal staining. The tree shrew was hydrodynamically injected with pHBV1.2 plasmid. At the various times, the sera were collected and in which ALT, HBsAg, anti-HBsAg and HBV DNA were examined by ELISA or real-time fluorescence quantitative PCR respectively. The tree shrews injected hydrodynamically with pHBV1.2 were detected the markers of HBV in their sera. The serum HBsAg and HBV DNA transiently increased, less than a week. Tree shrews treated with cyclophosphamide can not carry HBV longer than those without cyclophosphamide. Hydrodynamic-based injection via the great saphenous vein can be used to deliver the gene to hepatocyte of tree shrews. We preliminary generated a tree shrew model of hepatitis B virus expressing in the liver by the hydrodynamic injection. Our study indicated a new method for establishment of tree shrews models with hepatitis virus infection.
Based the technique of hydrodynamic gene delivery, we genearated the mouse model for monitoring IFN-βactivity and preliminary developed the tree shrew model for HBV study.
第一部分水动力转染方法建立IFN-β启动子调控荧光素酶表达小鼠模型
I型干扰素(Interferon ,IFN)在天然免疫反应中有着重要作用,其主要成员是IFN-α和IFN-β,干扰素最先被人们发现是因为它的抗病毒的作用,后来的研究发现它还参与器官发生、肿瘤的发展等生理病理学的机制。病毒的识别及蛋白信号级联反应的启动是引发抗病毒天然免疫的必要条件,病毒感染或是双链RNA、双链DNA都能够诱导I型干扰素的产生,随后I型干扰素通过自分泌或者旁分泌结合到细胞表面引发干扰素级联反应,通过正反馈诱发干扰素刺激基因ISGs的大量表达,这些基因编码一系列的抗病毒蛋白和炎性因子来阻止病毒的扩散避免宿主进一步的被感染。但是许多病毒在进化过程中逐渐产生一些逃避免疫的方式。一些细胞水平上有关干扰素的调节机制的研究丰富了天然免疫网络。但是在有着完整免疫力的活体动物内进行的研究相对较少。肝脏是蛋白合成和许多天然免疫成分活化的一个重要场所,许多免疫相关分子能够被肝细胞来源的信号活化,肝脏作为一个重要的免疫器官的说法在逐渐被人们接受。肝脏的天然免疫系统在肝炎病毒感染及其免疫致病中发挥何种效应,已经成为学者们研究的热点。
因此本研究构建了一种含有噬菌体整合酶识别位点attB的、由IFN-β启动子调控萤火虫荧光素酶表达的报告基因载体;先细胞水平通过荧光素酶检测和荧光成像来验证了该载体能够正常表达。然后通过水动力注射法将质粒转染至小鼠肝脏,并通过活体成像和Western blot证实了该质粒在小鼠肝脏的表达。进一步通过巢式PCR和活体荧光成像证实了表达载体在小鼠肝脏发生位点特异性整合。上述结果提示已成功建立IFN-β启动子调控荧光素酶表达小鼠模型。随后我们用经典的IFN-β的激活剂poly(I:C)来诱导小鼠模型肝脏中的荧光素酶表达,荧光素酶在6-8小时达到峰值。同时用ELISA法监测血清中的IFN-β,血清中的IFN-β的表达在时效上与荧光素酶表达一致,结果显示报告基因的表达能够指示IFN-β的活化。用不同剂量的poly(I:C)来激活小鼠肝脏的IFN-β,结果显示激活小鼠肝脏IFN-β最佳剂量为250μg/kg。已知HCV NS3/4A蛋白酶能够在人的细胞中阻断IFN-β信号通路,我们将HCV NS3/4A质粒水动力转染到小鼠肝脏,通过活体成像、血清IFN-β监测及Western blot检测,结果提示HCV NS3/4A蛋白酶能够在小鼠体内阻断IFN-β信号活化通路。
以上结果显示,我们建立了一种灵敏、稳定的在活体肝脏内评价IFN-β活性的模型体系,用此模型动物可以研究不同因素在体内对IFN-β启动子活性的调节作用,也可作为天然免疫中干扰素调节药物的评价模型。同时也有助于体内研究肝脏的天然免疫系统和肝炎病毒之间的相互作用。
第二部分水动力转染方法初步建立HBV转染树鼩模型
乙型肝炎病毒(HBV)是一种严重危害人类健康的嗜肝DNA病毒,全球约有1/4的人曾经感染过HBV,有约3.6亿慢性携带者。虽然大部分成年人在感染后病毒很快被清除,但婴幼儿感染HBV后却有很高的慢性化率。HBV慢性感染的机制至今未能明确,长期以来缺乏合适的动物模型阻碍了HBV的研究进展。HBV有严格的宿主特异性,已知的宿主有人,黑猩猩、和树鼩。黑猩猩等灵长类动物价格昂贵,实验还受到伦理学的限制。树鼩是一种生活在热带和亚热带地区的哺乳纲攀鼩类的小型动物,在生物进化上比啮齿类、猫、犬、兔等动物更接近灵长类。新陈代谢与解剖结构比啮齿类更接近人类,且易于驯养。因此,树鼩可能是应用于建立人类疾病动物模型的最佳动物之一。近年来在建立嗜肝病毒感染模型上有着较多的探索并取得了一定的进步。常规感染方法是用阳性血清或纯化的病毒颗粒接种树鼩,但是阳性率低,多为一过性感染。因此我们尝试用水动力转染HBV质粒的方法建立树鼩肝炎模型。
为了建立一种水动力注射将外源基因导入树鼩肝脏的方法,本研究摸索了水动力转染树鼩的适宜条件,确定了树鼩水动力注射以0.8ml/s速度注射100ml/kg的生理盐水为最佳条件。在确定了水动力转染的条件后用萤火虫荧光素酶和β-半乳糖苷酶(β-galactosidase,β-Gal)作为报告基因,通过树鼩大隐静脉将报告基因导入树鼩肝脏,通过活体成像和β-Gal染色观察到报告基因在肝脏特异性的表达,活体成像检测到肝脏中荧光光子数达到106～107。然后将pAAV/HBV1.2质粒用水动力转染的方法转入树鼩肝脏,检测到树鼩血清中ALT在注射后第1天达到峰值,3天后恢复到原来水平,实时荧光定量检测血清中HBV DNA在一周内一过性的升高,检测值在103～105,到第二周大部分树鼩血清HBV DNA降至检测线下。ELISA检测血清中HBsAg一过性升高,一周后大部分树鼩血清HBsAg转阴,部分树鼩在一周后出现anti-HBsAg。用环磷酰胺抑制树鼩免疫力后,水动力转染pAAV/HBV1.2质粒,血清标志物检测结果对照组和实验组未见有明显差别。水动力转染后树鼩血清标志物阳性率达到90%。由于树鼩不是模式化动物,树鼩之间个体差异大,所得数据差异也较大。
因此本研究结果显示大隐静脉水动力注射法能够作为外源基因导入树鼩肝脏的一种方法,用这种方法初步探索了树鼩的HBV转染模型。本研究可为建立树鼩感染肝炎病毒模型提供一种新的方法。
本研究以水动力转染技术作为平台,成功建立了IFN-β启动子调控荧光素酶表达小鼠模型,并用此技术初步探索了树鼩的HBV模型的建立。
Hydrodynamic delivery is a simple and effective non-viral method for intracellular delivery of exogenous substances in whole animals. This procedure employs a pressure generated by a rapid injection of large volume of solution into the vessel to expand the sinusoids and enhance the permeability of membrane for entrance of substance into cell. The gene delivery strategy is gradually popular for study in whole animal, including establishment of animal models, therapy of diseases, DNA immunization, evaluation of drug etc. The substance hydrodynamically delivered is not only DNA, but also protein, oligo nucleotides, genomic DNA and RNA. Based on the technique, we generated two kinds of animal model.
Part I Establishment of a mouse model with IFN-βpromoter-driven firefly luciferase reporter in the liver by hydrodynamic gene delivery
Type I IFNs, which is represented by IFN-αand IFN-β, play a critical role in the innate immune response. They were initially identified for their antiviral activity and effects on cells of the innate responses. Recognition of the invading virus by immune system and initiation of protein signaling cascades are prerequisites for triggering an effective anti-viral innate immune response. Type I IFNs can be induced by virus infection or by exposure to double-stranded RNA or DNA (dsRNA or dsDNA) in every cell type, and then binds to the cell surface receptors via autocrine or paracrine to trigger the IFN response signaling cascade which results in the expression of interferon-stimulated genes (ISGs). These genes encode a variety of antiviral proteins and inflammatory cytokines, leading to limit the virus spread and protect the host from further viral infection. But even so, most viruses can evade these host immune responses at various degrees. Researches targeting to the mechanism, regulation and application of type I IFN signaling pathways contribute to innate immune network. But few works have been carried out in animals, under a complex immune environment, to evaluate the type I IFN activation. The liver is a crucial site for proteins synthesis as well as metabolism. Many of the immunological components are triggered by signals from hepatocytes. Its function as a major immune organ is gradually appreciated. Many researches are gradually focusing on the effect producted by the innate immunological components in the liver during the infection of hepatitis virus.
So, we report a simple, sensitive and visualized method to measure activation of IFN-βin vivo. Mice received only a simple hydrodynamic injection of expression vectors of phage PhiC31o integrase and IFN-βpromoter-driven firefly luciferase reporter. The luciferase expression in mouse liver induced with synthesized dsRNA poly(I:C) can be monitored by bioluminescence imaging, which was consistent with the IFN-βin the sera. The optimal dose of poly(I:C) to induce luciferase expression is 250μg/kg. HCV NS3/4A protease not only inhibited IFN-βproduction, but also blocked the luciferase expression in mouse liver. We clearly demonstrated that our mouse model carried an IFN-βpromoter luciferase reporter gene is a sensitive and stable tool that can be applied to the evaluation of IFN-βpromoter activation under various conditions. The mouse model may not only help further understand the complex biological feature of the type I IFN system in liver, but also screen drug candidates for stimulator or inhibitor of IFN-βgene expression in vivo. It also contributed to the interaction mechanism between the innate immune system and hepatitis virus.
Part II Establishment of tree shrews models for HBV study by hydrodynamic gene delivery
Hepatitis B virus (HBV) has infected 25% of the world population. 360 million people were chronic infected. Although, the virus is cleared soon for most adults infected, there is a high rate of chronic infection in infants and young children. The mechanism of chronicity of HBV infection is still unclear because of the lack of suitable animal models. HBV has strict host specificity, It only infects some kinds of primates such as human, chimpanzees and tree shrews. However, some primates such as chimpanzee are an endangered species and expensive. Tree shrew lives in tropical and subtropical regions. It is closer to primates than rodents, cats, dogs, rabbits and other animals in biological evolution. Metabolism and anatomical structure are closer to humans than rodents, and easy to be domesticated. Therefore, Tree shrews can be used in many research fields. In recent years, tree shrew model of hepatotropic virus infection have made some progresses. HBsAg positive serum,or purified virus particles were commomly used to inoculate tree shrews for animal model generation. But the rate of infection is low. So we try to generate a tree shrews model of HBV transfection by hydrodynamic with plasmid pAAV/HBV1.2.
To establish a method for hydrodynamic-based gene delivery to tree shrews, we explored the conditions of the hydrodynamic transfection. The optimal injection speed is 0.8ml/s and volume is 100ml/kg. And the vectors containing firefly luciferase andβ-galactosidase reporter gene were hydrodynamically injected into tree shrew via the great saphenous vein. The reporter gene was only detected in the liver of tree shrew by bioluminescence imaging andβ-gal staining. The tree shrew was hydrodynamically injected with pHBV1.2 plasmid. At the various times, the sera were collected and in which ALT, HBsAg, anti-HBsAg and HBV DNA were examined by ELISA or real-time fluorescence quantitative PCR respectively. The tree shrews injected hydrodynamically with pHBV1.2 were detected the markers of HBV in their sera. The serum HBsAg and HBV DNA transiently increased, less than a week. Tree shrews treated with cyclophosphamide can not carry HBV longer than those without cyclophosphamide. Hydrodynamic-based injection via the great saphenous vein can be used to deliver the gene to hepatocyte of tree shrews. We preliminary generated a tree shrew model of hepatitis B virus expressing in the liver by the hydrodynamic injection. Our study indicated a new method for establishment of tree shrews models with hepatitis virus infection.
Based the technique of hydrodynamic gene delivery, we genearated the mouse model for monitoring IFN-βactivity and preliminary developed the tree shrew model for HBV study.
引文
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