【作者】 陈清烽；

【导师】 田志刚；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2008， 博士

【摘要】 在1999年9月份,当一个正在通过肝动脉给药,接受高滴度腺病毒载体治疗的病人死于病毒载体所引发的毒性反应后,人们对于腺病毒(Ad)载体用于临床基因治疗的理解发生了转变。当然,在临床治疗中所需的对腺病毒载体的连续性给药可能关系到机体对腺病毒的强烈反应,但是更重要的是,通过研究人们获得了更多的对腺病毒毒性的了解。我们都意识到,没有哪一种载体系统是完美无缺的。在看到腺病毒载体并不适于所有治疗应用的同时,我们也不会因此而忽略腺病毒载体那些相对于其他载体系统的显著优点:能够有效地感染静息或分裂中细胞,这是很多载体无法做到的;腺病毒能够在短时间内实现高水平的基因表达,这对于那些只需要短期高表达转基因就能产生足够治疗效应的应用意义重大。腺病毒载体潜在应用包括治疗性的血管生成作用,能够在中央神经系统(CNS)实施感染,以及在那些腺病毒的毒副作用也能产生帮助的治疗当中如作为肿瘤疫苗。当然毫无疑问的是,为了更广泛地应用腺病毒载体,解决其引起体内免疫应答的问题是当务之急。本次研究集中在腺病毒诱导的肝脏损伤,诱导体内肝脏抗病毒免疫抑制和黑色素瘤基因治疗方面,获得了如下的结果:1、新型高压注射重组腺病毒诱导肝损伤模型的建立目前,腺病毒载体能在肝脏中定位并引起肝脏炎症和损伤的研究甚多,但是通用的动物模型研究并不多。本部分研究的目的就是建立一种新的腺病毒诱导的爆发性肝炎模型,来促进对腺病毒诱导的自身免疫性肝炎的机制研究。我们采用了高压注射(注射体积占体重10%,并在7秒中内完成注射)腺病毒载体的方法,将腺病毒载体快速地特异地通过尾静脉传递到小鼠肝脏内部,观察后继的病毒感染引起的肝脏损伤。C57 BL/6小鼠尾静脉高压注射表达不同产物的腺病毒(AdLacZ,AdEGFP,AdsiNeg),通过流式细胞术分析肝脏内部淋巴细胞的比例和绝对数,血清转氨酶和H&E染色被用作评价肝脏损伤的指标。我们发现,高压注射腺病毒比普通注射诱导更加严重的肝脏炎症:大量的单个核细胞迅速浸润,尤其是肝脏NK细胞显著增加和活化;同时发现血清ALT转氨酶水平大幅升高:肝脏内出现多处坏死;IFN-γ在肝脏中的转录水平及在血清中蛋白水平上升。通过注射抗体预先清除NK细胞,可以显著减轻腺病毒诱导的肝脏损伤以及炎症因子IFN-γ的表达水平。这些数据表明腺病毒感染诱导了肝脏中NK细胞的浸润和活化,从而导致了肝细胞的损伤以及肝脏炎症的产生,NK细胞是该模型中病毒感染引发的早期炎症的主要效应细胞。总的来说,我们的工作展示了一个与人的急性肝脏感染相关的新的病毒肝炎模型,为研究腺病毒提供了一个实用有效的工具和平台。2、治疗性RNA干扰CX3CL1抑制腺病毒引起的肝脏损伤我们研究了趋化因子CX3CL1/fractalkine(FKN)在抗病毒感染免疫应答中的作用。FKN是一种CX3C类的趋化因子,有膜结合型和分泌型两种存在形式。大部分NK细胞,CD14~-单核细胞及部分的CD8~+T细胞表达相应受体CX3CR1。通过进一步研究以上构建的高压注射腺病毒诱导的肝脏损伤模型,我们发现CX3CL1在腺病毒感染的肝脏中是显著上调的,并且有意思的是同时发现浸润到炎症肝脏中的主要效应细胞NK细胞上相应受体的CX3CR1表达也显著上调。为了进一步证明二者关系,我们构建了表达重组小鼠CX3CL1的腺病毒进行注射,发现在肝脏内过表达CX3CL1大大增加了CX3CR1~+NK细胞的浸润和活化水平,血清中ALT转氨酶的水平及肝脏坏死程度也大幅升高。这表明腺病毒感染肝脏后诱导肝脏内CX3CL1的表达增加,CX3CL1与其受体CX3CR1相互作用在NK细胞的招募和活化以及IFN-γ的产生中行使重要的作用。因此,抑制CX3CL1的表达将可作为抗腺病毒炎症治疗的一个选择。实验中,运用高压注射表达CX3CL1特异性干扰小RNA的腺病毒进行了体内RNA干扰,发现病毒感染后CX3CL1在肝脏中的表达水平被明显抑制,相应地,NK细胞的浸润及肝脏损伤和炎症也显著下调。为了更好地验证CX3CL1的促炎症作用,我们在注射腺病毒之前用CX3CL1或CX3CR1的阻断抗体预先处理小鼠,结果显示肝脏中的炎症损伤及淋巴细胞浸润也能被很好地抑制。综上,该实验证明了CX3CL1-CX3CR1趋化因子系统在腺病毒诱导的肝脏损伤中的促炎症功能和重要性,并为体内基因治疗提供了一个新的靶点和手段。3、RNA干扰CX3CL1抑制黑色素瘤体内生长及机制研究虽然有报道称过表达的CX3CL1促进对肿瘤细胞的杀伤,CX3CL1行使的是抑制肿瘤生长的作用。而我们的研究经RT-PCR,Western Blotting以及免疫荧光检测发现小鼠黑色素瘤B16-F0细胞及一些肿瘤病人的实体瘤表达CX3CL1。进一步的流式细胞术抗体检测发现B16上CX3CL1为阳性,而ELISA检测发现B16的培养上清中无可溶性FKN的表达,这证明了B16表达的CX3CL1为膜结合型。因此我们猜测这种膜结合型的CX3CL1可能参与了肿瘤的发生过程。运用表达CX3CL1特异性干扰小RNA的腺病毒感染B16-F0黑色素瘤细胞,沉默了B16细胞上CX3CL1的表达。通过流式细胞分选技术筛选得到CX3CL1表达被有效抑制的B16细胞。在C57 BL/6小鼠上进行体内荷瘤的结果发现CX3CL1敲除鼠与对照组相比肿瘤的成瘤性大幅降低;生存曲线数据表明CX3CL1敲除组的小鼠的生存情况要显著优于对照组。对肿瘤切片加以研究发现,血管内皮细胞表达CX3CL1的受体CX3CR1;CX3CL1敲除组的肿瘤生长受到严重抑制且瘤体内部和周围只有少量的血管生成,而对照组肿瘤生长旺盛,并伴随着大量的肿瘤内血管分布。因此我们有理由认为肿瘤表达一定水平的CX3CL1来促进它们自身与体内表达CX3CR1的血管内皮细胞相互作用,加强肿瘤发生过程中对血管内皮的黏附及促肿瘤内部血管生成,从而促进了肿瘤的发生。更多还原

【Abstract】 In September 1999,the perceptions of the use of adenoviral(Ad)vectors for gene therapy were altered when a patient exposed via the hepatic artery to a high dose of adenoviral vector succumbed to the toxicity related to vector administration. Appropriately,concerns were raised about continued use of the Ad vector system and, importantly,there were increased efforts to more fully understand the toxicity.Today it is recognized that there is no ideal vector system,and that while Ad vectors are not suitable for all applications,the significant advantages over other vector systems including efficient transduction of a variety of cell types,both quiescent and dividing, make it optimal for certain applications.These include protocols where high levels of short-term expression are sufficient to provide a therapeutic benefit.Potential target applications include therapeutic angiogenesis,administration into immune-privileged sites such as the CNS,or treatments where the adjuvant effect of adenovirus can be of benefit such as cancer vaccines.Broader applicability of Ad vectors will require resolution of toxicity issues.The researches presented here focus on the adenovirus-induced acute liver injury,induction of in vivo hepatic immuno suppression against adenoviral infection and gene therapy of melanoma.1,Hydrodynamic injection of adenovirus to construct a novel liver injury model.So far,it has been widely reported that adenovirus vectors delivered by i.v. injection accumulate in the liver and induce liver injury.But few common animal models were established to study the adenovirus-induced liver injury.The purpose of our research is to construct a novel adenovirus-indued fulminant hepatitis model to study the mechanisms involved in the adenovirus-induced auto-immune hepatitis.We employed hydrodynamics-based injection(10%body weight and the tail vein injection was performed over less than 5 s)to deliver adenovirus vector into the mouse livers and observe the pathological changes after infection.C57 BL/6 mouse was hydrodynamic injection with different adenovirus vectors(AdLacZ,AdEGFP and AdsiNeg),and then assayed the proportion and total amount of hepatic lymphocytes by FACS.The serum ALT levels and pathological H&E staining of liver sections were used as indication of liver injury.We found that hydrodynamic injection of adenovirus vector induced much more severe liver inflammation than conventional i.v. injection.HP-injection of adenovirus preferentially led a substantial increase of lymphocytes in infected liver.Especially the hepatic NK cells were significantly increased and activated.It was also observed that the serum ALT levels were dramatically increased following severe liver necrosis.The transcript level in infected liver and the serum level of IFN-γwere both up-regulated.Depletion of NK/NKT cells or NK cells prevented mice from adenovirus vector-induced liver injury and down-regulate the expression of IFN-γ.All these data indicated that adenovirus infection induced the infiltration and activation of hepatic NK cells leading to liver injury and inflammation.So NK cells are the key effector cell in the early phase of adenovirus infection in this model.In conclusion,our work presents a novel viral hepatitis model associated with human acute liver viral infection and provides a efficient platform for studying adenovirus infection.2、Therapeutic RNA silencing of CX3CL1 prevent adenovirus induced liver injuryWe investigate the role of chemokine CX3CL1/fractalkine(FKN)involved in the anti-viral infection immunity.Fractalkine,the only member of CX3C chemokine containing both membrane-anchored and soluble forms,has both chemoattractant and cell-adhesive functions and is believed to be an important regulator of inflammatory response as an inducer of cellular infiltration,including induction of IFN-γ.Most of NK cells,CD14~+ monocytes and partial CD8~+ T cells express its receptor CX3CR1. By further studying the hydrodynamic infection of adenovirus induced liver injury model,we found that the expression of CX3CL1 in adenovirus infected liver was significantly upregulated,and interestingly,the CX3CR1 expression on the infiltrated NK cells was also up-regulated.To explore the relationship between them,we constructed adenovirus vector to express mouse recombinant CX3CL1(AdFKN). Injection of AdFKN induced the overexpression of CX3CL1 in mouse liver and dramatically promoted the infiltration and activation of CX3CR1~+ NK cells while the serum ALT levels and the liver necrosis were increased too.These results indicated that adenovirus infection induced the expression of hepatic CX3CL1,then NK cells were recruited and activated through the interaction between CX3CL1 and CX3CR1 following the production of IFN-γ.So,inhibition of CX3CL1 expression might be a good choice for eluding adenovirus vector immunity.We constructed adenovirus vector carrying mouse CX3CL1 specifc siRNA(AdsiFKN)to perform in vivo RNA interference.After hydrodynamic injection of AdsiFKN,the expression of CX3CL1 in infected mouse liver was greatly inhibited.At the meantime,the infiltration of NK cells and the liver inflammation were significantly down-regulated too.In order to further demonstrating the pro-inflammatory role of CX3CL1,the mice were pre-treated with anti-CX3CL1 or anti-CX3CR1 specific neutralizing antibody before virus injection.We found that the injury and inflammation of infected liver could be greatly suppressed too.Our findings suggest a strategy to prevent or alleviate adenovirus vector-induced acute liver injury by blocking CX3CL1/CX3CR1 interaction in adenovirus vector-based gene therapy.3、Down-regulation of surface fractalkine by RNA interference in B16 melanoma reduced tumor growth in miceIt is reported that membrane-bound form of CX3CL1/fractalkine(FKN)promotes cell-cell adhesion by binding with its unique receptor CX3CR1,and membrane fraetalkine works as an adhesion molecule for the communication between tumor cells and vascular endothelial cells.Here,we show that CX3CL1/fractalkine was expressed on both mouse and human solid tumors,and small interfering RNA-mediated knock down of fractalkine gene inhibited melanoma B16-F0 cells growth in vivo,which was correlated to the decreased angiogenesis around the tumor. Our findings for the first time suggest that membrane fractalkine may possibly promote tumor angiogenesis through its strong cell adhesion function and therefore serves as a potential target of tumors therapy including RNA interference.更多还原