【作者】 费晨；

【导师】 钱永华；

【作者基本信息】 西北农林科技大学 ， 细胞生物学， 2010， 博士

【摘要】 痘病毒可以感染人、哺乳动物和昆虫,造成皮肤损伤,引起发热性疾病和疱疹,甚至造成死亡(如天花病毒)。痘病毒感染可引起宿主强烈的免疫反应,诱导产生过量的细胞因子,造成“细胞因子爆发”或脓毒症等免疫病理症状。基于该病毒可引起强烈免疫反应等生物学特性,其被广泛应用于基因工程中,用于构建痘病毒表达载体以及癌症和HIV等免疫缺陷性疾病的活载体疫苗,还作为溶瘤病毒用于肿瘤的治疗。因此,对于痘病毒感染机理的研究不仅有助于该病毒感染的防治,而且可以更好地将其应用于基因工程以治疗其它疾病。基质金属蛋白酶(Matrix metalloproteinase, MMPs)是一类细胞内锌离子依赖的限制性内切酶,广泛参与许多生理和病理过程。其生物合成和活性受基因激活、酶原激活和基质金属蛋白酶组织抑制剂(Tissue inhibitors of metalloproteinase,TIMPs)抑制三个水平的调控。MMPs可通过降解细胞外基质和基底膜,调控细胞因子和趋化因子的活性,来促进病毒的扩散并参与病毒感染引起的宿主免疫反应,从而在HIV等病毒感染过程中发挥重要的调控作用。研究表明,病毒的种类不同,MMPs的作用、表达变化以及参与的关键MMPs也不同。迄今为止,未见关于痘病毒感染过程中MMPs的作用和表达变化的报道。本课题以痘苗病毒(Vaccinia virus, VV)、粘液瘤病毒(Myxoma virus, MV)和特纳河痘病毒(Tanapox virus, TPV)为对象,研究MMPs的活性与这三种病毒感染能力的关系,并进一步探讨参与病毒感染的关键MMPs及其表达调控规律。本研究从一个新的角度——MMPs生物学功能角度来阐述痘病毒的感染机制,不仅为痘病毒感染防治新方法的开发奠定理论基础,而且对于痘病毒在基因工程中的应用,以及对癌症和HIV等疾病的治疗方面具有重要意义。实验结果如下:1.抑制MMPs活性导致VV、MV和TPV的感染能力下降本研究选用11种人工合成的MMPs抑制剂,检测它们对VV、MV和TPV感染能力的影响;并采用MTT法检测了MMPs抑制剂对三种宿主细胞——人上皮癌细胞(Human epithelial carcinoma cells,HeLa)、绿猴肾细胞(Baby green monkey kidney cells, BGMK)和枭猴肾上皮细胞(Owl monkey kidney epithelial cells, OMK)的毒性。结果显示,MMPs抑制剂对VV、MV和TPV的感染能力具有抑制作用。其中,氯已定对三种痘病毒的均具有最显著抑制效果。而且,11种抑制剂在工作浓度下对HeLa、OMK和BGMK细胞均无毒性。因此,抑制MMPs活性导致VV、MV和TPV的感染能力下降。MMPs参与这三种痘病毒的感染,可能是其感染过程的重要影响因子。分析抑制剂的底物得出,可抑制病毒感染的抑制剂底物均包括MMP-2和/或MMP-9,因此,参与VV、MV和TPV感染的关键MMPs可能是MMP-2和/或MMP-9。2. MMPs的活性是VV、MV和TPV感染的重要影响因子底物剪切比色法结果表明在VV、MV和TPV的感染过程中,MMPs的酶活性显著升高。由此可得,VV、MV和TPV的感染过程中MMPs活性升高,而抑制MMPs活性导致VV、MV和TPV的感染能力下降,因此,MMPs的活性是VV、MV和TPV感染的重要影响因子。利用抑制剂XG076阻断MMPs酶原激活途径,对VV、MV和TPV的感染能力没有显著影响。因此,MMPs在VV、MV和TPV感染中的生物学功能是催化非依赖性的。3. MMP-9、MMP-2和TIMP-1、TIMP-2比例失衡可能是促进VV和MV感染的重要机制本研究应用实时荧光定量PCR和明胶酶谱法,检测VV和MV感染中,MMP-2和MMP-9的mRNA和蛋白质水平变化,以及TIMP-1和TIMP-2的m RNA水平变化。结果显示,VV和MV的感染提高了MMP-2和MMP-9 mRNA和蛋白水平的表达。同时,MMP-2和MMP-9的天然组织抑制剂——TIMP-1和TIMP-2的mRNA表达水平下降。因此,MMP-9、MMP-2和TIMP-1、TIMP-2表达水平的变化可导致MMPs和TIMPs之间的比例失衡,其可能是促进VV和MV感染的重要机制。综上所述,MMPs参与VV、MV和TPV的感染过程,是痘病毒感染的一个重要影响因子。在三种痘病毒感染过程中,MMP-2和MMP-9的mRNA和蛋白质表达上调,而TIMP-1和TIMP-2的mRNA表达下调,从而破坏了MMPs/TIMPs间的平衡,导致MMP-2和MMP-9表达相对过剩,可能是促进VV和MV感染的重要机制。因此,在VV、MV和TPV感染过程中,MMP-2和MMP-9作为重要的调节因子发挥了关键的作用,为将它们作为靶点防治痘病毒感染提供了试验依据。同时,本研究从MMPs的生物学功能角度更加深入地了解痘病毒的感染机理,为其在HIV感染和癌症治疗方面的应用提供更多的理论依据。更多还原

【Abstract】 The host range of poxvirus is very broad, including human, mammalian and insects. Poxvirus can affect skin tissue, cause febrile illnesses in human and animals with a prominent vesicular rash,even death, such as variola virus. The poxvirus infection can induce primary and adaptive immune responses, the symptoms associated with serious poxvirus infections are a result of a“cytokine storm”or sepsis and that this may be the underlying cause of pathology. As technical advances in engineering have resulted in the creation of numerous recombinant poxviruses for use as candidate vaccines, vectors for gene delivery, cancer immunotherapy and oncolytic agents in the treatment of cancer. Central to the rational design of these therapies is the need for detailed information about the immune interaction between poxvirus and hosts.MMPs play important roles in both physiological and pathological processes such as cancer metastasis and virus infection. The main MMPs involved in different virus replication and the modulation modes are different. Until now, the roles and expressiong of MMPs in poxvirus replication have not been reported. MMPs get involved in immune responses through breakdown of ECM, BM and modulate cytokine and chemokine to help immune cells arrive at inflammation sites through ECM. So, MMPs may play very important role in poxvirus replication.In this project, we studied on the procesure of vaccinia virus, myxoma virus and tanapox virus infection in their adaptive cells. The objectives of this study are determining the contribution of MMP activity to poxvirus virulence and the specific MMPs upregulated during poxvirus infection. This study can help us understand the mechanisms of poxvirus replication and find a new way for preventing poxvirus replication.1. Depression MMPs activity induce the suppressing of VV、MV and TPV replicationIn this study, a panel of eleven synthetic MMP inhibitors was tested the inhibitory effects on VV、MV and TPV replications. The effects of MMPs inhibitors on cell viability at their working concentrations were tested by standard tetrazole reduction (MTT) assay.The results showed, MMPs inhibitors have general inhibition effects on poxvirus infection. All three viruses’replications were suppressed remarkably by CHX in a dose-dependent manner. MTT results showed that there was no any visible cytotoxic effect on cell survival by any of the concentrations of inhibitors in all treatment groups compare to untreated cells. According to the substrates of MMPs inhibiors, the main MMPs involved in poxvirus infection may be MMP-2 or MMP-9.VV、MV and TPV replication can be suppressed by MMPs inhibitors in the absence of inhibitor-associated cytotoxicity. So, depression MMPs activity induce the suppressing of VV、MV and TPV replication, MMPs activity may play an important role in VV、MV and TPV replication.According to the substrates of MMPs inhibiors, the main MMPs involved in poxvirus infection may be MMP-2 or MMP-9.2. MMP activity is a main factor of VV、MV and TPV replicationThe MMP activity following poxvirus replication was evaluated by analyzing the activity in the serum free culture medium of cells using a colorimetric substrate cleavage assay. XG076 was applied to blockade of zymogen processing and test the influence of this procedure on poxvirus replication. The inhibition of zymogen activity by XG076 did not significantly alter poxvirus infection comparing with the untreated groups (XG076 untreated).MMP activity is a main factor of VV、MV and TPV replication. And, MMPs may exert biological effects in manners that are independent of their enzyme activation pathway.3. VV and MV infection are associated with imbalance of MMP-9, MMP-2, TIMP-1 and TIMP-2Real time PCR was applied to evaluate the mRNA levels of MMPs and TIMPs. Gelatin zymogaphy was applied to evaluate the protein levels of MMP-2 and MMP-9.The mRNA and protein levels of MMP-2 and MMP-9 were up regulated after VV and MV infection. At the same time, the mRNA levels of TIMP-1 and TIMP-2 were down regulated.Thus, VV and MV infection are associated with imbalance of MMP-9, MMP-2, TIMP-1 and TIMP-2.In summary, the MMP activity is a main factor of VV、MV and TPV replication. And, the expression of MMP-9 and -2 were up regulated at mRNA and protein levels, in contrast, the mRNA levels of TIMP-1 and -2 were down regulated. Poxvirus infection is associated with imbalance MMP-9, MMP-2, TIMP-1 and TIMP-2. The results suggest that MMPs, especially MMP-9 and MMP-2, may be a target molecule in the strategy of treatment of poxvirus replication and related diseases. This study can help us find a new way to prevent poxvirus infection, understand the mechanisms of poxvirus replication in order to apply poxvirus in gene enginerring and cancer immunotherapy and virotherapy.更多还原