杆状病毒早期感染对家蚕血淋巴细胞的影响以及宿主细胞的应激反应研究

【作者】 王冠；

【导师】 徐旭士；

【作者基本信息】 南京师范大学 ， 微生物学， 2004， 硕士

【摘要】 本实验以BmNPV-家蚕血淋巴细胞这一系统为研究对象，运用家兔免疫的方法获得病毒粒子的多克隆抗体再辅助二抗标记实现在In vivo的条件下标记病毒粒子，建立一个In vivo的条件下利用激光共聚焦技术实时研究病毒感染的新方法。运用BmNPV的病毒粒子实时采集的家蚕血淋巴细胞感染吸附后的一个小时计为0时间，激光共聚焦实时跟踪时间为1-3小时。跟踪研究病毒在感染早期对于宿主细胞的钙离子和线粒体的影响。 实验通过对正常与病毒感染细胞内以Fluo-3标记钙离子的荧光信号的实施跟踪，采集细胞范围内荧光信号数据，观察其变化过程。发现：在正常情况下家蚕血淋巴细胞内钙离子浓度的变化具有规则的波动性，振幅一致，周期较为恒定约为900s，且相邻的细胞具有相同的波动特征以及良好的同步性；在病毒感染的情况下，钙离子浓度总体上有所增加，且波动周期延长、浓度变化的灵敏度降低。说明病毒感染会影响正常条件下钙离子有规律的释放与聚集的过程，推测感染早期病毒携带的病毒编码的糖蛋白修饰质膜所造成的胞内一些细胞器(内质网、线粒体等)的膜通透性损伤性改变是胞内钙离子浓度增加，是钙离子聚集与释放过程失调的主要原因。病毒依赖细胞骨架的质-核运输过程也是钙离子浓度升高的一个因素。而相邻细胞钙离子浓度变化的一致性体现了通过钙离子而实现的细胞间信号传递过程。 实验通过与研究钙离子相同的病毒感染方式对正常与病毒感染细胞内以Rhodamine123标记线粒体的荧光信号的实施跟踪发现：线粒体的膜电位在细胞正常条件下也具有规则的波动性，周期恒定约为500s，但是在波形上与钙离子的波动有着比较大的差别，而在病毒感染的条件下线粒体的活性变化具有三种不同的紊乱形式。说明线粒体的膜电位可以反映线粒体膜上呼吸链的状态，即体现了线粒体功能状态以及活性。而其在病毒感染的条件下的三种不同的紊乱形式，体现了病毒与宿主细胞之间的相互作用的不同结果。推测为宿主细胞死亡、凋亡或为对病毒感染的可承受状态。对于三种状态的选择线粒体起了关键的作用。认为细胞内钙离子浓度的一段时间的升高易造成线粒体膜上钙离子超载，再加上线粒体自身受到的伤害，按照其损伤程度的不同，产生不同的结果，或损伤在可以承受的范围内则维持原有的状态，使得病毒得以持续感染；或损伤使得膜间质中的自由基，和一些凋亡前体肴片邓佬吠，尹2004届柳{全笋夕老戈产次厉毒早筋澎奕刀紧菌恤球巴绷糟劣多瀚以资 按金绷脸甜应减反应研泞释放至胞内引发对细胞的伤害以及凋亡的发生;或者损伤严重造成线粒体的活性过低，没有足够的能量引发凋亡，则细胞死亡。不同的结果体现了线粒体在凋亡过程中的重要性，以及病毒与宿主细胞的相互作用。 对感染不同时期淋巴细胞中三种保护性酶(CAT、户00、500)活力的测定结果为:病毒感染引起体内一系列保护酶活性的相应升高，而随感染的进行到后期酶活有所降低。说明在感染过程中线粒体受到损伤会产生大量自由基释放至胞内而激活了清除自由基的相关酶。 综合以上可认为病毒感染早期就会对宿主细胞的产能系统、信号传导系统、保护酶系统产生一定的影响，造成宿主细胞不同的应激反应。而宿主细胞的这些反应对病毒感染过程亦有一定的反作用。更多还原

【Abstract】 In this paper BmNPV and Bombyx mori hemolymph cells were used in the research of the interaction between virus and its host cells by the means of laser scanning confocal microscopy. And the muticloning antibody of virus and its second antibody were applied in the works using the method of immunity in rabbit for the sake of tagging virus in vivo. Then the new method which will be used in the research on the process of the infection has been found. The time while the host cells have been absorbed by virus for one hour was set the initial point. Then from this point the system would be tailed for 1~3 hours, in order to discover the sway of calcium ions and mitochondria causing by the early infection of the virus.In these experiment calcium ions in the cells was targeted by FIuo-3 under normal or infection circumstance. The data of the fluorescence signal were gathered to research the change. And we get that: under the normal circumstance the varieties of the calcium ion density mitochondria. Its cycle of wave is meaning about 900s. But when the cells were infected by virus, the calcium ion density had increased, and the wave had changed into turbulence, the cycle became longer; the change of the density became insensitive. The conclusion has been get that virus infection causes the change of the procession of the assembling and releasing of the calcium ion which fluctuating well-regulated. These would be speculated that some glycoprotein cording by virus decorate the membranes causing the damage of some organelle membranes (such as mitochondria and endoplasmic reticulum) and the change of the penetration ability of the membranes would cause the maladjustment of calcium ion. Furthermore the procession of the transport from cytoplasm to nucleolus may be one of the reasons which cause the increasing of the calcium ion density. On the other hand, the coherence of the wave of the calcium ion density in different cells shows that calcium ion could be concerned with the signal transduction among cells.In these experiment mitochondria was targeted by Rhodaminel23 under normal or infection circumstance. The data of the fluorescence signal were gathered to research the change. And we get that: under the normal circumstance the variety of the mitochondriamembrane potential has their own regulations and the same swing in different cells, the cycle is meaning about 500s. The wave of the membrane potential change in tree different ways. And there must be some relations with the interactions between the infection of the virus and the apoptosis of the host cells. In this procession, mitochondria are one of the key points to decide the infection’s result, productive infection, apoptosis, or death. Those could reflect the importance of mitochondria in apoptosis, and the interaction between virus and host cells.On the other hands, it was certified that the infection may cause the increasing of the activity of protection enzymes(such as CAT, POD, SOD), the reason may be that the infection cause the change of the mitochondria membrane, and some free radical were released from the mitochondria; CAT as the protection enzyme must be activated by the infection.It was proved that the early infection could lead to some damage of the energe systems, signal transduction systems and protection enzyme systems in the host, and cause the different reactions in host cells, and these reactions may cause some reaction to the virus.更多还原