臺灣博碩士論文加值系統

單純疱疹病毒第一型(HSV-1)為一種DNA病毒，根據血清學統計，世界上成年人口中有90%以上曾被此病毒感染過。此病毒可以在哺乳類動物的神經系統中複製與擴散，並造成致死性腦炎。然而，病毒的毒力致病機轉仍不清楚，先前的研究指出當小鼠感染了KOS病毒株，其半致死劑量 (LD50) 大於2 × 107 PFU。當小鼠感染另一病毒株294.1，其LD50為1.38 × 104 PFU。因此我設計實驗去了解這兩個病毒株在感染小鼠後，為何其毒力有如此大的差異。首先我分析這兩株病毒在周邊組織與神經組織生長複製的情形，結果顯示這兩個病毒複製的能力並無明顯差異，然而不論是在周邊或是神經組織，294.1這株病毒存在的時間都比KOS持久。其次，我也藉由分析血腦屏障(blood-brain barrier；BBB)的通透性來做為病毒引起腦部損害的指標。根據伊凡氏藍(Evans blue extraction assay)萃取法與M4 tracer法，顯示不論是在KOS或是294.1感染的腦部都表現出BBB通透性的增加。此外我選用一個只能在周邊組織複製而不能在神經組織複製之突變病毒株294.1△tk，來分析被該病毒感染的小鼠BBB的通透性之改變，結果發現該組小鼠的通透性並無增加，此結果確定了BBB通透性的增加是因為病毒在腦部複製所引起，然而免疫組織化學染色的結果說明了，病毒影響BBB的通透性並不是以直接地對內皮細胞或是其他構成BBB的細胞造成傷害，而是以一種間接的方式。許多的研究指出發炎反應的細胞激素(pro-inflammatory cytokines)會改變BBB的通透性，同時我發現到在病毒感染的腦部中有前發炎反應之細胞激素的產生。另外在BBB的通透性增加後，我發現病毒感染的腦部裡，免疫細胞有增加的現象，其中以單核球及巨噬細胞為主。同時在病理染色切片發現到294.1感染的小鼠腦部其神經細胞所受到的傷害都較KOS嚴重，根據上述的結果，294.1其毒力之所以會比KOS強，可能是因為本身病毒基因上的差異與所引起不同程度的免疫反應所導致。

Herpes simplex virus type 1 (HSV-1), a member of Herpesviridae with double-stranded DNA genome, is able to multiply and spread in multiple organs, including nervous system, to cause death. However, the pathogenesis of HSV virulence is not clearly understood. Previous and my studies showed that different HSV-1 strains varied in their virulence to cause death in mice. My results showed that these two viruses replicated to the same degree but 294.1 persisted longer than KOS in peripheral and nervous tissues. How these two viruses affect the infected brains was also investigated and compared. I found the permeability of blood-brain barrier (BBB) was altered in infected mice. The change of BBB permeability is due to viral replication in the brain because 294.1Δtk, which cannot replicate in nervous tissues, failed to open BBB. The expression of pro-inflammatory cytokines were detected in infected brains. The number of inflammatory cells increased and the major type of inflammatory cells switched from lymphocytes to monocyte/marcopgage in the brain after infection. The change of BBB permeability and degree of cytokine expression as well as inflammatory responses observed in 294.1-infected brains were greater than those in KOS-infected brains. Moreover, histological examination and Nissl staining showed the neuron damage and loss, particularly in 294.1-infected brains. Taken together, the higher virulence of 294.1 in mice might result from neuron damage in the CNS caused by both viral as well as immune activities.

CHINESE ABSTRACT………………………………………………….. V
ENGLISH ABSTRACT………………………………………………….. VII
LIST OF TABLES………………………………………………………… VIII
LIST OF FIGURES………………………………………………………. IX



INTRODUCTION………………………………………………………… 1
MATERIALS AND METHODS…………………………………………. 5
Viruses and cells……………………………………………………………………… 5
Infection of mice and tissue collection……………………………………………… 5
Assay of BBB permeability………………………………………………………….. 6
Immunohistochemistry staining…………………………………………………….. 7
Western blotting……………………………………………………………………... 8
Quantification of immune cells in HSV-1-infected mouse brain…………………. 8
Histological examination…………………………………………………………….. 9
Nissl staining…………………………………………………………………………. 9
TUNEL assay……………………………………………………………………… 10
Statistical analysis…………………………………………………………………. 10
RESULTS………………………………………………………………….. 11
HSV-1 strains varied in their virulence in mice in vivo…………………………… 11
Virus growth in peripheral tissues………………………………………………….. 11
Virus growth in nervous tissues…………………………………………………….. 12
Change of BBB permeability during infection…………………………………….. 12
Increase of immune cells and change of immune cell type in infected brains…… 15
Damages of HSV-1-infected brains…………………………………………………. 15
DISCUSSION……………………………………………………………… 17
REFERENCES……………………………………………………………. 23

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