腦脊髓液檢體中流感病毒與腸病毒之分子診斷及化學激素成份分析

Molecular diagnosis of influenza virus and enterovirus and analysis of chemokines in cerebrospinal fluid samples

王雅利(Ya-Li Wang)

指導教授 ： 林克亮 ； 陳志豪

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2005年)

https://doi.org/10.6834/CSMU.2005.00070

摘要

中樞神經系統的感染可能會造成腦膜炎、嚴重腦炎甚至死亡，病毒是造成無菌性腦膜炎的主要病因之一，其中又以腸病毒佔85 %最多，不同型的腸病毒可能導致嚴重程度不同的疾病，而流感病毒也曾報告與中樞神經系統感染有關，因其感染範圍廣且加上基因容易突變而具有爆發世界大流行的潛力，本研究中利用病毒培養、反轉錄 聚合酶連鎖反應、腸病毒診斷晶片等，檢測腦脊髓液中是否有腸病毒及流感病毒的存在。從文獻中發現病毒性腦膜腦炎病患腦脊髓液中有明顯的特定化學激素增多現象，例如：Yoshinori Ito的研究指出流感病毒造成的腦炎病人其腦脊髓液中IL-6增加，因此本實驗藉由酵素免疫吸附分析法檢測腦脊髓液中RANTES/CCL5、MIP-1β/CCL4、MCP-1/CCL2、GROα/CXCL1、IP-10/CXCL10、IL-8/CXCL8等六種化學激素的含量，並配合臨床檢驗數據、診斷和病毒檢查結果作比較及分析，為了確定反轉錄聚合酶連鎖反應產物為流感病毒的基因片段，也進一步將其非結構基因片段經選殖後作定序分析。本研究結果中流感病毒與腸病毒在腦脊髓液的反轉錄聚合酶連鎖反應陽性率分別為14 %及5 %，陽性病例以腦膜炎的臨床診斷最多，且其腦脊髓液中都可偵測到與病毒感染有關的GROα/CXCL1化學激素濃度，儘管濃度高低不一。此外，本研究結果中一名腸病毒培養、腸病毒反轉錄聚合酶連鎖反應及腸病毒診斷晶片皆為陽性的病例，其腦脊髓液中的六種化學激素含量都明顯增加，推測化學激素含量的多寡可能反映病毒量的高低。快速偵測腦脊髓液中的病毒有助於疾病的診治，將傷害降到最小，避免因未確知病原即使用化學療法產生的醫療浪費，本研究結果顯示反轉錄聚合酶連鎖反應可在病程的早期就得到陽性的結果且敏感性優於病毒培養 (P=0.004)，在臨床檢驗上值得廣泛應用，未來若能再快速細分病毒亞型，除了有助於流行病學的監控，也有助於鑑別診斷，對防疫機制的啟動更是相當重要。

關鍵字

腦脊髓液 ； 流感病毒 ； 腸病毒 ； 化學激素

並列摘要

Infection of the central nervous system may result in meningitis, severe encephalitis, or even death. Viruses are major causes of aseptic meningitis, and enterovirus account for 85% of the cases. Different enterovirus may trigger diseases of different severities. Influenza viruses have been reported to have to do with infection of the central nervous system. The fact that their scope of contagiousness is widespread and that their genes are prone to mutation make influenza viruses a potential bomb that may explode to worldwide infection. Through virus culture, reverse transcription polymerase chain reaction and enterovirus diagnosis chip, this study examines whether enterovirus and influenza viruses exist in cerebrospinal fluid. Review of literatures reveals that specific chemokines increases in the cerebrospinal fluid of patients who suffer virus-triggered meningitis. For example, Yoshinori Ito in his study indicates that IL-6 of the cerebrospinal fluid of virus-triggered meningitis patients rises. Therefore, this experiment examines the level of 6 chemokines- RANTES/CCL5, MIP-1β/CCL4, MCP-1/CCL2, GROα/CXCL1, IP-10/CXCL10 and IL-8/CXCL8 in cerebrospinal fluid through enzyme linked immunosorbent assay. The outcome is analyzed against clinical test data, diagnosis and virus examination for verification of the fact that the substance generated out of reverse transcription polymerase chain reaction is a genetic section of influenza viruses, as well as for sequencing of the Non-Structural genetic sequences following selective cultivation. In this study the “positive” rates of influenza viruses and enterovirus in cerebrospinal fluid of reverse transcription polymerase chain reaction are 14% and 5% respectively. Among those “positive” cases clinical diagnosis of meningitis predominates. In those cases, the presence of GROα/CXCL1 (which has to do with virus infection), though of different densities in cerebrospinal fluid can be detected. In addition, in this study all 6 chemokines of the cerebrospinal fluid of a “positive” case show significant increase in enterovirus cultivation, enterovirus reverse transcription polymerase chain reaction and enterovirus diagnosis chip. It is assumed that the level of chemokines reflects the content of the viruses. Speedy detection of viruses in cerebrospinal fluid helps diagnose and treat diseases, minimize the damage and avoid waste of medical resources due to the use of chemical treatment before the actual cause is verified. Outcomes of this study show that reverse transcription polymerase chain reaction is capable of detecting “positive” result early in the ailment and that its sensitivity is superior to that of virus cultivation (P=0.004). It is, therefore, an effective method for extensive clinical application. In the future our ability to further speedily distinguish the subtype of viruses will help us engage in effective epidemiology monitoring and discernible diagnosis. It is very important to the triggering of our epidemic prevention or control mechanism.

並列關鍵字

cerebrospinal fluid ； influenza virus ； enterovirus ； chemokines

參考文獻

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