| 摘要: | 熱休克蛋白(Heat shock protein；HSP)是一種細胞因外來壓力或溫度上升而表現。當中最常被研究的是HSP 90；它具有伴隨蛋白(chaperone)功能，可以保護新生成或是未成熟的蛋白，避免蛋白產生不正確的折疊。在先前的研究指出HSP90會參與病毒複製及複合體的形成，而HSP90有協助蛋白摺疊及蛋白降解的功能，並且會和co-chaperone形成複合體。日本腦炎病毒(Japanese encephalitis virus；JEV)歸屬黃病毒科(flavivirudae)，感染人類會導致中樞神經疾病，可能引發成腦膜炎。本研究的目的是探討HSP 90對於日本腦炎複製之影響。首先利用倉鼠腎細胞(BHK-21，baby hamster kidney-21)與人類腦胚胎瘤(TE671，human medulloblastma line)感染病毒後，以免疫螢光染色(immunofluorescence；IF)觀察發現JEV感染會造成HSP 90由細胞質轉位到核周圍，顯示HSP 90與JEV有關。使用兩種HSP 90抑制劑: 17-(Allylamio)-17-demethoxygeldanamycin(17AAG)與Novobiocin sodium salt，以不同濃度加入受JEV感染的TE671與BHK-21細胞，觀察抑制病毒複製的功效。其中在免疫螢光法觀察到加入17AAG後會減低HSP 90轉位的功能；17AAG對TE671細胞之細胞毒殺性以細胞存活試驗求得CC50為10.7μM；BHK-21的CC 50則為2.22μM，以MOI=0.05的JEV病毒液分別感染TE671與BHK-21，可以明顯地觀察到細胞病變現象(cytopathic effect；CPE)；收集感染48小時後的病毒上清液，在TE671細胞中17AAG在0.01μM的病毒產率為1.625x10^6pfu/ml；接著以BHK-21細胞作病毒斑試驗換算出處理17AAG在0.01μM的病毒產率為1.345x10^6 pfu/ml。
IC 50>0.01μM，以novobiocin處理和JEV感染TE671與BHK-21細胞，所得到在TE671的CC 50>100 μM，而BHK-21的CC 50=98.56μM；CPE也降低病毒生長；收集感染48小時後的病毒上清液，在TE671細胞中，novobiocin在50μM的病毒產率1.225x10^6 pfu/ml；以BHK-21作病毒斑試驗換算出novobiocin在50μM的病毒產率為0.7x10^6pfu/ml；並得到IC 50=23.12μM。從CPE現象及病毒產率來看，17AAG與novobiocin都有抑制其JEV生長的作用。將BHK-21與TE671分別以MOI=0.1、0.5的JEV感染，並加入17AAG(0.1μM)及novobiocin(50μM)收取感染後24/36小時之細胞蛋白，以西方墨點法分析，利用JEV(PK-1)多株抗體對感染後36小時以novobiocin處理的病毒表現蛋白的表現量有下降。但反之以17AAG所處裡的蛋白卻沒有顯著變化；這表示17AAG與novobiocin的作用機轉可能不同，因此之後利用即時定量聚合酶連鎖反應的方法分析比較加入抑制劑前後，病毒RNA含量的變化。證明加入17AAG可以抑制細胞內病毒RNA的轉譯，間接抑制病毒的複製作用。將17AAG與novobiocin兩種藥物共同處理也可以得到協同作用，有較好抗病毒效果可相對降低約達40 %。
Heat shock protein (HSP) is a cell due to external pressure or the temperature rise performance. The most studied in the family is HSP 90; which has chaperone can protect the newly generated or immature protein, to avoid incorrect folding protein production. In a previous study pointed out that HSP90 is involved in viral replication and complex formation, and HSP90 has assisted with protein folding and protein degradation function, and co-chaperone complex formation. Japanese encephalitis virus (JEV) vesting Flaviviridae, infection in humans can cause central nervous system disease that may lead to meningitis. The purpose of this study was to investigate the HSP 90 for the impact of Japanese encephalitis replication. Firstly hamster kidney cells (BHK-21) and human embryonic brain tumors (TE671) after infection, immunofluorescence staining (IF) observed that JEV infection can cause HSP 90 translocation from the cytoplasm to the peri- nucleus, showing HSP 90 and JEV was related. Using two HSP 90 inhibitors: 17 - (Allylamio)-17-demethoxygeldanamycin (17AAG) and Novobiocin sodium salt, adding different concentrations by JEV infection with TE671 and BHK-21 cells were observed inhibition of viral replication effect. Which was observed in immunofluorescence join 17AAG will reduce HSP 90 translocation function; 17AAG on TE671 cells cytotoxic to cell survival test was obtained CC50 =10.7μM; BHK-21 of the CC 50 was 2.22μM, in the JEV MOI = 0.05, TE671and BHK-21 cells were infected with virus, can be clearly observed cytopathic phenomena (cytopathic effect; CPE); collected after 48 hours of infection of the virus supernatant in the TE671 cells 17AAG 0.01μM the virus yield was 1.625x10 ^ 6pfu/ml; followed by BHK-21 cells for the virus plaque assay in 0.01μM 17AAG converted by treatment of the virus yield was 1.345x10 ^ 6 pfu / ml. IC 50> 0.01μM, processing and JEV infection with novobiocin and BHK-21 cells, TE671 obtained in the CC 50> 100 μM, and the BHK-21 CC 50 = 98.56μM; CPE also reduced viral growth; collection infection 48 hours, the virus supernatant was TE671 cells, novobiocin viral yield in 50μM 1.225x10 ^ 6 pfu / ml; in BHK-21 for the translation of viral plaque assay of the virus yield novobiocin at 50μM was 0.7x10 ^ 6pfu / ml; and with IC 50 = 23.12μM. Phenomenon from the CPE and viral yield point of view, 17AAG with novobiocin has inhibited JEV growth. The BHK-21 and TE671 respectively MOI = 0.1,0.5 of JEV infection and join 17AAG (0.1μM) and novobiocin (50μM) received after infection 24/36 hours of cell protein by Western blot analysis, using JEV (PK-1) polyclonal antibodies against infection 36 hours after treatment with novobiocin expression of viral protein expression has declined. But contrary to 17AAG which did not significantly change in the; This means 17AAG with novobiocin mechanism of action may be different, so the use of real-time quantitative polymerase chain reaction after the method of analysis and comparison of before and after adding inhibitors, viral RNA content changes.Join 17AAG can prove that inhibition of intracellular viral RNA translation, indirectly inhibit virus replication effect. Novobiocin 17AAG with the two drugs together on synergistic effect can be obtained, a better antiviral effect may be relatively reduced by about 40%. |
