臺灣博碩士論文加值系統

呼吸道融合病毒（Respiratory Syncytial Virus）是一具有套膜之病毒。分類學上屬於副黏液病毒科（Paramyxoviridae）之肺炎病毒屬（pneumovirus），臨床上易造成小孩下呼吸道感染疾病。RSV是一單股負性核糖核酸病毒（single strain-negative sense RNA virus），其病毒基因可製造出十種不同的蛋白，其中F和G蛋白都位於病毒外套膜上，是一種醣化蛋白。
1985年，利用單株抗體可將RSV分成A和B兩種亞群。在RSV十種蛋白中，G蛋白在不同亞群間變異性最大，A和B亞群間胺基酸序列僅具有53﹪相似性，各亞群內之病毒株間亦有相異存在，我們實驗室曾證實台北地區A亞群間胺基酸之差異為11%。為進一步瞭解台北地區RSV B亞群之核酸變異情形，我們針對1984至1998年間，台北地區之RSV B亞群之臨床分離株，以RT-PCR方法和核酸自動定序儀直接定出G蛋白質基因序列。結果顯示台北地區B亞群分離株彼此之間，其核酸相似性為93~99﹪，而胺基酸序列為87~99﹪。將核酸序列轉換成胺基酸序列作比較時，發現其變異位置主要發生於胺基酸序列80-154及198-300間，而胺基酸155-197為穩定區域，胺基酸變異較國外為大。另根據G蛋白質基因核酸序列，做出種系分析圖（phylogenic tree）。台北地區分離株大多數屬於相同cluster（cluster I），而1984年和1998年中，各有少數分離株（840085、840196、981480）屬另一cluster （cluster II），此一特殊基因型態分離株是否能再繼續流行，而取代原先族群，或是此特殊基因型是來自原始基因型，而有再次流行的可能性，則需長期分析來證明。
RSV在臨床上會造成呼吸道綜合病徵，支氣管炎、細小支氣管炎、肺炎等症狀，嚴重者甚至會造成死亡。通常在肺泡之感染所引起之症狀遠較支氣管感染引起之症狀嚴重，又A亞群在臨床上之症狀亦較B亞群嚴重。此種RSV在不同部位之症狀表現差異，及兩亞群RSV致病性之差異，是否與其細胞上產生細胞激素之情形有關?我們比較此兩亞群病毒感染肺泡上皮細胞（A549）及支氣管上皮細胞（BEAS-2B），細胞激素IL-8、IL-6及RANTES產生之情形。研究結果顯示，不論下呼吸道較上端細胞（支氣管，BEAS-2B）及深層部位（肺泡細胞，A549），A亞群產生IL-8、IL-6及RANTES均多於B亞群，此可解釋臨床上，A亞群感染造成之疾病比B亞群嚴重之因。又在肺泡細胞中，A、B兩亞群產生IL-8、IL-6及RANTES均較呼吸道上端細胞多，此也可說明RSV在肺泡部位引起之症狀較嚴重之可能原因。

Respiratory syncytial virus (RSV), an enveloped virus, is classified within the genus Pneumovirus of the family Paraxoviridae. RSV is the most important cause of lower respiratory tract infection in infants and vulnerable adults. The RSV genome is a single strand negative sense of RNA composed of approximately 15000 nucleotides that are transcribed into 10 major mRNA. Each of the mRNA encodes a major viral protein. Three of these proteins are transmembrane surface proteins ( G, F and SH).
On the basis of monoclonal antibody studies in 1985, RSV has been classified into two major subgroups, A and B. The G protein is the most variable gene product between RSV isolates. Sequence identity at the amino acid level is only 53% between the G protein of the prototype strains of subgroup A and B. Sequence variation has been observed among the G proteins of HRSV isolates of the same antigenic group. Our laboratory found that 11% sequence variation at amino acid level in Taipei RSV subgroup A isolates. To elucidate the variation of G gene of subgroup B RSV isolated in Taipei area, 43 strains of subgroup B from 1984-1998 were enrolled in this study. G protein genes of subgroup B RSV were amplified by RT-PCR. The nucleotide sequence of G gene was determined by dye termination autosequencing method. The identity of nucleotide and amino acid sequence of 43 RSV isolates in Taipei from 1984-1998 were compared and three subgroup B reference strain (8/60: 1960; CH18537: 1962; 9320: 1977) were used as reference. Nucleotide sequences homology and amino acid homology of 43 strains of subgroup B RSV were 93-99% and 87-99% respectively. The deduced amino acids sequence in cytoplasmic and transmembrane domains are conserved. Variation was found in two regions of extracellular domain; amino acid 80-154 and amino acid 198-300 region, separated by a highly conserved region (amino acid 155-197). The conserved region of G protein gene of subgroup B RSV in Taipei was smaller than the same region in strains isolated in other countries. The phylogenic tree of Taipei strains and some strains isolated in other countries was established by Neighbor-joining method. We find that most strains of Taipei isolates from 1984-1998 are grouped into one cluster (cluster I) and only three strains (840085, 840196, and 981480) are grouped into another cluster (cluster II).
RSV infection of the lower respiratory tract in infants typically causes acute bronchiolitis and pneumonia but may result in persistent abnormalities in pulmonary function and syndrome of wheezing and respiratory distress. Hospitalized and death causes are estimated very high in each year. Syndrome of infection of RSV in alveolar is more serious than in bronchus. Subgroup A RSV causes disease more severe than subgroup B virus causes. The correlations of clinical manifestation in different part of respiratory tract infected with different RSV subgroups are an interested issue. Dose cytokine production among these areas between RSV subgroup A and B infection plays another important role in the pathogenesis of RSV infection? In order to elucidate these questions, the IL-6, IL-8, and RANTES production in the human alveolar epithelial cell (A549), and human bronchial epithelial cell (BEAS-2B) infected with RSV A and B were compared. The results indicate that both subgroup A and B can induce all these three cytokines in A549 and BEAS-2B cells. In general, the production of cytokines by subgroup A and B was usually found higher in A549 than BEAS-2B. We compared the amount of cytokines production, RSV subgroup A produces more amounts of cytokine than subgroup B. This finding may explain the pathogenicity of subgroup A is higher than subgroup B.

英文摘要 ………….…………….………………………. 1
中文摘要 ……………………….……………………….. 4
緒論 …………………………….………………………... 6
研究材料及實驗方法…………………………….……… 15
研究材料 …………………..………………….……… 15
實驗方法 ………………………………..……………. 18
壹、 G蛋白質基因之分析 …………….……… 18
Ⅰ. 病毒繼代培養 ……………….…………18
Ⅱ. 病毒核酸之增幅 …………….…………. 20
Ⅲ. 核酸定序 ……………………..……………25
貳、 細胞激素之分析 ……………..………………. 28
Ⅰ. 病毒製備 ……………..…………………….28
Ⅱ. 實驗設計 …………..………………………31
Ⅲ. 細胞激素之測定 ..…………………………33
結果 ………………………..………………………. 35
壹、 G蛋白質基因之分析 …..……………………35
貳、 細胞激素之分析 …...………………………… 39
討論 ……………………………………………………. 43
壹、 G蛋白質基因之分析 ………………………….43
貳、 細胞激素之分析 ……..………………………. 47
圖表 …………………………………………………..50
參考文獻 …………………………………………..… 82

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