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研究生:徐富美
研究生(外文):Fu-Mei Hsu
論文名稱:以人類横紋肌肉瘤細胞培養模式探討肺炎黴漿菌對沙賓疫苗株之影響
論文名稱(外文):The Interference Of Sabin Vaccine Co-culture With Mycoplasma pneumoniae In Rhabdomyosarcoma Cells
指導教授:孔建民孔建民引用關係
指導教授(外文):Chien-Min Kung
學位類別:碩士
校院名稱:元培科技大學
系所名稱:醫學檢驗生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
畢業學年度:102
語文別:中文
論文頁數:69
中文關鍵詞:沙賓疫苗肺炎黴漿菌疫苗相關性脊髓灰白質炎
外文關鍵詞:Sabin vaccineMycoplasma pneumoniaevaccine-associated paralytic poliomyelitis
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沙賓疫苗為一種減毒的小兒麻痺病毒,用來預防小兒麻痺病毒感染。小兒麻痺病毒屬於小RNA病毒科(Picornaviridae)中腸病毒(Enterovi¬rus)之病毒。可引起急性無力肢體痲痺症(AFP),具高度傳染性。沙賓疫苗病毒株除了易在腸道增殖外,也會有不明原因產生突變或而回復到具有神經細胞毒性的野生株的機率,導致接受疫苗個體產生肢體麻痺症狀。另外需要重視的是台灣兒童容易感染到社區性非典型肺炎,尤其是肺炎黴漿菌的感染。本研究以細胞培養模式,欲探討肺炎黴漿菌與沙賓疫苗在共同感染下是否會改變細胞活性或誘導雙方基因產生變異。實驗中採用人類横紋肌肉細胞(RD)為培養細胞,分析受感染細胞活性及分別定序培養前後的肺炎黴漿菌P1及沙賓疫苗type 1之VP1基因型態。結果發現沙賓疫苗 type 1和肺炎黴漿菌共同培養時,沙賓疫苗疫苗株的複製能力會受影響,反而提升受感染細胞的存活率。沙賓疫苗 type 1 VP1基因分析中發現有2群在第3222個位置核苷酸分別為腺嘌呤(A)及胞嘧啶(C)不同的病毒株存在,但與沙賓疫苗病毒株type 1 VP1序列仍有99.9%的相似度。另外,肺炎黴漿菌P1基因在培養前後都沒有變化。結論,研究得知RD細胞在肺炎黴漿菌與沙賓疫苗共同感染下,肺炎黴漿菌可以降低沙賓疫苗對細胞的傷害力。另外,也發現在肺炎黴漿菌存在下,可抑制沙賓疫苗type 1 VP1基因第3222位置的腺嘌呤(A)轉為胞嘧啶(C)的傾向。沙賓疫苗type 1 VP1基因的單點變化是否具有臨床意義,仍需進一步分析。
Sabin vaccine (SabV) was an attenuated vaccine for preventing poliomyelitis, an acute paralytic flaccid disease caused by poliovirus. Poliovirus, an enterovirus belonging to the Picornaviridae family. It is a highly infectious disease. In rare case, vaccine recipients appeared paralysis after SabV administration. In Taiwan, Mycoplasma pneumoniae (Mp) is a common causative agent of community-acquired pneumonia, especially in children. This study was designed to investigate the CPE of infected cells and variations of SabV VP1 gene and Mp P1 gene when SabV co-culture with Mp in Rhabdomyosarcoma (RD) cells. We found that survival of SabV infected RD cell increased by co-cultured with Mp. Meanwhile, there are two SabV strains which had different nucleotides (Adenosine or Cytosine) on ‘3222’ site of VP1 gene by nucleotide sequencing. Both SabV VP1 nucleotides almost were same, onlyone nucleotide was difference. However, Adenosine had a tendency to arise when SabV co-culture with Mp in RD cells. Mp P1 genes had no change from test and control group. It needs future study to elucidate the interference of single nucleotide different of SabV VP1gene.
誌謝 I
中文摘要 II
英文摘要 III
目錄 IV
圖目錄 V
表目錄 VII
第一章 前言 1
第一節 研究背景與動機 1
第二節 研究目的 6
第二章 材料與方法 7
第一節 實驗材料 7
第二節 儀器設備 10
第三節 研究方法與步驟 11
第三章 結果 19
一. 細胞對照組、病毒株對照組、細菌對照組及實驗組培養結果 19
二. 細胞活性分析(MTT) 19
三. 基因分析 20
第四章 討論 22
參考文獻 55
一. 基因分析 55
二. 細胞活性分析(MTT) 59

一、 英文部份
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4 CDC, “Update on Vaccine-Derived Polioviruses --- Worldwide, July 2009--March 2011,” 2011, 60(25), pp846-850.
Update on Vaccine-Derived Polioviruses --- Worldwide, July 2009--March 2011
5 Chi CY., Tseng FC., Liu DP., Chang YW., Wu HC., Huang YF., Hwang KP., Hsu YW., Wang SM., Liu CC., Wu HS., Yang JY., Yang CF., Wang JR., Su IJ., “Investigations of clinical isolations of oral poliovirus vaccine strains between 2000 and 2005 in southern Taiwan,” J Clin Virol, 2009, 45(2), pp129-134.
6 Costa EV., Campos Rde M., Tavares FN., Grégio CR., Burlandy FM., Silva EE.,“A RT-PCR method for selective amplification and phenotypic characterization of all three serotypes of Sabin-related polioviruses from viral mixtures,” Mem Inst Oswaldo Cruz, 2012, 107(5), pp698-701.
7 Couderc T., Guédo N., Calvez V., Pelletier I., Hogle J., Colbère-Garapin F., Blondel B., “Substitutions in the capsids of poliovirus mutants selected in human neuroblastoma cells confer on the Mahoney type 1 strain a phenotype neurovirulent in mice,” J Virol, 1994, 68(12), pp8386-891.
8 Chourasia BK., Chaudhry R., Malhotra P., “Delineation of immunodominant and cytadherence segment(s) of Mycoplasma pneumoniae P1 gene,” BMC Microbiol, 2014, 14(1).
9 Dorigo-Zetsma JW., Wilbrink B., Dankert J., Zaat SA., “Mycoplasma pneumoniae P1 type 1- and type 2-specific sequences within the P1 cytadhesin gene of individual strains.” Infect Immun, 2001, 69(9), pp 5612-5618.
10 Fisseha M. , Göhlmann HW. H., Herrmann R., Krause DC. , “Identification and Complementation of Frameshift Mutations Associated with Loss of Cytadherence in Mycoplasma pneumonia,” Journal of Bacteriology, 1999, 181(14), pp 4404–4410.
11 Foy HM., Kenny GE., Cooney MK., Allan ID., “Long-term epidemiology of infections with Mycoplasma pneumoniae,” J Infect Dis, 1979, 139(6), pp 681-687.
12 GenBank: AY184219.1, “Human poliovirus 1 strain Sabin 1, complete genome,”2002.
Human poliovirus 1 strain Sabin 1, complete genome - Nucleotide - NCBI
13 GPEI, Global Polio Eradication Initiative, “ Economic Case for Eradicating Polio,” www.polioeradication.org/Portals/0/.../EconomicCase.pdf
14 GPEI, “Circulating vaccine-derived poliovirus (cVDPV) 2000-2013,” 2014.
Global Polio Eradication Initiative > Data and monitoring > Polio this week > Circulating vaccine-derived poliovirus,
15 GPEI, “FINANCIAL RESOURCE REQUIREMENTS 2013-2018,” 2014, WHO, pp3.
16 Hunt R., “VACCINES: Past Successes And Future Prospects,” 2010, VIROLOGY, CHAPTER EIGHT.
17 Hsieh YC., “Community-acquired Pneumonia Among Children in Tai-wan,”Pediatrics and Neonatology, 2013,54, pp 1-2.
18 Klausner JD., Passaro D., Rosenberg J., Thacker WL., Talkington DF., Werner SB., Vugia DJ., “Enhanced Control of an Outbreak of Mycoplasma pneumoniae with Azithromycin Prophylaxis,” J Infect Dis,1998,177, pp 161–166.
19 John TJ., “A developing country perspective on vaccine-associated paralytic poliomyelitis,” Bulletin of the WHO, 2004, 82 (1), pp 53-58.
20 Kashyap S., Sarkar M., “Mycoplasma pneumonia: Clinical features and management,” Lung India, 2010, 27(2), pp 75–85.
21 Kew OM., Sutter RW, Nottay BK., McDonough MJ., Prevots DR., Quick L., Pallansch MA.,“Prolonged replication of a type 1 vaccine-derived poliovirus in an immunodeficient patient,” J Clin Microbiol, 1998, 36(10), pp2893-2899.
22 Kew O., “Reaching the last one per cent: progress and challenges in global polio eradication.” Curr Opin Virol, 2012, 2(2), pp188-198.
23 Kew OM., Sutter RW., Gourville EM., Dowdle WR., Pallansch MA., “Vaccine-derived polioviruses and the endgame strategy for global polio eradication,” Annu Rev Microbiol, 2005, 59, pp587-635.
24 Liang X., Zhang Y., Xu W., Wen N., Zuo S., Lee LA., Yu J.,“An outbreak of poliomyelitis caused by type 1 vaccine-derived poliovirus in China,” J Infect Dis. 2006 ,194(5), pp 545-551.
25 McDermott Jr BM., “Poliovirus receptor recognition: Visualization, kinetics, and thermodynamics,” 2001, pp 65.
26 McAllister RM., Melnyk J., Finkelstein JZ., Adams EC Jr., Gardner MB., “Cultivation in vitro of cells derived from a human rhabdomyosarcoma,” Cancer,1969, 24(3), pp 520-526.
27 Nolevaux G., Bessaci-Kabouya K., Villenet N., Andréoletti L., Laplanche D., Carquin J., Abély M, De Champs C., Motte J.,“Epidemiological and clinical study of Mycoplasma pneumoniae respiratory infections in children hospitalized in a pediatric ward between 1999 and 2005 at the Reims University Hospital,” Arch Pediatr, 2008, 15(11), pp 1630-1636.
28 Razin S., Yogev D., Naot Y., “Molecular biology and pathogenicity of Mycoplasmas,” Microbiol Mol Biol Rev, 1998, 62(4), pp 1094-1156.
29 Robertson S. “Poliomyelitis,” WHO, 1993, Module 6:The Immunological Basis for Immunization Series, pp 2.
30 Scott B., “Eradication versus control: the economics of global infectious disease policies,” Bulletin of WHO ,2004, 82 (9)
31 Shimizu H., Thorley B., Paladin FJ., Brussen AK., Stambos V., Yuen L., Utama A., Tano Y., Arita M., Yoshida H., Yoneyama T., Benegas A., Roesel S., Pallansch M., Kew O., Miyamura T., “Circulation of Type 1 Vaccine-Derived Poliovirus in the Philippines in 2001,”J Virol, 2004, 78(24), pp 13512-13521.
32 Sillis M., “The limitations of IgM assays in the serological diagnosis of Mycoplasma pneumoniae infections,” J Med Microbiol,1990 ,33(4), pp253-258.
33 Talkington DF., Thacker WL., Keller DW., Jensen JS.,“Diagnosis of Mycoplasma pneumoniae infection in autopsy and open-lung biopsy tissues by nested PCR,” J Clin Microbiol, 1998 ,36(4), pp 1151-1153.
34 Thacker WL., Talkington DF., “Comparison of two rapid commercial tests with complement fixation for serologic diagnosis of Mycoplasma pneumoniae infections,” J Clin Microbiol, 1995, 33(5), pp 1212-1214.
35 Tukei PM. “Polio Eradication by the year 2000,” Afr J Health Sci, 1996, 3 (3), pp 65.
36 Ursi D, Ieven M, van Bever H, Quint W, Niesters HG, Goossens H.“Typing of Mycoplasma pneumoniae by PCR-mediated DNA fingerprinting,” J Clin Microbiol, 1994, 32(11),pp2873-2875.
37 MILLIGAN WH., FLETCHER RD., “Effect of Mycoplasma pneumoniae on Poliovirus Replication,” Infection and Immunity, 1975, pp 607-608.
38 WHO, “Polio laboratory manual,” 2004, 4th edition, pp 1-2.
39 WHO, “Poliomyelitis,”2014.
WHO | Poliomyelitis
40 Williamson J., Marmion BP., Worswick DA., Kok TW., Tannock G., Herd R., Harris RJ., “Laboratory diagnosis of Mycoplasma pneumoniae infection. 4. Antigen capture and PCR-gene amplification for detection of the Mycoplasma: problems of clinical correlation,” Epidemiol Infect. 1992, 109(3), pp 519-537.
41 Zhanga P., Muellerb S., Moraisa MC., Batora CM., Bowmana VD., Hafensteina S., Wimmerb E., Michael GR., “Crystal structure of CD155 and electron microscopic studies of its complexes with polioviruses,” PNAS, 2008, 105 (47), pp 18284–18289.
二、中文部份
1. 行政院衛生署疾病管制局,「小兒麻痺症」,2010。
2. 李秉穎,「感染科問題-肺炎」,2013。
肺炎 (Pneumonia)

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