本文選題：登革病毒 + 人胚肺二倍體細(xì)胞　； 參考：《北京協(xié)和醫(yī)學(xué)院》2012年碩士論文

【摘要】：登革病毒(dengue virus,DEN)屬于黃病毒科黃病毒屬中的一個(gè)血清型亞群,主要通過埃及伊蚊和白紋伊蚊等媒介昆蟲傳播,引起登革熱(dengue fever,DF)以及發(fā)病率和死亡率很高的登革出血熱(dengue hemorrhagic fever,DHF)和登革休克綜合征(dengue shock syndrome,DSS)。尤其近年來,由于全球氣候變暖、人口流動(dòng)頻繁等因素,登革熱在全世界的發(fā)病率提高了近30倍。在過去兩年中全球登革熱感染人群的數(shù)量持續(xù)增長(zhǎng),在東南亞中部和南部地區(qū)、美國(guó)以及南太平洋地區(qū),每年約8000萬人感染登革病毒,估計(jì)24000人死亡,超過100個(gè)國(guó)家的30億人受到威脅,已成為世界性的公共衛(wèi)生問題。迄今為止全球仍沒有獲準(zhǔn)上市的登革病毒疫苗,因此登革疫苗研發(fā)迫在眉睫。目前最有潛力的傳統(tǒng)減毒活疫苗候選株是由泰國(guó)沃特瑞德研究所(WRAIR)研發(fā)的以犬腎細(xì)胞和恒河猴胚肺細(xì)胞為基質(zhì)的疫苗[3],Vero細(xì)胞也被認(rèn)為是疫苗開發(fā)的候選基質(zhì),以人源性細(xì)胞制備的登革疫苗候選株還未見報(bào)道。因此研究登革病毒在人源性細(xì)胞上的適應(yīng)性對(duì)于登革病毒疫苗的研發(fā)具有重要意義。 人二倍體細(xì)胞系KMB17取自人胚胎肺組織,為醫(yī)學(xué)生物學(xué)研究所自建并經(jīng)衛(wèi)生部批準(zhǔn)用于甲型肝炎疫苗的大規(guī)模生產(chǎn)的細(xì)胞株.其安全性和細(xì)胞穩(wěn)定性都得到了廣泛驗(yàn)證。 本研究對(duì)中緬邊境采集的100份登革熱病人血清樣本進(jìn)行編號(hào)和基本信息登記后,通過登革病毒IgM. IgG抗體檢測(cè)試劑盒對(duì)血清樣本進(jìn)行抗體檢測(cè)。經(jīng)過統(tǒng)計(jì)和分析,在這些樣本中,Ig M/Ig G陰性的樣本僅占總樣本數(shù)的5%,Ig M陽性/Ig G陰性的樣本占總樣本數(shù)的26%,而Ig G陽性的樣本所占比例高達(dá)69%。樣本接種C6/36細(xì)胞擴(kuò)增后經(jīng)血凝效價(jià)測(cè)定,無能夠使新鮮鵝血紅細(xì)胞凝集的樣本。由于IgM/Ig G陽性樣本所占比例高達(dá)95%,要從中分離活病毒并進(jìn)行基因分析十分困難。通過與廣東省CDC合作,獲得了Ⅰ-Ⅳ型登革病毒8個(gè)中國(guó)株,將其分別在C6/36細(xì)胞上進(jìn)行毒種擴(kuò)增和滴度測(cè)定后,以衛(wèi)生部公布的登革病毒通用引物及Ⅰ～Ⅳ型登革病毒型特異性引物通過RT-PCR法進(jìn)行型別鑒定。經(jīng)鑒定的毒株以4.0MOI在人胚肺二倍體細(xì)胞KMB17上反復(fù)傳代,直至登革病毒能在KMB17細(xì)胞中引起顯著病變,然后進(jìn)行培養(yǎng)條件優(yōu)化,確定其最佳感染條件。將病變毒株在KMB17細(xì)胞中連續(xù)傳代10代,選育出良好的細(xì)胞適應(yīng)株,并經(jīng)過三輪噬斑純化。微量滴定法檢測(cè)1-10代病毒的感染性滴度,免疫熒光法檢測(cè)病毒純化株的抗原性,實(shí)時(shí)熒光定量法進(jìn)行增殖動(dòng)力學(xué)研究。病毒培養(yǎng)液經(jīng)蔗糖梯度離心和超速離心純化后,電鏡檢測(cè)病毒顆粒形態(tài)。結(jié)果顯示,將Ⅰ～Ⅳ型登革病毒中國(guó)株提取RNA進(jìn)行RT-PCR,能分別擴(kuò)增出511bp的登革病毒特異性基因和482bp、119bp、290bp和392bp的型特異性基因；毒株接種C6/36細(xì)胞收獲擴(kuò)增的病毒液,將此病毒液感染KMB17細(xì)胞并經(jīng)過反復(fù)傳代后,可使KMB17產(chǎn)生明顯的細(xì)胞病變(CPE)；將病毒適應(yīng)株在KMB17細(xì)胞上進(jìn)行10代連續(xù)傳代培養(yǎng),細(xì)胞病變速度逐漸增快,至第10代達(dá)到增殖高峰。分別經(jīng)三輪噬斑純化后篩選出四型純化毒株,免疫熒光法檢測(cè)感染KMB17細(xì)胞的純化株病毒的抗原性,結(jié)果均呈陽性,病毒經(jīng)純化后電鏡下顯示形態(tài)正常增殖動(dòng)力學(xué)研究顯示,登革病毒適應(yīng)株在KMB17細(xì)胞中接種后第3天已經(jīng)開始增殖,在第5-6天增殖最為迅速,在第7天病毒增殖達(dá)到最高峰。本研究選育出了能在KMB17細(xì)胞中穩(wěn)定增殖且病毒滴度較高的細(xì)胞適應(yīng)株,為下一步研究登革病毒滅活疫苗和減毒活疫苗奠定基礎(chǔ)。
[Abstract]:dengue virus ( DEN ) belongs to a serological subgroup of the Flaviviridae family of Flaviviridae , which is mainly transmitted through the media insects such as aegypti and aegypti , causing dengue fever ( DF ) as well as dengue hemorrhagic fever ( DHF ) and dengue shock syndrome ( DSS ) with high morbidity and mortality . Especially in recent years , the incidence of dengue fever has increased nearly 30 times worldwide due to global warming and frequent population movements . In the past two years , the number of dengue - infected people has continued to grow . In the central and southern regions of South - East Asia , more than 3 billion people in the United States and the South Pacific region have been threatened and have become world - sex public health problems . So far , the most potential candidate strains of attenuated live vaccine have not yet been reported . Therefore , the adaptability of dengue virus to human - derived cells is of great significance for research and development of dengue virus vaccines .

Human diploid cell line KMB17 is taken from human embryonic lung tissue and is established by the Institute of Medical Biology and approved by the Ministry of Health for large - scale production of hepatitis A vaccine . Its safety and cell stability are widely verified .

In this study , 100 dengue fever human serum samples collected at the Sino - Myanmar border were numbered and registered with basic information , and IgM was detected by dengue virus . In this paper , we have obtained 8 Chinese strains of dengue virus type 鈪，

本文編號(hào)：1823750