發(fā)布時(shí)間：2018-06-01 13:13

  本文選題：人偏肺病毒 + 標(biāo)準(zhǔn)株�。� 參考：《遵義醫(yī)學(xué)院》2012年碩士論文

【摘要】：第一部分關(guān)于人偏肺病毒標(biāo)準(zhǔn)株病毒適合度的研究 目的人偏肺病毒(Human metapneumovirus hMPV)是2001年首次被分離的呼吸道病毒病原,可導(dǎo)致各年齡組人群呼吸道感染,以兒童感染為主,嚴(yán)重者威脅生命造成死亡,不僅如此,hMPV感染還與哮喘發(fā)生、發(fā)展有關(guān)。但目前針對(duì)hMPV感染,尚無(wú)可靠疫苗和治療藥物。病毒適合度(virai fitness)信息的獲得,能夠?yàn)橐呙绲脑O(shè)計(jì),病毒感染后病情、病程評(píng)估,藥物選擇及疾病的預(yù)后,提供有重要參考價(jià)值的信息,然而目前世界范圍內(nèi)對(duì)hMPV適合度的研究未見(jiàn)報(bào)道。本研究通過(guò)hMPV兩亞型代表株NL/1/00(A亞型)與NL/1/99(B亞型)的體內(nèi)外競(jìng)爭(zhēng)性復(fù)制實(shí)驗(yàn),檢測(cè)不同標(biāo)準(zhǔn)株的相對(duì)適合度,對(duì)hMPV標(biāo)準(zhǔn)株適合度進(jìn)行評(píng)價(jià),提供國(guó)內(nèi)外首份hMPV標(biāo)準(zhǔn)株適合度的分析報(bào)告。 方法1.將分別代表A、B兩亞型的病毒株NL/1/00與NL/1/99以20：80、50：50、80：20混合成3個(gè)比例不同的混合病毒株。2個(gè)單獨(dú)病毒株及3個(gè)混合株以相同的感染量分別接種于生長(zhǎng)于6孔板中的各孔單層Vero-E6細(xì)胞,設(shè)置復(fù)孔及空白對(duì)照孔。病毒連續(xù)培養(yǎng)十幾代,每代培養(yǎng)4-5天后將病毒液均放置于-80℃冰箱速凍保存,部分用于傳代,部分用于提取病毒RNA,反轉(zhuǎn)錄成cDNA后,Real-Time PCR及核苷酸序列圖譜法檢測(cè)兩個(gè)病毒株所占比例變化。2.將6-8周,雌性BALB/c小鼠隨機(jī)分為6組,其中5組以滴鼻法分別接種上訴2個(gè)單獨(dú)病毒株及3個(gè)混合株,每組每只小鼠接種病毒量均為50ul1×109copies/ul,剩余1組設(shè)為空白對(duì)照組。于感染后第1,2,4,6,8,13,17,20天處死小鼠取肺組織,提取病毒RNA后,反轉(zhuǎn)錄成用cDNA,用Real-Time PCR與核苷酸序列圖譜法檢測(cè)兩個(gè)病毒株所占比例變化. 結(jié)果在動(dòng)物實(shí)驗(yàn)中,hMPV感染BALB/C小鼠后,可在小鼠體內(nèi)復(fù)制存活約3周,于第4天到達(dá)復(fù)制高峰后病毒量逐漸減少,3周后檢測(cè)不到病毒；20天內(nèi)3個(gè)混合組均以NL/1/99所占比例為高,表現(xiàn)出較強(qiáng)的生長(zhǎng)趨勢(shì)；空白對(duì)照組中未檢測(cè)到hMPV感染。在細(xì)胞體系中,病毒共傳代培養(yǎng)至12代,1-5代3組混合組均以NL/1/99占較高比例,6-8代以NL/1/00為主,第9代開(kāi)始NL/1/99消失,只剩NL/1/00單獨(dú)生長(zhǎng)；空白孔未檢測(cè)到hMPV感染。在單獨(dú)感染NL/1/00和NL/1/99的動(dòng)物及細(xì)胞體系中,均未檢測(cè)到除此病毒株外的其余hMPV類型。 結(jié)論動(dòng)物實(shí)驗(yàn)中NL/1/99表現(xiàn)出較強(qiáng)的競(jìng)爭(zhēng)復(fù)制優(yōu)勢(shì),與細(xì)胞系中前期趨勢(shì)一致。細(xì)胞體系中NL/1/00和NL/1/99生長(zhǎng)優(yōu)勢(shì)出現(xiàn)交替,與近些年來(lái)廣泛報(bào)道的hMPV兩亞型在人群中交替流行的現(xiàn)象相吻合。 第二部分人偏肺病毒A與B基因型對(duì)小鼠致病性的觀察 目的通過(guò)觀察人偏肺病毒(human metapneumovirus, hMPV)A和B亞型感染BALB/c小鼠后,小鼠在體重、病毒載量、病理及氣道反應(yīng)性等方面的改變,探討兩亞型病毒致病性上的差異,為hMPV的進(jìn)一步研究奠定基礎(chǔ)。 方法hMPV感染BALB/c動(dòng)物模型后,通過(guò)使用Real-time PCR方法檢測(cè)鼠肺內(nèi)病毒載量的變化,肺功能檢測(cè)儀監(jiān)測(cè)小鼠氣道反應(yīng)性的變化及組織系統(tǒng)評(píng)分判定病理改變情況,來(lái)觀測(cè)兩亞型致病性的差異。 結(jié)果hMPV兩亞型感染組,在體重的動(dòng)態(tài)監(jiān)測(cè)、病毒載量、肺組織病理改變及氣道反應(yīng)性上均無(wú)統(tǒng)計(jì)學(xué)意義的差異。 結(jié)論hMPV兩基因型感染BALB/c小鼠的致病性無(wú)差異。
[Abstract]:The first part is about the fitness of human standard strain of human partial Lev virus.
Human metapneumovirus hMPV is the first pathogen of respiratory tract virus isolated in 2001. It can cause respiratory infection in all age groups, mainly children infection, and serious people threaten life cause death. Not only that, but hMPV infection is also related to the development of asthma, but there is no reliable vaccine for hMPV infection at present. The acquisition of Virai fitness information can provide important reference information for the design of the vaccine, the condition of the virus infection, the course evaluation, the drug selection and the prognosis of the disease. However, the research on the fitness of hMPV has not been reported worldwide. This study represents the NL/1/00 by the hMPV two subtype. In vivo and in vitro competitive replication (A subtype) and NL/1/99 (B subtype), the relative fitness of different standard strains was detected, the fitness of hMPV standard strain was evaluated, and the analysis report on the fitness of the first hMPV standard strain at home and abroad was provided.
Method 1. A, B two subtype virus strain NL/1/00 and NL/1/99 were mixed with 20:80,50:50,80:20 into 3 separate virus strains and 3 mixed strains of mixed virus strains and 3 mixed strains were inoculated separately in the monolayer Vero-E6 cells, which grew in 6 orifice plates, and set up the compound holes and blank control holes. For more than ten generations, 4-5 days after each generation, the virus was stored in -80 C fridge for quick freezing, partly used for passage and part used to extract virus RNA. After reverse transcription to cDNA, Real-Time PCR and nucleotide sequence atlas were used to detect the proportion of two virus strains for 6-8 weeks, and female BALB/c mice were randomly divided into 6 groups, of which 5 groups were treated with nasal drip method. 2 separate virus strains and 3 mixed strains were inoculated respectively. Each group was inoculated with 50ul1 x 109copies/ul, the remaining 1 groups were set as blank control group. The lung tissue was killed on day 1,2,4,6,8,13,17,20 after infection. After the virus RNA was extracted, the reverse transcription was cDNA. Real-Time PCR and nucleotide sequence mapping method were used to detect two. A change in the proportion of the virus strains.
Results in the animal experiment, in the animal experiment, hMPV infected BALB/C mice for about 3 weeks. After the fourth day of replication peak, the amount of virus gradually decreased and the virus was not detected in 3 weeks. The proportion of the 3 mixed groups in the 20 days was high in the proportion of NL/1/99, showing a strong growth trend, and the hMPV infection was not detected in the blank control group. In the cell system, the virus was co cultured to 12 generations, and the 1-5 generation 3 groups of mixed groups accounted for a high proportion of NL/1/99, the 6-8 generation was mainly NL/1/00, the ninth generation began to disappear, only the NL/1/00 alone was left, and the blank hole was not detected by hMPV infection. The virus strains were not detected in the animals and cell systems of NL/1/00 and NL/1/99 alone. The rest of the hMPV type.
Conclusion in animal experiments, NL/1/99 showed a strong competitive replication advantage, which was consistent with the early trend of cell lines. The growth advantages of NL/1/00 and NL/1/99 appeared alternately in the cell system, which coincided with the phenomenon of the alternately popular hMPV two subtype in the population in recent years.
Observation on pathogenicity of second human partial lung viruses A and B genotypes in mice
Objective to explore the difference in the pathogenicity of the two subtype virus by observing the changes in weight, viral load, pathology and airway responsiveness of the human metapneumovirus (hMPV) A and B subtypes in BALB/c mice, and to lay a foundation for further research on hMPV.
Methods after hMPV infection of BALB/c animal model, the changes of viral load in the lungs were detected by the Real-time PCR method. The pulmonary function detector monitored the changes of airway responsiveness and the histological grading of the mice to determine the pathological changes of the two subtypes.
Results there was no significant difference in the dynamic monitoring of body weight, viral load, pathological changes of lung tissue and airway responsiveness in hMPV two subtype infection group.
Conclusion there is no difference in pathogenicity of hMPV two genotype infected BALB/c mice.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R725.6

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 郭麗雙;李凱軍;徐艷;張文莉;付英梅;張曉莉;;雙黃連凍干粉對(duì)革蘭陰性桿菌的體外抑制作用[J];吉林中醫(yī)藥;2011年06期

2 趙成琳;王言奎;羅兵;王玉婷;彭麗娜;;青島地區(qū)宮頸病變組織中HPV16 URR和E6基因突變分析[J];現(xiàn)代婦產(chǎn)科進(jìn)展;2011年05期

3 尹小毛;謝貝;羅春明;黃麗晶;蔡杏珊;李昕潔;劉志輝;;應(yīng)用MPT64檢測(cè)建立結(jié)核分枝桿菌藥敏新方法的初步探索[J];實(shí)驗(yàn)與檢驗(yàn)醫(yī)學(xué);2011年04期

4 羅予;李杰;劉娜;;RT-PCR檢測(cè)金黃色葡萄球菌[J];中國(guó)微生態(tài)學(xué)雜志;2011年08期

5 祝賀;王文嶺;李海濤;敖俊紅;楊蓉婭;;不同來(lái)源阿薩希毛孢子菌菌絲相體外誘導(dǎo)條件的探討[J];實(shí)用皮膚病學(xué)雜志;2011年02期

6 董春雷;朱長(zhǎng)太;;實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)技術(shù)檢測(cè)解脲脲原體生物群的研究[J];檢驗(yàn)醫(yī)學(xué)與臨床;2011年15期

7 陳文峰;張子平;程波;蘇惠春;;利凡諾體外抗白念珠菌活性的研究[J];皮膚病與性病;2011年04期

8 謝榮輝;朱函坪;黃瀟瀟;楊章女;徐芳;姚蘋(píng)蘋(píng);朱智勇;;腸道病毒EV71型浙江分離株HZ08全基因組序列分析[J];中國(guó)人獸共患病學(xué)報(bào);2011年08期

9 顧文珍;倪紅霞;張姝;易波;楊天池;賀天鋒;許國(guó)章;;寧波市腸道病毒71型分離株基因組特征研究[J];中國(guó)衛(wèi)生檢驗(yàn)雜志;2011年08期

10 宋春涵;高素華;何旭瑛;;宮頸癌組織中HPV16 URR基因多態(tài)性分析[J];國(guó)際檢驗(yàn)醫(yī)學(xué)雜志;2011年09期

相關(guān)會(huì)議論文 前10條

1 王宇清;;2006-2007年蘇州地區(qū)兒童呼吸道人類偏肺病毒感染的流行和臨床特征[A];蘇州市自然科學(xué)優(yōu)秀學(xué)術(shù)論文匯編(2008-2009)[C];2010年

2 寇宇;孫晝;于新芬;楊旭輝;李鈞;謝立;潘勁草;;一起嬰幼兒醫(yī)院內(nèi)獲得性麻疹病毒和人類偏肺病毒感染的調(diào)查[A];2011年浙江省醫(yī)學(xué)會(huì)醫(yī)學(xué)病毒學(xué)分會(huì)、醫(yī)學(xué)微生物與免疫學(xué)分會(huì)學(xué)術(shù)年會(huì)論文匯編[C];2011年

3 季偉;王宇清;;2005-2006年蘇州地區(qū)兒童呼吸道人類偏肺病毒感染的流行和臨床特征[A];蘇州市自然科學(xué)優(yōu)秀學(xué)術(shù)論文匯編(2008-2009)[C];2010年

4 張金輝;竇穎;佘薇薇;趙耀;劉平;趙曉東;;人類偏肺病毒F蛋白糖基化位點(diǎn)對(duì)其體內(nèi)外復(fù)制能力的影響[A];中華醫(yī)學(xué)會(huì)第十五次全國(guó)兒科學(xué)術(shù)大會(huì)論文匯編（上冊(cè)）[C];2010年

5 周向明;張解寧;鄭莉;金佩麗;苗正友;;嘉興地區(qū)1347例急性呼吸道感染住院兒童人類偏肺病毒感染的研究[A];2011年浙江省醫(yī)學(xué)會(huì)兒科學(xué)分會(huì)學(xué)術(shù)年會(huì)暨兒內(nèi)科疾病診治新進(jìn)展國(guó)家級(jí)學(xué)習(xí)班論文匯編[C];2011年

6 毛華偉;趙曉東;;人偏肺病毒融合蛋白在昆蟲(chóng)細(xì)胞中的表達(dá)[A];中華醫(yī)學(xué)會(huì)第十四次全國(guó)兒科學(xué)術(shù)會(huì)議論文匯編[C];2006年

7 張宗德;陳效友;段鴻飛;馬s，

本文編號(hào)：1964301