本文選題：白紋伊蚊　切入點(diǎn)：抗藥性　出處：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：一、研究背景白紋伊蚊是登革熱的重要傳播媒介,在我國(guó)大部分地區(qū)廣泛分布。登革熱近年來(lái)在我國(guó)頻繁暴發(fā)流行,由于目前沒(méi)有成熟的疫苗或藥物,對(duì)其預(yù)防控制主要是針對(duì)其傳播媒介進(jìn)行控制,特別是對(duì)白紋伊蚊成蚊化學(xué)殺蟲(chóng)劑的大量廣泛應(yīng)用成為避免疫情加重和擴(kuò)散的主要措施。溴氰菊酯為經(jīng)常使用的殺蟲(chóng)劑。然而在長(zhǎng)期的溴氰菊酯篩選壓力下,已造成白紋伊蚊對(duì)其耐受性的提高,且伴隨著一系列生理和生化以及基因方面的變化。國(guó)外已有報(bào)道,媒介按蚊產(chǎn)生抗藥性后,對(duì)瘧原蟲(chóng)的傳播能力受到影響;而白紋伊蚊對(duì)溴氰菊酯產(chǎn)生抗性后,對(duì)登革病毒的感染性和傳播性有無(wú)改變,目前尚不清楚。本文在實(shí)驗(yàn)室建立了白紋伊蚊對(duì)溴氰菊酯的抗性品系,并對(duì)其對(duì)登革病毒的易感性進(jìn)行了研究。二、研究目的1.應(yīng)用溴氰菊酯對(duì)白紋伊蚊幼蟲(chóng)進(jìn)行篩選,建立實(shí)驗(yàn)室抗性品系。2.對(duì)比白紋伊蚊敏感株和抗性株對(duì)登革病毒的易感性差異。三、實(shí)驗(yàn)方法1.抗性種群篩選應(yīng)用世界衛(wèi)生組織(WHO)推薦的蚊蟲(chóng)抗藥性測(cè)定方法(幼蟲(chóng)浸潰法)檢測(cè)白紋伊蚊抗性水平,測(cè)試種群的半數(shù)致死濃度(lethal concentration 50,LC50)。對(duì)1500只左右的四齡幼蟲(chóng)按LC50水平施加溴氰菊酯,存活個(gè)體繼續(xù)飼養(yǎng)至羽化,喂血,產(chǎn)卵。按此方法,對(duì)每一代都進(jìn)行溴氰菊酯加壓篩選。成蚊抗性水平按WHO成蚊接觸筒藥膜濾紙接觸法進(jìn)行測(cè)試,采用WHO標(biāo)準(zhǔn)的0.05%溴氰菊酯藥膜濾紙。2.C6/36細(xì)胞富集2型登革病毒,以106.83 TCID50/mL的病毒濃度經(jīng)口飼感染抗性株和敏感株白紋伊蚊。3.抗原抗體間接免疫熒光實(shí)驗(yàn)檢測(cè)2型登革病毒在中腸、卵巢、唾液腺的存在,初步檢測(cè)感染效果。4.感染后0/4/7/10天,進(jìn)行抗性株和敏感株白紋伊蚊中腸、卵巢、唾液腺的解剖、總RNA提取、2型登革病毒特異片段的逆轉(zhuǎn)、聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)擴(kuò)增、電泳以檢測(cè)登革病毒是否存在。中腸感染率=陽(yáng)性中腸樣本數(shù)/所檢測(cè)中腸的總樣本數(shù),作為病毒感染蚊蟲(chóng)的指標(biāo);卵巢感染率=陽(yáng)性卵巢樣本數(shù)/所檢測(cè)卵巢的總樣本數(shù),作為病毒在蚊蟲(chóng)體內(nèi)播散的指標(biāo);唾液腺感染率=陽(yáng)性唾液腺樣本數(shù)/所檢測(cè)唾液腺的總樣本數(shù),作為蚊蟲(chóng)潛在傳播病毒能力的指標(biāo)。實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)椒磻?yīng)(RT-qPCR)測(cè)定陽(yáng)性樣本中病毒含量。四.實(shí)驗(yàn)結(jié)果1.經(jīng)九代篩選之后,白紋伊蚊幼蟲(chóng)的對(duì)溴氰菊酯的抗性水平LC50由0.005mg/L 達(dá)到了 0.053mg/L,抗性系數(shù)(Resistance Ratio,RR)為 10.60,具有了中度抗性(WHO標(biāo)準(zhǔn))。十三代幼蟲(chóng)LCso為0.052mg/L,RR為10.40,且九到十三代RR均維持在10-11之間。2.第九代成蚊的生測(cè)死亡率為80%。十三代成蚊的生測(cè)死亡率為67%。3.熒光顯微鏡下可觀察到抗性株和敏感株感染后4/7/10天中腸、7/10天卵巢和唾液腺的綠色熒光。4.抗性株和敏感株中腸、卵巢、唾液腺感染后0/4/7/10天的RT-PCR檢測(cè)結(jié)果抗性株和敏感株0天時(shí),100%中腸可檢測(cè)到登革病毒,說(shuō)明成功使蚊蟲(chóng)攝入登革病毒。感染后4d、7d和10d,白紋伊蚊敏感株和抗性株的中腸陽(yáng)性率在92.75%～97.18%之間,二者在各時(shí)間點(diǎn)均無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。感染后4d,可在卵巢檢測(cè)到DENV-2,敏感株和抗性株的陽(yáng)性率分別為36.11%和38.89%。感染后7d和10d,兩種蚊蟲(chóng)的卵巢陽(yáng)性率均較4天時(shí)有顯著上升(P0.05),感染后7d達(dá)到84.7%和77.8%,感染后10d達(dá)到84.72%和86.11%,然而各時(shí)間點(diǎn)上二者卵巢陽(yáng)性率之間無(wú)明顯的統(tǒng)計(jì)學(xué)差異(P0.05)。蚊蟲(chóng)唾液腺呈陽(yáng)性的檢測(cè)時(shí)間點(diǎn)為感染后7d,此時(shí)敏感株和抗性株的唾液腺陽(yáng)性率分別為80.56%和83.33%,無(wú)顯著的統(tǒng)計(jì)學(xué)差異(P0.05)。感染后10d,兩感染率均無(wú)明顯升高。5.RT-qPCR檢測(cè)顯示,在0天時(shí)兩蚊株中腸和各個(gè)時(shí)間點(diǎn)卵巢的病毒含量有統(tǒng)計(jì)學(xué)差異(中腸 0 天,P=0.005;卵巢 4dpi,P=0.012;7dpi,P=0.001;10dpi P=0.006),在隨后的時(shí)間里病毒量的上升,卵巢中最為顯著,抗性株的病毒含量高于敏感株。兩蚊株唾液腺的病毒含量均隨時(shí)間上升,但并無(wú)顯著統(tǒng)計(jì)學(xué)差異。五.研究結(jié)論1.溴氰菊酯連續(xù)應(yīng)用于白紋伊蚊幼蟲(chóng)會(huì)使其幼蟲(chóng)及成蚊對(duì)其抗性水平上升,已建立穩(wěn)定實(shí)驗(yàn)室抗溴氰菊酯的中度抗性實(shí)驗(yàn)室品系。2.隨感染時(shí)間的延長(zhǎng),白紋伊蚊不同組織中登革病毒感染率的增長(zhǎng)不同�？剐灾旰兔舾兄曛g并無(wú)統(tǒng)計(jì)學(xué)差異。3.第0天的抗性蚊株中腸有較高的病毒載量,提示其攝血能力較強(qiáng)�？剐灾晖僖合佥^敏感株對(duì)2型登革病毒的易感性無(wú)顯著改變。抗性株卵巢病毒量高于敏感株,說(shuō)明抗性株卵巢更利于病毒的增殖。
[Abstract]:First, the research background of Aedes albopictus is an important medium for the spread of dengue fever, widely distributed in most areas of China. Dengue fever in China in recent years the frequent outbreaks, because there is no vaccine or medicine for the prevention and control of mature, mainly for its media control, especially the Aedes albopictus into a large number of widely used mosquito insecticide as main measures to avoid aggravation of epidemic and spread. Deltamethrin for frequently used pesticides. However, long-term selection pressure in deltamethrin, has caused the Aedes albopictus to improve its tolerance, and accompanied by a series of physiological and biochemical changes and genetic aspects. It has been reported that Anopheles resistance, impact on the spread of Plasmodium; and Aedes albopictus of deltamethrin resistance to infection after landing and spread of dengue virus has no change, It is not clear. This paper establishes Aedes albopictus to deltamethrin resistant strains in the laboratory, and the susceptibility to dengue virus on board were studied. Two objective: 1. application of deltamethrin to Aedes albopictus larvae were selected to establish different susceptibility to dengue virus strains of laboratory resistant strains of.2. sensitive and resistant strains of Aedes albopictus. Three experimental methods 1. resistant population screening application of WHO (WHO) method for the determination of resistance to the recommended mosquitoes (larvae dipping method) to detect Aedes albopictus population resistance level, half lethal concentration (lethal test concentration 50, LC50). Of the four instar larvae by only about 1500 the level of LC50 applied to deltamethrin, the survival of individuals continue to raise to eclosion, feeding blood, spawning. According to this method, for each generation of deltamethrin resistance level. Screened mosquito mosquito contact tube membrane filter press WHO The contact method was tested using WHO standard 0.05% deltamethrin membrane filter paper.2.C6/36 cell enrichment of dengue virus type 2 virus, with a concentration of 106.83 TCID50/mL by oral infection resistant and sensitive strains of Aedes albopictus.3. antigen antibody indirect immunofluorescence assay to detect dengue virus type 2 in the midgut, ovary, salivary gland, the initial detection of 0/4/7/10 infection days after.4. infection of resistant strains and sensitive strains of Aedes albopictus midgut, ovary, salivary gland anatomy, total RNA extraction, 2 dengue virus type specific fragment reverse polymerase chain reaction (RT-PCR) amplification, electrophoresis to detect dengue virus exists. The total sample mesenteronal infection rate = positive samples detected by midgut / midgut number as an indicator of mosquito virus infection; the total infection rate of ovarian samples = positive ovarian samples / ovarian detected number, as the virus in the mosquito body spread index; The total sample rate = the salivary gland of checking the number of positive samples of salivary gland / salivary gland infection, as potential mosquito spread the virus ability index. Real time fluorescence quantitative polymerase chain reaction (RT-qPCR) determination of content of virus positive samples. Four. Experimental results of 1. after nine generations of selection, resistance to deltamethrin in white LC50 Aedes albopictus larvae from 0.005mg/L to 0.053mg/L, the resistance coefficient (Resistance Ratio, RR) was 10.60, with moderate resistance (WHO). The thirteen generation larvae of LCso 0.052mg/L, RR is 10.40, and the nine to the thirteen generation of RR were maintained at 10-11.2. between ninth mosquito bioassay mortality was 80%. thirteen the mosquito bioassay for 67%.3. mortality was observed by fluorescent microscopy in sensitive and resistant strains were infected after 4/7/10 days 7/10 days of midgut, ovary and salivary gland green fluorescent.4. resistant strains and sensitive strains of midgut, ovary, salivary gland. The results of 0/4/7/10 days after dyeing of RT-PCR resistant strains and sensitive strains on day 0, 100% were detected by the dengue virus, indicating the success of the mosquito intake of dengue virus. After infection of 4D, 7d and 10d, Aedes albopictus in susceptible and resistant strains were positive rate in 92.75% to 97.18%, two in the at each time point were not statistically significant (P0.05). 4D after infection, can be detected in ovarian DENV-2 susceptible and resistant strains of the positive rates were 36.11% and 38.89%. after infection of 7D and 10d, the positive rate of ovarian two mosquito species were 4 days increased significantly (P0.05), 7d after infection reached 84.7% and 77.8% 10d after infection reached 84.72% and 86.11%, but no statistically significant difference between each time point of the two ovarian positive rate (P0.05). The mosquito salivary gland was positive for the detection time point after 7d infection, the susceptible and resistant strains of the salivary gland positive rate are 80.56% and 83.33%, No statistically significant difference (P0.05). 10d after infection, the infection rate was two and no significant increase of.5.RT-qPCR showed that on the 0 day there was significant difference in two strains of virus mosquito midgut and each time point of the ovary (midgut for 0 days, P=0.005; 7dpi, P=0.012; ovarian 4dpi, P=0.001; 10dpi, P=0.006) increased in the subsequent period of the amount of virus, the most significant in the ovary, the amount of virus resistant strains was higher than that of sensitive strains. Two strains of virus in mosquito salivary gland were increased, but there is no significant difference. Five. Conclusion 1. continuous application of deltamethrin on Aedes albopictus larvae of the larvae and adult mosquitoes the rise of the resistance level, has established a stable Laboratory of deltamethrin resistance in moderate resistant laboratory strain.2. with the infection time, Aedes albopictus in different tissues of dengue virus infection rate of growth. There is no unified different resistant and sensitive strains Meter resistant mosquito strains midgut difference.3. zeroth days have a high viral load, suggesting that the perturbation of blood ability. Resistant strains were sensitive to 2 strains of salivary gland dengue susceptibility to dengue virus had no significant change. The amount of virus resistant ovarian than susceptible strains, resistant strains of the ovary is more conducive to the proliferation of the virus.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R384.1

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相關(guān)期刊論文 前1條

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本文編號(hào)：1686776