本文關(guān)鍵詞： 水痘—帶狀皰疹病毒 實(shí)時(shí)熒光定量多聚核苷酸鏈?zhǔn)椒磻?yīng) 外周血單個(gè)核細(xì)胞 腦源性神經(jīng)營(yíng)養(yǎng)因子 S100B　出處：《西南醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景和目的:水痘-帶狀皰疹病毒(varicella zoster virus,VZV)感染后可導(dǎo)致水痘、帶狀皰疹(herpes zoster,HZ)及帶狀皰疹后遺神經(jīng)痛(postherpetic neuralgia,PHN)等VZV相關(guān)疾病的發(fā)生。在VZV相關(guān)疾病中對(duì)VZV DNA進(jìn)行檢測(cè),可作為感染、病毒血癥存在的依據(jù),以及對(duì)神經(jīng)損傷程度、病情及預(yù)后作出相應(yīng)的判斷。目前國(guó)內(nèi)對(duì)于外周血VZV DNA的檢測(cè),通常運(yùn)用實(shí)時(shí)熒光定量多聚核苷酸鏈?zhǔn)椒磻?yīng)(Real-timeQuantitativepolymerasechain reaction,qPCR)檢測(cè)試劑盒。但因其價(jià)格昂貴,靈敏度不高,故在臨床中尚不能推廣。所以本文的目的之一在于優(yōu)化DNA的提取方法,簡(jiǎn)化標(biāo)準(zhǔn)品的制作,設(shè)計(jì)特異性的引物,提高檢測(cè)方法的敏感性、簡(jiǎn)單性、快速性、特異性及經(jīng)濟(jì)性。本文的另一個(gè)目的在于利用我們建立的qPCR方法,檢測(cè)VZV相關(guān)疾病外周血病毒載量的差異;利用我們建立的qPCR方法,檢測(cè)HZ外周血單個(gè)核細(xì)胞(peripheral blood mono-nuclear cells,PBMC)、血漿病毒載量的差異性;利用酶聯(lián)免疫吸附測(cè)定(enzymelinkedimmunosorbent assay,ELISA)法,檢測(cè)VZV相關(guān)疾病神經(jīng)損傷評(píng)估因子腦源性神經(jīng)營(yíng)養(yǎng)因子(brain-derived neurotrophic factor,BDNF)、S100B蛋白水平。方法實(shí)驗(yàn)一:qPCR方法檢測(cè)HZ外周血患者VZV DNA的優(yōu)化選擇1.隨機(jī)抽取10例HZ患者,分別利用煮沸法、離心柱法提取PBMC中dna。2.采用巢式pcr制備標(biāo)準(zhǔn)品。3.利用上海之江vzv檢測(cè)試劑盒(qpcr方法)與本文建立的方法,分別對(duì)煮沸法及離心柱法提取的dna進(jìn)行pbmc中vzvdna的定量檢測(cè)。實(shí)驗(yàn)二:本文建立的qpcr方法在vzv相關(guān)疾病中的臨床應(yīng)用及檢測(cè)血漿bdnf、s100b1.vzv相關(guān)疾病vzvdna載量差異性的分析:收集10例水痘、31例hz、11例phn及20例健康志愿者,分別設(shè)為水痘組、hz組、phn組及正常組,采用本文建立的qpcr方法對(duì)pbmc中vzvdna載量進(jìn)行檢測(cè)。2.hz患者治療前、后pbmc與血漿vzvdna載量差異性的分析:在31例患者中,隨機(jī)抽取22例hz患者,分別檢測(cè)治療前及治療14天后pbmc、血漿中的vzvdna。3.vzv相關(guān)疾病神經(jīng)損傷評(píng)估因子的分析:采用elisa法檢測(cè)水痘組、hz組、phn組血漿中bdnf、s100b的水平。結(jié)果:實(shí)驗(yàn)一:1.標(biāo)準(zhǔn)品建立:巢式pcr所建立的標(biāo)準(zhǔn)品大小為385bp。2.提取方法比較:煮沸法、離心柱法分別聯(lián)合上海之江vzv檢測(cè)試劑盒(qpcr法),vzvdna的陽(yáng)性檢出率為10%、60%。離心柱法較煮沸法檢出率高。3.引物比較:離心柱法分別聯(lián)合上海之江vzv檢測(cè)試劑盒(qpcr法)和本文建立的方法陽(yáng)性檢出率均為60%,離心柱法下,二者陽(yáng)性檢出一致。故本文建立的qpcr優(yōu)化方法為:離心柱法+巢式pcr制作標(biāo)準(zhǔn)品+設(shè)計(jì)特異性引物。實(shí)驗(yàn)二:1.vzv相關(guān)疾病外周血pbmc中vzvdna陽(yáng)性檢出率、載量的比較:治療前水痘組、hz組、phn組陽(yáng)性檢出率分別為:80%、70.97%、36.36%。治療前pbmc水痘組的vzvdna載量高于hz組及phn組,hz組高于phn組,差異有統(tǒng)計(jì)學(xué)意義(z=-2.82,p0.05;z=-2.98,p0.05;z=-2.47,p0.05)。正常對(duì)照組vzvdna檢出均為陰性。2.hz組治療前后pbmc、血漿中vzvdna陽(yáng)性檢出率、載量的比較:在hz組隨機(jī)抽取的22例患者中,治療前pbmc、血漿中vzvdna的陽(yáng)性檢出率分別為:72.72%、63.64%,差異無(wú)統(tǒng)計(jì)學(xué)意義(χ2=1.33,df=1,p0.05)。治療后分別為47.06%、35.29%,無(wú)明顯的差異性(χ2=0.5,df=1,p0.05)。治療前、后pbmc中的vzvdna載量較血漿高,兩者有明顯的差異(z=-4.107,p0.05;z=-2.432,p0.05)。治療后pbmc、血漿中的vzvdna載量均較治療前降低,差異有統(tǒng)計(jì)學(xué)意義(z=-4.11,p0.05;z=-3.03,p0.05)。3.vzv相關(guān)疾病血漿bdnf的比較:與正常組比較,治療前血漿bdnf水痘組、hz組、phn組較高,差異有統(tǒng)計(jì)學(xué)意義(i-j=190.89,p0.05;i-j=232.49,p0.05;i-j=246.65,p0.05)。水痘組較hz組、phn組低,有明顯的差異性(i-j=-46.60,p0.05;i-j=-55.75,p0.05)。HZ組與PHN組無(wú)明顯的差異性(I-J=-14.15,P0.05)。4.VZV相關(guān)疾病S100B的比較:治療前血漿S100B水痘組、HZ組、PHN組均較正常組高,差異有統(tǒng)計(jì)學(xué)意義(I-J=126.55,P0.05;I-J=164.85,P0.05;I-J=159.94,P0.05),水痘組較HZ組、PHN組低,具有明顯的差異性(I-J=-38.30,P0.05;I-J=-33.38,P0.05),HZ組與PHN組無(wú)明顯的差異性(I-J=4.96,P0.05)。5.HZ組血漿BDNF、S100B比較:HZ組治療前、后BDNF、S100B均較正常組高,差異有統(tǒng)計(jì)學(xué)意義(I-J=232.50,27.38,I-J=164.84,21.46,P0.05),HZ組治療后BDNF、S100B較治療前明顯降低,差異有統(tǒng)計(jì)學(xué)意義(I-J=205.11,P0.05;I-J=143.39,P0.05)。結(jié)論:1.離心柱法提取DNA用于qPCR優(yōu)于煮沸法。2.巢式PCR制作的標(biāo)準(zhǔn)品具有更高的穩(wěn)定性。3.本文建立的qPCR方法操作簡(jiǎn)單,靈敏度高,更經(jīng)濟(jì),可作為優(yōu)先選擇。4.水痘患者PBMC中的VZV DNA陽(yáng)性檢出率、載量高于HZ及PHN患者,血漿BDNF、S100B低于HZ、PHN患者,提示水痘患者雖有較高的PBMC中VZV DNA載量,但神經(jīng)損傷較HZ、PHN患者輕。5.HZ患者PBMC與血漿中的VZV DNA陽(yáng)性率無(wú)明顯差異性,血漿更方便用于臨床篩查。
[Abstract]:BACKGROUND & OBJECTIVE : The detection of the DNA in peripheral blood mononuclear cells ( PBMC ) and human peripheral blood mononuclear cells ( PBMC ) by using the qPCR method , which can be used as the basis for the detection of the DNA in peripheral blood , and the sensitivity , simplicity , rapidity , specificity and economical efficiency of the detection method . The aim of this paper is to optimize the method of DNA extraction , to simplify the production of standard products , to design specific primers , to improve the sensitivity , simplicity , rapidity , specificity and economy of the detection method . the quantitative detection of vzvdna in pbmc by boiling method and centrifugal column method was carried out . The results were as follows : 1 . The positive detection rate of vzvdna in pbmc and plasma vzvdna was determined by using the method of qpcr . z = - 2.98 , p . 05 ; z = - 2.47 , p . 05 ) . The positive rates of vzvdna in pbmc and plasma were 72 . 72 % and 63 . 64 % , respectively , and no significant difference was found between the two groups ( 蠂2 = 1.33 , df = 1 , p . 05 ) . After treatment , 47.06 % and 35.29 % respectively , there was no significant difference ( 蠂2 = 0.5 , df = 1 , p0.05 ) . The vzvdna content in pbmc before and after treatment was higher than that in plasma ( z = - 4.107 , p0.05 ; z = - 2.432 , p . 05 ) . The levels of vzvdna in plasma of pbmc and plasma were lower before treatment ( z = - 4.11 , p0.05 ; z = - 3.03 , p . 05 ) . 3 . The plasma bdnf in the plasma bdnf group , hz group and phn group were higher in the treatment group than in the normal group ( i - j = 190.89 , p < 0.05 ; i - j = 232.49 , p < 0.05 ; i - j = 246.65 , p . 05 ) . There was a significant difference in phn group ( i - j = - 46.60 , p . 05 ; i - j = - 55.75 , p . 05 ) . 4.VZV鐩稿叧鐤劇梾S100B鐨勬瘮杈，

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