本文選題：細(xì)胞因子 + 酶聯(lián)免疫斑點(diǎn)試驗(yàn)��； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2010年碩士論文

【摘要】： 細(xì)胞因子(Cytokine)是由免疫細(xì)胞和某些非免疫細(xì)胞經(jīng)刺激而合成、分泌的一類具有廣泛生物學(xué)活性的小分子蛋白質(zhì),細(xì)胞因子的檢測可以用于多種疾病(感染、炎癥、自身免疫病等)的輔助診斷及疫苗接種后的細(xì)胞免疫功能評價(jià),對于研究病毒感染后病毒與宿主免疫系統(tǒng)相互作用機(jī)制也同樣具有重要意義。 細(xì)胞因子是一個(gè)龐大的體內(nèi)功能群體,其檢測方法很多。根據(jù)檢測原理和技術(shù)手段,可將細(xì)胞因子的檢測技術(shù)大致分為免疫學(xué)方法、生物學(xué)方法和分子生物學(xué)方法等三類。目前較為常用的有酶聯(lián)免疫斑點(diǎn)試驗(yàn)(ELISPOT)、熒光定量RT-PCR、細(xì)胞內(nèi)細(xì)胞因子檢測、酶聯(lián)免疫吸附(ELISA)等。 ELISPOT技術(shù)是在溶血空斑技術(shù)和ELISA技術(shù)基礎(chǔ)之上建立起來的現(xiàn)代免疫學(xué)檢測技術(shù),實(shí)現(xiàn)了對細(xì)胞因子進(jìn)行單細(xì)胞水平上的精確定量檢測。目前ELISPOT技術(shù)在全球尚未完全標(biāo)準(zhǔn)化,研究人員必須采用最佳匹配的包被抗體和檢測抗體、ELISPOT板、形成斑點(diǎn)的底物及其顯色時(shí)間等,因此在實(shí)際操作中需對各個(gè)因素進(jìn)行優(yōu)化方能建立較為穩(wěn)定的檢測體系。 熒光定量PCR技術(shù)是將特異的熒光基團(tuán)加入到PCR反應(yīng)體系中,根據(jù)對熒光信號的實(shí)時(shí)檢測并做出精確的定量分析,目前該技術(shù)在病原體檢測等方面已得到廣泛應(yīng)用。當(dāng)前熒光定量主要包括SYBR Green I和TaqMan探針法兩種。SYBR Green I是一種能夠于雙鏈DNA非特異結(jié)合的熒光染料,適用于所有引物的擴(kuò)增,方法簡便,成本較低,但熒光染料能夠與dsDNA結(jié)合,因此能與引物二聚體或者非特異性擴(kuò)增產(chǎn)物結(jié)合,需要和熔解曲線分析加以辨別去除影響。而TaqMan探針法是將帶有熒光標(biāo)記探針加入到PCR反應(yīng)體系中,對引物設(shè)計(jì)要求更高,但同時(shí)檢測的特異性更高,結(jié)果更為可靠。 近年來,豬細(xì)胞免疫的研究逐漸引起人們的重視。豬是一種重要的經(jīng)濟(jì)動(dòng)物。豬群易受多種傳染病侵害,豬瘟、豬繁殖與呼吸綜合征(即豬藍(lán)耳病)等都給世界各地的養(yǎng)豬業(yè)造成了極大的經(jīng)濟(jì)損失。了解病毒與豬免疫系統(tǒng)之間相互作用的機(jī)制,尤其是細(xì)胞免疫應(yīng)答機(jī)制,既有助于防治豬病毒性疾病,也有助于對新型疫苗誘發(fā)的免疫效應(yīng)進(jìn)行評價(jià)。此外,豬也是重要的實(shí)驗(yàn)動(dòng)物,豬在解剖、組織和生理生化等許多生物學(xué)指標(biāo)上與人類非常相似,被認(rèn)為是研究人類疾病最合適的實(shí)驗(yàn)動(dòng)物模型。豬作為實(shí)驗(yàn)動(dòng)物已常被用作異種移植排斥反應(yīng)的模型,因此對其免疫系統(tǒng)及免疫應(yīng)答情況的研究對于該方面的研究也具有重要意義。 在本研究中,我們著重研究了豬細(xì)胞因子的檢測方法。我們利用五指山小型豬這一實(shí)驗(yàn)用小型豬,建立了豬細(xì)胞因子的檢測方法,包括豬IFN-γ的ELISPOT檢測方法及豬IL-2、IL-4、IL-10的實(shí)時(shí)定量RT-PCR檢測方法。 在建立豬IFN-γ的ELISPOT檢測方法時(shí),我們選擇了PVDF膜96孔板,然后分別優(yōu)化了捕獲抗體和檢測抗體的濃度、上板細(xì)胞數(shù)目及刺激物濃度。結(jié)果顯示,當(dāng)捕獲抗體和檢測抗體的濃度分別為1：60、上板細(xì)胞數(shù)為2×105個(gè)／孔、刺激用病毒量為2×105TCID50時(shí),獲得了較為穩(wěn)定的檢測體系。 在建立豬IL-2、IL-4、IL-10的實(shí)時(shí)定量RT-PCR檢測方法時(shí),我們首先以五指山小型豬cDNA為模板,擴(kuò)增了豬IL-2、IL-4、IL-10的基因保守區(qū),將其分別克隆入T載體中,構(gòu)建了相應(yīng)的重組質(zhì)粒。然后將重組質(zhì)粒分別進(jìn)行10倍梯度稀釋,將其作為質(zhì)粒標(biāo)準(zhǔn)品構(gòu)建了標(biāo)準(zhǔn)曲線。結(jié)果顯示,各標(biāo)準(zhǔn)曲線的直線相關(guān)系數(shù)|r|均大于0.990,且擴(kuò)增效率在90%～105%之間。對該檢測方法進(jìn)行了方法學(xué)評價(jià),結(jié)果顯示,該檢測方法的敏感性較高,IL-4檢測下限達(dá)到10~0copies/μL, IL-2、IL-10檢測下限達(dá)到10~2copies/μL;重復(fù)性較好,批內(nèi)重復(fù)性與批間重復(fù)性分析變異系數(shù)基本都小于5%。這些評價(jià)結(jié)果都進(jìn)一步說明了我們所建立的定量RT-PCR檢測方法的可靠性。 在建立豬細(xì)胞因子檢測方法的基礎(chǔ)上,我們對其進(jìn)行了初步應(yīng)用。我們采用ELISPOT方法對PRRSV接種后第0、7、14、21天豬PBMC特異性分泌γ-干擾素情況進(jìn)行了測定,結(jié)果發(fā)現(xiàn),接種后各豬IFN-γ表達(dá)水平呈明顯上升趨勢,第21天時(shí)達(dá)到最高峰；未接種的各豬則表現(xiàn)出IFN-γ表達(dá)水平穩(wěn)定無上升趨勢。這說明豬感染高致病性PRRSV后PBMC分泌IFN-γ能力明顯增強(qiáng),高致病性PRRSV可誘導(dǎo)豬體內(nèi)效應(yīng)T細(xì)胞發(fā)揮抗感染的作用。 同時(shí),我們還對攻毒后細(xì)胞中IL-2、IL-4及IL-10 mRNA的表達(dá)情況進(jìn)行了分析。用建立的方法檢測高致病性PRRSV感染仔豬Taqman熒光定量PCR檢測各樣品中IL-2、IL-4、IL-10細(xì)胞因子mRNA表達(dá)量,同時(shí)擴(kuò)增β-actin作為內(nèi)參對照。采用雙標(biāo)準(zhǔn)曲線法進(jìn)行相對定量,根據(jù)測得的Ct值與標(biāo)準(zhǔn)曲線得到樣品中對應(yīng)的IL-2、IL-4、IL-10基因拷貝數(shù),以β-actin作內(nèi)參,分析各細(xì)胞因子mRNA的表達(dá)差異。結(jié)果顯示,攻毒后某些豬的各細(xì)胞因子水平變化較大,而對照組相對變化較小。下一步還需增加樣本數(shù)才可從中總結(jié)中變化規(guī)律。 本研究將有助于病毒感染后豬體內(nèi)免疫應(yīng)答的研究,對于豬作為實(shí)驗(yàn)動(dòng)物的比較醫(yī)學(xué)研究也同樣具有重要意義。
[Abstract]:Cell factor (Cytokine) is composed of certain non immune cells and immune cells by stimulation of synthesis of small molecules with a wide range of biological activity one kind of protein secretion, cytokine assays can be used for a variety of diseases (infection, inflammation, autoimmune diseases) evaluation of cellular immune function in diagnosis and vaccination. For the study of virus after virus infection and host immune system interaction mechanism also has important significance.
Cytokine is a huge function in vivo group, the detection method. According to the principle and technical means of detection, detection techniques of cytokines can be roughly divided into immunological methods, biological methods and molecular biological methods. Three kinds of common with enzyme-linked immune spot test (ELISPOT), fluorescence quantitative RT-PCR, intracellular cytokine detection, enzyme-linked immunosorbent assay (ELISA).
ELISPOT technology is the modern immunological detection technology built on hemolytic plaque technique and ELISA technology foundation, realizes the accurate quantitative single cell level detection of cytokines. The ELISPOT technology in the world is not yet standardized, researchers must use the best match of the coated antibody and detection antibody, ELISPOT plate form spots on the substrate and time, so in the actual operation of the various factors were optimized in order to establish the detection system more stable.
Fluorescence quantitative PCR technology is the specific fluorescent groups into the PCR reaction system, according to the real-time detection of the fluorescence signal and make accurate quantitative analysis, at present the technology in pathogen detection has been widely used. The fluorescence quantitative I and TaqMan including SYBR Green.SYBR Green I two probe method is a kind of to double stranded DNA from non fluorescent dye binding, amplification, suitable for all primer method is simple, low cost, but the fluorescent dye can bind with dsDNA, and therefore can two primer dimer or non-specific amplification product of the combination, and melting curve analysis to identify and remove the influence of probe method is TaqMan. TaqMan probe into the PCR reaction system, higher primer design requirements, but also the specificity of detection is higher, the result is more reliable.
In recent years, cellular immunity of pigs has gradually attracted people's attention. The pig is an important economic animal. Pigs are vulnerable to a variety of infectious diseases, swine fever, porcine reproductive and respiratory syndrome (i.e. PRRS) etc. to the pig industry worldwide has caused tremendous economic losses. Understanding the mechanism of interaction between the virus and swine immune system, especially the mechanism of cellular immune responses, both contribute to the prevention and treatment of swine viral diseases, also contribute to the immune effect of vaccine induced evaluation. In addition, the experimental animal pig is also important, pig in anatomy, tissue and physiological and biochemical indexes and many other biological and very human similar, is considered to be the experimental animal model of human disease. The most suitable pig as experimental animal has often been used as models of xenograft rejection, the immune system and immune responses to study The research in this area is also of great significance.
In this study, we focus on the detection of porcine cytokines. We use the experiment of Five Fingers Group pigs pigs was established for detection of porcine cytokines, including porcine IFN- gamma ELISPOT detection method and pig IL-2, IL-4, real time quantitative RT-PCR IL-10 measurement method.
In the ELISPOT to establish a method for detection of porcine IFN- gamma, we chose the PVDF film 96 hole plate, and then optimized the concentration of capture antibody and detection antibody, concentration in cell number and stimuli. The results showed that when the concentration of capture antibody and detection antibody were 1:60, on board the cell number is 2 * 105 / hole, stimulated by the amount of virus was 2 * 105TCID50, the detection system is stable.
In the establishment of porcine IL-2, IL-4, real-time quantitative RT-PCR detection method of IL-10, we first to Five Fingers Group swine cDNA as template, amplified porcine IL-2, IL-4, the conserved region of IL-10 gene was cloned into T vector. The recombinant plasmids were constructed. Then the recombinant plasmid was 10 times the dilution as the standard plasmid construct the standard curve. The results showed that the linear correlation coefficient |r| and the standard curve was greater than 0.990, and the amplification efficiency in 90% ~ 105%. The detection method of the evaluation method, results show that the sensitivity of the detection method, detection limit of IL-4 reached 10~0copies/ L, IL-2, detection limit of IL-10 reached 10~2copies/ mu L; good reproducibility, repeatability and intra batch reproducibility analysis of coefficient of variation are less than the results of these evaluation 5%. further illustrates the quantitative RT-PCR we established The reliability of the detection method.
Based on the detection of porcine cytokines, we conducted a preliminary application on it. We use the ELISPOT method to PRRSV 0,7,14,21 days after inoculation of porcine PBMC specific interferon gamma secretion was measured, results showed that the porcine IFN- gamma expression levels were significantly increased after inoculation and reached a peak of twenty-first each day; pigs without inoculation showed IFN- expression level stable upward trend. This shows that the pigs infected with highly pathogenic PRRSV PBMC IFN- secretion significantly enhanced ability of highly pathogenic PRRSV can induce pig in vivo effects of T cells play a role in anti infection.
At the same time, we also challenged IL-2 cells, the expression of IL-4 and IL-10 mRNA are analyzed. By using this method, the detection of highly pathogenic PRRSV infection in piglets Taqman fluorescence quantitative PCR detection of IL-2 and IL-4 in all samples, the expression of IL-10 cytokines mRNA, simultaneous amplification of beta -actin was used as a control. Using double standard curve the relative quantitative method, based on the measured Ct value and the standard curve was IL-2, corresponding to the IL-4 in the sample, IL-10 gene copy number, with -actin as reference beta, analysis of the differential expression of various cytokines mRNA. The results showed that the changes of cytokine levels in some pigs after challenged greatly, while the control group, the relative change small. The next step need to increase the number of samples can be summed up from the change law.
This study will contribute to the study of immune responses in pigs after virus infection. It is also of great significance to the comparative medical research of pigs as experimental animals.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 夏九鮮;李銳;周顯珍;唐阿英;;PRRSV感染不同時(shí)期IFN-γ表達(dá)量分析[J];現(xiàn)代農(nóng)業(yè)科技;2013年05期

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本文編號：1740351