猪细胞因子检测方法的建立与初步应用

发布时间：2018-04-12 15:37

本文选题：细胞因子 + 酶联免疫斑点试验　；参考：《中国人民解放军军事医学科学院》2010年硕士论文

【摘要】： 细胞因子(Cytokine)是由免疫细胞和某些非免疫细胞经刺激而合成、分泌的一类具有广泛生物学活性的小分子蛋白质,细胞因子的检测可以用于多种疾病(感染、炎症、自身免疫病等)的辅助诊断及疫苗接种后的细胞免疫功能评价,对于研究病毒感染后病毒与宿主免疫系统相互作用机制也同样具有重要意义。细胞因子是一个庞大的体内功能群体,其检测方法很多。根据检测原理和技术手段,可将细胞因子的检测技术大致分为免疫学方法、生物学方法和分子生物学方法等三类。目前较为常用的有酶联免疫斑点试验(ELISPOT)、荧光定量RT-PCR、细胞内细胞因子检测、酶联免疫吸附(ELISA)等。 ELISPOT技术是在溶血空斑技术和ELISA技术基础之上建立起来的现代免疫学检测技术,实现了对细胞因子进行单细胞水平上的精确定量检测。目前ELISPOT技术在全球尚未完全标准化,研究人员必须采用最佳匹配的包被抗体和检测抗体、ELISPOT板、形成斑点的底物及其显色时间等,因此在实际操作中需对各个因素进行优化方能建立较为稳定的检测体系。荧光定量PCR技术是将特异的荧光基团加入到PCR反应体系中,根据对荧光信号的实时检测并做出精确的定量分析,目前该技术在病原体检测等方面已得到广泛应用。当前荧光定量主要包括SYBR Green I和TaqMan探针法两种。SYBR Green I是一种能够于双链DNA非特异结合的荧光染料,适用于所有引物的扩增,方法简便,成本较低,但荧光染料能够与dsDNA结合,因此能与引物二聚体或者非特异性扩增产物结合,需要和熔解曲线分析加以辨别去除影响。而TaqMan探针法是将带有荧光标记探针加入到PCR反应体系中,对引物设计要求更高,但同时检测的特异性更高,结果更为可靠。近年来,猪细胞免疫的研究逐渐引起人们的重视。猪是一种重要的经济动物。猪群易受多种传染病侵害,猪瘟、猪繁殖与呼吸综合征(即猪蓝耳病)等都给世界各地的养猪业造成了极大的经济损失。了解病毒与猪免疫系统之间相互作用的机制,尤其是细胞免疫应答机制,既有助于防治猪病毒性疾病,也有助于对新型疫苗诱发的免疫效应进行评价。此外,猪也是重要的实验动物,猪在解剖、组织和生理生化等许多生物学指标上与人类非常相似,被认为是研究人类疾病最合适的实验动物模型。猪作为实验动物已常被用作异种移植排斥反应的模型,因此对其免疫系统及免疫应答情况的研究对于该方面的研究也具有重要意义。在本研究中,我们着重研究了猪细胞因子的检测方法。我们利用五指山小型猪这一实验用小型猪,建立了猪细胞因子的检测方法,包括猪IFN-γ的ELISPOT检测方法及猪IL-2、IL-4、IL-10的实时定量RT-PCR检测方法。在建立猪IFN-γ的ELISPOT检测方法时,我们选择了PVDF膜96孔板,然后分别优化了捕获抗体和检测抗体的浓度、上板细胞数目及刺激物浓度。结果显示,当捕获抗体和检测抗体的浓度分别为1：60、上板细胞数为2×105个／孔、刺激用病毒量为2×105TCID50时,获得了较为稳定的检测体系。在建立猪IL-2、IL-4、IL-10的实时定量RT-PCR检测方法时,我们首先以五指山小型猪cDNA为模板,扩增了猪IL-2、IL-4、IL-10的基因保守区,将其分别克隆入T载体中,构建了相应的重组质粒。然后将重组质粒分别进行10倍梯度稀释,将其作为质粒标准品构建了标准曲线。结果显示,各标准曲线的直线相关系数|r|均大于0.990,且扩增效率在90%～105%之间。对该检测方法进行了方法学评价,结果显示,该检测方法的敏感性较高,IL-4检测下限达到10~0copies/μL, IL-2、IL-10检测下限达到10~2copies/μL;重复性较好,批内重复性与批间重复性分析变异系数基本都小于5%。这些评价结果都进一步说明了我们所建立的定量RT-PCR检测方法的可靠性。在建立猪细胞因子检测方法的基础上,我们对其进行了初步应用。我们采用ELISPOT方法对PRRSV接种后第0、7、14、21天猪PBMC特异性分泌γ-干扰素情况进行了测定,结果发现,接种后各猪IFN-γ表达水平呈明显上升趋势,第21天时达到最高峰；未接种的各猪则表现出IFN-γ表达水平稳定无上升趋势。这说明猪感染高致病性PRRSV后PBMC分泌IFN-γ能力明显增强,高致病性PRRSV可诱导猪体内效应T细胞发挥抗感染的作用。同时,我们还对攻毒后细胞中IL-2、IL-4及IL-10 mRNA的表达情况进行了分析。用建立的方法检测高致病性PRRSV感染仔猪Taqman荧光定量PCR检测各样品中IL-2、IL-4、IL-10细胞因子mRNA表达量,同时扩增β-actin作为内参对照。采用双标准曲线法进行相对定量,根据测得的Ct值与标准曲线得到样品中对应的IL-2、IL-4、IL-10基因拷贝数,以β-actin作内参,分析各细胞因子mRNA的表达差异。结果显示,攻毒后某些猪的各细胞因子水平变化较大,而对照组相对变化较小。下一步还需增加样本数才可从中总结中变化规律。本研究将有助于病毒感染后猪体内免疫应答的研究,对于猪作为实验动物的比较医学研究也同样具有重要意义。
[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.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【引证文献】