本文選題：EV71感染 + STAT1基因敲除小鼠模型��； 參考：《北京協(xié)和醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的研究STAT1基因敲除小鼠對EV71的敏感性,對EV71動物模型的優(yōu)化提供數(shù)據(jù)基礎(chǔ)。方法利用LPS刺激STAT1基因敲除C57BL/6小鼠對模型免疫反應(yīng)穩(wěn)定性進(jìn)行檢測,并利用微珠免疫分析(CBA)方法對血清免疫相關(guān)因子定量分析;利用EV71感染10日齡STAT1基因敲除小鼠后,觀察感染癥狀及病理表現(xiàn),利用實時熒光定量檢測對肌肉病毒RNA定量分析。利用相同劑量、相同毒株的EV71感染10日齡ICR小鼠,與STAT1基因敲除小鼠感染結(jié)果進(jìn)行對比。結(jié)果LPS刺激后,STAT1基因敲除小鼠血清IL-1α、L-1β、IL-6、IL-10、IL-12、TNF-α、MIP-1α分泌水平下降,與野生型小鼠相比具有統(tǒng)計學(xué)意義(P0.05);EV71感染后,STAT1基因敲除小鼠病理損傷加重,骨骼肌中EV71抗原水平升高;STAT1基因敲除小鼠骨骼肌中病毒載量升高(P0.05);與野生型C57BL/6小鼠及ICR小鼠相比,STAT1基因敲除小鼠表現(xiàn)出更嚴(yán)重的癥狀與更高的死亡率。結(jié)論STAT1在抗EV71感染的免疫過程中扮演重要角色,STAT1基因敲除可以提高小鼠對EV71病毒的敏感性。目的建立土撥鼠肝炎病毒(Woodchuck Hepatitis B virus,WHV)感染3日齡土撥鼠的持續(xù)性感染動物模型,利用模型對靈芝孢子粉藥物治療肝炎及免疫功能調(diào)節(jié)作用進(jìn)行評價,為慢性肝炎的機制性研究以及抗肝炎病毒藥物評價建立動物模型基礎(chǔ)。方法核酸檢測:根據(jù)土撥鼠肝炎病毒表面抗原(WHsAg)設(shè)計引物,利用PCR方法,以WHVDNA全基因組質(zhì)粒梯度稀釋作為模板檢測引物擴增靈敏度,以混合健康土撥鼠血清的WHV病毒為模板檢測引物的特異性,并從中篩選靈敏度高、無非特異性擴增條帶及引物二聚體的引物;利用Real-time PCR方法,WHVDNA全基因組質(zhì)粒梯度稀釋作為標(biāo)準(zhǔn)曲線,對土撥鼠血清WHV進(jìn)行核酸定量檢測。表面抗原與核心抗體檢測:利用本實驗室免疫兔和蛋雞制備并純化的多克隆抗體,進(jìn)行土撥鼠血清中表面抗原WHsAg與核心抗體WHcAb檢測。細(xì)胞因子mRNA檢測:Trizol法提取土撥鼠肝臟mRNA,反轉(zhuǎn)錄為cDNA后使用Real-time PCR方法進(jìn)行檢測。結(jié)果核酸檢測:引物WHV N3 F與WHV N3 R,PCR檢測靈敏度可達(dá)1 ×104copies/mL,且電泳未見明顯非特異性條帶及引物二聚體;WHVDNA全基因組質(zhì)粒的熔點曲線峰值一致,Tm均值為82.18℃,病毒拷貝數(shù)與Real-time PCR Ct值的標(biāo)準(zhǔn)曲線R2值為0.992。土撥鼠血清病毒載量水平在感染后3個月開始高于檢測下限,感染后4-5個月達(dá)到血清病毒載量高峰后有所降低,維持在104copies/mL左右,并持續(xù)至感染8個月。雌性土撥鼠血清WHV病毒載量高值水平及高載量持續(xù)時間高于雄性土撥鼠。給予靈芝孢子粉藥物后土撥鼠血清AST檢測數(shù)值降低。與給藥組相比,對照組IL-4、IL-8、PD-L1、NKp46的mRNA含量有所增高。給藥組CD8的mRNA含量高于對照組。對給藥組與對照組土撥鼠病理檢測結(jié)果顯示給藥組土撥鼠肝臟炎癥減輕。結(jié)論本實驗利用3日齡土撥鼠感染土撥鼠肝炎病毒,建立了土撥鼠肝炎病毒持續(xù)性感染動物模型。對土撥鼠肝炎病毒持續(xù)性感染動物模型的病毒載量、血清學(xué)指標(biāo)、肝功能酶指標(biāo)及病理進(jìn)行了分析,為土撥鼠模型建立及肝炎防治研究提供了數(shù)據(jù)基礎(chǔ)。利用土撥鼠肝炎病毒持續(xù)性感染動物模型對靈芝孢子粉藥物抗肝炎及免疫調(diào)節(jié)功能進(jìn)行評價,發(fā)現(xiàn)其對病毒導(dǎo)致的土撥鼠肝炎有減輕作用。通過檢測分析靈芝孢子粉藥物對土撥鼠肝臟細(xì)胞因子mRNA含量的影響,發(fā)現(xiàn)靈芝孢子粉藥物在土撥鼠肝炎病毒持續(xù)性感染動物模型中可以提高細(xì)胞毒性T細(xì)胞的數(shù)量。
[Abstract]:Objective to study the sensitivity of STAT1 gene knockout mice to EV71 and to provide a data basis for the optimization of EV71 animal models. Methods using LPS to stimulate STAT1 gene knockout C57BL/6 mice were used to detect the stability of the model immune response and the quantitative analysis of serum immunization related factors by microbead immunoassay (CBA), and EV71 infected 10 days old S. After TAT1 knockout mice, the infection symptoms and pathological manifestations were observed and the quantitative analysis of RNA was detected by real-time fluorescence quantitative detection. The same dose, the EV71 infection of 10 day old ICR mice with the same strain was compared with the results of STAT1 gene knockout mice. The results of LPS knocking, STAT1 gene knockout mice serum IL-1 a, L-1 beta, IL-6. The levels of IL-10, IL-12, TNF-, and MIP-1 alpha decreased, compared with the wild type mice (P0.05). After EV71 infection, the pathological damage of the STAT1 knockout mice increased, the level of EV71 antigen in the skeletal muscle increased, and the viral load in the skeletal muscle of the STAT1 knockout mice increased (P0.05), compared with the wild type C57BL/6 mice and ICR mice. 1 gene knockout mice showed more serious symptoms and higher mortality. Conclusion STAT1 plays an important role in the immune process of anti EV71 infection. STAT1 gene knockout can increase the sensitivity of mice to EV71 virus. Objective to establish the persistent infection of Groundhog hepatitis virus (Woodchuck Hepatitis B virus, WHV) to infect 3 day old Groundhog. Animal model was used to evaluate the effect of Ganoderma lucidum spore powder on hepatitis and immune function, and to establish an animal model basis for the mechanism study of chronic hepatitis and the evaluation of anti hepatitis virus drug. Method nucleic acid detection was designed according to the primer of WHsAg of Groundhog hepatitis virus (HBV), and the PCR method was used to make WHVDNA whole. The gradient dilution of the genomic plasmid was used as a template to detect primer amplification sensitivity, and the specificity of the primers was detected with the WHV virus in the mixed healthy earth mouse sera as a template, and the sensitivity was high, not not the specific amplification strip and primer two primers, and the Real-time PCR method was used as the standard for the gradient dilution of the whole genome plasmid of WHVDNA as the standard. The quasi curve, the nucleic acid quantitative detection of WHV in the groundhog serum. Surface antigen and core antibody detection: using the polyclonal antibody prepared and purified from the rabbits and laying hens in our laboratory, the surface antigen WHsAg and the core antibody WHcAb in the groundhog serum were detected. The cytokine mRNA was detected by the Trizol method to extract the mRNA of the dialing rat liver, and the reverse transcription was C After DNA, Real-time PCR method was used to test the results. Results the nucleic acid detection: primers WHV N3 F and WHV N3 R, the sensitivity of PCR detection can reach 1 x 104copies/mL, and there is no obvious non specific band and primer two polymer. The standard curve R2 value of the 0.992. groundhog serum virus load level is higher than the lower detection limit after 3 months of infection. After 4-5 months of infection, it reaches the peak of the peak of serum viral load and maintains at about 104copies/mL, and continues to the infection for 8 months. The high level of the WHV virus load and the high load duration of the female groundhog sera are high. In male chidren. The serum AST detection value of Groundhog mice decreased after giving ganoderma spore powder. Compared with the drug group, the mRNA content of IL-4, IL-8, PD-L1 and NKp46 in the control group was higher. The mRNA content of CD8 in the drug group was higher than that of the control group. The pathological examination of the dialing group and the control group showed that the liver inflammation of the dialing group was reduced. In this experiment, the animal model of the persistent infection of the groundhog hepatitis virus was established by using the 3 day old earth chuck infected chuck hepatitis virus. The viral load, the serological index, the liver function enzyme index and the pathology of the animal model of the persistent infective animal model of the groundhog hepatitis virus were analyzed to provide the groundhog model and the study on the prevention and control of hepatitis. Based on the data basis, the anti hepatitis and immunomodulatory function of Ganoderma lucidum spore powder drugs were evaluated using the animal model of the persistent infection of the groundhog hepatitis virus, and it was found that it had a mitigate effect on the virus induced groundhog hepatitis. The effect of ganoderma spore powder on the content of cytokine mRNA in the liver of Groundhog was detected and analyzed, and Ganoderma lucidum was found. Sub powder drugs can increase the number of cytotoxic T cells in animal models of persistent infection of Groundhog virus.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R-332;R725.1

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