本文選題：支氣管哮喘 + IL-5��； 參考：《山西醫(yī)科大學(xué)》2006年碩士論文

【摘要】：背景：支氣管哮喘是臨床內(nèi)、兒科常見的，嚴(yán)重危害健康的慢性疾病，近年其發(fā)病率、死亡率逐年增高，嚴(yán)重威脅人類健康，因此日益受到世界各國的重視。對其發(fā)病機(jī)制已有多方面的探討，但仍有許多尚未明了。文獻(xiàn)研究表明，淋巴細(xì)胞中Th1／Th2細(xì)胞的比例失衡，如Th1減少，Th2增加是支氣管哮喘的重要發(fā)病機(jī)制，這一失衡可引起Th2類細(xì)胞因子如：IL-5等產(chǎn)生過多，Th1類細(xì)胞因子如IFN-γ產(chǎn)生減少。越來越多的證據(jù)表明，IL-5可激活嗜酸性粒細(xì)胞(EOS)并誘導(dǎo)EOS肺部浸潤，從而引發(fā)受累器官(肺、支氣管)的慢性炎癥。因此，以IL-5為靶點(diǎn)的抗哮喘主動免疫基因治療具有良好的應(yīng)用前景。 目的：制備人IL-5 DNA疫苗(hIL-5)，鑒定其在真核細(xì)胞有效表達(dá)后，用該疫苗免疫小鼠，通過hIL-5誘導(dǎo)抗小鼠IL-5的交叉反應(yīng)來打破小鼠免疫系統(tǒng)對自身IL-5的免疫耐受，產(chǎn)生抗自身IL-5抗體。 方法：我們從Invivogen公司購回分別含有人和小鼠IL-5全段基因的克隆質(zhì)粒載體PORF9-hIL5和PORF9-mIL5，用Premier Primer 5.0軟件輔助設(shè)計PCR引物，通過PCR技術(shù)將人和小鼠IL-5基因擴(kuò)增。然后利用基因工程技術(shù)，通過酶切、連接反應(yīng)，將目的基因構(gòu)建于真核表達(dá)質(zhì)粒pcDNA3.1(+)中，在體外轉(zhuǎn)染真核細(xì)胞(COS-1)，，通過Western blot的檢測方法其證實(shí)有效表達(dá)后，在大腸桿菌中擴(kuò)增并回收大量質(zhì)粒，用這些真核表達(dá)質(zhì)粒作為DNA疫苗，肌肉注射免疫小鼠，利用Western blot方法檢測是否產(chǎn)生抗小鼠自身IL-5的體液免疫反應(yīng)。 結(jié)果：通過酶切、測序鑒定證實(shí)構(gòu)建了正確的含人IL-5(hIL-5)和小鼠IL-5(mIL-5)的真核表達(dá)重組質(zhì)粒。利用Western blot方法鑒定均能夠在真核細(xì)胞COS-1中有效表達(dá)相應(yīng)的蛋白質(zhì)分子。并且大量制備和純化了這二個DNA質(zhì)粒和相應(yīng)的空對照質(zhì)粒，用這三種質(zhì)粒及生理鹽水分別免疫小鼠，在第四次免疫后一周收集外周血，Western blot檢測發(fā)現(xiàn)hIL-5質(zhì)粒免疫小鼠血清中有抗小鼠IL-5的特異性抗體存在，而mIL-5質(zhì)粒、空質(zhì)粒及生理鹽水對照組免疫的小鼠血清中沒有檢測到抗小鼠IL-5的特異性抗體存在。 結(jié)論：利用與哮喘相關(guān)的異種同源基因疫苗(人IL-5 DNA疫苗)來打破免疫耐受，誘導(dǎo)產(chǎn)生種與種之間的交叉免疫反應(yīng)，為將來研究哮喘的防治奠定堅實(shí)的理論基礎(chǔ)。
[Abstract]:Background: bronchial asthma is a common and serious chronic disease in pediatrics. In recent years, the incidence and mortality rate of bronchial asthma are increasing year by year, which is a serious threat to human health, so it has been paid more and more attention to all over the world. Many aspects of its pathogenesis have been discussed, but many are still unclear. Literature studies have shown that the imbalance in the proportion of Th1/Th2 cells in lymphocytes, such as the decrease of Th1 and the increase of Th2, is an important pathogenesis of bronchial asthma. This imbalance can cause the production of excessive Th1 cytokines such as Th2 cytokines such as: IL-5, such as IFN- 緯, and decrease the production of Th1 type cytokines such as IFN- 緯. There is increasing evidence that IL-5 activates eosinophil eosinophils and induces pulmonary infiltration in EOS, leading to chronic inflammation in the affected organs (lungs, bronchi). Therefore, anti-asthma active immunotherapy with IL-5 as target has a good application prospect. Aim: to prepare human IL-5 DNA vaccine hIL-5 and identify its expression in eukaryotic cells. Mice were immunized with this vaccine. The cross-reaction of anti-mouse IL-5 was induced by hIL-5 to break the immune tolerance of mouse immune system to autogenic IL-5 and to produce anti-IL-5 antibody. Methods: the cloned plasmids PORF9-hIL5 and PORF9-mIL5 containing human and mouse IL-5 gene were purchased from Invivogen Company respectively. PCR primers were designed with Premier Primer 5.0 software, and the IL-5 gene of human and mouse were amplified by PCR technique. Then the target gene was constructed in eukaryotic expression plasmid pcDNA3.1 () by restriction endonuclease digestion and ligation reaction, and transfected into eukaryotic cells in vitro. The expression of the target gene was confirmed by Western blot assay. A large number of plasmids were amplified and recovered from Escherichia coli. These eukaryotic expression plasmids were used as DNA vaccine to immunize mice intramuscularly. The Western blot method was used to detect whether humoral immune reaction against IL-5 in mice was produced. Results: the recombinant plasmid containing human IL-5 (hIL-5) and mouse IL-5mIL-5 (mIL-5) was constructed by restriction endonuclease digestion and sequencing. Western blot method was used to identify the effective expression of protein molecules in eukaryotic COS-1. The two DNA plasmids and the corresponding blank control plasmids were prepared and purified in large quantities. Mice were immunized with these three plasmids and normal saline respectively. A week after the fourth immunization, the peripheral blood samples were collected to detect the presence of specific antibodies against mouse IL-5 in the sera of mice immunized with hIL-5 plasmid, while the mIL-5 plasmid was found to exist in the sera of mice immunized with hIL-5 plasmids. No specific antibodies against IL-5 were detected in serum of mice immunized with empty plasmids and saline control groups. Conclusion: the heterologous gene vaccine related to asthma (human IL-5 DNA vaccine) was used to break the immune tolerance and induce cross-immunoreaction between species, which laid a solid theoretical foundation for the future study on the prevention and treatment of asthma.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392

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