F蛋白的表达及其诱导免疫耐受的初步研究

发布时间：2019-02-19 18:17

【摘要】： 肝脏疾病严重影响人类健康,而肝癌是世界范围内最常见的恶性肿瘤之一,其致死率仅次于肺癌、胃癌、结肠癌而居第四位,病毒性肝炎、肝纤维化、肝硬化等都是肝癌主要的致病原因。目前肝移植是举世公认的治疗一些终末期肝病最为有效的手段。但肝移植后由于免疫抑制剂的大量使用,可造成慢性。肾衰、心血管病、突发性高血压及恶性肿瘤等严重并发症,极大地影响了病人的生存质量。因此肝移植后的主要目标就是在最佳化和最小化使用免疫抑制剂的基础上获得免疫耐受,提高病人生存质量。肝移植免疫耐受的机制有多种假说,F蛋白能够诱导免疫耐受是近年新提出的一种理论。F蛋白是肝细胞膜上和胞浆内特有表达蛋白,由非MHC基因编码,据报道同种肝移植模型受者血清中均可检测到这种抗原,但其作用还不清楚。 本文采用原核系统表达F蛋白。我们对本实验室预先保存的菌种E.coliBL21(DE3)pLysS表达和纯化,并进行SDS-PAGE和Western blot,证实该菌种含有目的蛋白——F蛋白。我们利用该菌种进行表达并改善纯化条件,低温诱导可得到大量可溶性的目的蛋白,用1/2×Wash buffer可得到较高浓度的目的蛋白,用于后续的动物实验。我们将20只SD大鼠随机分为4组,接受不同的处理,Ⅰ组一次性胸腺注射0.2ml PBS做为阴性对照;Ⅱ组足垫注射含1mg用相同体积弗氏完全佐剂乳化的F蛋白,一周后足垫注射含1mg用相同体积弗氏不完全佐剂乳化的F蛋白,每周1次,连续3次;Ⅲ组一次胸腺内注射4mg F蛋白,每叶2mg;Ⅳ组一次腹腔注射环磷酰胺100mg/kg做为阳性对照。7天后处死动物,留取外周血及胸腺标本,用TUNEL试剂盒对胸腺标本进行检测,判断细胞凋亡情况。用IL-10试剂盒检测外周血清IL-10水平。结果显示F蛋白能诱导胸腺淋巴细胞的凋亡,并促进IL-10的分泌。其机制可能是当胸腺接种F蛋白后,胸腺作为一种特殊器官,在被异基因抗原修饰后能产生克隆清除,进而通过对T细胞发育和成熟的影响,诱导低免疫应答状态的发生。 F蛋白在肝病早期诊断与治疗、肝损伤程度的判断以及肝移植免疫耐受方面的研究都具有重要的应用价值。用化学提纯的方法很难得到大量F蛋白,这就阻碍了其他研究的进行。因此,本文采用基因工程方法获得大鼠肝脏F蛋白,解决其来源问题。初步的实验说明F蛋白具有免疫原性,很可能会增加肝移植后的免疫耐受,F蛋白具有免疫反应性,大剂量或反复小剂量免疫可诱导出受体抗原特异性淋巴细胞凋亡。可能在MHC抗原之外,存在其它的抗原系统参与肝移植的免疫反应、决定移植后果。为下一步的移植打下基础。
[Abstract]:Liver diseases seriously affect human health, and liver cancer is one of the most common malignant tumors in the world, its fatality rate is second only to lung cancer, gastric cancer, colon cancer and occupies the fourth place, viral hepatitis, liver fibrosis. Liver cirrhosis is the main cause of liver cancer. At present, liver transplantation is recognized as the most effective treatment of some end-stage liver diseases. However, due to the large use of immunosuppressive agents after liver transplantation, chronic liver transplantation can be caused. Serious complications, such as renal failure, cardiovascular disease, sudden hypertension and malignant tumor, greatly affect the patient's quality of life. Therefore, the main goal of liver transplantation is to optimize and minimize the use of immunosuppressants on the basis of immune tolerance, improve the quality of life of patients. There are many hypotheses about the mechanism of tolerance in liver transplantation. The theory that F protein can induce immune tolerance is a new theory in recent years. F protein is a special expression protein on liver cell membrane and cytoplasm, which is encoded by non-MHC gene. It is reported that this antigen can be detected in the sera of the recipients of liver allotransplantation model, but its effect is not clear. In this paper, the prokaryotic system was used to express F protein. We expressed and purified E.coliBL21 (DE3) pLysS from the prepreserved strain in our laboratory, and confirmed by SDS-PAGE and Western blot, that the strain contained F protein. We used the strain to express and improve the purification conditions. A large amount of soluble target protein could be obtained by low temperature induction, and a high concentration of target protein could be obtained by using 1 / 2 脳 Wash buffer for subsequent animal experiments. We randomly divided 20 SD rats into 4 groups and received different treatments. Group I was given 0.2ml PBS as negative control. Group 鈪，

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