【摘要】： 肝臟疾病嚴(yán)重影響人類健康,而肝癌是世界范圍內(nèi)最常見的惡性腫瘤之一,其致死率僅次于肺癌、胃癌、結(jié)腸癌而居第四位,病毒性肝炎、肝纖維化、肝硬化等都是肝癌主要的致病原因。目前肝移植是舉世公認(rèn)的治療一些終末期肝病最為有效的手段。但肝移植后由于免疫抑制劑的大量使用,可造成慢性。腎衰、心血管病、突發(fā)性高血壓及惡性腫瘤等嚴(yán)重并發(fā)癥,極大地影響了病人的生存質(zhì)量。因此肝移植后的主要目標(biāo)就是在最佳化和最小化使用免疫抑制劑的基礎(chǔ)上獲得免疫耐受,提高病人生存質(zhì)量。肝移植免疫耐受的機(jī)制有多種假說,F蛋白能夠誘導(dǎo)免疫耐受是近年新提出的一種理論。F蛋白是肝細(xì)胞膜上和胞漿內(nèi)特有表達(dá)蛋白,由非MHC基因編碼,據(jù)報(bào)道同種肝移植模型受者血清中均可檢測(cè)到這種抗原,但其作用還不清楚。 本文采用原核系統(tǒng)表達(dá)F蛋白。我們對(duì)本實(shí)驗(yàn)室預(yù)先保存的菌種E.coliBL21(DE3)pLysS表達(dá)和純化,并進(jìn)行SDS-PAGE和Western blot,證實(shí)該菌種含有目的蛋白——F蛋白。我們利用該菌種進(jìn)行表達(dá)并改善純化條件,低溫誘導(dǎo)可得到大量可溶性的目的蛋白,用1/2×Wash buffer可得到較高濃度的目的蛋白,用于后續(xù)的動(dòng)物實(shí)驗(yàn)。我們將20只SD大鼠隨機(jī)分為4組,接受不同的處理,Ⅰ組一次性胸腺注射0.2ml PBS做為陰性對(duì)照;Ⅱ組足墊注射含1mg用相同體積弗氏完全佐劑乳化的F蛋白,一周后足墊注射含1mg用相同體積弗氏不完全佐劑乳化的F蛋白,每周1次,連續(xù)3次;Ⅲ組一次胸腺內(nèi)注射4mg F蛋白,每葉2mg;Ⅳ組一次腹腔注射環(huán)磷酰胺100mg/kg做為陽(yáng)性對(duì)照。7天后處死動(dòng)物,留取外周血及胸腺標(biāo)本,用TUNEL試劑盒對(duì)胸腺標(biāo)本進(jìn)行檢測(cè),判斷細(xì)胞凋亡情況。用IL-10試劑盒檢測(cè)外周血清IL-10水平。結(jié)果顯示F蛋白能誘導(dǎo)胸腺淋巴細(xì)胞的凋亡,并促進(jìn)IL-10的分泌。其機(jī)制可能是當(dāng)胸腺接種F蛋白后,胸腺作為一種特殊器官,在被異基因抗原修飾后能產(chǎn)生克隆清除,進(jìn)而通過對(duì)T細(xì)胞發(fā)育和成熟的影響,誘導(dǎo)低免疫應(yīng)答狀態(tài)的發(fā)生。 F蛋白在肝病早期診斷與治療、肝損傷程度的判斷以及肝移植免疫耐受方面的研究都具有重要的應(yīng)用價(jià)值。用化學(xué)提純的方法很難得到大量F蛋白,這就阻礙了其他研究的進(jìn)行。因此,本文采用基因工程方法獲得大鼠肝臟F蛋白,解決其來源問題。初步的實(shí)驗(yàn)說明F蛋白具有免疫原性,很可能會(huì)增加肝移植后的免疫耐受,F蛋白具有免疫反應(yīng)性,大劑量或反復(fù)小劑量免疫可誘導(dǎo)出受體抗原特異性淋巴細(xì)胞凋亡。可能在MHC抗原之外,存在其它的抗原系統(tǒng)參與肝移植的免疫反應(yīng)、決定移植后果。為下一步的移植打下基礎(chǔ)。
[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，

本文編號(hào)：2426740