發(fā)布時(shí)間：2018-01-21 16:02

  本文關(guān)鍵詞： 細(xì)胞間粘附分子-1 單克隆抗體 雜交瘤細(xì)胞 格雷夫斯病　出處：《天津醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 細(xì)胞間粘附分子1 (intercellular adhesion molecule-1 ICAM-1)是一種細(xì)胞表面單鏈糖蛋白,參與抗原識(shí)別、補(bǔ)體結(jié)合、細(xì)胞粘附功能�？扇苄约�(xì)胞間粘附分子1 (soluble intercellular adhesion molecule-1 sICAM-1)為細(xì)胞表面ICAM-1脫落所形成,其表達(dá)是導(dǎo)致Graves'病發(fā)生的重要因素之一,并且其血清水平對(duì)于判斷Graves'病的停藥和復(fù)發(fā)具有重要意義。利用純品ICAM-1免疫小鼠制備得到抗ICAM-1的單克隆抗體,為進(jìn)一步治療Graves'病奠定了基礎(chǔ),將會(huì)有一定的經(jīng)濟(jì)意義和臨床應(yīng)用前景。 目的：1.利用純品ICAM-1免疫小鼠制備得到具有生物活性的抗ICAM-1的單克隆抗體。 2.利用抗ICAM-1的單克隆抗體,治療Graves'病模型小鼠。 方法：1.用純品ICAM-1來免疫動(dòng)物。將純品ICAM-1蛋白與佐劑混合腹腔注射BALB/C小鼠三次,每次間隔大約3周,取小鼠內(nèi)眥靜脈血測(cè)免疫效價(jià),取效價(jià)高的小鼠尾靜脈注射加強(qiáng)免疫一次,三天后取小鼠脾細(xì)胞,與骨髓瘤細(xì)胞以5：1的數(shù)量混合,在PEG(polyethylene glycol,聚乙二醇)作用下進(jìn)行細(xì)胞融合。一周后取細(xì)胞生長(zhǎng)孔上清經(jīng)ELISA檢測(cè)抗體分泌情況,將陽(yáng)性分泌孔擴(kuò)大培養(yǎng)并進(jìn)行亞克隆。三次亞克隆后,將單克隆孔細(xì)胞擴(kuò)大培養(yǎng),進(jìn)行雜交瘤細(xì)胞及單克隆抗體的鑒定,同時(shí)采用動(dòng)物體內(nèi)誘生的方法大量制備單克隆抗體。最后用Protein G SepharoseTM 4 Fast Flow(簡(jiǎn)稱protein G)純化腹水得到抗ICAM-1的單克隆抗體。 2.腹腔注射抗ICAM-1的單克隆抗體治療Graves'病模型小鼠：6只,10ug/只,一共注射3次,每次間隔24小時(shí),3天后檢測(cè),比較治療前后小鼠血清T4和TRAb(以刺激性抗體TSAb為主)含量的變化,評(píng)價(jià)治療效果。 結(jié)果：1.利用純品ICAM-1免疫小鼠三次,經(jīng)三次基礎(chǔ)免疫后小鼠血清中的抗體效價(jià)可達(dá)到1：10000。融合后一周左右,雜交瘤細(xì)胞可生長(zhǎng)至1/3-1/2培養(yǎng)孔面積時(shí),取上清液經(jīng)ELISA檢測(cè)抗體分泌情況,篩選出4個(gè)陽(yáng)性孔,陽(yáng)性O(shè)D405值均約2.0。將陽(yáng)性孔擴(kuò)大培養(yǎng)后進(jìn)行三次亞克隆化,獲得單克隆,擴(kuò)大培養(yǎng)后進(jìn)行雜交瘤細(xì)胞及單克隆抗體的鑒定。雜交瘤細(xì)胞染色體分析發(fā)現(xiàn)染色體數(shù)目眾數(shù)為98-104。數(shù)目上接近兩種親代細(xì)胞染色體數(shù)目的總和,結(jié)構(gòu)上多數(shù)為端著絲點(diǎn)染色體,還有少數(shù)亞中部著絲點(diǎn)染色體。4株細(xì)胞分泌的單克隆抗體的免疫球蛋白類別均為IgG1型。動(dòng)物體內(nèi)誘生的腹水中抗體效價(jià)均可達(dá)1:200000, Protein G純化腹水上清效價(jià)均可達(dá)1：200000,純化后的單抗蛋白濃度為1.2 mg/ml,并均可與ICAM-1特異性結(jié)合。 2.利用抗ICAM-1的單克隆抗體治療Graves'病模型小鼠6只,比較治療前后小鼠血清：1)T4治療前為36.63±3.64,治療后為20.12±2.15,兩者比較為t=7.945,p=0.001,差別具有統(tǒng)計(jì)學(xué)意義；2)TRAb以刺激性抗體TSAb為主,治療前為2.02±0.27,治療后為0.54±0.14,兩者比較為t=11.388,p=0,差別具有統(tǒng)計(jì)學(xué)意義。 結(jié)論：1.成功地制備出4株抗ICAM-1的單克隆抗體,分別為：B9,F1,C11,D6. 2利用抗ICAM-1的單克隆抗體治療Graves'病模型小鼠,有一定的治療效果,為進(jìn)一步的研究奠定了基礎(chǔ)。
[Abstract]:Intercellular adhesion molecule 1 (intercellular adhesion molecule-1 ICAM-1) is a cell surface glycoprotein scFv, involved in antigen recognition, complement binding, cell adhesion function. Soluble intercellular adhesion molecule 1 (soluble intercellular adhesion molecule-1 sICAM-1) for cell surface ICAM-1 shedding formed, its expression is one of the important factors leading to Graves'disease, and the serum level of Graves' has an important significance for the judgment of disease withdrawal and relapse. Preparation of monoclonal antibodies against ICAM-1 using purified ICAM-1 immunized mice, which laid the foundation for the further treatment of Graves'disease, there will be some economic significance and Prospect of clinical application.
Objective: 1. the monoclonal antibodies with bioactive anti ICAM-1 were obtained from mice immunized with pure ICAM-1.
2. the anti ICAM-1 monoclonal antibody was used to treat the Graves'disease model mice.
Methods: 1. with pure ICAM-1. The purified ICAM-1 immunized animal protein and adjuvant intraperitoneal injection of BALB/C mice three times, each time interval of about 3 weeks, the mice medial venous blood samples showed high titers, were injected into tail vein of mice three days after booster immunization, spleen cells and bone marrow. The number of tumor cells in mixed 5:1, PEG (polyethylene glycol, polyethylene glycol) under the action of cell fusion. The cell growth by Kong Shangqing ELISA for detecting antibody secretion after a week, the positive hole secretion expanding culture and subcloned. Three sub cloning, cell culture expansion monoclonal antibody hybridoma, were identified. Cells and monoclonal, while using the method of animal in vivo induced a large number of monoclonal antibody. Finally Protein SepharoseTM 4 Fast G Flow (protein G) was purified from ascites monoclonal anti ICAM-1 Antibodies.
2. intraperitoneal injection of anti ICAM-1 monoclonal antibody was used to treat Graves'disease model mice: 6, 10ug/, only 3 times, 3 hours interval, 24 hours after interval. After 3 days, the changes of serum T4 and TRAb levels were evaluated before and after treatment.
Results: 1. using three pure ICAM-1 immunized mice, the antibody titer was three times of immunization in serum can be a week to reach 1:10000. after fusion, hybridoma cells can grow to 1/3-1/2 medium pore area, the supernatant was detected by ELISA antibody secretion, screened 4 positive holes, positive OD405 value all about 2.0. will be positive hole after culture expansion subcloning three times, obtained after identification of monoclonal expansion culture and monoclonal antibody hybridoma cell. Chromosome analysis of hybridoma cells showed the number of number of number of 98-104. close to two kinds of parent cell chromosome number sum, most of them were telocentric and there are a few submetacentric with monoclonal antibody acrocentric chromosome.4 strain cells of the immunoglobulin class was IgG1. The animal in vivo induced ascites antibody titer can reach 1 200000, the titer of purified ascites supernatant of Protein G can reach 1:200000, and the purified McAb protein concentration is 1.2 mg/ml, and it can be specifically combined with ICAM-1.
2. the use of anti ICAM-1 monoclonal antibody in the treatment of Graves'disease model of 6 mice were compared before and after treatment in serum of mice: 1) T4 before treatment was 36.63 + 3.64, 20.12 + 2.15 after treatment, compared to t=7.945, p=0.001, the difference was statistically significant; 2 TRAb) to stimulate antibody TSAb, before treatment was 2.02 after treatment was 0.54 + 0.27, + 0.14, compared to t=11.388, p=0, the difference was statistically significant.
Conclusion: 1. 4 monoclonal antibodies against ICAM-1 were successfully prepared, which were B9, F1, C11, D6., respectively.
2 the treatment of Graves'disease model mice by using anti ICAM-1 monoclonal antibody has certain therapeutic effect, which lays a foundation for further research.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

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