本文選題：ICOS + 分子��； 參考：《蘇州大學(xué)》2003年博士論文

【摘要】： ICOS(Inducible co-stimulator)是最近發(fā)現(xiàn)的CD28家族的新分子，主要表達(dá)在活化的T細(xì)胞上，與表達(dá)在B細(xì)胞、巨噬細(xì)胞或樹(shù)突狀細(xì)胞表面的相應(yīng)配體GL50分子相互作用，在機(jī)體的再次免疫階段、炎癥反應(yīng)、自身免疫性疾病、腫瘤免疫和移植免疫中發(fā)揮了重要作用，展示了其潛在的應(yīng)用前景。然而，ICOS-GL50在機(jī)體免疫調(diào)節(jié)中的分子機(jī)制尚待進(jìn)一步闡明，更重要的是，目前對(duì)ICOS-GL50的功能研究主要集中在小鼠。鑒此，本研究在國(guó)內(nèi)率先采用基因工程技術(shù)克隆了全長(zhǎng)的人ICOS基因和胞外段cDNA，構(gòu)建了ICOS轉(zhuǎn)基因細(xì)胞，并在大腸桿菌中成功表達(dá)了具有生物學(xué)活性的ICOS可溶性重組蛋白，對(duì)其生物學(xué)功能作了初步探討。利用ICOS轉(zhuǎn)基因細(xì)胞作免疫原，成功獲得兩株鼠抗人ICOS單克隆抗體，并對(duì)其中一株的生物學(xué)特性作了鑒定。在此基礎(chǔ)上，分析了ICOS-GL50在DC成熟與體液免疫中的作用，并進(jìn)一步研究了ICOS-GL50信號(hào)對(duì)多發(fā)性骨髓瘤細(xì)胞體內(nèi)外生物學(xué)行為的作用及其可能機(jī)制。 一、人ICOS分子的表達(dá)及其生物學(xué)活性研究 1、人ICOS的基因克隆及其在大腸桿菌中的表達(dá) 采用RT-PCR法從活化的人T細(xì)胞中擴(kuò)增了ICOS全長(zhǎng)基因，裝入T-vector克隆載體。重組質(zhì)粒T-ICOS經(jīng)測(cè)序確證后，進(jìn)而PCR擴(kuò)增ICOS胞外段編碼cDNA，即可溶性的ICOS基因片段，并克隆入表達(dá)載體pET-28a。構(gòu)建的重組表達(dá)載體pET-ICOS經(jīng)酶切和序列分析證實(shí)后，轉(zhuǎn)化BL-21大腸桿菌，在1～5mmol／L IPTG誘 人Icos分子的表達(dá)與單克隆抗體的研制及其生物學(xué)功能的研究 中文摘要 導(dǎo)下，重組蛋白以包涵體方式在BL一21菌株中誘導(dǎo)表達(dá)。分離的包涵體經(jīng)變性、復(fù) 性和FPLC分離純化后即獲得了較高純度的ICOS重組蛋白。 2、L929一ICOS轉(zhuǎn)基因細(xì)胞的建立 合成全長(zhǎng)ICOS(包括引導(dǎo)膚序列)特異性引物，引物中分別引入刀。朋Hl與Ec口R I酶切位點(diǎn)，以T-ICOS質(zhì)粒為模板進(jìn)行PCR反應(yīng)。PCR產(chǎn)物經(jīng)刀口腳Hl與Ec口Rl 雙酶切后裝入逆轉(zhuǎn)錄病毒表達(dá)載體pGEZ艦A一介nn，，測(cè)序正確的重組表達(dá)載體 pGEZ一ICOS與輔助病毒載體pHIT6O和pHIT456混合后，脂質(zhì)體法轉(zhuǎn)染包裝細(xì)胞293T 細(xì)胞，以獲得具有感染能力的重組逆轉(zhuǎn)錄病毒，后者再感染L929細(xì)胞于6孔板中， 在zcocin(500林留ml)加壓篩選獲得抗性轉(zhuǎn)基因細(xì)胞的基礎(chǔ)上，采用免疫熒光標(biāo)記 和流式細(xì)胞儀分選，即獲得了高表達(dá)ICOS的轉(zhuǎn)基因L929細(xì)胞。 3、ICOS蛋白抑制Daudi細(xì)胞的生長(zhǎng)和增殖 我們檢測(cè)了部分人的惡性淋巴瘤細(xì)胞株的GL50表達(dá)，初步結(jié)果表明，Daudi、 HL6O細(xì)胞高表達(dá)該分子，其陽(yáng)性表達(dá)率分別為99.1%、80.1%。為了觀察Daudi細(xì) 胞高表達(dá)GLSO分子的生物學(xué)意義，將L929一ICOS轉(zhuǎn)基因細(xì)胞、可溶性ICOS分別與 Daudi細(xì)胞體外共培養(yǎng)后，分析了Daudi細(xì)胞的生長(zhǎng)抑制及凋亡效應(yīng)。當(dāng)培養(yǎng)24小 時(shí)，顯微鏡下兩組均見(jiàn)Daudi細(xì)胞聚集成團(tuán)，細(xì)胞中出現(xiàn)顆粒，折光性和立體感減 弱。同時(shí)，Daudi細(xì)胞的表型也明顯發(fā)生了變化，導(dǎo)致粘附分子CD54、趨化因子CXCR4 下調(diào)，F(xiàn)as表達(dá)水平有不同程度的升高。通過(guò)細(xì)胞計(jì)數(shù)、流式細(xì)胞儀分析證實(shí) L929一ICOS轉(zhuǎn)基因細(xì)胞對(duì)腫瘤生長(zhǎng)的具有顯著的抑制作用，并能促使細(xì)胞凋亡，可 溶性ICOS重組蛋白也有類(lèi)似的作用，并且對(duì)Daudi促凋亡的效應(yīng)高于轉(zhuǎn)基因細(xì)胞。 另外，研究還發(fā)現(xiàn)，大多數(shù)高表達(dá)GL50分子腫瘤細(xì)胞，同時(shí)也異常表達(dá)了B7、CD40 分子，它們之間有無(wú)內(nèi)在聯(lián)系?這種現(xiàn)象與惡性B細(xì)胞的生物學(xué)行為有何關(guān)聯(lián)?值 得我們進(jìn)一步的研究。 綜合本研究結(jié)果:我們成功地在大腸桿菌中表達(dá)出ICOS重組蛋白，并構(gòu)建了高 表達(dá)膜型ICOS分子的轉(zhuǎn)基因細(xì)胞，對(duì)其生物學(xué)功能研究表明，ICOS轉(zhuǎn)基因細(xì)胞和 重組蛋白均能直接并顯著地抑制惡性淋巴瘤Daudi的體外增殖，疇導(dǎo)其凋亡，故重 組ICOS蛋白在惡性B細(xì)胞腫瘤的生物治療中可能具有潛在的運(yùn)用價(jià)值。 人IcOS分子的表達(dá)與單克隆抗體的研制及其生物學(xué)功能的研究中文摘要 二、ICoS一GL5o在DC成熟及T細(xì)胞依賴(lài)性B細(xì)胞產(chǎn)生抗體中的作用 1、ICoS一GL50在誘導(dǎo)DC分化成熟中的作用 樹(shù)突狀細(xì)胞(DC)是體內(nèi)己知功能最強(qiáng)的專(zhuān)職性抗原遞呈細(xì)胞(妙C)，能攝取、 加工及遞呈抗原，并能刺激未致敏T細(xì)胞的增殖和活化，從而啟動(dòng)機(jī)體的特異性免 疫應(yīng)答。對(duì)骨髓、臍血來(lái)源的CD34+造血干細(xì)胞的研究發(fā)現(xiàn)，CD34+造血干細(xì)胞不 表達(dá)GL50，但是經(jīng)過(guò)TNF一。、GM一CSF刺激后，可以在12h內(nèi)誘導(dǎo)上調(diào)表達(dá)GL50， 值得注意的是，GL50先于CD80/C D86被誘導(dǎo)表達(dá)，提示GL50可能是單核向DC 成熟發(fā)展過(guò)程的早期分化指標(biāo)之一。目前僅了解DC表達(dá)GL50，但是對(duì)于其是否在 DC的分化成熟以及增殖方面起作用，至今還沒(méi)有報(bào)道。為了探索GL50在DC上表 達(dá)的意義，我們用ICOS轉(zhuǎn)基因細(xì)胞和可溶性ICOS蛋白初步研究了其在DC的成熟 中的作用，研究結(jié)果顯示:在經(jīng)GM一CSF+IL一4誘導(dǎo)的同時(shí)加入ICOS蛋白，可以誘 導(dǎo)單核細(xì)胞向不成熟的DC分化過(guò)程中上調(diào)CD40、CD83，在低劑量CD40 mAb進(jìn) 一步的刺激下，可以發(fā)育成為成熟的DC，值得關(guān)注的是，成熟的DC細(xì)胞的CXCR4、 CDla、CDI lb下調(diào)，而CD45RA上調(diào)。我們推測(cè)ICOS一GL50早期激發(fā)后，有可能 使不成熟的DC向Ix型的DC
[Abstract]:ICOS (Inducible co-stimulator) is a recently discovered new molecules of CD28 family, mainly expressed in activated T cells, and its expression in B cells, the interaction of macrophages or dendritic cells on the surface of the corresponding ligand GL50 molecules in the body of the second immune stage, inflammation, autoimmune diseases, has played an important role in tumor immunity and transplantation immunity, demonstrating its potential applications. However, the molecular mechanism of ICOS-GL50 in immune regulation remains to be further elucidated, more importantly, the functional study of ICOS-GL50 mainly concentrated in mice. In view of this, this study first in China using genetic engineering technology to clone full length people ICOS gene and extracellular cDNA, constructed the ICOS transgenic cells, and expressed in E.coli successfully ICOS with biological activity of soluble recombinant protein, the biological function of the early To use ICOS as immunogen. Transgenic cells, successfully obtained two strains of mouse anti human ICOS monoclonal antibody, and the biological characteristics of one strain were identified. Based on the analysis of the role of ICOS-GL50 in DC maturation and humoral immunity, and further study the role of ICOS-GL50 signaling in multiple myeloma in vitro and in vivo biological behavior and its possible mechanism.
Expression and biological activity of human ICOS molecule
1, gene cloning of human ICOS and its expression in Escherichia coli
Using RT-PCR amplified the full-length ICOS gene from human T cell activation, cloned into T-vector plasmid. The recombinant plasmid T-ICOS by sequencing, and PCR amplification of ICOS extracellular fragment encoding cDNA gene fragment of ICOS, can be dissolved, and cloned into the expression vector pET-ICOS recombinant expression confirmed by enzyme digestion and sequence analysis vector pET-28a. was constructed and transformed into Escherichia coli BL-21. In 1 ~ 5mmol / L induced IPTG
Study on the expression of human Icos molecules and the development of monoclonal antibodies and their biological functions
Chinese abstract
Under the guidance of the recombinant protein, the expression of the inclusion body was induced in the BL 21 strain. The isolated inclusion body was denatured and reproduced.
High purity recombinant protein of ICOS was obtained after separation and purification of sex and FPLC.
The establishment of 2, L929 ICOS transgenic cells
The specific primers of the full length ICOS (including the guided skin sequence) were synthesized. The primers were introduced into the primers. Hl and Ec mouth R were introduced.
I enzyme cutting site and T-ICOS plasmid as template for PCR reaction of.PCR products via Hl and Ec mouth Rl
Double enzyme was loaded into retroviral vector pGEZ, A a NN, and the correct recombinant expression vector was sequenced
PGEZ 1 ICOS is mixed with pHIT6O and pHIT456, an auxiliary viral vector, and transfected 293T by liposome
Cells to obtain recombinant retrovirus infection ability, which infected L929 cells in 6 well plates,
Immunofluorescence markers were used on the basis of zcocin (500 Lin stay ml) pressure screening for resistant transgenic cells.
With the flow cytometry, the transgenic L929 cells with high expression of ICOS were obtained.
3, ICOS protein inhibits the growth and proliferation of Daudi cells
We detected the expression of GL50 in some human lymphoma cell lines, and the preliminary results showed that Daudi,
The positive expression rate of HL6O cells was 99.1%, respectively, and 80.1%. was used to observe Daudi fine.
The biological significance of high expression of GLSO molecules, L929 1 ICOS transgenic cells, soluble ICOS and
After co culture of Daudi cells in vitro, the growth inhibition and apoptosis effect of Daudi cells were analyzed. When 24 small cells were cultured, the cells were cultured.
At the time, two groups of Daudi cells were clustered together under the microscope, and there were particles, refraction and stereotyping in the cells.
At the same time, the phenotype of Daudi cells also changed significantly, leading to the adhesion molecule CD54, chemokine CXCR4
Down regulation, the expression level of Fas is elevated in varying degrees. By cell count, flow cytometry analysis confirmed
L929 - ICOS transgenic cells have a significant inhibitory effect on tumor growth and can induce apoptosis.
The recombinant protein of soluble ICOS also has a similar effect, and the effect of Daudi on apoptosis is higher than that of transgenic cells.
In addition, the study also found that most of the high expression of GL50 molecular tumor cells, but also abnormal expression of B7, CD40
Are there any intrinsic connections between them? What is the correlation between this phenomenon and the biological behavior of malignant B cells?
We'll have to study further.
Combined with the results of this study, we successfully expressed ICOS recombinant protein in Escherichia coli and constructed a high level of recombinant protein.
Transgenic cells expressing the membrane type ICOS molecules, and their biological function studies show that the ICOS transgenic cells and the cells are genetically modified.
The recombinant protein can directly and significantly inhibit the proliferation of malignant lymphoma Daudi in vitro, and the domain leads to its apoptosis.
Group ICOS protein may have potential application value in the biological treatment of malignant B cell tumor.
The expression of human IcOS molecules and the development of monoclonal antibodies and their biological functions
Two, the role of ICoS 1 GL5o in the production of antibodies in DC mature and T cell dependent B cells
The role of 1, ICoS 1 GL50 in the induction of DC differentiation and maturation
Dendritic cells (DC) are the best known specific antigen presenting cells (C) that have the strongest known function in the body.
Processing and presenting antigen, and stimulating the proliferation and activation of the non sensitized T cells, thus triggering the specific immunity of the body.
A study of CD34+ hematopoietic stem cells derived from bone marrow and umbilical cord blood found that CD34+ hematopoietic stem cells do not
GL50 is expressed, but after TNF 1. After GM CSF stimulation, the expression of GL50 can be up-regulated in 12h.
It is worth noting that GL50 was induced earlier than CD80/C D86, suggesting that GL50 may be mononuclear to DC
One of the early differentiation indicators of the mature development process. At present, only DC is known to express GL50, but whether it is in it or not
DC plays a role in differentiation and proliferation and has not yet been reported. In order to explore GL50 on the DC table
To the significance of this, we preliminarily studied the maturation of DC by using ICOS transgenic cells and soluble ICOS protein.
The results of the study show that the addition of ICOS protein at the same time as GM CSF+IL 1 4 can be induced.
In the process of immature DC differentiation, mononuclear cells increase CD40, CD83, and enter low dose CD40 mAb.
A step of stimulation, can develop into a mature DC, and it is noteworthy that the CXCR4 of the mature DC cells,
CDla, CDI LB down, and CD45RA up-regulated. We speculate that after ICOS GL50 early excitation, it is possible
Make the immature DC to Ix type DC

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2003
【分類(lèi)號(hào)】：R392.1

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 吳明方;;運(yùn)動(dòng)與Th細(xì)胞和ICOS分子研究新進(jìn)展[J];北京體育大學(xué)學(xué)報(bào);2005年12期

本文編號(hào)：1740492