發(fā)布時間：2018-02-26 23:04

  本文關(guān)鍵詞： B7-H4 單克隆抗體 單核細(xì)胞 腫瘤 基因工程抗體　出處：《蘇州大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： B7-H4分子是新近發(fā)現(xiàn)的B7家族又一負(fù)性協(xié)同刺激分子。盡管其mRNA在淋巴和非淋巴組織中呈廣泛性表達(dá),但由于受到轉(zhuǎn)錄后水平的嚴(yán)格調(diào)控,B7-H4蛋白僅在正常組織中呈現(xiàn)微弱的表達(dá)。越來越多的研究發(fā)現(xiàn),在腫瘤組織如卵巢癌、肺癌、乳腺癌、腎癌和腺樣囊性癌等的腫瘤細(xì)胞以及腫瘤相關(guān)巨噬細(xì)胞中異常高表達(dá)B7-H4分子。在B7-H4信號的作用下,腫瘤周圍的CD3+T細(xì)胞數(shù)量顯著減少,活化CTL的增殖受到明顯抑制,機(jī)體抵抗腫瘤的免疫應(yīng)答大為削弱。與此同時,B7-H4分子還能抑制腫瘤自身的凋亡,促進(jìn)上皮細(xì)胞惡性轉(zhuǎn)化,為腫瘤免疫逃逸提供了必要的條件。臨床研究發(fā)現(xiàn),腫瘤細(xì)胞B7-H4的表達(dá)與臨床和病理學(xué)特征息息相關(guān)。高表達(dá)B7-H4的患者往往腫瘤惡性程度高,預(yù)后結(jié)果差。此外,在腫瘤患者血清中高水平可溶性B7-H4分子的存在,還為腫瘤患者的臨床診斷,尤其是卵巢癌的早期發(fā)現(xiàn),提供了新的檢測指標(biāo)和依據(jù)。為此,繼負(fù)性協(xié)同刺激分子B7-H1,B7-H4成為研究腫瘤免疫逃逸和臨床診斷治療的新熱點。 本研究旨在以高表達(dá)B7-H4的基因轉(zhuǎn)染細(xì)胞為抗原,應(yīng)用細(xì)胞融合技術(shù),研制鼠抗人B7-H4單克隆抗體,為進(jìn)一步探求B7-H4分子在腫瘤免疫逃逸中的生物學(xué)特性和意義提供有效的研究手段,進(jìn)而為臨床診斷、預(yù)后和治療開拓新的道路。 一.鼠抗人B7-H4分子功能性單克隆抗體的研制及生物學(xué)特性的鑒定 【目的】研制鼠抗人B7-H4單克隆抗體,為探討人B7-H4分子在免疫細(xì)胞上的表達(dá)特性及其介導(dǎo)的負(fù)性信號對人T淋巴細(xì)胞抑制作用的效應(yīng)提供必備的研究手段。 【方法】以高表達(dá)人B7-H4分子的基因轉(zhuǎn)染細(xì)胞株293T/B7-H4為免疫原,常規(guī)免疫BALB/c小鼠,采用B淋巴細(xì)胞雜交瘤技術(shù)進(jìn)行細(xì)胞融合,并以該基因轉(zhuǎn)染細(xì)胞293T/B7-H4作為陽性篩選細(xì)胞,以轉(zhuǎn)空質(zhì)粒的對照細(xì)胞293T/Mock作為陰性對照細(xì)胞。經(jīng)間接免疫熒光標(biāo)記和流式細(xì)胞術(shù)分析、反復(fù)篩選和多次克隆化培養(yǎng),獲得能特異分泌鼠抗人B7-H4分子單克隆抗體的雜交瘤細(xì)胞株;采用細(xì)胞核染色體計數(shù)、Ig亞型快速定性試紙法、競爭結(jié)合抑制以及Western blot等實驗對獲得的雜交瘤細(xì)胞株及單克隆抗體進(jìn)行生物學(xué)特性的鑒定;用間接免疫熒光法初步分析單抗對正常人外周血淋巴細(xì)胞表面B7-H4蛋白的識別作用以及單核-樹突狀細(xì)胞誘導(dǎo)過程中B7-H4分子表達(dá)的變化。向293T/B7-H4與T細(xì)胞的混合反應(yīng)體系中加入單抗3C8,用3H-TdR摻入法檢測單抗對B7-H4信號抑制活化T細(xì)胞增殖的阻斷效應(yīng)。 【結(jié)果】成功獲得了1株持續(xù)、穩(wěn)定分泌鼠抗人B7-H4單克隆抗體的雜交瘤細(xì)胞株3C8,細(xì)胞核型分析顯示,雜交瘤細(xì)胞株的染色體數(shù)目在100條以上,超過小鼠B細(xì)胞和SP2/0細(xì)胞的染色體數(shù),為兩者融合體細(xì)胞;快速定性試紙分析顯示,這株單抗輕鏈為κ鏈,重鏈為IgG1亞類; Western blot分析結(jié)果顯示單抗3C8能與B7-H4分子特異性結(jié)合,形成陽性條帶;單抗識別的抗原表位分析結(jié)果表明,單抗3C8與商品化抗體H74識別的抗原表位較為接近,而與科室已有的一株鼠抗人B7-H4單抗1G7的抗原識別表位完全不同。流式細(xì)胞儀檢測結(jié)果表明,單抗3C8能檢測到健康人外周血CD14+單核細(xì)胞上B7-H4的表達(dá),而在CD4+、CD8+、CD19+和CD56+淋巴細(xì)胞上則均未檢測到B7-H4分子。此外,在單核-樹突狀細(xì)胞的誘導(dǎo)過程中,IL-4能下調(diào)B7-H4的表達(dá),而LPS刺激imDC后則能上調(diào)表達(dá)B7-H4,GM-CSF對B7-H4表達(dá)的影響不明顯。3H-TdR結(jié)果顯示,單抗3C8能有效地阻斷B7-H4信號對活化T細(xì)胞增殖的抑制效應(yīng)。 【結(jié)論】成功獲得了1株持續(xù)、穩(wěn)定分泌鼠抗人B7-H4阻斷型單克隆抗體的雜交瘤細(xì)胞株,其不同的抗原表位為進(jìn)一步研制可溶性B7-H4分子ELISA試劑盒奠定了良好的基礎(chǔ)。同時,健康人外周血CD14+單核細(xì)胞上B7-H4表達(dá)的變化以及單抗3C8對B7-H4信號地阻斷作用,為進(jìn)一步研究機(jī)體免疫自穩(wěn)和免疫調(diào)節(jié)開拓了道路。 二.B7-H4分子在腫瘤細(xì)胞中表達(dá)特性的初步研究 【目的】探尋腫瘤細(xì)胞和腫瘤微環(huán)境中B7-H4分子的表達(dá)特性,為進(jìn)一步研究B7-H4與腫瘤免疫逃逸及其相互關(guān)系開拓新的途徑。 【方法】采用間接免疫熒光標(biāo)記法和流式細(xì)胞術(shù)分析不同來源人腫瘤細(xì)胞株以及腫瘤患者胸、腹水中CD14+單核細(xì)胞膜表面B7-H4分子的表達(dá);采用細(xì)胞免疫組化分析B7-H4分子在腫瘤(肺癌)細(xì)胞株中的分布;Western blot鑒定腫瘤和腫瘤微環(huán)境中B7-H4蛋白的表達(dá)形式和糖基化修飾水平;同時運用RT-PCR技術(shù)檢測B7-H4分子在mRNA水平上的不同剪接形式,探求不同表達(dá)形式的B7-H4分子與腫瘤逃逸的相關(guān)關(guān)系。 【結(jié)果】在多株腫瘤細(xì)胞,尤其是上皮性腫瘤細(xì)胞株(如:肺癌、胃癌)表面存在B7-H4分子的表達(dá)。腫瘤患者胸、腹水中的單核細(xì)胞不同于健康人外周血單一的B7-H4+單核細(xì)胞,存在B7-H4+和B7-H4-兩個群體。細(xì)胞免疫組化實驗顯示,腫瘤細(xì)胞株胞漿中也存在大量的B7-H4分子。Western blot結(jié)果表明,腫瘤細(xì)胞株、腫瘤浸潤性單核細(xì)胞以及健康人外周血單核細(xì)胞中B7-H4分子量大小均在34KD左右,分子量較小,而RT-PCR結(jié)果則顯示,在這些腫瘤和腫瘤相關(guān)的細(xì)胞中沒有檢測到B7-H4分子剪接體的存在�！窘Y(jié)論】多株腫瘤細(xì)胞以及腫瘤患者胸、腹水中CD14+單核細(xì)胞雖然都以正常大小的B7-H4cDNA轉(zhuǎn)錄本為主,但其表達(dá)的B7-H4蛋白分子量較小,糖基化水平不高。低糖基化水平B7-H4分子在腫瘤和腫瘤微環(huán)境中所發(fā)揮的調(diào)控作用及機(jī)制值得關(guān)注和進(jìn)一步深入探討。此外,腫瘤患者胸、腹水中CD14+B7-H4+和CD14+B7-H4-是否分別代表著具有不同免疫調(diào)節(jié)功能的單核巨噬細(xì)胞亞群仍有待進(jìn)一步探明。 三.抗人B7-H4單克隆抗體3C8可變區(qū)基因的克隆及嵌合輕、重鏈基因的拼接 【目的】在已獲得的功能型單抗3C8的基礎(chǔ)上,克隆和拼接嵌合輕、重鏈基因,為抗體人源化構(gòu)建和改造鋪平道路。 【方法】根據(jù)抗體保守的框架區(qū)和前導(dǎo)序列設(shè)計簡并引物,采用RT-PCR技術(shù)克隆出單抗3C8的輕重鏈可變區(qū);應(yīng)用生物信息學(xué)方法對所擴(kuò)增的序列進(jìn)行CDR區(qū)的分析和二級結(jié)構(gòu)定位、天然信號肽預(yù)測、抗體可變區(qū)二級拓?fù)浣Y(jié)構(gòu)分析以及三維模擬。采用TP-PCR法將分析正確的鼠源性抗體可變區(qū)基因分別與人IgG1和κ鏈恒定區(qū)進(jìn)行拼接,形成嵌合輕、重鏈基因。 【結(jié)果】成功克隆到了含有信號肽序列的鼠抗人B7-H4單克隆抗體輕、重鏈可變區(qū)基因;經(jīng)生物信息學(xué)分析,抗體輕鏈可變區(qū)由V1-117*01和J1*01兩個亞群基因重排后形成,而重鏈可變區(qū)則由V1-14*01和J4*01以及D2-2*01三個亞群基因重排后形成。其輕、重鏈信號肽切割位點分別位于SSS-DS和VHS-EV,在第19-20個氨基酸之間。二級拓?fù)浣Y(jié)構(gòu)和三維模擬顯示,所獲序列除信號肽區(qū)為α-螺旋外,其他部分主要由β-折疊構(gòu)成,抗體CDR區(qū)位于反向平行的β-折疊之間,暴露在整個蛋白的表面,可與抗原相互結(jié)合。經(jīng)PCR拼接獲得的嵌合輕、重鏈基因大小分別約為750bp和1500bp,與理論值相一致。 【結(jié)論】抗體可變區(qū)基因的成功克隆及拼接,可變區(qū)CDR區(qū)域、二級拓?fù)浣Y(jié)構(gòu)分析以及三維結(jié)構(gòu)的模擬,為進(jìn)一步展開抗體3C8的人源化以及基于DNA點突變技術(shù)的抗體親和力改變實驗打下了堅實的基礎(chǔ)。 綜上所述,本項研究成功獲得了1株能穩(wěn)定分泌特異性鼠抗人B7-H4分子的新型功能型單抗,它能有效阻斷B7-H4信號;其抗體可變區(qū)基因的克隆及嵌合輕、重鏈的拼接為繼續(xù)開展抗體人源化和靶向B7-H4分子的腫瘤免疫治療提供了必要的物質(zhì)基礎(chǔ)。此外,多株腫瘤細(xì)胞以及腫瘤患者胸、腹水中CD14+單核細(xì)胞雖然都以正常大小的B7-H4cDNA轉(zhuǎn)錄本為主,但其表達(dá)的B7-H4蛋白分子量較小,糖基化水平不高。低糖基化B7-H4分子在健康人外周血和腫瘤患者胸、腹水中CD14+單核細(xì)胞上表達(dá)強(qiáng)度的差異,也將為我們探索機(jī)體免疫自穩(wěn)和腫瘤免疫逃逸提供新的線索。
[Abstract]:B7 - H4 molecule is a newly discovered B7 family and a negative co - stimulatory molecule . Although its mRNA is widely expressed in lymph and non - lymphoid tissues , B7 - H4 protein can be expressed weakly in tumor cells such as ovarian cancer , lung cancer , breast cancer , renal cancer and cystic carcinoma . The purpose of this study was to develop murine anti - human B7 - H4 monoclonal antibody with high expression of B7 - H4 gene - transfected cells . To further explore the biological characteristics and significance of B7 - H4 molecule in tumor immune escape , this study provides a new way for clinical diagnosis , prognosis and treatment . 1 . Preparation and characterization of murine anti - human B7 - H4 functional monoclonal antibody Objective To study the anti - human B7 - H4 monoclonal antibody of murine anti - human B7 - H4 , and provide necessary research methods for the study of the expression of B7 - H4 molecules on immune cells and the effect of negative signals mediated by human B7 - H4 on human T lymphocytes . A hybridoma cell line with monoclonal antibody against B7 - H4 in human peripheral blood was obtained by means of indirect immunofluorescence and flow cytometry . The results showed that monoclonal antibody 3C8 could inhibit the expression of B7 - H4 in human peripheral blood CD14 + monocytes . Conclusion The monoclonal antibody of B7 - H4 monoclonal antibody against human B7 - H4 has been successfully developed . The different epitopes of the hybridoma cell line have established a good foundation for further development of the soluble B7 - H4 molecule ELISA kit . At the same time , the expression of B7 - H4 in the peripheral blood CD14 + monocytes in healthy human peripheral blood and the blocking effect of monoclonal antibody 3C8 on B7 - H4 signal have been successfully developed , which has opened the way for further study on the immune homeostasis and immune regulation of the organism . Preliminary study on the expression of B7 - H4 molecule in tumor cells Objective To explore the expression of B7 - H4 molecules in tumor cells and tumor microenvironment , and explore new ways to further study B7 - H4 and tumor immune escape and their correlation . Methods : The expression of B7 - H4 and B7 - H4 in tumor and ascites were analyzed by indirect immunofluorescence and flow cytometry . The expressions of B7 - H4 in tumor and tumor microenvironment were analyzed by immunohistochemistry . Western blot was used to identify the expression of B7 - H4 protein in tumor and tumor microenvironment . Western blot was used to identify the expression of B7 - H4 protein in tumor and tumor microenvironment . The expression of B7 - H4 and B7 - H4 in peripheral blood mononuclear cells of tumor cells , tumor infiltrating mononuclear cells and healthy human peripheral blood mononuclear cells showed that the molecular weight of B7 - H4 in tumor cell lines , tumor infiltrating mononuclear cells and healthy human peripheral blood mononuclear cells was less than 34KD , and the molecular weight of B7 - H4 was low . III . Cloning of anti - human B7 - H4 monoclonal antibody 3C8 variable region gene and splicing of chimeric light and heavy chain genes Objective : To clone and splice the chimeric light and heavy chain genes on the basis of the obtained functional monoclonal antibody 3C8 , and pave the way for the construction and transformation of humanized antibody humanized antibody . According to the conserved framework region and leader sequence of antibody , the monoclonal antibody 3C8 heavy chain variable region was cloned by RT - PCR technique , and the amplified sequence was analyzed by using bioinformatics method , secondary structure localization , natural signal peptide prediction , secondary topological structure analysis of antibody variable region and three - dimensional simulation . The light weight and heavy chain signal peptide cleavage sites were composed of V1 - 117 * 01 and J4 * 01 and D2 - 2 * 01 three sub - group genes . The light weight and heavy chain signal peptide cleavage sites were located between 19 - 20 amino acids . Conclusion The successful cloning and splicing , CDR region , secondary topological structure analysis and 3D structure simulation of antibody variable region gene can provide a solid foundation for further developing humanized antibody 3C8 and antibody affinity change experiment based on DNA point mutation technology . In conclusion , a novel functional monoclonal antibody capable of stably secreting specific mouse anti - human B7 - H4 molecule is successfully obtained in this study , which can effectively block the B7 - H4 signal , and it can effectively block the B7 - H4 signal , and the splicing of the heavy chain is a necessary material basis for the continuation of antibody humanized and targeted B7 - H4 molecule tumor immunotherapy .

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392;R730.3

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