【摘要】：作為新近發(fā)現(xiàn)的共信號(hào)分子，B7-H3在T細(xì)胞免疫應(yīng)答中的作用還存在較大爭議，這也提示該分子功能具有多樣性。與功能相比，B7-H3表達(dá)譜比較明確。該膜分子可在腫瘤組織以及誘導(dǎo)活化的單核巨噬細(xì)胞表達(dá)，且與臨床進(jìn)展密切相關(guān)。如前所述，共信號(hào)分子通常存在膜性以及可溶性兩種形式，那么是否也存在可溶性B7-H3，有何臨床意義？除可以調(diào)控T細(xì)胞免疫應(yīng)答外，最近發(fā)現(xiàn)B7-H3也可調(diào)控其他組織細(xì)胞的生物學(xué)行為，那么B7-H3是否可以參與調(diào)控單核-巨噬細(xì)胞生物學(xué)功能，其機(jī)制如何？B7-H3在多種腫瘤組織呈異常高表達(dá)且與臨床進(jìn)展密切相關(guān)，那么腫瘤微環(huán)境中髓系來源細(xì)胞是否也表達(dá)該分子，表達(dá)的臨床意義及作用機(jī)制如何？針對上述問題，本論文開展如下研究： 第一部分：可溶性B7-H3的生物學(xué)特性及其檢測的臨床意義 目的：研制sB7-H3定量檢測試劑盒，鑒定人體內(nèi)是否存在sB7-H3，并分析其生物學(xué)特征及其檢測的臨床意義。 方法：利用自主研發(fā)的抗人B7-H3單抗4H7和21D4雙夾心法配制sB7-H3ELISA檢測試劑盒。在此基礎(chǔ)上，檢測不同年齡階段健康人血清（血漿）中sB7-H3含量。檢測外周血免疫細(xì)胞如樹突細(xì)胞、淋巴細(xì)胞、單核細(xì)胞以及長期細(xì)胞株等膜型以及培養(yǎng)上清中可溶性B7-H3表達(dá)水平，并利用金屬蛋白酶抑制劑進(jìn)行干預(yù)，分析sB7-H3產(chǎn)生的可能機(jī)制。利用Western-blotting實(shí)驗(yàn)分析濃縮細(xì)胞培養(yǎng)上清中sB7-H3分子量，并利用流式細(xì)胞技術(shù)，分析該濃縮天然sB7-H3是否可與體外活化的T淋巴細(xì)胞結(jié)合，分析該天然形式sB7-H3是否為功能片段。篩查不同疾病狀態(tài)下sB7-H3表達(dá)水平，分析該可溶性分子表達(dá)的臨床意義。 結(jié)果：利用抗B7-H3單抗4H7為包被抗體，生物素標(biāo)記單抗21D4為檢測抗體，成功構(gòu)建sB7-H3ELISA試劑盒。該試劑盒檢測區(qū)間為27-20,000pg/ml，線性檢測區(qū)間為20-1,600pg/ml。準(zhǔn)確性，靈敏度都達(dá)到同類產(chǎn)品國際水平。利用該試劑盒，我們發(fā)現(xiàn)人體內(nèi)存在天然形式的sB7-H3，且不同年齡階段水平不盡相同，以臍帶血水平最高，成年后則無明顯變化。檢測健康人外周血單個(gè)核細(xì)胞以及體外培養(yǎng)的樹突細(xì)胞，我們發(fā)現(xiàn)健康人外周血免疫細(xì)胞幾乎都檢測不到膜型B7-H3表達(dá)，但是在體外誘導(dǎo)活化的T淋巴細(xì)胞、單核細(xì)胞以及樹突細(xì)胞則能同時(shí)檢測到膜型B7-H3以及sB7-H3表達(dá)。因此，我們推測膜B7-H3可能是sB7-H3來源。金屬蛋白酶抑制試驗(yàn)中也證實(shí)MMPs參與了sB7-H3表達(dá)調(diào)控。Western-blotting證實(shí)該可溶性分子大小約為16.5kDa。競爭抑制試驗(yàn)表明該天然形式可溶性分子且可與活化T細(xì)胞結(jié)合，證明其為具有生物學(xué)活性的功能片段。臨床檢測發(fā)現(xiàn)，sB7-H3在細(xì)菌引起的炎癥性疾病患者體內(nèi)水平顯著升高，而在病毒性感染患者sB7-H3則未見明顯變化。 結(jié)論：成功構(gòu)建可以定量分析sB7-H3的ELISA試劑盒，證實(shí)人體內(nèi)存在天然形式sB7-H3，其來源為金屬蛋白酶剪切而來的膜型B7-H3，在細(xì)菌性感染患者體內(nèi)明顯升高，具有臨床檢測意義。 第二部分：B7-H3協(xié)同TLR2/TLR4信號(hào)通路促進(jìn)膿毒癥炎癥反應(yīng)的機(jī)制 目的：研究共信號(hào)分子B7-H3對單核巨噬細(xì)胞的調(diào)控作用，分析該分子在炎癥應(yīng)答中的作用機(jī)制。 方法：臨床標(biāo)本分析膿毒癥患者外周血淋巴細(xì)胞及單核細(xì)胞上B7-H3R表達(dá)，并分析B7-H3R表達(dá)的調(diào)控機(jī)制。在此基礎(chǔ)上，利用小鼠B7-H3Ig融合蛋白以及抗小鼠B7-H3阻斷型單抗，研究炎癥反應(yīng)條件下B7-H3對骨髓來源巨噬細(xì)胞的調(diào)控作用。構(gòu)建TLR-2以及TLR4基因過表達(dá)工程細(xì)胞株293，分析B7-H3發(fā)揮生物學(xué)作用是否依賴于TLR2/4信號(hào)。此外，構(gòu)建內(nèi)毒素動(dòng)物模型，利用抗B7-H3抗體進(jìn)行體內(nèi)干預(yù)，分析B7-H3體內(nèi)炎癥應(yīng)答中的生物學(xué)作用及對疾病進(jìn)展的影響。 結(jié)果：臨床標(biāo)本檢測發(fā)現(xiàn)sB7-H3可以與單核巨噬細(xì)胞上潛在受體結(jié)合；進(jìn)一步研究發(fā)現(xiàn)該分子以協(xié)同作用方式，增強(qiáng)LPS及BLP誘導(dǎo)的炎癥反應(yīng)，促進(jìn)單核巨噬細(xì)胞分泌炎性細(xì)胞因子IL-6以及TNF-α。作用機(jī)制研究表明B7-H3協(xié)同作用依賴于TLR-2/4信號(hào)通路并可顯著促進(jìn)NF-κb活性。體內(nèi)實(shí)驗(yàn)也證實(shí)阻斷B7-H3抑制炎癥應(yīng)答，并可顯著延長膿毒癥小鼠生存時(shí)間。 結(jié)論：本研究首次提出在膿毒癥患者體內(nèi)B7-H3的效應(yīng)細(xì)胞為單核巨噬細(xì)胞，其在炎癥應(yīng)答中的生物學(xué)作用主要表現(xiàn)為依賴TLR信號(hào)調(diào)控固有免疫應(yīng)答促進(jìn)炎性細(xì)胞因子分泌。 第三部分：組織浸潤B7-H3+MDSC的生物學(xué)特性及其在肺癌免疫逃逸中的作用 目的：分析腫瘤浸潤部位B7H3+HLA-DR-CD14+MDSC表達(dá)特征、臨床意義以及生物學(xué)功能。 方法：收集手術(shù)切除的非小細(xì)胞肺癌癌灶部位、癌遠(yuǎn)端組織標(biāo)本以及對應(yīng)外周血標(biāo)本60例，組織標(biāo)本經(jīng)過膠原酶消化處理、Ficoll分離獲得單個(gè)核細(xì)胞；外周血標(biāo)本直接溶血，檢測組織以及血標(biāo)本中HLA-DR-CD14+MDSC表達(dá)；分析B7協(xié)同刺激分子在該類細(xì)胞上的表達(dá)特征，結(jié)合病例資料分析其臨床意義，通過體內(nèi)外實(shí)驗(yàn)比較分析B7-H3-以及B7-H3+MDSC在表型、臨床意義以及生物學(xué)功能等方面的差異。 結(jié)果：（1）與健康對照相比，非小細(xì)胞肺癌患者外周血HLA-DR-CD14+MDSC比例顯著升高(P0.01)，B7-1、B7-2、B7-H1、B7-H2、B7-H3以及B7-H4都沒有顯著差異（P0.05）；（2）HLA-DR-CD14+MDSC比例在腫瘤浸潤部位較癌遠(yuǎn)端組織顯著升高(P=0.02)，B7-H3在癌灶部位HLA-DR-CD14+MDSC上較癌遠(yuǎn)端組織表達(dá)顯著升高（P0.01），其他B7家族協(xié)同刺激分子均無統(tǒng)計(jì)學(xué)差異（P0.05）；（3）更為有意義的發(fā)現(xiàn)是，以B7-H3單抗圈門可以將HLA-DR-CD14+MDSC分為兩個(gè)亞群：B7H3+以及B7H3-HLA-DR-CD14+MDSC；（4）結(jié)合病史，比較分析兩類MDSC的臨床意義，結(jié)果發(fā)現(xiàn)僅B7-H3+MDSC與腫瘤分期以及淋巴結(jié)轉(zhuǎn)移密切相關(guān)（P0.05）；（5）以流式細(xì)胞分選技術(shù)從浸潤肺癌組織分離獲得高純度B7H3+以及B7-H3-MDSC兩亞群細(xì)胞，并比較其對CD3+T功能的影響，結(jié)果發(fā)現(xiàn)兩亞群細(xì)胞均能有效抑制T細(xì)胞體外增殖（P0.01），但兩亞群之間并不存在顯著差異（P0.05）；（6）體內(nèi)實(shí)驗(yàn)證實(shí)，，未剔除B7-H3+MDSC組荷瘤小鼠較剔除組腫瘤生長更快（P0.05）；分析兩亞群對Treg體外擴(kuò)增的影響，結(jié)果發(fā)現(xiàn)B7-H3+較B7-H3-MDSC更為有效擴(kuò)增Treg（P0.05）。（7）荷瘤小鼠實(shí)驗(yàn)進(jìn)一步證實(shí)腫瘤微環(huán)境可以誘導(dǎo)B7-H3+MDSC，且發(fā)現(xiàn)B7-H3在介導(dǎo)MDSC誘生Treg中發(fā)揮了部分作用。 結(jié)論：鑒定了一類以B7H3+表達(dá)為特征的新型MDSC亞群，并證實(shí)該新型MDSC亞群可通過誘生Treg在肺癌腫瘤免疫逃逸中發(fā)揮重要作用。
[Abstract]:As a newly discovered co-signal molecule, the role of B7-H3 in the T-cell immune response is also controversial, which also suggests that the molecular function has diversity. The expression profile of B7-H3 is clear compared with the function. The membrane molecules can be expressed in tumor tissue and in the induction of activated mononuclear macrophages, and are closely related to clinical progress. As previously mentioned, the co-signal molecules typically have both membrane and soluble forms, and whether soluble B7-H3 is also present, and what is the clinical significance? In addition to the regulatory T-cell immune response, it has been recently found that B7-H3 can also regulate the biological behavior of other tissue cells, and can B7-H3 be involved in the regulation of the biological function of mononuclear-macrophage, and how is its mechanism? The expression of B7-H3 is highly expressed in a variety of tumor tissues and is closely related to clinical progress. In view of the above problems, the present paper carries out the following research: The first part: the biological characteristics of soluble B7-H3 and its clinical significance The purpose of this study was to develop a kit for quantitative detection of sB7-H3, to identify the presence of sB7-H3 in human body and to analyze its biological characteristics and its detection. Methods: sB7-H3ELISA was prepared by self-developed anti-human B7-H3 monoclonal antibody 4H7 and 21D4 double-sandwich method the kit is used for detecting the sB7 in the serum (plasma) of the healthy people of different age groups on the basis of the detection kit, -H3 content. The expression level of soluble B7-H3 in peripheral blood immune cells, such as dendritic cells, lymphocytes, monocytes, and long-term cell lines, was detected, and the expression levels of soluble B7-H3 in the supernatant were measured, and the production of sB7-H3 was analyzed using a metalloprotease inhibitor. The molecular weight of sB7-H3 in the supernatant of the concentrated cell culture was analyzed by Western-blotting, and the flow cytometry was used to analyze whether the concentrated natural sB7-H3 could be combined with the in vitro activated T-lymphocytes to analyze whether the natural form sB7-H3 the expression level of sB7-H3 in different disease states is screened, and the soluble molecular expression is analyzed Results: The anti-B7-H3 monoclonal antibody 4H7 was used as the coating antibody, and the biotin-labeled monoclonal antibody 21D4 was used as the detection antibody and the sB7-H3E was successfully constructed. the detection interval of the kit is 27-20,000 pg/ ml, the linear detection interval is 20-1, 00 pg/ ml. The accuracy and the sensitivity are the same. The international level of the products. With the kit, we find that the human body has the natural form of sB7-H3, and the levels of different ages are different, and the umbilical cord blood is the highest and the adult The peripheral blood mononuclear cells of healthy people and the dendritic cells cultured in vitro were detected. We found that the peripheral blood immune cells of healthy people were almost undetectable to the expression of the membrane-type B7-H3, but they were induced in vitro. The membrane-type B7-H3 and s can be detected simultaneously with T-lymphocytes, monocytes, and dendritic cells B7-H3 expression. Therefore, we speculate that the membrane B7-H3 may be s Source of B7-H3. MMPs were also confirmed to be involved in sB7 in the metalloprotease inhibition test -H3 expression regulation. Western-blotting confirmed that the size of the soluble molecule was about 16.5 kDa. The competition inhibition test shows that the native form of soluble molecule and can be combined with the activated T cell to demonstrate that it is biological The clinical test found that sB7-H3 was significantly elevated in the body of patients with inflammatory disease caused by bacteria, whereas in patients with viral infection, sB7-H3 Conclusion: The ELISA kit for quantitative analysis of sB7-H3 was successfully constructed to confirm the presence of the natural form of sB7-H3 in the human body, the source of which was the membrane-type B7-H3, which was cut from the metalloprotease, and was significantly elevated in the patients with bacterial infection. And the second part: B7-H3 and the TLR2/ TLR4 signal path. The purpose of the mechanism of promoting the inflammatory response of sepsis is to study the effect of co-signaling molecule B7-H3 on the regulation and control of mononuclear macrophages. The mechanism of the role of the molecule in the inflammatory response. Methods: The expression of B7-H3R on peripheral blood lymphocytes and monocytes in patients with sepsis was analyzed by clinical specimens. The expression of B7-H3R was analyzed by using B7-H3Ig fusion protein and anti-mouse B7-H3 blocking monoclonal antibody, and the expression of B7-H in the condition of inflammatory reaction was studied by using B7-H3Ig fusion protein and anti-mouse B7-H3 blocking monoclonal antibody. 3. To construct TLR-2 and TLR4 gene overexpressing engineering cell line 293 and analyze B7-H3 to play a role in the control of bone marrow-derived macrophages. Whether the effect is dependent on the TLR2/4 signal. In addition, an endotoxemia animal model is constructed, in-vivo intervention with an anti-B7-H3 antibody, in the analysis of the in vivo inflammatory response in the B7-H3 The results showed that sB7-H3 could be combined with the potential receptors on single-core macrophages. The further study found that the molecule was co-active, enhanced the inflammatory response induced by LPS and BLP, and promoted the secretion of mononuclear macrophages. The role of B7-H3 is dependent on TLR-2/4. The signal pathway can also significantly promote the activity of NF-3b. In vivo experiments also confirm that the blocking of the B7-H3 inhibition of inflammation should A. The survival time of the mice with sepsis can be significantly prolonged. Conclusion: The first time that the effect cells of B7-H3 in the patients with sepsis are mononuclear macrophages, their biological effects in the inflammatory response are mainly dependent on the TLR letter. Regulation of innate immune response to regulate the secretion of inflammatory cytokines. Part III: Tissue infiltration of B7-H3 + M The Biological Characteristics of DSC and Its Role in the Immune Escape of Lung Cancer: Analysis of the Tumor Infiltrating Site B7H3 + HLA-DR-CD 14 + MDSC expression, clinical significance and biological function. Methods:60 cases of non-small cell lung cancer, the distal tissue specimen of the cancer and the corresponding peripheral blood were collected. The tissue specimens were digested with collagenase and the Ficoll was isolated to obtain a single core cell. The peripheral blood samples were directly hemolyzed and the test group was detected. Expression of HLA-DR-CD14 + MDSC in woven and blood specimens, and analysis of the expression profiles of B7-co-stimulatory molecules on such cells. The clinical significance of B7-H3-and B7-H3 + was analyzed by in-vivo and out-of-vivo experiments. Results: (1) The proportion of HLA-DR-CD14 + MDSC in peripheral blood of non-small cell lung cancer was significantly higher than that of healthy control (P0.01), and there was no significant difference between B7-1, B7-2, B7-H1, B7-H2, B7-H3 and B7-H4 (P0.05); (2) HLA-DR-CD14 + MDSC The expression of B7-H3 on HLA-DR-CD14 + MDSC was significantly higher (P = 0.02), and there was no statistical difference between the other B7 family and the other B7 family (P0.05). (3) It was found that the B7-H3 McAb could divide the HLA-DR-CD14 + MDSC into two subpopulations: B7H3 + and B7. H3-HLA-DR-CD14 + MDSC; (4) The clinical significance of two types of MDSCs was compared with the medical history. The results showed that only B7-H3 + MDSC was closely related to tumor stage and lymph node metastasis (P0.05). (5) High-purity B7H3 + and B7-H were obtained by flow cytometry. The results showed that both subpopulations could effectively inhibit the in vitro proliferation of T cells (P 0.01), but there was no significant difference between the two subpopulations (P0.05). (6) In vivo experiments, no B7-H3 was removed. The effect of the two subpopulations on the in vitro amplification of the mice in the + MDSC group was found to be faster (P <0.05), and the results showed that the B7-H3 + was higher than that of the control group. The B7-H3-MDSC was more effective in the amplification of Treg (P0.05). (7) The experimental study of tumor-bearing mice further confirmed that the tumor microenvironment could induce B7-H3 + MDSC and Conclusion: A new type of MDSC subpopulation characterized by the expression of B7H3 + is identified and the new MD is confirmed.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

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