人共信号分子B7-H3在炎症反应及肺癌肿瘤免疫逃逸中的作用机制研究
发布时间:2019-05-27 00:08
【摘要】:作为新近发现的共信号分子,B7-H3在T细胞免疫应答中的作用还存在较大争议,这也提示该分子功能具有多样性。与功能相比,B7-H3表达谱比较明确。该膜分子可在肿瘤组织以及诱导活化的单核巨噬细胞表达,且与临床进展密切相关。如前所述,共信号分子通常存在膜性以及可溶性两种形式,那么是否也存在可溶性B7-H3,有何临床意义?除可以调控T细胞免疫应答外,最近发现B7-H3也可调控其他组织细胞的生物学行为,那么B7-H3是否可以参与调控单核-巨噬细胞生物学功能,其机制如何?B7-H3在多种肿瘤组织呈异常高表达且与临床进展密切相关,那么肿瘤微环境中髓系来源细胞是否也表达该分子,表达的临床意义及作用机制如何?针对上述问题,本论文开展如下研究: 第一部分:可溶性B7-H3的生物学特性及其检测的临床意义 目的:研制sB7-H3定量检测试剂盒,鉴定人体内是否存在sB7-H3,并分析其生物学特征及其检测的临床意义。 方法:利用自主研发的抗人B7-H3单抗4H7和21D4双夹心法配制sB7-H3ELISA检测试剂盒。在此基础上,检测不同年龄阶段健康人血清(血浆)中sB7-H3含量。检测外周血免疫细胞如树突细胞、淋巴细胞、单核细胞以及长期细胞株等膜型以及培养上清中可溶性B7-H3表达水平,并利用金属蛋白酶抑制剂进行干预,分析sB7-H3产生的可能机制。利用Western-blotting实验分析浓缩细胞培养上清中sB7-H3分子量,并利用流式细胞技术,分析该浓缩天然sB7-H3是否可与体外活化的T淋巴细胞结合,分析该天然形式sB7-H3是否为功能片段。筛查不同疾病状态下sB7-H3表达水平,分析该可溶性分子表达的临床意义。 结果:利用抗B7-H3单抗4H7为包被抗体,生物素标记单抗21D4为检测抗体,成功构建sB7-H3ELISA试剂盒。该试剂盒检测区间为27-20,000pg/ml,线性检测区间为20-1,600pg/ml。准确性,灵敏度都达到同类产品国际水平。利用该试剂盒,我们发现人体内存在天然形式的sB7-H3,且不同年龄阶段水平不尽相同,以脐带血水平最高,成年后则无明显变化。检测健康人外周血单个核细胞以及体外培养的树突细胞,我们发现健康人外周血免疫细胞几乎都检测不到膜型B7-H3表达,但是在体外诱导活化的T淋巴细胞、单核细胞以及树突细胞则能同时检测到膜型B7-H3以及sB7-H3表达。因此,我们推测膜B7-H3可能是sB7-H3来源。金属蛋白酶抑制试验中也证实MMPs参与了sB7-H3表达调控。Western-blotting证实该可溶性分子大小约为16.5kDa。竞争抑制试验表明该天然形式可溶性分子且可与活化T细胞结合,证明其为具有生物学活性的功能片段。临床检测发现,sB7-H3在细菌引起的炎症性疾病患者体内水平显著升高,而在病毒性感染患者sB7-H3则未见明显变化。 结论:成功构建可以定量分析sB7-H3的ELISA试剂盒,证实人体内存在天然形式sB7-H3,其来源为金属蛋白酶剪切而来的膜型B7-H3,在细菌性感染患者体内明显升高,具有临床检测意义。 第二部分:B7-H3协同TLR2/TLR4信号通路促进脓毒症炎症反应的机制 目的:研究共信号分子B7-H3对单核巨噬细胞的调控作用,分析该分子在炎症应答中的作用机制。 方法:临床标本分析脓毒症患者外周血淋巴细胞及单核细胞上B7-H3R表达,并分析B7-H3R表达的调控机制。在此基础上,利用小鼠B7-H3Ig融合蛋白以及抗小鼠B7-H3阻断型单抗,研究炎症反应条件下B7-H3对骨髓来源巨噬细胞的调控作用。构建TLR-2以及TLR4基因过表达工程细胞株293,分析B7-H3发挥生物学作用是否依赖于TLR2/4信号。此外,构建内毒素动物模型,利用抗B7-H3抗体进行体内干预,分析B7-H3体内炎症应答中的生物学作用及对疾病进展的影响。 结果:临床标本检测发现sB7-H3可以与单核巨噬细胞上潜在受体结合;进一步研究发现该分子以协同作用方式,增强LPS及BLP诱导的炎症反应,促进单核巨噬细胞分泌炎性细胞因子IL-6以及TNF-α。作用机制研究表明B7-H3协同作用依赖于TLR-2/4信号通路并可显著促进NF-κb活性。体内实验也证实阻断B7-H3抑制炎症应答,并可显著延长脓毒症小鼠生存时间。 结论:本研究首次提出在脓毒症患者体内B7-H3的效应细胞为单核巨噬细胞,其在炎症应答中的生物学作用主要表现为依赖TLR信号调控固有免疫应答促进炎性细胞因子分泌。 第三部分:组织浸润B7-H3+MDSC的生物学特性及其在肺癌免疫逃逸中的作用 目的:分析肿瘤浸润部位B7H3+HLA-DR-CD14+MDSC表达特征、临床意义以及生物学功能。 方法:收集手术切除的非小细胞肺癌癌灶部位、癌远端组织标本以及对应外周血标本60例,组织标本经过胶原酶消化处理、Ficoll分离获得单个核细胞;外周血标本直接溶血,检测组织以及血标本中HLA-DR-CD14+MDSC表达;分析B7协同刺激分子在该类细胞上的表达特征,结合病例资料分析其临床意义,通过体内外实验比较分析B7-H3-以及B7-H3+MDSC在表型、临床意义以及生物学功能等方面的差异。 结果:(1)与健康对照相比,非小细胞肺癌患者外周血HLA-DR-CD14+MDSC比例显著升高(P0.01),B7-1、B7-2、B7-H1、B7-H2、B7-H3以及B7-H4都没有显著差异(P0.05);(2)HLA-DR-CD14+MDSC比例在肿瘤浸润部位较癌远端组织显著升高(P=0.02),B7-H3在癌灶部位HLA-DR-CD14+MDSC上较癌远端组织表达显著升高(P0.01),其他B7家族协同刺激分子均无统计学差异(P0.05);(3)更为有意义的发现是,以B7-H3单抗圈门可以将HLA-DR-CD14+MDSC分为两个亚群:B7H3+以及B7H3-HLA-DR-CD14+MDSC;(4)结合病史,比较分析两类MDSC的临床意义,结果发现仅B7-H3+MDSC与肿瘤分期以及淋巴结转移密切相关(P0.05);(5)以流式细胞分选技术从浸润肺癌组织分离获得高纯度B7H3+以及B7-H3-MDSC两亚群细胞,并比较其对CD3+T功能的影响,结果发现两亚群细胞均能有效抑制T细胞体外增殖(P0.01),但两亚群之间并不存在显著差异(P0.05);(6)体内实验证实,,未剔除B7-H3+MDSC组荷瘤小鼠较剔除组肿瘤生长更快(P0.05);分析两亚群对Treg体外扩增的影响,结果发现B7-H3+较B7-H3-MDSC更为有效扩增Treg(P0.05)。(7)荷瘤小鼠实验进一步证实肿瘤微环境可以诱导B7-H3+MDSC,且发现B7-H3在介导MDSC诱生Treg中发挥了部分作用。 结论:鉴定了一类以B7H3+表达为特征的新型MDSC亚群,并证实该新型MDSC亚群可通过诱生Treg在肺癌肿瘤免疫逃逸中发挥重要作用。
[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.
【学位授予单位】:苏州大学
【学位级别】:博士
【学位授予年份】:2012
【分类号】:R392
[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.
【学位授予单位】:苏州大学
【学位级别】:博士
【学位授予年份】:2012
【分类号】:R392
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