NKp30配体B7-H6的诱导表达与NK细胞抗癌效应
发布时间：2018-08-03 10:52
【摘要】：NK细胞是重要的抗肿瘤效应细胞,通过其表面的活化性受体识别肿瘤细胞表面对应的配体而行使杀伤功能。肿瘤细胞表达的这些配体由自身基因编码,在应激条件下过表达在细胞表面,因而被称为应激诱导的识别(Stress induced recognition)。NKp30是新近发现的重要的NK细胞活化性受体,B7-H6是已知的唯一一种膜蛋白形式表达的NKp30配体,B7-H6仅表达在肿瘤细胞和肿瘤细胞系表面,在正常组织细胞表面不表达,具有肿瘤特异性,因而对B7-H6的研究具有重要的意义。为了研究B7-H6在肿瘤细胞中的应激表达及其调节,我们制备了抗B7-H6多克隆抗体。我们从Ho8910细胞系中克隆扩增得到了B7-H6胞外段编码序列,构建了B7-H6-6His表达载体。然后在大肠杆菌中表达、利用Ni2+柱亲和层析得到了B7-H6-6His融合蛋白。用B7-H6-6His蛋白免疫兔,获得了B7-H6抗血清。经过protein G亲和层析及B7-H6抗原特异性亲和层析,我们得到了抗B7-H6多克隆抗体,并验证了其特异性。为了探讨肿瘤治疗对肿瘤细胞B7-H6表达及其对NK细胞抗肿瘤功能的影响,我们选用多种肿瘤治疗手段、试剂处理肿瘤细胞,用定量PCR、Western blot及流式细胞术方法检测细胞内B7-H6 mRNA、蛋白质以及细胞表面B7-H6分子表达情况。结果发现化疗药物顺铂、5-氟尿嘧啶,137Cs辐照,热休克处理以及TNF-α处理均上调细胞B7-H6分子表达,同时NK细胞杀伤肿瘤细胞效应增强且为B7-H6依赖的,敲低细胞表面B7-H6分子表达导致NK细胞对肿瘤细胞的杀伤效应降低。全反式视黄酸(ATRA)被用于临床肿瘤治疗,研究发现,全反式视黄酸下调肿瘤细胞表面B7-H6分子的表达,但是细胞总B7-H6表达上调,提示可能是翻译后调控机制发挥作用。为此我们采用免疫共沉淀实验发现CIN85与B7-H6有相互作用,且C1N85参与细胞表面B7-H6的表达下调,敲低CIN85表达后,细胞表面B7-H6分子表达增强。通过GST-pull down实验,我们发现CIN85能够与B7-H6胞内段直接结合。但是CIN85如何调控细胞表面B7-H6表达需要进一步探究。我们的研究显示B7-H6是压力诱导表达的跨膜蛋白,常规肿瘤治疗手段能够通过上调B7-H6表达来增强NK细胞抗肿瘤功能；同时我们也发现肿瘤细胞通过CIN85依赖的途径下调其表面B7-H6的表达,具体机制有待于进一步研究。我们所得到的结果提示B7-H6可能成为一个潜在的肿瘤治疗靶点。
[Abstract]:NK cells are important antitumor effector cells. NK cells perform killing function by recognizing the corresponding ligands on the surface of tumor cells through the activated receptors on the surface of NK cells. These ligands expressed by tumor cells are encoded by their own genes and overexpressed on the surface of cells under stress. Therefore, known as stress-induced recognition of (Stress induced recognition). NKp30 is an important newly discovered NK cell activating receptor (B7-H6), which is the only known membrane protein expressed NKp30 ligand B7-H6 only expressed on the surface of tumor cells and tumor cell lines. The study of B7-H6 is of great significance because it is not expressed on the surface of normal tissue and has tumor specificity. In order to study the stress expression and regulation of B7-H6 in tumor cells, anti-B7-H6 polyclonal antibodies were prepared. We cloned and amplified the extracellular coding sequence of B7-H6 from Ho8910 cell line and constructed B7-H6-6His expression vector. Then expressed in Escherichia coli, B7-H6-6His fusion protein was obtained by Ni2 affinity chromatography. B7-H6 antiserum was obtained by immunizing rabbits with B7-H6-6His protein. After protein G affinity chromatography and B7-H6 antigen specific affinity chromatography, we obtained anti B7-H6 polyclonal antibody and verified its specificity. In order to investigate the effect of tumor therapy on the expression of B7-H6 and the anti-tumor function of NK cells, we used a variety of tumor therapy methods to treat tumor cells with reagents. The expression of B7-H6 mRNAs, proteins and B7-H6 molecules on cell surface were detected by quantitative PCR Western blot and flow cytometry. The results showed that the chemotherapeutic drug cisplatin 5-fluorouracil 137Cs irradiation, heat shock treatment and TNF- 伪 treatment all upregulated the expression of B7-H6 molecules, while NK cells had enhanced cytotoxicity and were B7-H6 dependent. The expression of B7-H6 molecules on the knockout cells reduced the cytotoxicity of NK cells to tumor cells. All trans retinoic acid (ATRA) has been used in clinical tumor therapy. It has been found that all trans retinoic acid down-regulates the expression of B7-H6 molecules on tumor cells, but the total B7-H6 expression is up-regulated, suggesting that the mechanism of posttranslational regulation may play a role. For this reason, we found that CIN85 interacted with B7-H6 by immunoprecipitation, and that C1N85 was involved in down-regulation of B7-H6 expression on cell surface, and B7-H6 molecule expression on cell surface increased after CIN85 expression was lowered. Through GST-pull down experiments, we found that CIN85 can bind directly to the intracellular segment of B7-H6. However, how CIN85 regulates the expression of B7-H6 on cell surface needs further study. Our study shows that B7-H6 is a transmembrane protein expressed under stress. Conventional tumor therapy can enhance the anti-tumor function of NK cells by upregulating the expression of B7-H6. At the same time, we also found that tumor cells down-regulate the expression of B7-H6 through CIN85 dependent pathway, the specific mechanism needs further study. Our results suggest that B7-H6 may be a potential target for tumor therapy.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R73-3
本文编号：2161519
[Abstract]:NK cells are important antitumor effector cells. NK cells perform killing function by recognizing the corresponding ligands on the surface of tumor cells through the activated receptors on the surface of NK cells. These ligands expressed by tumor cells are encoded by their own genes and overexpressed on the surface of cells under stress. Therefore, known as stress-induced recognition of (Stress induced recognition). NKp30 is an important newly discovered NK cell activating receptor (B7-H6), which is the only known membrane protein expressed NKp30 ligand B7-H6 only expressed on the surface of tumor cells and tumor cell lines. The study of B7-H6 is of great significance because it is not expressed on the surface of normal tissue and has tumor specificity. In order to study the stress expression and regulation of B7-H6 in tumor cells, anti-B7-H6 polyclonal antibodies were prepared. We cloned and amplified the extracellular coding sequence of B7-H6 from Ho8910 cell line and constructed B7-H6-6His expression vector. Then expressed in Escherichia coli, B7-H6-6His fusion protein was obtained by Ni2 affinity chromatography. B7-H6 antiserum was obtained by immunizing rabbits with B7-H6-6His protein. After protein G affinity chromatography and B7-H6 antigen specific affinity chromatography, we obtained anti B7-H6 polyclonal antibody and verified its specificity. In order to investigate the effect of tumor therapy on the expression of B7-H6 and the anti-tumor function of NK cells, we used a variety of tumor therapy methods to treat tumor cells with reagents. The expression of B7-H6 mRNAs, proteins and B7-H6 molecules on cell surface were detected by quantitative PCR Western blot and flow cytometry. The results showed that the chemotherapeutic drug cisplatin 5-fluorouracil 137Cs irradiation, heat shock treatment and TNF- 伪 treatment all upregulated the expression of B7-H6 molecules, while NK cells had enhanced cytotoxicity and were B7-H6 dependent. The expression of B7-H6 molecules on the knockout cells reduced the cytotoxicity of NK cells to tumor cells. All trans retinoic acid (ATRA) has been used in clinical tumor therapy. It has been found that all trans retinoic acid down-regulates the expression of B7-H6 molecules on tumor cells, but the total B7-H6 expression is up-regulated, suggesting that the mechanism of posttranslational regulation may play a role. For this reason, we found that CIN85 interacted with B7-H6 by immunoprecipitation, and that C1N85 was involved in down-regulation of B7-H6 expression on cell surface, and B7-H6 molecule expression on cell surface increased after CIN85 expression was lowered. Through GST-pull down experiments, we found that CIN85 can bind directly to the intracellular segment of B7-H6. However, how CIN85 regulates the expression of B7-H6 on cell surface needs further study. Our study shows that B7-H6 is a transmembrane protein expressed under stress. Conventional tumor therapy can enhance the anti-tumor function of NK cells by upregulating the expression of B7-H6. At the same time, we also found that tumor cells down-regulate the expression of B7-H6 through CIN85 dependent pathway, the specific mechanism needs further study. Our results suggest that B7-H6 may be a potential target for tumor therapy.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R73-3
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1 曹国帅;NKp30配体B7-H6的诱导表达与NK细胞抗癌效应[D];中国科学技术大学;2016年
，本文编号：2161519
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