發(fā)布時(shí)間：2018-08-03 10:52

【摘要】：NK細(xì)胞是重要的抗腫瘤效應(yīng)細(xì)胞,通過其表面的活化性受體識(shí)別腫瘤細(xì)胞表面對(duì)應(yīng)的配體而行使殺傷功能。腫瘤細(xì)胞表達(dá)的這些配體由自身基因編碼,在應(yīng)激條件下過表達(dá)在細(xì)胞表面,因而被稱為應(yīng)激誘導(dǎo)的識(shí)別(Stress induced recognition)。NKp30是新近發(fā)現(xiàn)的重要的NK細(xì)胞活化性受體,B7-H6是已知的唯一一種膜蛋白形式表達(dá)的NKp30配體,B7-H6僅表達(dá)在腫瘤細(xì)胞和腫瘤細(xì)胞系表面,在正常組織細(xì)胞表面不表達(dá),具有腫瘤特異性,因而對(duì)B7-H6的研究具有重要的意義。為了研究B7-H6在腫瘤細(xì)胞中的應(yīng)激表達(dá)及其調(diào)節(jié),我們制備了抗B7-H6多克隆抗體。我們從Ho8910細(xì)胞系中克隆擴(kuò)增得到了B7-H6胞外段編碼序列,構(gòu)建了B7-H6-6His表達(dá)載體。然后在大腸桿菌中表達(dá)、利用Ni2+柱親和層析得到了B7-H6-6His融合蛋白。用B7-H6-6His蛋白免疫兔,獲得了B7-H6抗血清。經(jīng)過protein G親和層析及B7-H6抗原特異性親和層析,我們得到了抗B7-H6多克隆抗體,并驗(yàn)證了其特異性。為了探討腫瘤治療對(duì)腫瘤細(xì)胞B7-H6表達(dá)及其對(duì)NK細(xì)胞抗腫瘤功能的影響,我們選用多種腫瘤治療手段、試劑處理腫瘤細(xì)胞,用定量PCR、Western blot及流式細(xì)胞術(shù)方法檢測(cè)細(xì)胞內(nèi)B7-H6 mRNA、蛋白質(zhì)以及細(xì)胞表面B7-H6分子表達(dá)情況。結(jié)果發(fā)現(xiàn)化療藥物順鉑、5-氟尿嘧啶,137Cs輻照,熱休克處理以及TNF-α處理均上調(diào)細(xì)胞B7-H6分子表達(dá),同時(shí)NK細(xì)胞殺傷腫瘤細(xì)胞效應(yīng)增強(qiáng)且為B7-H6依賴的,敲低細(xì)胞表面B7-H6分子表達(dá)導(dǎo)致NK細(xì)胞對(duì)腫瘤細(xì)胞的殺傷效應(yīng)降低。全反式視黃酸(ATRA)被用于臨床腫瘤治療,研究發(fā)現(xiàn),全反式視黃酸下調(diào)腫瘤細(xì)胞表面B7-H6分子的表達(dá),但是細(xì)胞總B7-H6表達(dá)上調(diào),提示可能是翻譯后調(diào)控機(jī)制發(fā)揮作用。為此我們采用免疫共沉淀實(shí)驗(yàn)發(fā)現(xiàn)CIN85與B7-H6有相互作用,且C1N85參與細(xì)胞表面B7-H6的表達(dá)下調(diào),敲低CIN85表達(dá)后,細(xì)胞表面B7-H6分子表達(dá)增強(qiáng)。通過GST-pull down實(shí)驗(yàn),我們發(fā)現(xiàn)CIN85能夠與B7-H6胞內(nèi)段直接結(jié)合。但是CIN85如何調(diào)控細(xì)胞表面B7-H6表達(dá)需要進(jìn)一步探究。我們的研究顯示B7-H6是壓力誘導(dǎo)表達(dá)的跨膜蛋白,常規(guī)腫瘤治療手段能夠通過上調(diào)B7-H6表達(dá)來增強(qiáng)NK細(xì)胞抗腫瘤功能；同時(shí)我們也發(fā)現(xiàn)腫瘤細(xì)胞通過CIN85依賴的途徑下調(diào)其表面B7-H6的表達(dá),具體機(jī)制有待于進(jìn)一步研究。我們所得到的結(jié)果提示B7-H6可能成為一個(gè)潛在的腫瘤治療靶點(diǎn)。
[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- 偽 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.
【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R73-3

【相似文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 曹國帥;NKp30配體B7-H6的誘導(dǎo)表達(dá)與NK細(xì)胞抗癌效應(yīng)[D];中國科學(xué)技術(shù)大學(xué);2016年

，

本文編號(hào)：2161519