白介素-2受体α（IL-2Rα）在NK/T细胞淋巴瘤中过表达及诱导化疗耐药的机制研究

Natural killer/T cell lymphoma is highly aggressive, featured by resistance to common chemotherapy drugs, and the prognosis is very poor. High level of interleukin-2 receptor α (IL-2Rα) expression was demonstrated in NKTL patients, and the level of IL-2Rα was significantly related to the treatment responses and prognosis, which suggested that IL-2Rα may be involved in the pathogenesis of NKTL and contributed to the chemoresistance of NKTL. In this study, NKTL cell lines (SNK-6, SNT-8, NK-92, KHYG-1) and LMP-1/pcDNA3.1-transfected cell lines (NK-92, KHYG-1) will be used to detect the role of LMP-1 and NF-κB in the expression mechanism of IL-2Rα in Epstein-Barr virus positive NKTL. Then, using SNK-6, NK-92, IL-2Rα/pcDNA3.1-transfected SNK-6 and NK-92 cell lines, we will investigate the mechanism of proliferation induced by IL-2Rα through soft agar colony formation assay, cell cycle analysis, and detection of cell cycle related proteins. Furthermore, these cell lines will be used to determine the resistance ability to common chemotherapy drugs such as doxorubicin, gemcitabine, oxaliplatin, and asparaginase, and we will investigate the chemoresistance mechanisms mediated by IL-2Rα through detection of apoptosis and anti-apoptosis related proteins, multidrug resistance related genes and proteins, asparagine synthetase, glutamine synthetase, status of mTOR pathway and autophagy system. Finally, anti-IL-2Rα antibody will be used to inhibit the mechanisms mentioned above, and we will explore the potential role of targeted therapy against IL-2Rα.

NK/T细胞淋巴瘤（NKTL）侵袭性强，对化疗耐药，患者预后差。研究表明NKTL高表达白介素-2受体α (IL-2Rα)，且与预后密切相关，推测IL-2Rα在NKTL的发生发展及化疗耐药中起到重要作用。本研究采用LMP-1/pcDNA3.1质粒转染的NK-92及KHYG-1细胞系，联合NF-κB抑制剂来检测IL-2Rα的表达情况，探讨IL-2Rα的表达机制及其与EB病毒的关系；然后采用IL-2Rα/pcDNA3.1质粒转染的SNK-6及NK-92细胞系，通过克隆形成实验、细胞周期分析、周期相关蛋白检测，探讨IL-2Rα促细胞增殖的机制；MTT实验检测上述细胞系对常见化疗药物的敏感性，通过检测凋亡及抗凋亡相关蛋白的表达、多药耐药相关基因的表达及mTOR通路、自噬系统的活化情况，探讨IL-2Rα介导化疗耐药的机制，并应用IL-2Rα抗体抑制上述作用机制，探讨IL-2Rα靶向治疗的潜在应用价值。

NK/T细胞淋巴瘤（NKTL）是一种与EB病毒密切相关的高侵袭性淋巴瘤，常表现为对化疗耐药，患者预后较差。我们既往的研究表明NKTL细胞表面高表达白介素-2受体α (IL-2Rα)，患者血清中可溶性IL-2Rα显著升高，且与患者的治疗反应和预后密切相关，推测IL-2Rα在NKTL的发生发展及化疗耐药中起到重要作用。本项研究中，我们通过western blot，qRT-PCR，和ELISA的方法均证实IL-2Rα在NKTL细胞株SNK-6（LMP-1强阳性）中的表达水平显著高于人NK细胞株NK-92（LMP-1弱阳性）。我们将载有LMP-1基因的慢病毒转染至NK-92细胞株，使其过表达LMP-1后，发现MAPK/NF-κB通路相关蛋白表达上调，且IL-2Rα的基因及蛋白表达水平均显著增加；采用MAPK/NF-κB通路相关蛋白抑制剂抑制下游通路后，IL-2Rα表达显著下调，提示EB病毒的致癌基因LMP-1通过激活MAPK/NF-κB通路进而介导了NKTL中IL-2Rα的过表达。之后我们采用载有IL-2Rα基因的慢病毒转染NK-92和SNK-6细胞株，转染后的NK-92细胞和SNK-6细胞增殖速度显著增加，细胞周期分析显示G0/1期细胞显著减少，S期细胞显著增加，而细胞凋亡不受影响；western blot分析提示cyclin A, B, D,以及CDK1, 4参与了IL-2Rα过表达细胞的细胞周期调节。最后我们将转染IL-2Rα后的SNK-6细胞株暴露于常见的化疗药物（吉西他滨，阿霉素和左旋门冬酰胺酶），与对照SNK-6细胞相比，过表达IL-2Rα使得所有三种化疗药物的IC50值均显著增高，提示IL-2Rα过表达介导了NKTL的化疗耐药；联用抗IL-2Rα抗体能够显著降低IC50值，提示其可以逆转NKTL的化疗耐药。因此，靶向IL-2Rα的治疗或许可以改善复发难治NKTL患者的治疗效果及预后。