1、硕士学位论文 I 慢病毒介导的稳定表达 GSTM5的人支气管上皮细胞株的建立及其核转位分析 摘 要 目的 ： 构建稳定表达 谷胱甘肽 S 转移酶 mu5（ GSTM5） 的人支气管上皮16HBE 细胞株， 探讨 诱导 GSTM5 核转位的关键因素 。方法 ： 构建表达 GSTM5 基因的重组慢病毒载体（ PLVX-puro-GSTM5-SBP-3*flag-N 、PLVX-puro-3*flag-SBP-GSTM5-C）并制备出相应的慢病毒，感染人支气管上皮16HBE 细胞后，经嘌呤霉素筛选获得 稳转株 ；实时荧光定量 PCR 和蛋白印迹法（ Western-Blot）分别检测细胞株中 GST
2、M5 的 mRNA、蛋白表达水平；以 TNF-（ 10 ng/mL） 刺激稳转株 0.5 小时，共聚焦荧光显微镜检测 GSTM5 核转位。结果 ： 成 功 构 建 稳 定 表 达 GSTM5 人 支 气 管 上 皮 细 胞 株（ 16HBE-GSTM5-SBP-3*flag-N、 16HBE-3*flag-SBP-GSTM5-C）； 荧光共聚焦显微镜显示稳转株在 TNF- 刺激 诱导后， GSTM5 由胞浆转入胞核（核转位），以16HBE-GSTM5-SBP-3*flag-N 稳转株更显著。结论 ： TNF- 炎症刺激可诱导 稳定表达 GSTM5 的 人支气管 上皮细胞株中 GSTM5 发生核
3、转位，可能与其 N 端含有非经典核定位信号密切相关。 急性肺损伤（ Acute Lung Injury, ALI）的病理生理过程是炎症反应与氧化应激过度激活的过程，而且炎症反应与氧化应激密切相关 1。炎症失衡诱发了氧化相关基因的过度表达及活性氧过量生成，继发氧化应激损伤，加重了肺结构细胞凋亡 2, 3。在探讨 TNF-诱导肺泡 II 型上皮细胞的烟酰胺腺嘌呤二核苷酸磷酸氧化酶 1 （ NADPH oxidase 1, Nox1） 基因转录调控过程中，发现 TNF-刺激后，肺泡 II 型上皮细胞内 Nox 家族及 ROS 表达明显上调 4，同时 GSTM5摘要 II 表达上调，并从胞浆转移入胞核
4、，与 Nox1 启动子结合 5。 GSTM5 属于 GST 家族，具有催化氧化还原反应的活性 6，我们前期研究也证实 GSTM5 能影响 P38、NF- B 等激酶活化，参与细胞炎症反应、凋亡等生理过程的调节 7。我们推测，在 ALI 的发生、发展过程中， GSTM5 对炎症反应及其继发的氧化应激过程有具有调控作用，其机制与 Nox 基因表达密切相关。 GSTM5 对氧化应激的调控需要其发生核转位，但 GSTM5 没有核定位信号， 不是经典的转录因子。 第一种解释是 GSTM5 可能与其他有核定位信号的分子发生相互作用后核转位，第二种是 GSTM5 可能 含有非经典的核定位信号。其是否能在炎症
5、刺激诱导下入核（核转位），是研究其非酶学（转录）活性的关键。 为了使 GSTM5 基因能够在 16HBE 细胞核中稳定表达，本实验构建慢病毒介导的稳定表达 GSTM5 的人支气管上皮细胞株。相比其它方法， 慢病毒感染因具有随机整合到细胞基因组的特性，细胞株的建立更容易，荧光素酶基因的表达更稳定 8，还能确保携带的外源基因不发生改变。慢病毒另一优点是可以同时感染有分裂能力细胞和无分裂能力细胞，是基因治疗载体的理想方案 9，保证了其在在多种细胞中的应用。本研究所构建的稳定表达 GSTM5 的人支气管上皮细胞株，经酶切、 RT-qPCR、 WB 验证，从基因、蛋白方面均证明稳转株构建成功，可用于下一
6、步研究 GSTM5 结构、功能、相互作用蛋白等实验。另外，所构建的载体中有 SBP、 3*flag 标签，可用于后续的串联亲和纯化TAP-MS，以便分析 GSTM5 的相互作用蛋白，为探究其功能提供基础。 本实验构建慢病毒介导的稳定表达 GSTM5 的人支气管上皮细胞株，经 TNF-刺激诱导后，荧光共聚焦显微镜显示 C、 D 两组细胞株所表达的 GSTM5 都有不同程度的入核。当标签位于 GSTM5 的 C 端 (N 端游离 ) 时（ C 组16HBE-GSTM5-SBP-3*flag-N ），其核内荧光强度较另一个稳转株（ D 组16HBE-3*flag-SBP-GSTM5-C）大，提示 G
7、STM5 的核转位与 N 端结构域密切相关。当标签位于 GSTM5 的 N 端时入核减少，可能与 GSTM5 的 N 端序列被标签封闭相关。 Miho Kawakatsu 等人发现 GSTpi 的线粒体定位序列与其 N 端相硕士学位论文 III 关，其 195-208aa则对其核定位有重要作用，并证实其为一种非经典核定位信号（ NLS） 10。经搜索 expasy 等数据库表明， GSTM5 分 N 端和 C 端结构域，分别是 1-88aa 和 90-207aa。我们推测， GSTM5 的 N 端结构域含有非经典核定位信号，对其核转位有重要作用。在后续的研究中，本课题组将分别构建GV230-G
8、STM51-88aa-GFP 及 GV230-GSTM5 90-207aa-GFP 载体，进一步研究这两个结构域对 GSTM5 的核转位影响。此外，免疫荧光实验显示，在 TNF-刺激诱导前后对照组细胞株（ B 组 16HBE-SBP-3*flag）核内外均有较强绿色荧光表达，可能与 SBP、 3*flag 标签分子量小，可自由弥散进入细胞核相关。 综上所述， 本 实验 成功建立了慢病毒介导的稳定表达 GSTM5 的人支气管上皮细胞株，同时证实在 TNF-诱导的炎症过程中 GSTM5 存在核转位，并与其 N 端含有非经典核定位信号密切相关。本研究发现为后续 ALI 炎症氧化失衡导致的肺结构细胞损
9、伤调控机制的研究奠定基础。 关键词 ：谷胱甘肽 S 转移酶 mu 5 重组慢病毒表达载体 16HBE 核转位 TNF-硕士学位论文 i The Establishment of 16HBE Cell Stably Expressing GSTM5 Mediated by Lentivirus and the Mechanism of GSTM5 Nuclear Translocation ABSTRACT 1 Backgroud Acute respiratory distress syndrome (ARDS) is a complex clinical syndrome, which is
10、 characterized by complicated pathology and pathogenesis1. Excessive inflammatory responses triggered oxidative stress , inducing the increase of endogenous reactive oxygen species (ROS) generation, and surpass the removal ability of the antioxidant system2. So that the body in the state of progress
11、ive oxidative stress , lead to lung structure cell injury. Glutathione S transferase combined with glutathione, is a multifunctional enzyme superfamily， which catalytic oxidation, and cooperate with other antioxidant enzymes in cells to scavenge active oxygen and protect cells from oxidative damage3
12、. Glutathione S transferase Mu 5 (GSTM5) is a member of the GST family. The previous study found that, the transcription and expression of NOX1 in 16HBE were significantly increased after stimulation of TNF- cells8. Also， there are Interaction between GSTM5 and NOX1 promoter4， which causes our atten
13、tion to GSTM5. 2 objectives This study intends to construct the human GSTM5 gene lentiviral expression vector and to build human bronchial epithelial cell line stably expressing GSTM5. To ABSTRACT ii observe GSTM5 nuclear translocation under cell inflammatory state with TNF simulation, which lays a
14、foundation for further study the function of GSTM5 in transcription regulation. 3 methods 3.1 To construct the human GSTM5 gene lentiviral expression vector ：PLVX-puro-GSTM5-SBP-3*flag-N 、 PLVX-Puro-3*flag-SBP-GSTM5-C. 3.2 To build human bronchial epithelial cell lines stably expressing GSTM5. 3.3 T
15、o verify cell lines by enzyme digestion, RT-qPCR and WB. 3.4 To observe GSTM5 nuclear translocation under cell inflammatory state with TNF simulation. 4 Results human bronchial epithelial cell line stably expressing GSTM5, verified by enzyme digestion, RT-qPCR and WB, were proved successfully constr
16、ucted.When GSTM5 is located in the N terminal of tags (16HBE- GSTM5-SBP-3*flag-N), the nucleus fluorescence intensity was higher than another cell line (16HBE-3*flag-SBP-GSTM5-C) after stimulation of TNF- alpha. 5 conlusions The pathophysiological process of acute lung injury (Acute Lung Injury, ALI
17、) is the process of excessive activation of inflammatory response and oxidative stress, and inflammation is closely related to oxidative stress5.The imbalance of inflammation induces the over expression of oxidative related genes and the generation of reactive oxygen species, and the secondary oxida
18、tive stress injury, which aggravates the apoptosis of lung structure cell 6, 7.In the study of TNF- induced II nicotinamide adenine dinucleotide phosphate oxidase 1 (NADPH oxidase 1, Nox1) gene transcription process, we found that after TNF- stimulation, the expression of Nox family and ROS were up-
19、regulated in type II alveolar epithelial 硕士学位论文 iii cells, 8 while GSTM5 up-regulated and transferred from cytoplasm to the nucleus, and bound to the Nox1 promoter 4. GSTM5 belongs to the GST family, which has the activity of catalytic redox reaction. 3 Our previous studies have confirmed that GSTM5
20、 can affect the activation of P38, NF- kappa B and other kinase, and participate in the regulation of cell inflammatory response, apoptosis and other physiological processes 9 We speculate that GSTM5 plays a regulatory role in the process of ALI, and its mechanism is closely related to the expressio
21、n of Nox1 gene. In order to stabilize the expression of GSTM5 gene in 16HBE cells, we constructed a lentiviral vector mediated human bronchial epithelial cell line stably expressing GSTM5.Compared with other methods, the lentivirus infection can randomly integrate target gene into the cell genome, t
22、he establishment of the cell line is easier, and the luciferase gene expression is more stable 10, but also ensure that the exogenous gene does not change. Another advantage of lentivirus is that it can infect both mitotic and non dividing cells, which ensure its application in a variety of cells an
23、d it is the ideal solution for gene therapy.11 What we constructed in this research , human bronchial epithelial cell line stably expressing GSTM5, verified by enzyme digestion, RT-qPCR and WB, from the gene and protein, were proved successfully constructed, which can be used for the next step study
24、 on the structure ,function, interacting protein of GSTM5.In addition, there are SBP and 3*flag tags in the constructed vector, which can be used in tandem affinity purification - mass spectrum ,(TAP-MS) in order to analyze the interaction proteins of GSTM5 and provide the basis for exploring its fu
25、nction. Because GSTM5 does not have a classical nuclear localization sequence, our primary task is to prove that it can transport into nucleus directly or indirectly after TNF- stimulation. The constructed human bronchial epithelial cell lines stably ABSTRACT iv expressing GSTM5, were tested by fluo
26、rescence confocal microscopy after stimulation of TNF- alpha. It showed that when GSTM5 is located in the N terminal of tags (16HBE- GSTM5-SBP-3*flag-N), the nucleus fluorescence intensity was higher than another cell line (16HBE-3*flag-SBP-GSTM5-C), suggesting that nuclear translocation is closely
27、related.to N terminal domain of GSTM5.When GSTM5 is located at the C terminal of the tag (16HBE-3*flag-SBP-GSTM5-C), the nucleus fluorescence intensity is lower than another cell lines, which may related to the N terminal sequence being blocked by the tag. Miho Kawakatsu et al. Found that the mitoch
28、ondrial localization sequence of GSTpi was related to its N terminal 12, and that 195-208aa played an important role in the nuclear localization of GSTpi, and confirmed that it was a non classical nuclear localization signal (NLS). After searching ExPASY and other databases, it show that GSTM5can be
29、 devided into two part, N and C terminal, namely 1-88aa and 90-207aa. In view of this, we believe that the N terminal domain of GSTM5 contains a non classical nuclear localization signal, plays an important role in its nuclear translocation. To further study on the mechanism of nuclear translocation
30、 of the N terminal domain, our research group will construct vectors GV230-GSTM51-88aa-GFP and GV230-GSTM55 90-207aa-GFP respectively. In summary, we constructed a lentiviral vector mediated human bronchial epithelial cell line stably expressing GSTM5. We also confirmed that GSTM5 translocate to nuc
31、leus in cell inflammation induced by TNF- alpha, and its N terminal is closely related with the non-classical nuclear localization signal. This study laid the foundation for the study of the regulation mechanism of lung cells damage induced by inflammation Oxidative imbalance in ALI. Keywords: Gluta
32、thione S-transferase M5； lentiviral vector； 16HBE； nuclear translocation ； TNF- alpha 硕士学位论文 v 目 录 摘 要 . I ABSTRACT . I 前 言 . 1 第一章 构建重组慢病毒表达载体 PLVX-puro-GSTM5-SBP-3*flag- N、 PLVX-puro-3*flag-SBP-GSTM5-C 及慢病毒制备 . 5 前言 .5 1 材料和方法 .5 2 结果 .20 3 讨论 .26 第二章 16HBE-GSTM5 稳定细胞株构建与验证 . 28 前言 .28 1 材料和方法 .29 2 结果 .40 3 讨论 .42 第三章 GSTM5 的核定位分析 . 46 前言 .46 1 材料和方法 .46 2 结 果 .49 3 讨论 .51 全文总结 . 54 中英文缩写词简表 . 63 致 谢 . 64
