Non-small cell lung cancer (NSCLC) is a disease related to inflammation. Proinflammatory cytokines such as interleukin 17 (IL-17) can induce cancer cell proliferation, metastasis and immune escape. Although NSCLC immune escape is partly due to the interaction between PD-1 and PD-L1 and PD-L1 expression can be upregulated in cancer cells upon stimulation with IL-17, the underlying mechanism of IL-17-triggered PD-L1 gene transcription in NSCLC cells remains elusive. RT‒PCR, real-time PCR, and IB were used to assess the levels of PD-L1, MEF2C, and TRIM31 in NSCLC tissues as well as in IL-17–stimulated H1299 or PC9 cells. Bioinformatics analysis, luciferase assays, and ChIP were utilized to investigate the transcriptional mechanism of the PD-L1 gene. Co-IP/IB was used to examine the interaction between MEF2C and PD-L1, including MEF2C ubiquitination. IHC staining was carried out to analyse the expression of IL-17RA, MEF2C, TRIM31, and PD-L1 in NSCLC tissue arrays. The corresponding plasmids were constructed and identified. An isograft model was used to verify the findings in vitro. PD-L1, MEF2C and TRIM31 expression levels were increased in NSCLC tissues and NSCLC cells exposed to IL-17. Mechanistically, MEF2C could bind to the − 778 to -475 nt and − 336 to -97 nt regions of the PD-L1 promoter. TRIM31 could mediate MEF2C K63-linked polyubiquitination at Lys 25, increasing MEF2C recruitment to the PD-L1 promoter and PD-L1 gene transcription. MEF2C, TRIM31 or PD-L1 gene silencing effectively suppressed MEF2C K63-linked polyubiquitination, PD-L1 induction and NSCLC growth in mice inoculated with Lewis lung cancer (LLC) cells transfected with the corresponding shRNA and treated with IL-17. IL-17 induces PD-L1 gene transcription in NSCLC cells through TRIM31-dependent MEF2C K63-linked polyubiquitination.

The cancer cell metastasis is a major death reason for patients with non-small cell lung cancer (NSCLC). Although researchers have disclosed that interleukin 17 (IL-17) can increase matrix metalloproteinases (MMPs) induction causing NSCLC cell metastasis, the underlying mechanism remains unclear. In the study, we found that IL-17 receptor A (IL-17RA), p300, p-STAT3, Ack-STAT3, and MMP19 were up-regulated both in NSCLC tissues and NSCLC cells stimulated with IL-17. p300, STAT3 and MMP19 overexpression or knockdown could raise or reduce IL-17-induced p-STAT3, Ack-STAT3 and MMP19 level as well as the cell migration and invasion. Mechanism investigation revealed that STAT3 and p300 bound to the same region (−544 to −389 nt) of MMP19 promoter, and p300 could acetylate STAT3-K631 elevating STAT3 transcriptional activity, p-STAT3 or MMP19 expression and the cell mobility exposed to IL-17. Meanwhile, p300-mediated STAT3-K631 acetylation and its Y705-phosphorylation could interact, synergistically facilitating MMP19 gene transcription and enhancing cell migration and invasion. Besides, the animal experiments exhibited that the nude mice inoculated with NSCLC cells by silencing p300, STAT3 or MMP19 gene plus IL-17 treatment, the nodule number, and MMP19, Ack-STAT3, or p-STAT3 production in the lung metastatic nodules were all alleviated. Collectively, these outcomes uncover that IL-17-triggered NSCLC metastasis involves up-regulating MMP19 expression via the interaction of STAT3-K631 acetylation by p300 and its Y705-phosphorylation, which provides a new mechanistic insight and potential strategy for NSCLC metastasis and therapy.

目的:研究人胚肾293T(HEK 293T,简称293T)细胞中外源性肿瘤坏死因子受体相关因子(tumor necrosis factor receptor-associated factor 6,TRAF6)与 Krüppel样因子5(Krüppel-like factor 5,KLF5)的结合及TRAF6多聚泛素化修饰KLF5 的方式和修饰的位点.方法:将构建的Flag-TRAF6、HA-KLF5、泛素过表达质粒、shTRAF6小干扰质粒和TRAF6 C70A位点突变质粒行不同组合转染293T细胞48 h.用免疫沉淀(immunoprecipitation,IP)和免疫印迹(immunoblotting,IB)实验检查TRAF6与KLF5的结合以及KLF5 K63或K48多聚泛素化水平.此外,构建KLF5全部赖氨酸突变的质粒,分别与TRAF6质粒共转染293T细胞.用前述IP/IB检测KLF5 K63连接的多聚泛素化修饰,并确定KLF5 K63泛素化修饰的位点.结果:293T细胞中TRAF6能与KLF5结合;TRAF6过表达和基因沉默或TRA6酶活性缺失能相应上调或下调KLF5 K63的多聚泛素化;KLF5被TRAF6 K63多聚泛素化修饰的位点是其第99位和第100位的赖氨酸.结论:TRAF6能与KLF5相互作用,并对KLF5-K99和K100进行K63多聚泛素化修饰.

Rat Thy-1 nephritis (Thy-1N) is an experimental model for studying human mesangioproliferative glomerulonephritis (MsPGN), and its pathological features are glomerular mesangial cell (GMC) proliferation and extracellular matrix (ECM) accumulation. Although we have confirmed that renal lesions of Thy-1N rats are sublytic C5b-9-dependent, and ECM accumulation is related to tissue inhibitor of matrix metalloproteinase (TIMP) inhibiting matrix metalloproteinase (MMP) activity, whether sublytic C5b-9 can induce TIMP production by GMC in Thy-1N rat and the underlying mechanism remains unclear. In the study, we proved that the expressions of TIMP3, krϋppel-like transcription factor 5 (KLF5) and tumor necrosis factor receptor-associated factor 6 (TRAF6) were simultaneously up-regulated both in the renal tissues of Thy-1N rats (in vivo) and in the GMC exposed to sublytic C5b-9 (in vitro). Further mechanism exploration discovered that KLF5 and TRAF6 as two upstream molecules could induce TIMP3 gene transcription through binding to the same region i.e., -1801nt to -1554nt (GGGGAGGGGC) and -228nt to -46nt (GCCCCGCCCC) of TIMP3 promoter. In the process, TRAF6 mediated KLF5 K63-linked ubiquitination at K99 and K100 enhancing KLF5 nuclear localization and binding to TIMP3 promoter, augmenting its gene activation. Furthermore, the experiments in vivo exhibited that silencing KLF5, TRAF6 or TIMP3 gene could markedly lessen renal KLF5 K63-linked ubiquitination or TIMP3 induction, ECM accumulation and other pathological changes of Thy-1N rats. Besides, the positive expressions of above-mentioned these proteins and ECM accumulation and their correlation in the renal tissues of MsPGN patients were also demonstrated. Overall, our findings implicate that KLF5 and TRAF6 play a promoting role in sublytic C5b-9-triggered TIMP3 gene transcription and expression, which might provide a novel mechanistic insight into rat Thy-1N and human MsPGN.

目的:检查人胶质母细胞瘤(glioblastoma,GBM)组织和细胞系中ZBTB3的表达,并探讨ZBTB3对GBM细胞增殖和克隆形成的影响及其调控机制.方法:通过GEPIA2数据库分析GBM患者肿瘤组织中ZBTB3的表达情况.RT-PCR、qPCR和Western blot检测GBM细胞系(U251、U373、U87)中ZBTB3的mRNA和蛋白表达水平,筛选出ZBTB3表达最高的U87细胞.CCK-8和克隆形成实验检测沉默ZBTB3基因对U87细胞增殖和克隆形成的影响.用p38MAPK、AMPK、Akt1抑制剂处理U87细胞后,Western blot 检测 p38MAPK、AMPK、Akt1 的磷酸化水平,RT-PCR、qPCR 和 Western blot 测定 ZBTB3 的 mRNA 和蛋白表达水平,CCK-8和克隆形成实验检测细胞增殖和克隆形成.结果:GBM患者肿瘤组织中ZBTB3的表达显著高于正常组织.U251、U373和U87细胞中均可见ZBTB3的表达,其中U87细胞表达最高.沉默ZBTB3基因能明显抑制U87细胞的增殖和克隆形成.抑制AMPK既能显著降低U87细胞ZBTB3的表达水平,又可明显减弱U87细胞增殖和克隆形成.结论:GBM组织和细胞系中ZBTB3的表达显著上调,GBM细胞中AMPK活化并上调ZBTB3基因的表达,促进GBM细胞的增殖和克隆形成.

目的:探讨长链非编码RNA01518(long intergenic non-protein coding RNA 01518,LINC01518)基因过表达或沉默对白介素(interleukin,IL)-17诱导非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞增殖的影响.方法:Western blot检测NSCLC 细胞系(PC9、H1299 和 H1975)IL-17 受体 A(IL-17 receptor A,IL-17RA)的表达后,用 IL-17 刺激 H1299 和 PC9 细胞不同时间,利用CCK-8实验测定细胞的增殖水平.LncRNA芯片筛查IL-17刺激H1299和PC9细胞3h后LncRNA上调的结果,从中选取某些上调的LncRNA进行RT-PCR和Real-time PCR验证.此外,将构建的pcDNA3.1/LINC01518或shLINC01518质粒转染H1299细胞,用CCK-8、EdU和克隆形成实验检查细胞的增殖情况.结果:3种NSCLC细胞系均有IL-17RA的表达.实验发现,IL-17刺激H1299和PC9细胞后能提高其增殖水平,同时受刺激的细胞内LINC01518的上调最为显著,且过表达LINC01518可提高H1299细胞的增殖水平,而沉默LINC01518基因则能抑制IL-17诱导的细胞增殖.结论:LINC01518能促进IL-17诱导的H1299细胞增殖.

Interleukin-6 (IL-6) overproduction has been considered to contribute to inflammatory damage of glomerular mesangial cells (GMCs) in human mesangial proliferative glomerulonephritis (MsPGN) and its rat model called Thy-1 nephritis (Thy-1N). However, the regulatory mechanisms of IL-6 expression in GMCs upon sublytic C5b-9 timulation remain poorly understood. We found that Krüppel-like factor 4 (KLF4) bound to the IL-6 promoter (−618 to −126 nt) and activated IL-6 gene transcription. Furthermore, lysine residue 224 of KLF4 was acetylated by p300/CBP-associated factor (PCAF), which was important for KLF4-mediated transactivation. Moreover, lysine residue 5 on histone H2B and lysine residue 9 on histone H3 at the IL-6 promoter were also acetylated by PCAF, which resulted in an increase in IL-6 transcription. Besides, NF-κB activation promoted IL-6 expression by elevating the expression of PCAF. Overall, these findings suggest that sublytic C5b-9-induced the expression of IL-6 involves KLF4-mediated transactivation, PCAF-mediated acetylation of KLF4 and histones, and NF-κB activation in GMCs.

目的:构建大鼠S100钙结合蛋白A8(S100A8)基因启动子荧光素酶报告质粒,并在HEK-293T中检查过表达性别决定区Y框蛋白7(SOX7)基因对S100A8启动子活性的影响,同时筛选可能的SOX7结合元件.方法:采用PCR技术扩增大鼠S100A8启动子全长,经双酶切后连接到pGL3-basic中,命名为pGL3-S100A8-FL.将pGL3-S100A8-FL与前期构建的pIRES2-SOX7质粒共转染HEK-293T,再测定荧光素酶活性.此外,运用JASPAR预测S100A8启动子区可能包含的SOX7结合元件,并依此构建4个启动子截短质粒(即pGL3-S100A8-1～4).将pGL3-S100A8-FL和pGL3-S 100A8-1～4分别与pIRES2-SOX7共转染HEK-293T,检查荧光素酶活性.接着构建SOX7结合元件突变的S100A8启动子质粒(即pGL3-S100A8-M),与pIRES2-SOX7转染HEK-293T,检测其荧光素酶活性.结果:将pGL3-S100A8-FL与pIRES2-SOX7共转染HEK-293T,发现过表达SOX7可显著增加pGL3-S100A8-FL启动子活性.将pGL3-S100A8-FL和pGL3-S100A8-1～4分别与pIRES2-SOX7共转染HEK-293T,发现pGL3-S100A8-4启动子活性显著低于pGL3-S100A8-FL和pGL3-S100A8-1～3,提示SOX7与S100A8启动子结合元件可能位于-200～+51 nt区域内.将-86～-57 nt元件突变质粒(pGL3-S100A8-M)或pGL3-S100A8-FL与pIRES2-SOX7共转HEK-293T,发现pGL3-S100A8-M启动子活性显著低于pGL3-S100A8-FL.提示SOX7可能与S100A8启动子-86～-57 nt元件结合.结论:成功构建大鼠S100A8基因启动子全长、截短和突变荧光素酶报告质粒,并初步确定S100A8启动子区的SOX7结合元件.

目的:探讨GCN5调控sublytic C5b-9刺激大鼠肾小球系膜细胞(glomerular mesangial cell,GMC)诱导Cyclin D1基因表达以及GCN5乙酰化修饰SOX9转录因子的作用.方法:先用Western blot检测Thy-1肾炎(Thy-1 nephritis,Thy-1N)大鼠的肾组织和sublytic C5b-9刺激的GMC中GCN5、SOX9和Cyclin D1的表达.然后分别将pIRES2-GCN5过表达和shGCN5小干扰质粒或将这些质粒与Cyclin D1启动子质粒行不同组合转染GMC,用荧光素酶报告基因实验和real-time PCR、Western blot检测过表达或沉默GCN5后对Cyclin D1表达的影响.同时用免疫共沉淀(Co-IP)和Western blot检测sublytic C5b-9刺激或分别转染GCN5过表达和酶活性突变质粒(△GCN5)的GMC中SOX9的乙酰化修饰.结果:Thy-1N大鼠肾组织和sublytic C5b-9刺激的GMC中GCN5、SOX9、CyclinD1蛋白水平均明显上调,而过表达GCN5基因能上调CyclinD1的启动子活性、mRNA和蛋白表达;敲低GCN5表达则得到了相反的结果.此外,sublytic C5b-9刺激和过表达GCN5基因促进了SOX9的乙酰化修饰,而沉默GCN5后则降低了SOX9的乙酰化水平.结论:GCN5可上调GMC中CyclinD1的表达,并促进SOX9的乙酰化修饰.

目的:探讨C5a对非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞增殖和迁移的影响及其潜在的分子机制.方法:RT-PCR和Western blot检测人正常支气管上皮细胞系BEAS-2B和3种NSCLC细胞系(H1703、PC9、H1299)中C5a受体(C5aR)的表达;CCK-8和划痕实验检测C5a对PC9细胞增殖和迁移的影响;Western blot检查C5a刺激PC9细胞后蛋白激酶Akt1、细胞外信号调节激酶1/2 (extracellular signal-regulated kinase,ERK1/2)和蛋白激酶C-α (protein kinase C-α,PKC-α)表达及磷酸化水平;用Akt1抑制剂Perifosine和ERK1/2抑制剂U0126分别处理PC9细胞后再给予C5a刺激,Western blot检测Akt1和ERK1/2的表达和磷酸化及其上下游调控关系,CCK-8和划痕实验检测Perifosine和U0126对细胞增殖和迁移的影响.结果:PC9细胞C5aR的表达水平显著高于其他细胞;C5a可明显促进PC9细胞的增殖和迁移能力;C5a刺激PC9细胞后,可显著增强Akt1和ERK1/2的磷酸化;Akt1和ERK1/2抑制剂均能明显降低C5a刺激PC9细胞后引起的细胞增殖和迁移,且Akt1抑制剂不仅能减弱Akt1的磷酸化,还能减弱ERK1/2的磷酸化,而ERK1/2抑制剂则仅能阻断其自身的磷酸化.结论:C5a刺激NSCLC细胞后可能通过激活Akt1-ERK1/2通路促进细胞的增殖和迁移.

目的:探讨大鼠核因子-κB (nuclear factor-κB,NF-κB)p65亚基在细胞内过表达和其活性变化对于扰素调节因子-8(interferon regulatory factor-8,IRF-8)基因启动的影响,并初步筛查IRF-8启动子上可能的p65结合元件.方法:采用聚合酶链式反应(polymerase chain reaction,PCR)技术扩增大鼠p65基因蛋白编码区(complete sequence coding,CDS)序列,将其插入到空载pIRES2-EGFP质粒中,构建大鼠野生型(wild type,WT)p65过表达质粒(pIRES2-p65 WT).在此基础上,将p65第535位丝氨酸(serine,S)突变为天冬氨酸(aspartic,D)或丙氨酸(alanine,A),分别构建p65持续活化突变型质粒(pIRES2-p65 S535D)和p65显性负性突变型质粒(pIRES2-p65 S535A).之后应用生物信息学软件预测IRF-8基因启动子区p65的结合元件,并据此构建IRF-8启动子全长(full-length,FL)和3个截短的荧光素酶报告质粒,即pGL3-IRF-8-FL(-1 892～+174 nt)、pGL3-IRF-8-1(-1 360～+174 nt)、pGL3-IRF-8-2(-752～+174 nt)和pGL3-IRF-8-3(-68 +174 nt).将上述质粒行不同组合共转染人胚肾293T(humanembryonic kidney 293T,HEK-293T)细胞,Western blot和荧光素酶实验分别检查p65的表达和IRF-8的启动子活性,并分析IRF-8启动子区可能的p65结合元件.结果:菌液PCR及测序证实上述质粒构建成功.分别将pIRES2-p65 WT、pIRES2-p65S535D、pIRES2-p65 S535A和pGL3-IRF-8-FL共转染HEK-293T,发现过表达pIRES2-p65 WT或pIRES2-p65 S535D均可明显增加IRF-8启动子活性,且以pIRES2-p65 S535D更为显著;而过表达pIRES2-p65 S535A后,IRF-8启动子活性无明显变化.将pGL3-IRF-8-FL、pGL3-IRF-8-1～3和pIRES2-p65 S535D共转染HEK-293T后发现,pGL3-IRF-8-3的启动子活性显著低于pGL3-IRF-8-FL、pGL3-IRF-8-1和pGL3-IRF-8-2,提示大鼠IRF-8启动子-752～68 nt区域可能存在p65结合元件.结论:在HEK-293T细胞内过表达野生型或持续活化突变型p65可显著促进IRF-8基因的启动,且p65与IRF-8启动子的结合元件可能位于-752～-68 nt部位.

HEADING AIMS:LncRNA HOXB-AS3 is proved as an oncogene in tumors. Herein, we determine the function and mechanism of HOXB-AS3 in epithelial ovarian cancer (EOC) cells.MATERIALS AND METHODS:Chi-square test, Kaplan-Meier (KM) analysis and Cox regression analysis were used to analyze the clinicopathological features of HOXB-AS3 in EOC patients. CCK8, transwell and wound healing assay were used to test the function of HOXB-AS3. Luciferase reporter assay, western blot and glycolysis rate assay were used for further mechanistic studies.KEY FINDINGS:HOXB-AS3 was abundantly expressed in EOC tissues, and higher levels of HOXB-AS3 in EOC patients were significantly associated with disease status and overall survival status. EOC patients with high levels of HOXB-AS3 had strikingly shorter disease-free survival (DFS) and overall survival (OS) times than those with low levels. HOXB-AS3 also might as an independent prognostic factor. Further study revealed knockdown of HOXB-AS3 significantly inhibited the proliferation, invasion and migration of EOC cells. Mechanistic investigations suggested that knockdown of HOXB-AS3 could decrease lactate dehydrogenase A (LDHA) expression and the extracellular acidification rate (ECAR) by sponging miR-378a-3p.SIGNIFICANCE:To our knowledge, this is the first study to suggest that HOXB-AS3 could crosstalk with miRNA in the cytoplasm and alter glycolysis in cancer cells. Our results improve our understanding of the mechanism of HOXB-AS3 and suggest that HOXB-AS3 can act as a predictor of OS and a target for EOC therapies.

为了研究翻转课堂教学模式在医学免疫学教学中的可行性及其效果,对超敏反应章节的教学进行了翻转课堂的实践,同时采用问卷调查和与学生座谈的方式对这种教学方法的效果进行分析.结果 显示,学生普遍喜欢此种教学方法,并对其教学效果给予了较高的评价,认为翻转课堂能够激发学习兴趣,充分发挥自主学习的优势,促进学习中的沟通与交流.课堂实践表明,翻转课堂教学模式在医学免疫学的教学中有较高的应用价值.

Interleukin 17 (IL-17), as a pro-inflammatory cytokine, is up-regulated in the sera and tumor tissues of glioma patients; however the effects of IL-17 on glioma proliferation and migration remain unclear. In this study, the roles of IL-17 in the proliferation and migration of glioma cells and their potential mechanisms were determined. The results showed that IL-17 could not only enhance the proliferation and migration of cultured glioma cells (in vitro), but also promote the tumor formation of glioma cells in BALB/c nude mice (in vivo). Mechanical exploration revealed that IL-17 stimulation could increase the phosphorylation levels of Akt1 and NF-κB-p65 in glioma cells, and knockdown or inhibition of PI3K, Akt1 and NF-κB-p65 could also reduce the IL-17-induced proliferation and migration of the glioma cells. Moreover, PI3K/Akt1 was the upstream regulator of NF-κB-p65 activation in IL-17-incubated glioma cells. Furthermore, the inhibition of PI3K, Akt1 and NF-κB-p65 markedly suppressed the tumor formation of glioma cells induced by IL-17. Together, these data indicate that IL-17 can promote the proliferation and migration of glioma cells via PI3K/Akt1/NF-κB-p65 activation, and these findings might provide a new insight into glioma pathogenesis.

临床医学专业"5+3"一体化是我国培养卓越医生的一种新模式.鉴于医生在诊治疾病时,需要应用逻辑思维与推理,故"5+3"临床专业医学生的培养环节中,增加科研思维与实践的训练,提高其解决问题的能力极为重要.作者结合自身课题组带教"5+3"临床医学生的经验,介绍了在基础医学学习阶段对这些本科生进行科研思维和科研能力训练的方法,同时还对目前培养环节中存在的一些问题进行了分析和思考.

医学免疫学理论性强,概念抽象,知识点相互渗透、相互依存,具有难理解、难记忆和难应用之特点.在本科生免疫学教学实践中摸索出一种将传统教学、网络教学和PBL教学联合应用、并能取长补短的三位一体的教学方法.该教学法实施后已取得了初步成效,激发了学生学习兴趣,提高了免疫学教学质量.

目的:构建大鼠巨噬细胞炎性蛋白-1α (macrophage inflammatory protein-1α,MIP-1α)基因启动子(全长和截短)荧光素酶报告质粒,并观察在人胚肾细胞(HEK-293T)中过表达干扰素调节因子-8(interferon regulatory factor-8,IRF-8)对MIP-1α基因启动活性的影响,同时筛选其可能的IRF-8结合元件.方法:采用PCR技术,扩增出大鼠MIP-1α基因启动子序列,将MIP-1α基因启动子插入到荧光素酶报告基因载体pGL3-basic中,构建MIP-1α基因启动子全长荧光素酶报告质粒(pGL3-MIP-1α-FL).将上述pGL3-MIP-1α-FL和本课题组已构建的大鼠IRF-8过表达质粒(pIRES2-IRF-8)共转染HEK-293T细胞,检测细胞内荧光素酶活性,确定IRF-8对MIP-1α基因的启动作用.同时,应用生物信息学软件预测MIP-1α基因启动子上IRF-8的结合元件,并据此构建3个MIP-1α基因启动子截短的荧光素酶报告质粒(pGL3-MIP-1α-1～3).将上述MIP-1α基因启动子全长和各截短的荧光素酶报告质粒和IRF-8过表达质粒共转染HEK-293T细胞,再测定荧光素酶活性,初步确定IRF-8的结合元件.结果:菌液PCR及核酸测序证实,上述pGL3-MIP-1α-FL(-1 400～+94 nt)质粒构建成功.将pGL3-MIP-1α-FL和pIRES2-IRF-8共转染HEK-293T后发现,过表达IRF-8可显著增加MIP-1α基因启动子活性.应用生物信息学软件预测发现MIP-1α基因启动子上IRF-8的结合元件(-1 157～-1 144nt、-740～-734 nt、-683～-670 nt、-365～-359 nt、-249～-236 nt),并据此构建3个MIP-1α基因启动子截短的荧光素酶报告质粒,即pGL3-MIP-1α-1(-453～+94 nt)、pGL3-MIP-1α-2(-352～+94 nt)和pGL3-MIP-1α-3(-3～+94 nt).将pGL3-MIP-1α-FL、pGL3-MIP-1α-1～3和pIRES2-IRF-8共转染HEK-293T后发现,pGL3-MIP-1α-3的启动活性显著低于pGL3-MIP-1α-FL、pGL3-MIP-1α-1和pGL3-MIP-1α-2.提示IRF-8可能结合在大鼠MIP-1α基因启动子的-352～-3 nt区域的IRF-8结合元件(-249～-236 nt)上.结论:本实验成功构建了大鼠MIP-1α基因启动子全长及截短荧光素酶报告质粒,并初步筛查出IRF-8在MIP-1α基因启动子上的可能结合元件,为后续研究奠定了基础.

目的:检查信号转导与转录激活因子3 (signal transducer and activator of transcription 3,STAT3)对人胶质瘤细胞程序性死亡配体1 (programmed death-ligand 1,PD-L1)基因转录和表达的影响,同时筛选PD-L1基因启动区可能的STAT3结合元件.方法:培养U251细胞,给予STAT3抑制剂(BP-1-102)或DMSO处理,9h后用流式细胞术检查细胞表面PD-L1蛋白的表达情况.将PD-L1基因启动子全长荧光素酶报告质粒(pGL3-PD-L1-FL)转染U251细胞,再给予BP-1-102或DMSO处理,9h后检查细胞内荧光素酶活性.将PD-L1基因启动子全长和各截短(pGL3-PD-L1-1～4)荧光素酶报告质粒和STAT3过表达质粒(pC-MV-STAT3)共转染HEK293细胞,再测定荧光素酶活性,初步确定STAT3的结合区域.应用生物信息学软件预测PD-L1基因启动子上STAT3的结合元件,并开展结合元件突变实验确定有效性.结果:BP-1-102可明显下调U251细胞中PD-L1的基因转录和蛋白表达.此外,将pGL3-PD-L1-FL、pGL3-PD-L1-1～4和pCMV-STAT3共转染HEK293细胞后,pGL3-PD-L1-4的启动活性显著低于其他质粒,提示STAT3可能结合于PD-L1基因启动子-200～0 nt区域.生物信息学软件预测发现,此区域含有2个STAT3的结合元件,分别位于-194～-184 nt和-135～-125 nt部位.进一步研究揭示突变-194～-184 nt和-135～-125 nt元件均可显著下调pGL3-PD-L1-FL的荧光素酶活性,联合突变更为明显.结论:本研究成功筛查出STAT3在人PD-L1基因启动子上的可能结合区域,为后续研究胶质瘤细胞中STAT3相关的PD-L1基因转录调控奠定了基础.

目的:检查特异性蛋白1 (specificity protein 1,SP1)在Thy-1肾炎(Thy-1 nephritis,Thy-1N)大鼠肾组织中和亚溶解型C5b-9 (sublytic C5b-9)刺激的大鼠肾小球系膜细胞(glomerular mesangial cells,GMCs)中的表达情况,并探讨SP1表达上调对sublytic C5b-9促大鼠GMCs增殖的作用.方法:复制大鼠Thy-1N模型,同时用sublytic C5b-9刺激体外培养的大鼠GMCs,分别用real-time PCR和Western blot检查Thy-1N大鼠肾组织(体内)和sublytic C5b-9刺激的大鼠GMCs(体外)中SP1基因的mRNA和蛋白表达水平及其表达时相.与此同时,构建SP1基因的表达质粒(pEGFP-N 1/SPl-His)和发夹状小干扰RNA质粒(SP1shRNA,shSPl).将上述质粒分别转染大鼠GMCs(同时设对照质粒转染组),再给予或不给予sublytic C5b-9刺激.用real-time PCR和Western blot检查SP1表达量,并行CCK-8实验检查GMCs的增殖情况.结果:在Thy-1N大鼠肾组织和sublytic C5b-9刺激的大鼠GMCs中,SP1 mRNA和蛋白表达水平均显著上调,且其体内外表达时相较为一致.体外过表达SP1可显著促进GMCs增殖,而沉默SP1可明显抑制sublytic C5b-9诱导的GMCs增殖反应.结论:Sublytic C5b-9刺激大鼠GMCs后能通过上调SP1表达促进GMCs增殖.