Correction for 'Association between low-sodium salt intervention and long-term blood pressure changes is modified by ENaC genetic variation: a gene-diet interaction analysis in a randomized controlled trial' by Hao Sun et al., Food Funct., 2023, 14, 9782-9791, https://doi.org/10.1039/D3FO02393A.

Objective： To observe roproductive hormone levels in varicocele patients during a cycle (6 years) of Wu Yun Liu Qi, and explore whether the cycle had effect on the roproductive hormone levels. Methods： Data of roproductive hormone levels in varicocele patients from 2015 to 2020 were analyzed retrospectively. FSH、LH、T、PRL、E2 levels and T/E2 ratio were compared among the six years. According to Chinese sexagenary cycle heavenly stems and earthly branches of each year from 2015 to 2020 its yunqi characteristics were determined. Results: Totally data of 848 cases of varicocele patients were collected from 2015 to 2020. Among which, in 2015 (Yiwei year) there were 57 cases, in 2016 (Bingshen year) 83 cases, in 2017 (Dingyou year ) 133 cases, in 2018(Wuxu year) 156 cases, in 2019(Sihai year) 274 cases, and in 2020(Gengzi year) 145 cases. The levels of FSH、LH、PRL、T were not diferrent statistically from the six years except individual year. However, the level of E2 in 2016 when the Yunqi was Shao Yang Xiang Huo Si Tian and Jue Yin Feng Mu Zai Quan obviously higher than other years excpet 2018( All P< 0.05). And T/E2 ratio was lower in 2016 than other years except 2018 and 2020( All P< 0.05). Conclusions: Shi Xiang factors of Wu Yun Liu Qi had effect on roproductive hormone levels in varicocele patients, showing by higher E2 level in Yinshen year when the Shi Xiang factors may have bad effect on human fertility.

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.

Mycobacterium tuberculosis 6-kDa early secretory antigenic target (ESAT-6) is a dominant target antigen for cell-mediated immunity in the early phase of tuberculosis. The fms-like tyrosine kinase 3 ligand (FL) that induces potent immune response has been used as an adjuvant in vaccine development. In this study, a new recombinant plasmid (pIRES-epitope-peptides-FL) encoding three T cell epitopes of ESAT-6 and FL was constructed, and the immunogenicity of the DNA vaccine was assessed in C57BL/6 mice immunized with the plasmid DNA vaccine. Additionally, a strategy of intramuscular injection with the DNA vaccine (prime) and intranasal administration of the epitope peptides (boost) was employed to induce higher immune reaction of the mice. The results showed that mice vaccinated with the recombinant plasmid DNA vaccine and boosted with the peptides not only increased the levels of Th1 cytokines (IFN-γ and IL-12), the number of IFN-γ(+) T cells and activities of cytotoxic T lymphocytes as well as IgG, but also enhanced protection against Mycobacterium tuberculosis challenge. In conclusion, these data indicate that the novel recombinant pIRES-epitope-peptides-FL plasmid is a useful DNA vaccine for preventing Mycobacterium tuberculosis infection.

Mesangioproliferative glomerulonephritis (MsPGN) is a common human kidney disease. Rat Thy-1 nephritis (Thy-1N) is an animal model widely used for the study of MsPGN. Thy-1N is not only sublytic C5b-9-dependent, but also related to pro-inflammatory cytokine production and macrophage (Mφ) accumulation in rat renal tissues. In this study, we found that the expression or phosphorylation of chemokine CCL3/4, CD68 (Mφ marker), IRF-8, PKC-α and NF-κB-p65 (p65) were all up-regulated both in the renal tissues of Thy-1N rats (in vivo) and in the glomerular mesangial cells (GMCs) upon sublytic C5b-9 stimulation (in vitro). Further experiments in vitro revealed that the phosphorylated PKC-α (p-PKC-α) could promote p65 phosphorylation, and then p-p65 enhanced IRF-8 expression through binding to IRF-8 promotor (-591 ~ -582 nt and -299 ~ -290 nt). Additionally, up-regulation or silencing of IRF-8 gene promoted or reduced CCL3/4 production, and then regulated Mφ chemotaxis. The underlying mechanism involved in IRF-8 binding to CCL3 promoter (-249 ~ -236 nt), which resulted in CCL3 gene transcription. The experiments in vivo showed that knockdown of renal PKC-α, p65, IRF-8 and CCL3/4 genes could inhibit CCL3/4 production, Mφ accumulation, GMC proliferation and proteinuria of Thy-1N rats. Furthermore, p-PKC-α, p-p65, IRF-8, CCL3/4 expression and Mφ accumulation were also increased in the renal tissues of MsPGN patients. Collectively, these findings indicate that sublytic C5b-9 induces CCL3/4 production and Mφ accumulation via PKC-α/p65/IRF-8 axis, and finally aggravates the pathological changes of MsPGN.

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.

Glomerular mesangial cell (GMC) proliferation is a histopathological alteration in human mesangioproliferative glomerulonephritis (MsPGN) or in animal models of MsPGN, e.g., the rat Thy‐1 nephritis (Thy-1N) model. Although sublytic C5b-9 assembly on the GMC membrane can trigger cell proliferation, the mechanisms are still undefined. We found that sublytic C5b-9-induced rat GMC proliferation was driven by extracellular signal‐regulated kinase 1/2 (ERK1/2), sry-related HMG-box 9 (SOX9), and Cyclin D1. Here, ERK1/2 phosphorylation was a result of the calcium influx-PKC-α-Raf-MEK1/2 axis activated by sublytic C5b-9, and Cyclin D1 gene transcription was enhanced by ERK1/2-dependent SOX9 binding to the Cyclin D1 promoter (−582 to −238 nt). In addition, ERK1/2 not only interacted with SOX9 in the cell nucleus to mediate its phosphorylation at serine residues 64 (a new site identified by mass spectrometry) and 181 (a known site), but also indirectly induced SOX9 acetylation by elevating the expression of general control non-repressed protein 5 (GCN5), which together resulted in Cyclin D1 synthesis and GMC proliferation. Moreover, our in vivo experiments confirmed that silencing these genes ameliorated the lesions of Thy‐1N rats and reduced SOX9 phosphorylation, acetylation and Cyclin D1 expression. Furthermore, the renal tissue sections of MsPGN patients also showed higher phosphorylation or expression of ERK1/2, SOX9, and Cyclin D1. In summary, these findings suggest that sublytic C5b-9-induced GMC proliferation in rat Thy-1N requires SOX9 phosphorylation and acetylation via enhanced Cyclin D1 gene transcription, which may provide a new insight into human MsPGN pathogenesis.

Rat Thy-1 nephritis (Thy-1N) is an animal model of human mesangioproliferative glomerulonephritis (MsPGN), accompanied by glomerular mesangial cell (GMC) proliferation and extracellular matrix (ECM) deposition. Although sublytic C5b-9 formed on GMC membrane could induce cell proliferation, the mechanism is still unclear. In this study, we first demonstrated that the level of SRY related HMG-BOX gene 9 (SOX9), general control nonderepressible 5 (GCN5), fibroblast growth factor 1 (FGF1) and platelet-derived growth factor α (PDGFα) was all elevated both in the renal tissues of Thy-1N rats (in vivo) and in the GMCs (in vitro) with sublytic C5b-9 stimulation. Then, we not only discovered that sublytic C5b-9 caused GMC proliferation through increasing SOX9, GCN5, FGF1 and PDGFα expression, but also proved that SOX9 and GCN5 formed a complex and combined with FGF1 and PDGFα promoters, leading to FGF1 and PDGFα gene transcription. More importantly, GCN5 could mediate SOX9 acetylation at lysine 62 (K62) to enhance SOX9 binding to FGF1 or PDGFα promoter and promote FGF1 or PDGFα synthesis and GMC proliferation. Besides, the experiments in vivo also showed that FGF1 and PDGFα expression, GMC proliferation and urinary protein secretion in Thy-1N rats were greatly reduced by silencing renal SOX9, GCN5, FGF1 or PDGFα gene. Furthermore, the renal tissues of MsPGN patients also exhibited positive expression of these genes mentioned above. Collectively, our findings indicate that GCN5, SOX9 and FGF1/PDGFα can form an axis and play an essential role in sublytic C5b-9-triggered GMC proliferation, which might provide a novel insight into the pathogenesis of Thy-1N and MsPGN.

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:探讨转录因子Krüppel样因子5(Krüppel-like factor,KLF5)调控sublytic C5b-9刺激大鼠肾小球系膜细胞(glomerular mesangial cell,GMC)合成促炎因子白细胞介素(interleukin,IL)-36α的作用.方法:首先,培养大鼠GMC,体外用sublytic C5b-9刺激GMC后,不同时间点行反转录聚合酶链式反应(reverse transcription polymerase chain reaction,RT-PCR)和蛋白质印迹(Western blot)检查KLF5、IL-36α mRNA和蛋白水平的变化.接着,构建KLF5的过表达(pIRES2-KLF5)及发夹状小干扰RNA(shKLF5)质粒.将pIRES2-KLF5转染GMC或在shKLF5转染GMC后再用sublytic C5b-9刺激,行RT-PCR和Western blot检查过表达或沉默KLF5基因后对GMC合成IL-36α的影响.同时用荧光素酶报告实验检查过表达或沉默KLF5基因对IL-36α启动子活性的影响.结果:用sublytic C5b-9刺激GMC,能显著上调KLF5和IL-36α的mRNA和蛋白表达,且KLF5的表达时相早于IL-36α.过表达KLF5能上调IL-36α的生成,而沉默KLF5基因后再行sublytic C5b-9刺激,由GMC产生的IL-36α则显著下降.sublytic C5b-9刺激GMC或过表达KLF5基因均可提高IL-36α启动子的活性,而沉默KLF5基因则能明显减低sublytic C5b-9上调IL-36α启动子的活性.结论:KLF5的表达对sublytic C5b-9诱导GMC合成IL-36α有促进作用.

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.

Rat Thy-1 nephritis (Thy-1N) is an experimental mesangial proliferative glomerulonephritis (MsPGN) for studying human MsPGN. Although sublytic C5b-9 complex formation on glomerular mesangial cells (GMCs) and renal MCP-1 and RANTES production in rats with Thy-1N have been proved, the role and mechanism of MCP-1 or RANTES synthesis in GMCs induced by sublytic C5b-9 are poorly elucidated. In this study, we first found the expression of transcription factor (KLF6), co-activator (KAT7) and chemokines (MCP-1 and RANTES) was all up-regulated both in renal tissue of Thy-1N rats (in vivo) and in sublytic C5b-9-induced GMCs (in vitro). Further in vitro experiments revealed that KLF6 bound to MCP-1 promoter (-297 to -123 nt) and RANTES promoter (-343 to -191 nt), leading to MCP-1 and RANTES gene transcription. Meanwhile, KAT7 also bound to the same region of MCP-1 and RANTES promoter in a KLF6-dependent manner, and KLF6 was acetylated by KAT7 at lysine residue 100, which finally promoted MCP-1 and RANTES expression. Moreover, our in vivo experiments discovered that knockdown of renal KAT7 or KLF6 gene obviously reduced MCP-1 and RANTES production, GMCs proliferation, ECM accumulation, and proteinuria secretion in Thy-1N rats. Collectively, our study indicates that sublytic C5b-9-induced MCP-1 and RANTES synthesis is associated with KAT7-mediated KLF6 acetylation and elevated KLF6 transcriptional activity, which might provide a new insight into the pathogenesis of rat Thy-1N and human MsPGN.

目的:研究沉默大鼠肾组织中通用控制核苷酸合成5 (general control nonderepressible 5,GCN5)基因、性别决定区Y框蛋白9(SRY related HMG box-9,SOX9)基因对Thy-1肾炎(Thy-1 nephritis,Thy-1N)大鼠肾组织内转化生长因子-β1 (transforming growth factor-β1,TGF-β1)生成的影响.方法:分别用慢病毒(lentivirus,LV)包装GCN5和SOX9发夹状小干扰RNA(shRNA),即制备LV-shGCN5和LV-shSOX9重组病毒.然后行大鼠肾动脉灌注术将LV-shGCN5和LV-shSOX9分别导入大鼠肾脏,再经尾静脉注射兔抗大鼠胸腺细胞抗血清(anti-thymocyte serum,ATS)复制Thy-1N模型.在注射ATS后3h,取大鼠肾组织,用RTPCR和Western blot检查各组大鼠肾组织中GCN5和SOX9的mRNA及蛋白表达水平,以验证干扰效果及GCN5、SOX9对TGF-β1产生的影响.结果:利用肾动脉灌注术将LV-shGCN5或LV-shSOX9重组病毒导入大鼠肾组织后,不仅能有效沉默相应的靶基因,而且还能下调TGF-β1的表达.结论:沉默大鼠肾组织中GCN5或SOX9基因后能显著抑制Thy-1N大鼠肾内TGF-β1的生成.

Mesangioproliferative glomerulonephritis (MsPGN) is characterized by the proliferation of glomerular mesangial cells (GMCs) and accumulation of extracellular matrix (ECM), followed by glomerulosclerosis and renal failure of patients. Although our previous studies have demonstrated that sublytic C5b‐9 complex formed on the GMC membrane could trigger GMC proliferation and ECM expansion of rat Thy‐1 nephritis (Thy‐1N) as an animal model of MsPGN, their mechanisms are still not fully elucidated. In the present studies, we found that the levels of response gene to complement 32 (RGC‐32), myeloid zinc finger 1 (MZF1), phosphorylated extracellular signal‐regulated kinase 5 (phosphorylated ERK5, p‐ERK5), F‐box only protein 28 (FBXO28) and TNF receptor‐associated factor 6 (TRAF6) were all markedly up‐regulated both in the renal tissues of rats with Thy‐1N (in vivo) and in the GMCs upon sublytic C5b‐9 stimulation (in vitro). Further in vitro experiments revealed that up‐regulated FBXO28 and TRAF6 could form protein complex binding to ERK5 and enhance ERK5 K63‐ubiquitination and subsequent phosphorylation. Subsequently, ERK5 activation contributed to MZF1 expression and MZF1‐dependent RGC‐32 up‐regulation, finally resulting in GMC proliferative response. Furthermore, the MZF1‐binding element within RGC‐32 promoter and the functions of FBXO28 domains were identified. Additionally, knockdown of renal FBXO28, TRAF6, ERK5, MZF1 and RGC‐32 genes respectively markedly reduced GMC proliferation and ECM production in Thy‐1N rats. Together, these findings indicate that sublytic C5b‐9 induces GMC proliferative changes in rat Thy‐1N through ERK5/MZF1/RGC‐32 axis activated by the FBXO28‐TRAF6 complex, which might provide a new insight into MsPGN pathogenesis.

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.

Department of Pathology and Pathophysiology, Faculty of Basic Medical Sciences, Kunming Medical University, Kunming 650500, People’s Republic of China; Department of Physiology, School of Basic Medical Sciences, Wuhan University School of Medicine, Wuhan 430071, People’s Republic of China; Department of Neurosurgery, Renmin Hospital of Wuhan University, Wuhan 430060, People’s Republic of China; Department of Physiology, School of Basic Medical Sciences, Hubei University of Medicine, Shiyan, Hubei 442000, People’s Republic of China; School of Pharmacy, Wuhan University, Wuhan 430071, People’s Republic of China; Department of Neurology, The Central Hospital of Wuhan, Tongji Medical College of Huazhong University of Science & Technology, Wuhan 430013, People’s Republic of China; Institute of Neuroregeneration & Neurorehabilitation, Collaborative Innovation Center for Brain Science, Department of Neurosurgery of the Affiliated Hospital, Qingdao University, Qingdao 266071, People’s Republic of China

目的:探讨甘氨酸在大鼠实验性脑出血中的作用及其机制.方法:成年SD大鼠经左侧纹状体输注自体血100 μL,造模后1h侧脑室注射甘氨酸(0.2～3.0 mg/kg),甘氨酸注射前0.5 h注射Akt抑制剂Ⅳ(100 μmol/L,2μL).结果:脑出血早期血肿周围脑组织PTEN表达增加;甘氨酸降低PTEN表达,增加AKT磷酸化(Ser473)水平;甘氨酸治疗减少脑出血后死亡的神经元数量,减轻血脑屏障损伤及脑水肿,减小血肿面积,改善神经行为学评分;而Akt抑制剂Ⅳ部分阻断了甘氨酸的保护作用.结论:首次证实甘氨酸通过PTEN/Akt信号通路减轻大鼠脑出血损伤.

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.