15-脂氧合酶-1表达与活性调节及其机制

5-脂氧合酶-1(15-LOX-1)可将亚油酸、花生四烯酸分别代谢为13-羟基十八碳二烯酸和15-羟基二十碳四烷酸。15-LOX-1及其代谢产物在肿瘤的发生发展过程中起着重要作用。15-LOX-1在机体内的分布具有组织特异性,其表达量和酶活性受其基因结构、细胞质Ca2+及自身自杀性灭活的调节;在不同肿瘤组织中其表达活性也不同。IL-4,IL-13,组蛋白脱乙酰化酶抑制剂、非甾体类抗炎药及DNA甲基转移酶抑制剂等多种生物分子能够在体外诱导15-LOX-1表达。     

L1型金属β-内酰胺酶的原核表达及特性研究

目的：对临床分离的嗜麦芽窄食单胞菌所产L1型金属β-内酰胺酶基因进行原核表迭，并检测多种β-内酰胺类抗生素对重组L1酶的稳定性。方法：PER扩增L1酶的编码基因，将其亚克隆入pUCm—T载体，测序后将L1基因克隆至表达栽体pET-41a（＋）后在大肠埃希菌BL21（DE3）中表达，分光光度计测定多种β-内酰胺类抗生素对L1型金属β-内酰胺酶的稳定性。结果：本实验中的L1酶与国外同类酶的氨基酸同源性为92．44％。重组L1酶水解青霉素及碳青霉烯类抗生素的能力接近，氨曲南及头孢他啶对L1酶高度稳定。结论：对L1型金属B内酰胺酶的克隆测序及成功表达为进一步研究该酶的分子生物学特性奠定了基础。抗生素对L1酶的稳定性研究为指导临床合理应用抗生素提供了科学的理论依据。     

白三烯对微循环的影响及其临床意义

白三烯对微循环的影响及其临床意义附图白三烯的生理功能戴顺龄＊白细胞三烯（Ｌｅｕｋｏｔｒｉｅｎｅｓ，ＬＴｓ，白三烯）是广泛存在于机体的一簇具有高度生物活性的低分子磷脂化合物，是花生四烯酸经脂氧酶代谢途径而生成的衍生物，含有不同羟基的二十碳四烯酸。自从１...     

环氧化酶-2在胃癌发生发展中的作用研究进展

胃癌是严重危害人类生命健康的恶性肿瘤，传统的治疗方法不足以降低其致死率。探索胃癌新的防治措施及其发生发展的分子机制一直为人们所关注。环氧化酶-2（cyclooxygenase-2，cox4）是分解花生四烯酸、生成各种内源性前列腺素（PG）过程中重要的诱生型限速酶。近年来的研究发现，COX-2在胃癌中的表达明显增高，而抑制COX-2表达有可能预防和逆转胃癌的发生，表明COX-2在胃癌的发生发展过程中起重要作用，     

胱硫醚-γ-裂解酶在三种微血管内皮细胞上的表达

目的鉴定微血管内皮细胞上是否表达生成硫化氢（H2s）的酶,为深入研究硫化氢的心血管作用提供新线索。方法原代培养大鼠心脏微血管内皮细胞（CMEC）,设计相应引物后用RT—PCR的方法检测胱硫醚-1-裂解酶（CSE）和胱硫醚-β-成酶（CBS）基因转录情况,进一步用兔抗大鼠CSE和CBS多克隆抗体对CMEC进行蛋白水平的免疫荧光染色。此外对另两种微血管内皮细胞株RF／6A和bEnd．3也检测了H2S生成酶的表达。结果三种微血管内皮细胞都扩增出CSE基因的特异条带,而CBS基因都未扩增出相应产物。CMEC的免疫荧光染色显示CSE抗体染色为阳性,CBS抗体染色为阴性。结论首次发现了微血管内皮细胞上存在CSE的表达,提示内皮细胞可以通过生成硫化氢调节心血管活动。     

表面修饰羟基磷灰石修复骨缺损骨形态发生蛋白-2的表达

目的了解精氨酸-甘氨酸-天冬氨酸多肽表面修饰的羟基磷灰石（hydroxyapatite,HA）修复节段性骨缺损局部骨形态发生蛋白-2（bone morphogenefic protein-2，BMP-）的表达。方法以骨髓基质干细胞（marrow stromal cels，MSCs）复合Arg-Gly-Asp（RGD）多肽表面修饰的HA或单纯材料培养制备组织工程骨，选择60只新西兰白兔。制作15mm长的桡骨节段性骨缺损模型，根据植入不同的材料分为A、B、C、D组。A组：骨缺损区植入MSCs复合RGD多肽表面修饰的HA培养制备的组织工程骨；B组：骨缺损区植入MSCs复合HA培养制备的组织工程骨；C组：骨缺损区植入RGD多肽表面修饰的HA；D组：骨缺损区植入HA。术后4周取材，行修复区局部BMP-2免疫组化分析。结果术后4周各组骨缺损区均有新骨生成，修复区局部BMP-2表达水平依次为：A〉B〉C〉D（P〈0．05）。结论RGD多肽表面修饰对以HA为支架材料组织工程骨的修复作用有明显优化作用。     

N6-甲基腺苷甲基化修饰对肿瘤PD-L1表达调控的研究进展

随着对肿瘤免疫逃逸机制的深入研究, 以PD-1/PD-L1抑制剂为代表的免疫检查点抑制剂极大地改变了包括非小细胞肺癌在内的多种晚期癌症患者的治疗前景, 在癌症治疗中取得了突破性进展。在肿瘤发生发展过程中, PD-L1的表达受多种机制调控, 包括:基因组学改变、表观遗传学调控、转录后加工和蛋白翻译后修饰等。越来越多的研究表明, N6-甲基腺苷甲基化是真核信使RNA中常见的RNA转录后修饰, 在细胞分化、组织发育以及肿瘤的发生发展过程中起着重要作用。N6-甲基腺苷甲基化水平由甲基转移酶、去甲基化酶和甲基化识别酶调节, 现就上述3种酶的异常修饰对肿瘤PD-L1表达的调控作一综述。     

胆管癌中m7G甲基化调节因子相关预后风险模型的构建及临床意义

目的 表观遗传重编程在胆管癌（CCA）进展中起关键作用,m7G甲基化修饰是转录后调控中最常见的碱基修饰形式之一,其通过调节肿瘤相关基因的表达在增殖、侵袭和转移等多种肿瘤相关的生物学活动中发挥着巨大作用。实验研究是探索m7G甲基化调节因子构建的相关修饰模式及其对CCA临床预后及治疗效果的评估。方法 分析基于癌症基因组图谱（TCGA）和基因表达综合数据库（GEO）中27个m7G甲基化调节因子[Argonaute2蛋白（AGO2）,细胞质脆性X智力迟钝蛋白1 (FMR1)相关蛋白1 (CYFIP1),脱帽mRNA2 (DCP2)、清道夫脱帽酶（DCPS）,真核翻译起始因子3(EIF3)亚基D(EIF3D),真核翻译起始因子4A1(EIF4A1),真核翻译起始因子4E(EIF4E),真核翻译起始因子4E家族成员1B(EIF4E1B),真核翻译起始因子4E家族成员2(EIF4E2),真核翻译起始因子4E家族成员3(EIF4E3),真核翻译起始因子4伽玛3(EIF4G3),宝石核细胞器相关蛋白5(GEMIN5),干扰素诱导蛋白5(IFIT5),La核糖核蛋白1(LARP1),Sm样蛋白（LSM1）...     

15-HETE与ERK1/2在大鼠缺氧性肺动脉收缩中的作用

目的:为揭示缺氧性肺血管收缩机制,探讨细胞外信号调节激酶1/2 (ERK1/2)是否参与15-羟基二十碳四烯酸（15-HETE）收缩缺氧大鼠肺动脉的过程以及15-HETE对 ERK1/2活性的影响。 方法: 将大鼠置于氧气分数为12.0％的低氧箱中连续9 d形成缺氧模型。完整取出心肺，在显微镜下分离直径0.8-1.0 mm肺动脉剪为3 mm长的动脉环在组织浴槽内进行张力研究。比较15-HETE给药前后肺动脉环张力变化；用ERK1/2上游激酶抑制剂U0126孵育肺动脉环，比较U0126孵育前后15-HETE对缺氧性肺动脉环的收缩作用；机械法去除动脉环内皮，再比较U0126孵育前后15-HETE的收缩作用。酶法分离培养大鼠肺动脉平滑肌细胞。Western blotting方法检测15-HETE作用时间（5-90 min）及浓度（10^-9-10^-6 mol/L）对 ERK1/2 的表达及活性的影响。 结果: 15-HETE对缺氧大鼠肺动脉环有收缩作用，呈浓度-效应关系，与正常对照组比较差异显著（P<0.05）；内皮完整和内皮去除的肺动脉环，U0126孵育前后，15-HETE的缩血管作用都受到抑制，差异显著（均为P<0.05）。Western bloting结果显示，15-HETE明显增强肺动脉血管平滑肌细胞ERK1/2的活性，随时间延长而降低，随浓度增加而增加，但对ERK1/2的蛋白表达无影响。 结论:15-HETE能上调大鼠肺动脉血管平滑肌细胞ERK1/2的活性，提示ERK1/2的活化是15-HETE收缩慢性缺氧大鼠肺动脉的一个重要环节。     

5-脂氧合酶及其激活蛋白在大鼠腹腔巨噬细胞中的表达

自三烯类是一类花生四烯酸(AA)的5-脂氧合酶(5-lo)代谢通路的产物,具有多种生物学活性,与炎性疾病、肿瘤和老年性痴呆等有密切的关系。5-lo是这一通路的关键酶,可催化AA转变为5-氢过氧化二十碳四烯酸(5-HPETE),并进一步使其转化为白三烯A4(LTA4)。5-lo的活性除了依赖钙离子和ATP外,还必须有5-脂氧合酶激活蛋白(FLAP)的存在。5-lo和5-脂氧合酶激活蛋白(flap)都不是看家基因。5-lo和flap表达于中性粒细胞、单核巨噬细胞和肥大细胞等细胞中。人外周血单核细胞在培养到第6 d后,表现     

Reduced 15-lipoxygenase-2 immunostaining in prostate adenocarcinoma: correlation with grade and expression in high-grade prostatic intraepithelial neoplasia

Arachidonic acid (AA) metabolites are implicated in the oncogenesis of several tumors, including prostate cancer. 15-Lipoxygenase-2 (15-LOX-2) is a novel AA-metabolizing enzyme with a limited tissue distribution, which includes prostate, lung, skin, and cornea. Previous studies have shown that 15-LOX-2 is present in benign prostate secretory cells and reduced in prostate adenocarcinoma and that production of the 15-LOX-2 metabolite 15S-hydroxyeicosatetraenoic acid is reduced in malignant compared with benign prostate. The objective of this study was to determine the frequency with which 15-LOX-2 immunostaining is reduced in prostate carcinoma and to correlate reduced expression with tumor differentiation (grade) and other pathologic parameters in radical prostatectomy specimens. Paraffin immunoperoxidase with a polyclonal antibody specific for 15-LOX-2 was performed on tumors and benign portions from 70 cases, and the percentage of tumor immunostaining for 15-LOX-2 was assessed. Whereas uniform 15-LOX-2 immunostaining was observed in secretory cells of benign glands, it was markedly reduced or absent in most adenocarcinomas: 23 of 70 tumors showed completely absent 15-LOX-2 immunostaining, and 45 of 70 cases showed negative immunostaining in more than 50% of the tumor. The extent of reduced 15-LOX-2 immunostaining correlated with tumor differentiation, with retained expression particularly in Gleason score 5 tumors versus a significant reduction of 15-LOX-2 in higher-grade tumors (mean +/- SD tumor 15-LOX-2 positive: Gleason score 5 = 67%+/-30%, Gleason score 6 = 16%+/-30%, Gleason score 7 = 23%+/-28%, Gleason score > or =8 = 41%+/-46%). In 16 cases with multifocal tumors or different foci of the same tumor with different grades, the higher-grade foci had significantly reduced 15-LOX-2 expression compared with the lower-grade foci. In peripheral zone tumors without complete loss of 15-LOX-2 expression, there was a significant inverse relationship between 15-LOX-2 immunostaining and tumor volume. There was not a significant correlation between 15-LOX-2 immunostaining and serum PSA or pathologic stage. In a subset of 27 cases, 15-LOX-2 expression in high-grade prostatic intraepithelial neoplasia (HGPIN) glands was significantly reduced compared with benign glands. These data show that in contrast to the uniform expression of 15-LOX-2 in differentiated secretory cells of benign prostate, reduced 15-LOX-2 is a common alteration in prostate carcinoma, and this correlates with tumor cell differentiation. That reduced expression is seen in HGPIN suggests that this may be an early alteration in carcinoma development.     

15-Lipoxygenase-2 (15-LOX-2) Is Expressed in Benign Prostatic Epithelium and Reduced in Prostate Adenocarcinoma

Human 15S-lipoxygenase-2 (15-LOX-2) is a recently identified lipoxygenase that has approximately 40% sequence identity to the known human 5S-, 12S-, and 15S-lipoxygenases. 15-LOX-2 has a limited tissue distribution, with mRNA detected in prostate, lung, skin, and cornea, but not in numerous other tissues, including peripheral blood leukocytes. In the current study, we have characterized the distribution of 15-LOX-2 in the human prostate by immunohistochemistry, demonstrated the ability of benign prostate tissue to form 15S-hydroxyeicosatetraenoic acid (15S-HETE) from exogenous arachidonic acid (AA), and begun characterizing possible alterations in 15-LOX-2 in prostate adenocarcinoma. Incubation of benign prostate tissue with [14C]AA resulted in formation of [14C]15-HETE, as determined by reverse- and straight-phase high-performance liquid chromatography. 15-HETE was the major AA metabolite formed. By immunohistochemistry, 15-LOX-2 is located in secretory cells of peripheral zone glands and large prostatic ducts and somewhat less uniformly in apical cells of transition and central zone glands. 15-LOX-2 was not detected in the basal cell layer, stroma, ejaculatory ducts, seminal vesicles, or transitional epithelium. Immunostaining of 18 radical prostatectomy specimens showed a loss of 15-LOX-2 in the majority of prostate adenocarcinomas; 14 of 18 cases showed loss of 15-LOX-2 in >25% of the tumor (mean, 74.9% negative for 15-LOX-2; range, 38.9% to 100%). Incubation of paired pure benign and pure malignant prostate tissue from the same radical prostatectomies showed that 15-HETE formation was markedly reduced (>90%) or undetectable in incubations of prostate adenocarcinoma. 15-LOX-2 is a novel human lipoxygenase with a limited tissue distribution that is strongly expressed in benign prostate glandular epithelium and lost to a variable degree in the majority of prostate adenocarcinomas.     

The role of 15-LOX-1 in colitis and colitis-associated colorectal cancer

Introduction

Chronic inflammation is known to be mechanistically linked to the development of cancer. This article reviews and discusses the role of 15-lipoxygenase-1 (15-LOX-1) in the resolution of colitis and prevention of colitis-associated colorectal cancer.

Discussion

15-LOX-1 is an inducible and highly regulated enzyme in cells that play an important role in the production of lipid signaling mediators from linoleic acid and arachidonic acid. Together, these acids and 15-LOX-1 are the driving force for the resolution of acute and chronic inflammation in normal cells. Widespread inflammation can progress from local inflammation to ulcerative colitis, tumorigenesis, and finally invasive, metastatic, or benign colon cancer. Thus, reversing inflammation will halt the proliferation of cancerous cells. Decreased expression of 15-LOX-1 may lead to the development of colitis-associated colorectal cancer and colorectal cancer.

Conclusion

n-3 Polyunsaturated fatty acids are potent anti-inflammatory and pro-resolution products of 15-LOX-1 that can potentially prevent colitis-associated colorectal cancer and colorectal cancer.

     

A strategy for promoting bone regeneration by inducible expression of 15-LOX-1

Repairing large bone defects is a major orthopaedic problem, with current treatments being constrained by the regenerative capacity of human tissue. Current methods for repairing bone defects include osteogenesis, osteoconduction, osteoinduction, and tissue engineering; however, the cumulative effect of these methods is, as of yet, rather unsatisfactory. Recent research has demonstrated that knockout of the cell cycle inhibitor p21, which works as a switch to control regenerative capacity, can promote cell proliferation and appendage regeneration. The enzyme 15-lipoxygenase type 1 (15-LOX-1), which is involved in arachidonic and linoleic acid metabolism, has the potential to down-regulate the expression of p21. Therefore, we hypothesise that the construction of a new bone substitute that expresses 15-LOX-1 locally will promote osteoblast proliferation through inhibition of p21, resulting in an effective and inducible method for promoting bone regeneration.     

IL-15 mediates antigen-induced neutrophil migration by triggering IL-18 production

We have investigated the mechanisms underlying IL-15-induced neutrophil migration into inflamed tissues. IL-15 induced neutrophil migration to the peritoneal cavity in mice in a time- and dose-dependent manner. The cell migration was not induced in IL-18-/-, MIP-1alpha (CCL3)-/-, TNFR1-/- or 5-LOX-/- mice but was normal in IFN-gamma-/- mice. IL-15-induced neutrophil migration was inhibited by anti-MIP-2 (CXCL2) antibody or MK886 (leukotriene synthesis inhibitor). IL-18-induced neutrophil migration was also dependent on TNFR1, MIP-1alpha, MIP-2 and leukotriene. Consistent with this observation, IL-15 induced IL-18 production, and IL-15 or IL-18 injection induced the production of MIP-2, MIP-1alpha, TNF-alpha and LTB4. In an antigen-specific inflammation model, ovalbumin (OVA)-induced neutrophil migration was completely inhibited by soluble IL-15Ralpha (sIL-15Ralpha) or anti-MIP-2 antibody. Furthermore, cell migration was absent in IL-18-/-, MIP-1alpha-/-, TNFR1-/-, or 5-LOX-/- mice. OVA challenge induced the release of MIP-2, MIP-1alpha, TNF-alpha and LTB4 in the peritoneal cavity in an IL-15- and IL-18-dependent manner. We also found that neutrophils from the peripheral blood and synovial fluid of patients with rheumatoid arthritis produced substantial amounts of IL-18 and LTB4 following activation by IL-15. Together, these results demonstrate that IL-15 plays an important role in antigen-induced neutrophil migration during inflammation, triggering a sequential OVA, IL-15, IL-18, MIP-2, MIP-1alpha, TNF-alpha, LTB4 and neutrophil migration signaling cascade.     

Differential regulation of interleukins IL-13 and IL-15 by ovarian steroids, TNF-alpha and TGF-beta in human endometrial epithelial and stromal cells

Based on the endometrial spatial and temporal expression of interleukins (ILs) IL-13 and IL-15 during the normal menstrual cycle, we hypothesized that ovarian steroids and non-steroidal factors regulate their expression in a cell-specific manner. To test this hypothesis and determine IL-13/IL-15 actions, we used endometrial epithelial (EEC) and stromal (ESC) cells isolated and cultured under defined conditions. We confirmed the expression of IL-13 and IL-15 in these cells and further demonstrated that 17beta estradiol (E2), medroxyprogesterone acetate (MPA) and their combination differentially regulated their mRNA expression and protein production in a time- and cell-specific manner (P < 0.05). We also showed that tumour necrosis factor-alpha (TNF-alpha; 10 and 25 ng/ml) and transforming growth factor-beta (TGF-beta; 1 and 5 ng/ml), cytokines with inflammatory and immune regulatory functions in a cell- and dose-dependent manner regulate the expression of IL-13 and IL-15 (P < 0.05). Functionally, IL-13 and IL-15 1-100 ng/ml displayed a limited mitogenic activity towards EEC and ESC; however, they regulated the expression of TNF receptor type 1 (TNFR) mRNA and soluble protein in a cell-specific manner (P < 0.05). We conclude that ovarian steroids, TNF-alpha and TGF-beta act as key regulators of endometrial IL-13 and IL-15 expression which act locally regulating TNFR expression in a cell-specific manner. Based on these findings, we conclude that IL-13/IL-15, either alone or through their interactions with other cytokines, influence the outcome of endometrial inflammatory/immune responses during the normal menstrual cycle, and due to their altered expression may extend these processes in dysfunctional bleeding and endometriosis.     

Regulation of 15-lipoxygenase isozymes and mucin secretion by cytokines in cultured normal human bronchial epithelial cells

OBJECTIVE AND DESIGN: To study the effects of IL-4, IL-13, IL-1beta, IL-8 and TNFalpha on 15-lipoxygenase(15-LO) isozyme expression and mucin secretion in normal human bronchial epithelial cells. MATERIAL AND METHODS: The effects of IL-4, IL-13, IL-1beta, IL-8 and TNFalpha on 15-LO isoenzyme mRNA and protein expression, total 15-LO enzyme activity and mucin secretion were examined in cultures of normal human bronchial epithelial cells. In addition, in order to determine whether the observed effects on mucin secretion were due to lipoxygenase (LO) products, the effect of the non-selective LO inhibitor nordihydroguaiaretic acid was examined. RESULTS: IL-4 and IL- 13 selectively enhanced 15-LOa mRNA and protein levels, and total 15-LO enzyme activity. In contrast, no changes were observed in 15-LOb mRNA or protein levels. IL-4 and IL-13 both reduced mucin secretion in this cell type, however the non-selective LO inhibitor nordihydroguaiaretic acid had no effect on this action of IL-4. CONCLUSIONS: These data demonstrate that IL-4 and IL-13 selectively regulate the expression of the 15-LOa isozyme. However, 15LOa products do not mediate the IL-4-induced reduction in mucin secretion observed in this cell type.