Expression and function of interleukin-11 and its receptor alpha in the human endometrium

The interleukin-11 (IL-11) receptor alpha has an important function in decidualization of mouse endometrial stroma but the function of IL-11 and its receptor in the human endometrium remains unknown. The mRNA for IL-11 and its receptor alpha in human endometrial tissue samples were analysed by semi-quantitative RT-PCR and RNase protection assays respectively. The proteins were detected in frozen endometrial tissue samples by immunofluorescence. The effect of heparin-binding epidermal growth factor (HB-EGF) on secretion of IL-11 by cultured endometrial stromal cells was assessed by enzyme-linked immunosorbent assay. The proliferative potential of IL-11 in endometrial stromal cells was assessed by [(3)H]thymidine uptake. IL-11 and its receptor alpha mRNAs and proteins were detected in the endometrium throughout the cycle. Distinct patterns of localization of the ligand and receptor were observed. HB-EGF induced IL-11 secretion by cultured stromal cells, and IL-11 induced [(3)H]thymidine uptake by these cells. Our data suggest that IL-11-receptor interactions may perform different functions in the human endometrium at different stages of the cycle, and that secretion of IL-11 is modulated by local growth factors.     

Expression and regulation of human alveolar macrophage-derived interleukin-1 receptor antagonist.

The alveolar macrophage (AM) is the sentinel immune cell of the distal airspace of the lung. These mononuclear phagocytic cells represent the major host defense against inhaled environmental agents. When activated, the AM has the capacity to release reactive oxygen and arachidonic acid metabolites and produce a number of cytokines, such as interleukin-1 (IL-1). This latter cytokine has pleiotropic effects on a variety of cells and has been implicated as one of the preeminent mediators of acute inflammation. Recently, an IL-1 receptor antagonist (IRAP) has been isolated, purified, and cloned from peripheral blood monocytes (PBM) stimulated with either adherent IgG (adhIgG) lipopolysaccharide (LPS), or phorbol myristate acetate. IRAP acts as a true receptor antagonist without agonist activity. We postulated that the AM would be a significant cellular source of IRAP from the lung. To test this hypothesis, normal human AM were immediately isolated or stimulated in a dose-dependent fashion with either LPS or adhIgG. For comparison, PBM were also isolated and treated in a similar manner. PBM expressed steady-state IRAP mRNA by Northern blot analysis only in response to LPS or adhIgG. In contrast, AM were found to express significant levels of antigenic IRAP by Western blot analysis, immunostaining, and specific ELISA, and express steady-state levels of IRAP mRNA under unstimulated culture conditions. Moreover, LPS or adhIgG failed to induce AM-derived IRAP antigen generation over unstimulated control.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of interleukin-1 and interleukin-1 receptor antagonist by human rheumatoid synovial tissue macrophages.

Interleukin-1 (IL-1) has protean effects in the pathogenesis of rheumatoid arthritis (RA). These effects include production of prostaglandins and collagenase from rheumatoid fibroblasts as well as upregulation of adhesion molecule expression on these cells. IL-1 can activate monocytes and neutrophils, as well as promote the growth of fibroblasts and endothelial cells. Recently, a novel interleukin-1 receptor antagonist protein (IRAP) has been isolated, purified, cloned, and expressed, which may modulate the effects of IL-1. In this study, we present data demonstrating that macrophages isolated from human RA synovial tissues express both IL-1 and IRAP genes. In addition, RA synovial tissue macrophages and lining cells display IL-1 and IRAP antigenic expression by immunohistochemistry. In contrast, osteoarthritis synovial tissues, as compared to RA, have fewer IL-1 and IRAP-positive macrophages. Thus, the production of IL-1 balanced by IRAP may affect the joint destruction found in these diseases.     

重组人白细胞介素1受体拮抗蛋白荧光质粒在软骨细胞的表达

背景：白细胞介素1受体拮抗蛋白能延缓骨性关节炎进程,通过转基因方法可以使白细胞介素1受体拮抗蛋白表达的增加。 目的：观察重组人重组人白细胞介素1受体拮抗蛋白荧光质粒的构建及经脂质体转染软骨细胞的表达情况。 方法：双酶切法切取重组人白细胞介素1受体拮抗蛋白的c-DNA片段,通过T4DNA连接酶连接到pEGFP-C1载体上。体外分离培养兔关节软骨细胞,然后用构建的重组人白细胞介素1受体拮抗蛋白质粒经脂质体转染软骨细胞,通过荧光显微镜观察转基因的表达和荧光定量PCR检测其表达。 结果与结论：获得重组人pEGFP-C1-IL-1Ra真核表达载体质粒,酶切及测序结果证明表达质粒的DNA序列完全正确。荧光显微镜观察有绿色荧光蛋白表达,荧光定量PCR鉴定证实转染的软骨细胞基因得到表达。     

Expression of prolactin-releasing peptide and its receptor in the human decidua

Human decidua and decidualized endometrial cells produce prolactin (PRL). Several growth factors and cytokines have been shown to regulate decidual PRL release, but a specific PRL-releasing substance remains to be characterized. Prolactin-releasing peptide (PrRP) is a peptide isolated from the brain and distinguished by its potent and specific stimulation of PRL release by cultured pituitary cells. Here, we demonstrate that human decidua expresses immunoreactive PrRP as well as the mRNAs encoding PrRP and its receptor. First trimester deciduas were obtained from women undergoing elective termination of pregnancy. Tissue specimens were stained by immunohistochemistry using a rabbit anti-human PrRP-31 antibody, and PrRP was localized in both epithelial cells of the decidual glands and in stromal cells, with diffuse distribution and no special relation with the neighbourhood of blood vessels. In primary cultures of decidual stromal cells, PrRP and PrRP receptor gene expression were detected using RT-PCR, and the identity of the PCR products was further confirmed by restriction enzyme digestion. The effect of PrRP on decidual PRL release was also evaluated, and there was a significant increase in PRL production (135 +/- 4% of control levels, P < 0.05) after incubation of decidual stromal cells with synthetic PrRP. The expression of PrRP and PrRP receptor in human decidual cells and the ability of PrRP to induce PRL secretion by cultured decidual cells suggests that this peptide may be a novel local modulator of decidual PRL release.     

人白介素24腺病毒载体构建及其生物学活性分析

目的：克隆人白介素24基因，构建其腺病毒载体，获取病毒重组子，并研究其生物学括性。方法：用密执毒素（Mezerein）诱导HeLa细胞表达IL-24，通过RT-PCR获取IL-24 cDNA，将其亚克隆至pAdTrack-CMV载体，经PmeⅠ线性化后，与腺病毒的骨架载体pAdEasy-Ⅰ在BJ5183菌中同源重组，HEK293细胞包装扩增，PCR、Western blot鉴定。AdIL-24处理宫颈癌CaSki细胞，通过MTT细胞存活实验检测其活性；Hoeehst33342染色、流式细胞仪检测凋亡；WesternNot检测Bax、Bd。2、p53蛋白的表达。结果：获取人IL-24的cDNA，序列与GeneBank公布序列完全一致，成功构建腺病毒载体，AdIL-24对CaSki细胞具有抑制生长、促进凋亡的作用，并上调Bax、p53蛋白表达，下调Bcl-2蛋白表达。结论：成功构建人IL-24的重组腺病毒载体，其病毒重组子具有生物活性。     

Expression of orexin A and its receptor 1 in the human prostate

The peptides orexin A (OXA) and orexin B, deriving from the cleavage of the precursor molecule prepro‐orexin, bind two G‐coupled transmembrane receptors, named as receptor 1 (OX1R) and receptor 2 for orexin, showing different affinity‐binding properties. First discovered in the rat hypothalamus, orexins and their receptors have been also found in many peripheral tissues where they exert neuroendocrine, autocrine and paracrine functions. Because inconclusive data on their localization in the mammalian prostate are reported, the aim of this study was to investigate the presence of prepro‐orexin, OXA and OX1R in the human normal and hyperplastic gland. Immunohistochemistry revealed the localization of both OXA and OX1R in the cytoplasm of the follicular exocrine epithelium of all tested normal and hyperplastic prostates. Positive immunostaining was mainly observed in the basal cells of the stratified epithelium, and only rarely in the apical cells. The expression of mRNAs coding for prepro‐orexin and OX1R and of proteins in the tissues was also ascertained by polymerase chain reaction and Western blotting analysis, respectively. In order to gain insights into the functional activity of OXA in the prostate, we administered different concentrations of OXA to cultured prostatic epithelial cells PNT1A. We first demonstrated that PNT1A cells express OX1R. The addition of OXA did not affect PNT1A cell proliferation, while it enhanced cAMP synthesis and Ca²⁺ release from intracellular storage. Overall, our results definitely demonstrate the expression of OXA and OX1R in the human prostate, and suggest an active role for them in the metabolism of the gland.     

人肝肿瘤细胞选择性白细胞介素-7剪接变异体cDNA的克隆和序列测定

目的:寻找人肝肿瘤细胞选择性的白细胞介素-7(IL-7)剪接变异体。方法:运用自行设计的IL-7引物,采用逆转录聚合酶链反应(RT-PCR)技术,分离正常肝组织、原发肝癌组织、培养肝癌细胞株IL-7mRNA,再对电泳所获条带进行克隆、测序。结果:正常肝组织、原发肝癌组织、培养肝癌细胞株(BEL-7402和SMMC-7721)都能检测到IL-7mRNA,而且从肝癌组织、培养肝癌细胞株分离出1条新带,经亚克隆及测序,证实该条带系人IL-7基因cDNA第4外显子缺乏所致。结论:肝癌组织及培养的肝癌细胞株存在选择性IL-7剪接变异体。该变异体的发现,为肝癌病人的免疫调节紊乱以及肝癌病人的临床免疫治疗提供了新的研究方向。     

Expression and regulation of interleukin-33 in human monocytes

Interleukin‐33 (IL‐33) is an IL‐1 family cytokine that has a role in regulating T helper type 2 cytokines and mast cell development. Expression of IL‐33 is also associated with chronic inflammatory conditions such as rheumatoid arthritis. However, there is little information regarding IL‐33 in myeloid cell immune responses, which are important in immunity and inflammation. We therefore investigated the expression, intracellular location and regulation of myeloid cell IL‐33 by lipopolysaccharide (LPS) from Escherichia coli and the periodontal pathogen Porphyromonas gingivalis. We detected IL‐33 messenger RNA in the human promonocytic cell line THP‐1, in monocytes derived from these cells and in primary human monocytes. However, IL‐33 was not expressed in primary monocyte‐derived dendritic cells. Stimulation of monocytes with E. coli LPS (Toll‐like receptor 4 agonist) and LPS from P. gingivalis (Toll‐like receptor 2 agonist) up‐regulated IL‐33 at both the messenger RNA and protein levels but IL‐1β and tumour necrosis factor‐α had no effect. The IL‐33 protein was mainly found in the cytoplasm of monocytes with no evidence of nuclear translocation in stimulated cells. Furthermore, no IL‐33 secretion was detected after stimulation with LPS and/or ATP. These data indicate that the function, if any, of IL‐33 in activated monocytes is primarily intracellular. Interestingly, immunofluorescence analysis indicated that IL‐33 was sequestered in the nucleus of monocytes undergoing apoptosis but released into the extracellular milieu by LPS‐stimulated cells in which necrosis had been induced by freeze–thawing. Therefore, this endorses the view that IL‐33 may function as an ‘alarmin’ and have a role in signalling cellular damage and inflammatory disease pathogenesis through release from damaged or necrotic cells.     

白细胞介素-6及其受体在卵巢组织中的表达

本研究通过检测白细胞介素-6（Interleukin-6,IL-6）及其受体在正常卵巢和多囊卵巢综合征（Polycystic ovary syndrome,PCOS）患者卵巢中的表达,测定其在血清中的含量,初步研究IL-6在卵巢组织的分布情况,探索其在卵泡形成发育中的作用及其对PCOS患者排卵障碍的影响。     

Expression of recombinant human interleukin-8 and its purification using a single buffer system

Chemokines, a class of small secreted proteins, direct immune cells to their target sites and play an important role in chronic inflammations and allergies. To study their interactions with their cellular receptors or potential inhibitors large quantities of chemokines are required. Here we present a fast and efficient strategy to purify the human chemokine interleukin-8 (IL-8, CXCL8). The chemokine is expressed with a pelB-leader peptide that is cleaved off its N-terminus by an endogenous bacterial peptidase. This yields wild-type 72aa IL-8 with a serine at its N-terminus. IL-8 is recovered in the soluble fraction after lysis while pelB-IL8 fusion protein remains in the pellet. Interleukin-8 is purified via cation exchange chromatography and heparin affinity chromatography using a single inexpensive buffer system. No dialysis or membrane filtration steps are required and the final protein fractions may be used without any desalting steps. The use of 0.5% Triton X-114 in the lysis buffer leads to low endotoxin levels in the resulting protein. The protein can be eluted from the gel filtration column with a variety of buffers and is ready to be used in binding assays and activity assays.     

FHL1在人胰腺癌族中的表达及其与胰腺癌的侵袭及转移的相关性研究

目的:探讨FHL1蛋白在胰腺癌组织中的表达及其与胰腺癌临床病理特征的关系.方法:利用Western blot检测62例胰腺癌手术切除标本与其相对应的癌旁胰腺组织中FHL1蛋白的表达.用Log-rank检验分析FHL1蛋白表达与预后的关系.结果:FHL1的蛋白表达在胰腺癌中与对应癌旁胰腺组织相比下降明显(0.197±0.042 vs. 0.508±0.272,P<0.01).同时,FHL1在胰腺癌中的表达与临床分期、病理分级、淋巴结转移、肿瘤累及范围、远处转移均有明显差异(P<0.05),与高表达FHL1组相比,低表达FHL1组患术后生存时间明显缩短(P<0.01).结论:FHL1的异常表达很可能与胰腺癌的侵袭、转移等密切相关,而这种异常表达又影响了胰腺癌患的预后.     

Modelling of fish interleukin-1 and its receptor

This paper presents original data regarding: the three-dimensional modelling of rainbow trout (Oncorhynchus mykiss) and sea bass (Dicentrarchus labrax) IL-1beta, the modelling of trout IL-1 receptor type I (IL-1RI) and the modelling of trout IL-1beta bound to its receptor. The 3D models of trout and sea bass IL-1beta molecules were predicted by comparison with those already available of human and mouse, and, in both cases, a structure consisting of a beta-trefoil fold was obtained, a motif well conserved during evolution of IL-1beta-related proteins. Moreover, a model for the rainbow trout IL-1 receptor alone and complexed with IL-1beta was predicted and compared to the murine model. Both ligand-receptor complex models were compared with the known crystal structure of the human IL-1beta/IL-1R complex. A cross-interaction of trout IL-1beta with the mouse receptor was also simulated. Such analysis and the predicted interaction energies calculated from the models have helped to explain the different biological efficacies of mammalian and fish IL-1beta molecules in assays based on mammalian target cells. The obtained data are discussed on the basis of evolutionary and applied perspectives.     

重组hIL-10在毕赤酵母X-33的表达及其生物活性鉴定

目的:用毕赤酵母表达hIL-10并探索合适的表达条件,为IL-10生物制品研发及临床应用奠定基础。方法:以0.5%甲醇诱导酵母X-33表达rhIL-10,用SDS-PAGE和Western blot分析鉴定rhIL-10,比较不同温度、不同pH值和有无PMSF条件下rhIL-10的表达水平。用ELISA法测定培养液rhIL-10含量,用Bradford法测定培养液总蛋白含量,计算rhIL-10占总蛋白的比例。以淋巴细胞转化试验分析rhIL-10的生物学活性。结果:SDS-PAGE后可见42 kD左右蛋白条带;Western blot也检测到42kD左右特异性蛋白质条带。pH值为6.0、温度在28℃并加入抑制剂时rhIL-10表达量最高值为20 mg/L,同时段培养液中总蛋白质含量为50 mg/L,rhIL-10占总蛋白比例为40%。在外周血淋巴细胞转化试验中,rhIL-10抑制了淋巴细胞的增殖。结论:hIL-10能在毕赤酵母高效表达,表达产物有较好生物活性;发酵条件对rhIL-10的表达水平影响大,改善发酵条件能促进rhIL-10的表达。