组蛋白乙酰化对A549和BEAS-2B细胞哮喘易感基因ADAM33的影响

目的:研究丁酸钠、曲古抑菌素A(trichostatin A,TSA)及腺病毒E1A相关的300 kD蛋白(adenoviral E1A binding protein of 300 kD,P300)介导的组蛋白乙酰化在人非小细胞肺癌A549细胞及人支气管上皮BEAS-2B细胞中对哮喘易感的解整合素金属蛋白酶33(a disintegrin and metalloproteinase 33,ADAM33)基因表达的影响。方法:将A549细胞及BEAS-2B细胞分别分为丁酸钠对照组(双蒸水处理)和1、2.5、5 mmol/L丁酸钠组,TSA对照组(0.1%二甲基亚砜处理)和0.2、0.4、0.8μmol/L TSA组,按照组别分别予以相应处理;另将BEAS-2B细胞分为对照组(转染P300突变质粒)和P300组(转染P300表达质粒);采用双荧光素酶报告基因法分析ADAM33启动子活性的变化,实时荧光定量PCR(quantitative real time-PCR,qRT-PCR)检测ADAM33 mRNA表达,蛋白质印迹法检测ADAM33蛋白表达。结果:在人非小细胞肺癌A549细胞中,与对照组相比,1 mmol/L丁酸钠组及0.2μmol/L TSA组ADAM33基因启动子活性明显降低(P<0.01);在BEAS-2B细胞中,与对照组相比,1 mmol/L丁酸钠组及0.2μmol/L TSA组ADAM33基因启动子活性、mRNA及蛋白相对表达水平明显降低(P<0.05或P<0.01)。加大丁酸钠、TSA药物浓度,ADAM33表达无显著差异。在人支气管上皮BEAS-2B细胞中,与对照组相比,P300组ADAM33启动子活性、mRNA和蛋白相对表达水平明显降低(P<0.05)。结论:丁酸钠、TSA通过组蛋白乙酰化降低人非小细胞肺癌A549细胞和人支气管上皮BEAS-2B细胞中ADAM33表达,P300通过组蛋白乙酰化降低人支气管上皮BEAS-2B细胞中ADAM33表达。

Transplantation of human placental chorionic plate-derived mesenchymal stem cells for repair of neurological damage in neonatal hypoxic-ischemic encephalopathy

Neonatal hypoxic-ischemic encephalopathy is often associated with permanent cerebral palsy,neurosensory impairments,and cognitive deficits,and there is no effective treatment for complications related to hypoxic-ischemic encephalopathy.The therapeutic potential of human placental chorionic plate-derived mesenchymal stem cells for various diseases has been explored.However,the potential use of human placental chorionic plate-derived mesenchymal stem cells for the treatment of neonatal hypoxic-ischemic encephalopathy has not yet been investigated.In this study,we injected human placental chorionic plate-derived mesenchymal stem cells into the lateral ventricle of a neonatal hypoxic-ischemic encephalopathy rat model and observed significant improvements in both cognitive and motor function.Protein chip analysis showed that interleukin-3 expression was significantly elevated in neonatal hypoxic-ischemic encephalopathy model rats.Following transplantation of human placental chorionic plate-derived mesenchymal stem cells,interleukin-3 expression was downregulated.To further investigate the role of interleukin-3 in neonatal hypoxic-ischemic encephalopathy,we established an in vitro SH-SY5Y cell model of hypoxic-ischemic injury through oxygen-glucose deprivation and silenced interleukin-3 expression using small interfering RNA.We found that the activity and proliferation of SH-SY5Y cells subjected to oxygen-glucose deprivation were further suppressed by interleukin-3 knockdown.Furthermore,interleukin-3 knockout exacerbated neuronal damage and cognitive and motor function impairment in rat models of hypoxic-ischemic encephalopathy.The findings suggest that transplantation of hpcMSCs ameliorated behavioral impairments in a rat model of hypoxic-ischemic encephalopathy,and this effect was mediated by interleukin-3-dependent neurological function.

Regulation of interleukin 33/ST2 signaling of human corneal epithelium in allergic diseases

AIM:To identify the function of ST2 and explore the role of IL-33/ST2 signaling in regulating the pro-allergic cytokine production in human corneal epithelial cells (HCECs). METHODS:Human corneal tissues and cultured primary HCECs were treated with IL-33 in different concentrations without or with different inhibitors to evaluate the expression, location and signaling pathways of ST2 in regulating production of pro-allergic cytokine and chemokine. The expression of mRNA was determined by reverse transcription and real time PCR, and protein production was measured by enzyme-linked immunosorbent assay (ELISA), immunohistochemical and immunofluorescent staining. ST2 protein was detected in donor corneal epithelium, and ST2 signal was enhanced by exposure to IL-33. ·RESULTS:IL-33 significantly stimulated production of pro-allergic cytokines thymic stromal lymphopoietin (TSLP) and chemokine (CCL2, CCL20, CCL22) in HCECs at both mRNA and protein levels. These stimulated productions of pro-allergic mediators by IL-33 were blocked by ST2 antibody or soluble ST2 protein(P 【0.05). Interestingly, the IκB-α inhibitor BAY11-7082 or NF-κB activation inhibitor quinazoline blocked NF-κB p65 protein nuclear translocation, and also suppressed the productions of these pro-allergic cytokines and chemokine induced by IL-33. CONCLUSION:These findings demonstrate that IL-33/ ST2 signaling plays an important role in regulating IL-33 induced pro-allergic responses. IL-33 and ST2 could become novel molecular targets for the intervention ofallergic diseases in ocular surface.

Study of recombinant human interleukin-12 for treatment of complications after radiotherapy for tumor patients

AIM To evaluate the treatment effects of recombinant human interleukin-12(rh IL-12) on radiotherapy complications, such as severe myelosuppression or pancytopenia, the decline or imbalance of immune function, etc.METHODS The patients received high-dose and short-course precise radiotherapy, such as Cyber knife and image-guided radiotherapy(IGRT), which can cause myelosuppression or pancytopenia and immune function decline within a short time. One-hundred subjects were enrolled in the study, and 50 were randomized to a treatment group which used rh IL-12 and 50 were randomized to a control group which used symptomatic and supportive therapy after radiotherapy. The 50 subjects in the treatment group were further divided into five subgroups and intervenedwith rh IL-12 at a dose of 50, 100, 150, 200 or 250 ng/kg respectively. The dose-effect relationship was observed. RESULTS Rh IL-12 significantly attenuated the decrease of peripheral blood cells in the treatment group, and immune function was improved after treatment. Due to the different radiation doses, there was a fluctuation within 12 h after treatment but mostly showing an increasing trend. As to the clinical manifestations, 2 patients in the 250 ng/kg subgroup showed low fever after administration, 1 patient in the 200 ng/kg subgroup and 2 patients in the 250 ng/kg subgroup showed mild impairment of liver function during the observation period.CONCLUSION Rh IL-12 has effective therapeutic and protective effects on complications following radiotherapy, such as the decline of blood cells, myelosuppression and the decline or imbalance of immune function, which indicated good prospects for development and application.

High efficient mammalian expression and secretion of a functional humanized single-chain Fv/human interleukin-2 molecules

AIM： To construct and produce a recombinant bispecific humanized single-chain Fv （sFv） /Interleukin-2 （IL-2） fusion protein by using mammalian cells. METHODS： The sFv/IL-2 protein was genetically engineered, and transfected to mammalian cells to determine whether the mammalian protein folding machinery can produce and secrete active sFv/IL-2 with high efficiency. RESULTS： The fusion protein was constructed and high efficiently expressed with yields up to 102 ±4.2 mg/L in culture supernatant of the stably transfected 293 cell line. This recombinant fusion protein consisted of humanized variable heavy （VH） and light （VL） domains of monoclonal antibody （mAb） 520C9 directed against the human HER-2/neu （c-erbB2） proto-oncogene product p185, and human IL-2 connected by polypeptide linker. The fusion protein was shown to retain the immunostimulatory activities of IL-2 as measured by IL- 2-dependent cell proliferation and cytotoxicity assays. In addition to its IL-2 activities, this fusion protein also possessed antigen-binding specificity against p185, as determined by indirect ELISA using p185 positive SKOV 3ip1 cells. CONCLUSION： The large-scale preparation of the recombinant humanized sFv antibody/IL-2 fusion protein is performed with 293 cells. The recombinant humanized sFv antibody/IL-2 fusion protein may provide an effective means.of targeting therapeutic doses of IL-2 to p185 positive tumors without increasing systemic toxicity or immunogenicity.

Up-regulation interleukin-6 and interleukin-8 by activated protein C in lipopolysaccharide-treated human umbilical vein endothelial cells

Objective: To investigate the effect of activated protein C (APC) on inflammatory responses in human umbilical vein endothelial cells (HUVEC) stimulated with lipopolysaccharide (LPS). Methods: The second passage of collagenase digested HUVEC was divided into the following groups: serum free medium control group (SFM control), phosphate buffer solution control group (PBS control), LPS group with final concentration of 1 μg/ml (LPS group), APC group with final concentration of 7 μg/ml, Pre-APC group (APC pretreatment for 30 min prior to LPS challenge), and Post-APC group (APC administration 30 min after LPS challenge). Supernatant was harvested at 0, 4, 8, 12 and 24 h after LPS challenge. Interleukin-6 (IL-6) and Interleukin-8 (IL-8) levels were analyzed with ELISA. Cells were harvested at 24 h after LPS challenge, and total RNA was extracted. Mes-senger RNA levels for IL-6 and IL-8 were semi-quantitatively determined by RT-PCR. Results: Compared with control group, IL-6 and IL-8 levels steadily increased 4 to 24 h after LPS stimulation. APC treatment could increase LPS-induced IL-6 and IL-8 production. The mRNA levels of IL-6 and IL-8 exhibited a similar change. Conclusion: APC can further increase the level of IL-6 and IL-8 induced by LPS. The effect of these elevated cytokines is still under investigation.

Recombinant human interleukin-11 for treatment of chemotherapy-induced thrombocytopenia in patients with gastrointestinal cancer

Objective： To evaluate the efficacy and safety of recombinant human interleukin-11 （rhIL-11） for the chemotherapy-induced thrombocytopenia in patients with gastrointestinal cancer. Methods： It was an opened and non-randomized controlled clinical study. When the platelet counts was under 75 × 10^9/L after chemotherapy, rhlL-11 was administered 25 μg/（kg·d） as a daily SC injection last for 7-14 days, or discontinued when platelet counts 〉 100 × 10^9/L. Results： Seventysix patients were enrolled into this study. The treatment group and the control group had thirty-eight cases, respectively. The mean recovery time to PLT ≥ 100 × 10^9/L was 8.1 days in treatment group, while in control group was 12.2 days （P 〈 0.01）. Moreover, the mean recovery time from PLT 〈_ 50 × 10^9/L to 〉 100 × 10^9/L was 8.9 days in treatment group, while in control group was 12.9 days （P 〈 0.05）. There was a statistical difference between the two groups. Major side effects included edema, fever, articular muscle soreness, but they were all mild and well tolerable. Conclusion： rhIL-11 can be safely and effectively used for the treatment of chemotherapy-induced thrombocytopenia in patients with gastrointestinal cancer.

IL-33和炎症细胞因子在结肠腺癌中的表达及相关性

目的基于人类蛋白质图谱(HPA)数据库探讨细胞因子白细胞介素33(IL-33)和炎症细胞因子在结肠腺癌(COAD)中的蛋白质表达差异及相关性。方法COAD患者IL-33和炎症细胞因子的表达通过TIMER 2.0数据库进行分析,为了进一步验证,借助HPA生物信息学分析和免疫组化,检测了15例健康人和47例COAD患者结肠组织IL-33和炎症细胞因子(TNF、IL-4、IL-6和TGF-β1)的蛋白表达水平,并且基于免疫组织化学的蛋白表达数据分析COAD患者结肠组织IL-33与炎症细胞因子的相关性。结果HPA数据库中提取出相应条件下的所有样本,通过免疫组织化学和生物信息学分析IL-33和炎症细胞因子(TNF、IL-4、IL-6和TGF-β1)的差异表达,确定了四种促炎和抗炎细胞因子,IL-33表达与TNF相关性,具体结果显示:HPA024426、CAB078469、CAB078477、CAB023406和CAB000361抗体,分别检测COAD结肠组织IL-33、TNF、IL-4、IL-6和TGF-β1阳性细胞的表达,COAD癌变结肠组织IL-33和TNF的表达升高,而IL-4和IL-6的表达降低,且IL-33与TNF的表达呈正相关性,差异有统计学意义(P<0.05)。此外,TIMER 2.0数据库进一步证实了COAD患者IL-33和炎症细胞因子的表达水平上调。结论结肠组织IL-33调控COAD炎症促进其发生、发展,特异性靶向IL-33可能是COAD免疫治疗的有效新策略。

Neuronal changes in the retinal ganglion cell layer following recombinant human interleukin-2 intravitreal injection in a rat model of chronically elevated intraocular pressure

Intraperitoneal injection of recombinant human interleukin-2（rhIL-2）inhibits neuronal apoptosis in the chronic ocular hypertension retinal ganglion cell layer.Intravitreous injection was performed on retinal ganglion cells in a Wistar rat model of chronically elevated intraocular pressure to observe the effects of LY294002 and AG490 on retinal ganglion cell survival,macrophage activation,and PI3K/Akt and JAK/STAT activation.The number of retinal ganglion cells in the rhIL-2 treatment group was much greater than in the normal control and phosphate-buffered saline groups.Western blot analysis revealed low Akt and STAT3 protein expression in the retina after 3-hour intravitreous injections of rhIL-2.However,protein expression was increased at 12 hours,but decreased again at 24 hours,with very low expression at 96 hours.LY294002 and AG490,which are inhibitors of the PI3K/Akt and JAK/STAT3 signal pathways,prevented upregulation of Akt and STAT3 protein expression in the retina,respectively.Intravitreous injection of rhIL-2 exhibited neuroprotective effects by decreasing retinal ganglion cell layer damage in a rat model of chronic glaucoma.These results suggest that intravitreal injection of rhIL-2 could induce the PI3K/Akt and JAK/STAT3 signaling pathways to protect retinal ganglion cells in chronically elevated intraocular pressure models.

Artificial nerve graft constructed by coculture of activated Schwann cells and human hair keratin for repair of peripheral nerve defects

Studies have shown that human hair keratin(HHK) has no antigenicity and excellent mechanical properties. Schwann cells, as unique glial cells in the peripheral nervous system, can be induced by interleukin-1β to secrete nerve growth factor, which promotes neural regeneration. Therefore, HHK with Schwann cells may be a more effective approach to repair nerve defects than HHK without Schwann cells. In this study, we established an artificial nerve graft by loading an HHK skeleton with activated Schwann cells. We found that the longitudinal HHK microfilament structure provided adhesion medium, space and direction for Schwann cells, and promoted Schwann cell growth and nerve fiber regeneration. In addition, interleukin-1β not only activates Schwann cells, but also strengthens their activity and increases the expression of nerve growth factors. Activated Schwann cells activate macrophages, and activated macrophages secrete interleukin-1β, which maintains the activity of Schwann cells. Thus, a beneficial cycle forms and promotes nerve repair. Furthermore, our studies have found that the newly constructed artificial nerve graft promotes the improvements in nerve conduction function and motor function in rats with sciatic nerve injury, and increases the expression of nerve injury repair factors fibroblast growth factor 2 and human transforming growth factor B receptor 2. These findings suggest that this artificial nerve graft effectively repairs peripheral nerve injury.

Breast cancer in schizophrenia could be interleukin-33-mediated

Recent epidemiological and genetic studies have revealed an interconnection between schizophrenia and breast cancer.The mutual underlying pathophysiological mechanisms may be immunologically driven.A new cluster of molecules called alarmins may be involved in sterile brain inflammation,and we have already reported the potential impact of interleukin-33(IL-33)on positive symptoms onset and the role of its soluble trans-membranes full length receptor(sST2)on amelioration of negative symptoms in schizophrenia genesis.Furthermore,these molecules have already been shown to be involved in breast cancer etiopathogenesis.In this review article,we aim to describe the IL-33/suppressor of tumorigenicity 2(ST2)axis as a crossroad in schizophreniabreast cancer comorbidity.Considering that raloxifene could be tissue-specific and improve cognition and that tamoxifen resistance in breast carcinoma could be improved by strategies targeting IL-33,these selective estrogen receptor modulators could be useful in complementary treatment.These observations could guide further somatic,as well as psychiatric therapeutical protocols by incorporating what is known about immunity in schizophrenia.

Upregulation of stromal cell-derived factor-1 alpha/CXCR4 axis-induced migration of human neural progenitors by tumor necrosis factor-alpha and interleukin-8

BACKGROUND： Studies of several animal models of central nervous system diseases have shown that neural progenitor cells （NPCs） can migrate to injured tissues. Stromal cell-derived factor 1 alpha （SDF-la）, and its primary physiological receptor CXCR4, have been shown to contribute to this process. OBJECTIVE： To investigate migration efficacy of human NPCs toward a SDF-1α gradient, and the regulatory roles of tumor necrosis factor-α （TNF-α） and interleukin-8 （IL-8） in SDF-1α/CXCR4 axis-induced migration of NPCs. DESIGN, TIME AND SETTING： An in vitro, randomized, controlled, cellular and molecular biology study was performed at the Laboratory of Department of Cell Biology, Medical College of Soochow University between October 2005 and November 2007. MATERIALS： SDF-1α and mouse anti-human CXCR4 fusion antibody were purchased from R＆D Systems, USA. TNF-αwas purchased from Biomyx Technology, USA and IL-8 was kindly provided by the Biotechnology Research Institute of Soochow University. METHODS： NPCs isolated from forebrain tissue of 9 to 10-week-old human fetuses were cultured in vitro. The cells were incubated with 0, 20, and 40 ng/mL TNF-α, or 0, 20, and 40 ng/mL IL-8, for 48 hours prior to migration assay. For antibody-blocking experiments, cells were further pretreated with 0, 20, and 40 μg/mL mouse anti-human CXCR4 fusion antibody for 2 hours. Subsequently, the transwell assay and CXCR4 blockade experiments were performed to evaluate migration of human NPCs toward a SDF-1α gradient. Serum-free culture medium without SDF-1α served as the negative control. MAIN OUTCOME MEASURES： The transwell assay was performed to evaluate migration of human NPCs toward a SDF-1α gradient, which was blocked by fusion antibody against CXCR4. In addition, CXCR4 expression in human NPCs stimulated by TNF-α and IL-8 was measured by flow cytometry. RESULTS： Results from the transwell assay demonstrated that SDF-1α was a strong chemoattractant for human NPCs （P 〈 0.01）, and 20 ng/mL produced the highest levels of migration. Anti-human CXCR4 fusion antibody significantly blocked the chemotactic effect （P 〈 0.05）. Flow cytometry results showed that treatment with TNF-α and IL-8 resulted in increased CXCR4 expression and greater chemotaxis efficiency of NPCs towards SDF-1α（P 〈 0.01）. CONCLUSION： These results demonstrated that SDF-la significantly attracted NPCs in vitro, and neutralizing anti-CXCR4 antibody could block part of this chemotactic function. TNF-α and IL-8 increased chemotaxis efficiency of NPCs towards the SDF-1αgradient by upregulating CXCR4 expression in NPCs.

Links between donor macrosteatosis,interleukin-33 and complement after liver transplantation

BACKGROUND As prevalence of nonalcoholic fatty liver disease increases in the population,livers with steatosis will continue to infiltrate the donor pool.Safe utilization of these extended criteria grafts is paramount given the increased risk associated with their use in transplantation.Prognostic factors that can predict liver dysfunction immediately after transplantation with macrosteatotic grafts are lacking.AIM To understand the relationship between interleukin-33(IL-33)and complement in recipients immediately following liver reperfusion as a marker of liver dysfunction.METHODS Cohort consisted of patients who received a liver transplant from September 2016–September 2019 at our institution.Clinical variables were retrospectively extracted from the electronic medical record.Back-table donor biopsies were obtained with donor steatosis percentage retrospectively determined by a boardcertified pathologist.Blood samples were available immediately following liver transplantation.Quantification of plasma IL-33 and complement proteins,C3a and C5a,were determined by enzyme-linked immunosorbent assay.For mRNA expression,RNA was extracted from donor biopsies and used against a 780 gene panel.RESULTS Cohort consisted of 99 donor and recipients.Donor median age was 45 years and 55%male.Recipients had a median age of 59 years with 62%male.The main etiologies were alcoholic hepatitis,nonalcoholic steatohepatitis,and hepatocellular carcinoma.Median MELD-Na at transplant was 21.Donors were grouped based on moderate macrosteatosis(≥30%).Recipients implanted with moderate macrosteatotic grafts had significantly higher peak alanine aminotransferase/aspartate aminotransferase(P<0.001 and P<0.004),and increased incidence of early allograft dysfunction(60%compared to 18%).Circulating IL-33 levels were significantly elevated in recipients of≥30%macrosteatotic grafts(P<0.05).Recipients with detectable levels of circulating IL-33 immediately following reperfusion had significantly higher alanine aminotransferase/aspartate aminotransferase(P<0.05 and P<0.01).Activated complement(C3a and C5a)were elevated in recipients implanted with moderate macrosteatotic grafts.RNA expression analysis of donor biopsies revealed moderate steatotic grafts upregulated genes inflammatory processes while downregulated hepatocyte-produced complement factors.CONCLUSION Circulating IL-33 and activated complement levels immediately following liver reperfusion in recipients of moderate macrosteatotic grafts may identify which patients are at risk of early allograft dysfunction.

RESPONSES OF HUMAN FETAL SPLENOCYTES AND THYMOCYTES TO INTERLEUKIN-2: LAK ACTIVITY AND PROLIFERATION

Using cytotoxicity and thymidine uptake assays, we investigated the effects of human recombinant in-terleukin-2 (rIL-2) on the induction of lympholine-activated killer (LAK) activity and cellular proliferation in splenocytes and thymocytes from human fetuses (18-22 weeks). We observed that fetal splenocytes and thymocytes incubated with low doses of rIL-2 (10-100 U ml) developed broad antitumor activity (LAK activity) although the kinetics and magnitudes of the responses were different. It indicated the LAK precursors are present in fetal spleen and thymus. Further, rIL-2 induced a strong proliferative response in splenocytes, but not in thymocytes. On the basis of the findings, we conclude that the responses of fetal splenocytes and thymocytes to IL-2 are different.

Current status and prospects of interleukin-33 in lung area immunity

Interleukin (IL) 33 is a key cytokine in type II immune and airway diseases. It is abundantly expressed in lung epithelial cells and plays an important role in both innate and adaptive immunity. In innate immunity, IL-33 responds promptly to produce an immune response that maintains homeostasis. In adaptive immunity, IL-33 interacts with various immune cells. At the same time, IL-33 also plays an important role in chronic inflammation of the airway and its remodeling. This article reviews the relevant biological knowledge of IL-33 and its research progress in lung immunity, and discusses the related issues of IL-33 as a lung immune test site and therapeutic target.

类风湿关节炎合并肺间质病变的临床特点及与白介素33及其受体人基质裂解素2的相关性研究

目的探讨类风湿关节炎合并肺间质病变(RA-ILD)患者的临床、实验室特征及与细胞因子白介素33(IL-33)及其受体人基质裂解素2(ST2)的相关性,为RA-ILD的早期诊断提供依据。方法收集我院风湿免疫科初诊为类风湿关节炎(RA)患者114例,进一步分为RA-ILD组(21例)和RA组(93例),观察各组的临床症状及实验室指标,另选取健康体检者30例为对照组。酶联免疫吸附实验(ELISA)法测定以上各组参加者的血清IL-33及其受体ST2水平。结果 (1)RA-ILD在RA患者中的发生率为18.4%(21/114)。(2)与RA组相比,RA-ILD组患者年龄较大,病程较长,晨僵时间长,压痛关节数、肿胀关节数均较多,关节功能分级和手X线分期较严重,差异有统计学意义(P<0.01);且C-反应蛋白(CRP)、红细胞沉降率(ERS)、类风湿因子(RF)、抗环瓜氨酸多肽抗体(ACPA)、疾病活动度评分(DAS28)也均较高,差异有统计学意义(P<0.05);两病例组肺活量(VC)、用力肺活量(FVC)、最大呼气中段流量(MMF)和一氧化碳弥散量(DLCO)四项指标间差异有统计学意义(P<0.01)。(3)与对照组相比,两病例组血清IL-33及ST2水平均增高,差异有统计学意义(P<0.01);且RA-ILD组高于RA组,差异有统计学意义(P<0.01)。(4)IL-33水平与RF、ACPA滴度呈正相关(r值分别为0.817、0.550,P<0.01);与DLCO水平呈负相关(r=-0.801,P<0.01)。结论 RA-ILD易发生于年龄偏大、病程较长的RA患者中,且与疾病活动度及ACPA有关;血清IL-33及ST2参与了RA的发病过程,且可能与RA-ILD的发病有关;IL-33水平增高可能为RA预后不良的因素。

支气管哮喘患者血清IL-33、IL-35水平及相关性分析

目的 研究支气管哮喘患者血清IL-33、IL-35的表达水平,分析IL-33、IL-35间相关性.方法 采用酶联免疫法（ELISA）检测81例非细菌性感染所致中-重度支气管哮喘急性发作期患者治疗前及80名正常对照者血清IL-35、IL-33水平,采用血细胞分析仪检测白细胞数（WBC）、中性粒细胞绝对值（N）、嗜酸性粒细胞绝对值（EOS）.细胞培养实验分为空白对照组、细胞刺激素组和IL-35刺激组,ELISA法检测其培养上清IL-33浓度.统计分析支气管哮喘患者急性发作期与正常对照组血清中IL-35、IL-33、WBC、N、EOS,并分析IL-35、IL-33间的相关性.结果 ①哮喘组血WBC、N及EOS计数均高于正常对照组[（7.82±2.93）×109/L vs.（6.38±2.14）×109/L;（5.63±1.47）×109/L vs.（4.57±1.22）×109/L;（0.43±0.27）×109/L vs.（0.22±0.15）×109/L;P＜0.001].②哮喘组血清IL-33水平较正常对照组明显升高[（217.26±52.31）ng/L vs.（105.43±31.64）ng/L],IL-35水平较对照组明显降低[（156.71±24.29）ng/L vs.（311.62±35.28）ng/L],差异有统计学意义（P＜0.001）;总体IL-35与IL-33水平呈负相关（r=-0.803,P＜0.05）.③哮喘组经过IL-35刺激IL-33水平与仅细胞刺激素相比更低[（15.08±1.92）ng/L vs.（57.33±15.62）ng/L],差异有统计学意义（P＜0.01）.结论 IL-35、IL-33均参与了支气管哮喘的发病,支气管哮喘发作期存在各细胞因子间调节失衡.

IL-33蛋白及抗IL-33全人源单链抗体的表达、纯化及鉴定

目的表达IL-33蛋白及抗IL-33全人源天然单链抗体并进行纯化及鉴定。方法前期采用噬菌体展示技术从文库容量为108的全人源天然抗体文库筛选了抗IL-33全人源单链抗体,并将筛选的抗IL-33基因转入表达载体p LY16中。在前期工作的基础上,进行了以下研究:表达并纯化人IL-33蛋白,将构建的IL-33/PET102/D-TOPO质粒转化到大肠杆菌BL21中,表达后用Ni-NTA树脂纯化、浓缩再进行SDS-PGE电泳和Western blot验证。将抗IL-33 sc Fv/p LY16重组质粒转化入大肠杆菌DH5αF’中,应用PCR技术挑选阳性克隆并进行验证,IPTG诱导可溶性表达,Ni-NTA树脂纯化,SDS-PGE电泳和Western blot验证。结果 IL-33/PET102/D-TOPO质粒测序结果显示,插入的外源基因为人全长IL-33 c DNA。表达的IL-33融合蛋白相对分子质量为45 000左右。Western blot结果显示表达的蛋白为人IL-33蛋白。对从天然抗体文库中筛选的抗IL-33单链抗体进行测序,结果显示序列大小为750 bp左右,为开放阅读框,各序列之间有个别氨基酸的差异,说明为不同的单链抗体。ELISA结果显示抗IL-33单链抗体与IL-33有一定的结合能力。Western blot结果证实,表达的抗体为抗IL-33抗体。结论成功表达并纯化了IL-33抗原、抗IL-33全人源天然单链抗体。

角蛋白K18及其33、52位丝氨酸磷酸化在HBV感染肝脏疾病中的表达及意义

目的探索K18磷酸化ser33和ser52水平与HBV感染肝脏疾病的关系。方法应用组织化学免疫荧光法检测HBV感染后不同进程肝组织中K18及其磷酸化ser33和ser52的表达及其相对亚细胞定位;Western blot方法检测HBV感染后不同进程肝组织中K18及其磷酸化ser33和ser52水平。结果组织化学免疫荧光结果显示K18及其磷酸化Ser33和Ser52在所有肝组织都有表达,K18的表达没有明显差异,K18磷酸化Ser33和Ser52在正常细胞中表达较弱,位于细胞膜周围,在慢性HBV肝病时弥漫分散于细胞浆中且表达增强,重型肝炎表达最高;Western blot结果显示与对照相比,K18的Ser52水平被显著诱导但在不同程度肝脏疾病的表达无显著差异,重型肝炎时,K18的Ser33水平被显著诱导。结论K18高度磷酸化可能是HBV感染后肝脏疾病病情进展的标志。

HELF中IL-33/ST2L-TRAF-6信号通路的激活对其增殖、活化及胶原合成的影响

目的观察rhIL-33对人胚肺成纤维细胞增殖及其间充质细胞标记物的影响,探讨IL-33/ST2L-TRAF信号通路在肺纤维化形成过程中的作用。方法培养HELF细胞,PCR检测IL-33受体ST2L m RNA;不同浓度梯度的rh IL-33刺激细胞,MTT法检测不同时间点（24、48、72 h）IL-33对HF细胞增殖的影响;Real-time PCR方法检测IL-33刺激HELF后不同时间点（0、6、12、24、48、72 h）细胞标志性基因α-SMA m RNA、Vimentin m RNA、collagn I m RNA及TRAF-6 m RNA的变化;Western blot法检测细胞标志性蛋白α-平滑肌肌动蛋白（α-SMA）、波形蛋白（Vimentin）、I型胶原（collagen I）及关键性信号分子TRAF-6与下游信号分子ERK1/2、JNK、NF-kappa B等的改变。结果 IL-33能促进HELF的增殖,10 ng/ml的IL-33促增殖作用最强,且72 h最为明显;随着IL-33刺激细胞时间的逐渐延长,在0～72 h内α-SMA、Vimentin、collagen I在基因与蛋白水平均先上调后下调,信号分子TRAF-6、ERK1/2、JNK、NF-kappa B（P65）等亦呈现先上调后下调的趋势。结论 IL-33能促进HELF细胞增殖、活化及合成胶原,IL-33/ST2L-TRAF-6通路在此过程中起了关键作用,尤其是在病变早期炎症过程中作用最为明显。