人sCD40L-Ig重组腺病毒载体的构建、表达及功能检测

目的:构建含有分泌型人胞外区CD40L融合蛋白(sCD40L-Ig)基因的重组腺病毒载体,确定其表达和功能学意义。方法:通过PCR获得人源IgGFc和sCD40L基因并予以连接,将其插入到腺病毒穿梭质粒pAdTrack-CMV中,构建重组质粒pAdTrack-sCD40L-Ig。将其与pAdEasy-1-BJ5183菌行同源重组后,用293细胞包装,通过观察绿色荧光蛋白(GFP)和Western blot分析等方法鉴定重组腺病毒,并进行双向混合淋巴细胞反应(MLR)以确定其功能。结果:所构建的sCD40L-Ig基因的重组腺病毒,经酶切和PCR鉴定正确。原代腺病毒的滴度达到2.69×1011pfu/L,并有相对分子质量(Mr)为61×103的目的蛋白表达。MLR显示,重组腺病毒对淋巴细胞的增殖有抑制作用。结论:成功地构建了含有sCD40L-Ig基因的重组腺病毒,并对MLR有抑制作用。     

GPR30/EGFR在多囊卵巢综合征患者卵巢组织中的表达

目的:探讨雌激素膜受体G蛋白偶联受体30(GPR30)及表皮生长因子(EGFR)在多囊卵巢综合征(PCOS)患者卵巢组织中的表达。方法:选取2012~2015年在青岛市妇女儿童医院生殖中心行手术治疗的PCOS患者25例,以及同期因良性卵巢肿瘤行剥除术的25例患者为对照组。采用实时荧光定量PCR技术检测卵巢组织中ERα、ERβ、GPR30及EGFR mRNA的表达水平;Western blot法检测卵巢组织中ERα、ERβ、GPR30及EGFR蛋白表达;免疫组化法检测卵巢组织中ERα、ERβ、GPR30及EGFR蛋白的表达定位。结果:PCOS卵巢组织中GPR30及EGFR mRNA和蛋白表达水平均显著高于对照组,差异均有统计学意义(P0.05)。始基卵泡及初级卵泡中,GPR30蛋白主要定位表达于卵母细胞胞浆与胞膜以及颗粒细胞的细胞核,始基卵泡卵母细胞表达明显高于初级卵泡卵母细胞,较对照组GPR30在PCOS组卵母细胞及颗粒细胞中表达均升高;EGFR蛋白主要表达于颗粒细胞的细胞核,在PCOS组表达升高。结论:GPR30及EGFR在PCOS患者的卵泡发育障碍过程中可能具有重要作用。     

淋巴细胞共刺激信号和凋亡信号异常与RA免疫效应亢进的研究

目的：探究T淋巴细胞表面多种细胞信号分子所介导的细胞活化或凋亡信号在RA患者免疫功能紊乱中的作用。方法：采用流式细胞术检测RA患者外周血T细胞亚群及其表面共刺激分子cD154（cD40L）、CD30和凋亡受体CD95（Fas）的表达。结果：RA患者外周血T细胞亚群偏移，CD4^＋T细胞增加，CD8^＋T细胞减少；共刺激分子CD154在CD4^＋和CD8^＋T细胞上的表达均上调，但CD30分子的表达均降低，并以CD4^＋T细胞降低更为明显。同时，凋亡受体CD95分子在T细胞亚群上的表达均明显增加。结论：RA患者T淋巴细胞表面多种信号分子表达异常，共同导致了RA患者免疫功能紊乱。     

CLINICOPATHOLOGICAL,IMMUNOLOGICAL AND GENETIC STUDIES OF CD30+ ANAPLASTIC LARGE CELL LYMPHOMA OF B-CELL TYPE; ASSOCIATION WITH EPSTEIN–BARR VIRUS IN A JAPANESE POPULATION

The clinicopathological features, the immunophenotype, and the presence of Epstein–Barr virus (EBV)-associated genomes and gene products were examined in 17 cases of CD30⁺ anaplastic large cell lymphoma (ALCL) of B-cell type. Microscopically, the 17 cases were divided into ten cases of the monomorphic type and seven cases of the pleomorphic type. EBV was detected in 6 of 17 cases (38 per cent) by RNA in situ hybridization (ISH) with EBV-encoded RNA (EBER1). EBER1⁺ cases consisted of two cases (20 per cent) of the monomorphic type and four cases (57 per cent) of the pleomorphic type. The five EBER1⁺ cases showed clonality of the EBV genome by Southern blotting, consistent with the presence of EBV in a monoclonal proliferation. The EBV-encoded latent membrane protein 1 (LMP1) was found in all six EBER1⁺ cases and EBV-encoded nuclear antigen 2 (EBNA2) was present in two cases by immunohistochemistry. No expression of LMP1 or EBNA2 was observed in the EBER1⁻ cases. The EBER1⁺ cases had a tendency for a more favourable prognosis than the EBER1⁻ cases. It is concluded that EBV has an association with CD30⁺ ALCL of B-cell type in the Japanese population studied, and especially with the large pleomorphic type. EBV infection may play a pathoaetiological role and may influence clinical behaviour.     

Serum levels of soluble CD30 in chronic hepatitis B virus infection

There is evidence that both cellular and humoral components of the immune response are required for viral clearance to occur in chronic hepatitis B. Recent studies demonstrated that CD30 molecule, a member of the tumour necrosis factor superfamily of membrane cytokine receptors, is expressed on, and released as a soluble molecule (sCD30) by activated T cells producing T helper 2 (Th2) cytokines, which modulate antibody responses. To better characterize the immunoregulatory mechanisms in chronic hepatitis B virus (HBV) infection, sCD30 values were evaluated by an ELISA in 90 hepatitis B surface (HBsAg)-positive patients with chronic hepatitis, selected on the basis of active viral replication and biochemical activity. At presentation abnormal levels (>20 U/ml) of sCD30 were detected in 57 (63%) out of 90 patients with chronic hepatitis B, and median value was significantly higher in this group of patients compared with that of healthy HBsAg carriers (26.7 versus 10.5 U/ml, P < 0.000 05) and with normal controls (26.7 versus 3 U/ml, P < 0.000 01). Sequential studies of chronic hepatitis B did confirm the association of raised sCD30 levels with the active phase of the illness. On the other hand, a significant decrease was noted when sCD30 levels at diagnosis and after termination of HBV replication and biochemical remission of hepatitis were compared in 10 untreated patients (median, 28 U/ml at entry versus 8 U/ml at remission, P < 0.01) and in six patients responding to interferon-alpha therapy (median, 29.5 U/ml at entry versus 6 U/ml at remission, P < 0.05). The high serum sCD30 levels reported during the active phase of HBsAg-positive chronic hepatitis suggest a certain degree of immune competence of these patients, at least with respect to a Th2-type response. These data are in agreement with recent serologic surveys showing that most chronic hepatitis B patients do demonstrate ongoing humoral immune response to HBV antigens, using novel immunoassays designed to detect antibody in the presence of excess serum viral antigen. Th2 functions that mainly promote humoral immunity to HBV antigens may be critical, in association with a competent virus-specific cytotoxicity, for efficient termination of HBV replication in chronic hepatitis B.     

Effect of intrajejunal elemental diet perfusion on local secretion of soluble CD4 and CD8

The effects of nutrients on the mucosal immune system are poorly understood. The aim of this work was to study the cellular mucosal immune response to intrajejunal perfusion of an elemental diet (ED) or a control (C) electrolyte solution by measuring jejunal secretion of soluble CD4 (sCD4) and sCD8. sCD4 and sCD8 are markers of helper/inducer and suppressor/cytotoxic regulatory functions of T cells, respectively. A four lumen tube with a proximal occluding balloon at the angle of Treitz was used for jejunal perfusion in seven healthy volunteers (mean age 23 years). The length of the test segment was 40 cm. The jejunum was successively perfused with C for 80 min and then with ED containing 21.3g/l of free amino acids and 104.2g/l of oligosaccharides for 100 min. Jejunal fluid and serum concentrations of sCD4 and sCD8 were measured and their jejunal outputs calculated. When compared with C perfusion, jejunal perfusion with the ED resulted in a significant increase of sCD8 but not sCD4 jejunal secretion rates. sCD8 jejunal values increased early after ED perfusion and stayed at roughly the same level during the perfusion. Serum concentrations of sCD4 and sCD8 were not modified during ED perfusion. These data support the hypothesis that ED suppresses the immunologic tone of the gut, which could explain its beneficial effect in the management of intestinal inflammatory disease.     

Activated T cell subsets in human type 1 diabetes: evidence for expansion of the DR+ CD30+ subpopulation in new-onset disease

An important limitation in T cell studies of human autoimmune (type 1) diabetes is lack of direct access to cells infiltrating the pancreas. We hypothesized that cells recently released from the pancreas into the blood might express a characteristic combination of markers of activation. We therefore examined the recently activated circulating T cell population [CD3+, human leucocyte antigen D-related (HLA-DR+)] using cytokine production and 10 additional subset markers [CD69, CD25, CD122, CD30, CD44v6, CD57, CD71, CCR3 (CD193), CCR5 (CD195) or CXCR3 (CD183)], comparing newly diagnosed adult (ND) (age 18-40 years) patients (n=19) to patients with diabetes for >10 years [long-standing (LS), n=19] and HLA-matched controls (C, n=16). CD3+ DR+ cells were enriched by two-step immunomagnetic separation. No differences in basal or stimulated production of interleukin (IL)-4, IL-10, IL-13 or interferon (IFN)-gamma by CD3+ DR+ enriched cells were observed between the different groups of subjects. However, among the CD3+ DR+ population, significant expansions appeared to be present in the very small CD30+, CD69+ and CD122+ subpopulations. A confirmatory study was then performed using new subjects (ND=26, LS=15), three-colour flow cytometry, unseparated cells and three additional subset markers (CD38, CD134, CD4/CD25). This confirmed the expansion of the CD3+ DR+ CD30+ subpopulation in ND subjects. We conclude that a relative expansion in the T cell subpopulation with the activated phenotype CD3+ DR+ CD30+ is seen in the peripheral blood of subjects with newly diagnosed type 1 diabetes. This subpopulation represents less than 0 x 7% of circulating T cells and may provide a rich source of disease-specific T cells that can be isolated from blood.     

miR-30a对实验性自身免疫性脑脊髓炎模型小鼠发病的作用

目的:探讨在体过表达miR-30a对实验性自身免疫性脑脊髓炎(EAE)模型小鼠脱髓鞘病变的作用。方法:构建含稳定表达miR-30a的shRNA慢病毒载体,以含有空载体的慢病毒为对照(LV-ctrl)。雌性C57BL/6小鼠随机分为正常对照组(normal)、对照慢病毒(LV-ctrl)组和miR-30a慢病毒(LV-miR-30a)组,2组慢病毒经尾静脉注射,注射7 d后用MOG35-55多肽免疫小鼠。Kono 5分法检测发病情况,快蓝(LFB)染色及透射电镜检测脊髓脱髓鞘程度及髓鞘结构的变化。结果:LV-ctrl组小鼠在MOG_(35-55)多肽免疫的第10天开始发病,到第21天,其Kono评分为2.57±0.79,LV-miR-30a组小鼠的Kono评分显著低于LV-ctrl组小鼠;miR-30a处理可显著提高小鼠体质量增长率,同时LFB评分也较LV-ctrl组显著增加;髓鞘超微结构显示LV-ctrl组小鼠脊髓髓鞘板层松解且稀疏,而LV-miR-30a组小鼠的髓鞘结构相对完好,髓鞘厚度显著大于LV-ctrl组。结论:过表达miR-30a可以明显减缓EAE的发病,减轻脊髓脱髓鞘程度。     

急性冠脉综合征患者血清可溶性CD40L水平的临床研究

目的:探讨可溶性白细胞分化可溶性抗原40配体(sCD40L)和超敏C反应蛋白(hs-CRP)在急性冠脉综合征(ACS)患者发病及预后中的临床意义及机制。方法:根据急性冠脉综合征诊断标准选择86例患者,分为两组:正常对照组(NCHD)17例,ACS组69例,其中不稳定心绞痛(UA)58例,急性心肌梗死(AMI)11例,入院当天采用ELISA测定血清sCD40L浓度和hs-CRP水平。随诊2个月,观察急性心血管事件发生率。结果:ACS组明显高于对照组(P<0.01);AMI组略高于UA组但无统计学意义(P>0.05);sCD40L和或hs-CRP升高组心血管事件较正常组增多。结论:急性冠脉综合征患者早期外周血清sCD40L和hs-CRP水平明显升高,提示CD40/CD40L系统与ACS的发生有关,并与炎症因子C反应蛋白协同,对动脉粥样硬化斑块不稳定性起着重要作用。     

Induction of interleukin-10 in the T helper type 17 effector population by the G protein coupled estrogen receptor (GPER) agonist G-1

Interleukin-10 (IL-10) is a potent suppressor of the immune system, commonly produced by CD4(+) T cells to limit ongoing inflammatory responses minimizing host damage. Many autoimmune diseases are marked by large populations of activated CD4(+) T cells within the setting of chronic inflammation; therefore, drugs capable of inducing IL-10 production in CD4(+) T cells would be of great therapeutic value. Previous reports have shown that the small molecule G-1, an agonist of the membrane-bound G-protein-coupled estrogen receptor GPER, attenuates disease in an animal model of autoimmune encephalomyelitis. However, the direct effects of G-1 on CD4(+) T-cell populations remain unknown. Using ex vivo cultures of purified CD4(+) T cells, we show that G-1 elicits IL-10 expression in T helper type 17 (Th17) -polarized cells, increasing the number of IL-10(+) and IL-10(+) IL-17A(+) cells via de novo induction of IL-10. T-cell cultures differentiated in the presence of G-1 secreted threefold more IL-10, with no change in IL-17A, tumour necrosis factor-α, or interferon-γ. Moreover, inhibition of extracellular signal-regulated kinase (but not p38 or Jun N-terminal kinase) signalling blocked the response, while analysis of Foxp3 and RORγt expression demonstrated increased numbers of IL-10(+) cells in both the Th17 (RORγt(+)) and Foxp3(+) RORγt(+) hybrid T-cell compartments. Our findings translated in vivo as systemic treatment of male mice with G-1 led to increased IL-10 secretion from splenocytes following T-cell receptor cross-linking. These results demonstrate that G-1 acts directly on CD4(+) T cells, and to our knowledge provide the first example of a synthetic small molecule capable of eliciting IL-10 expression in Th17 or hybrid T-cell populations.