树突细胞相关C型凝集素-1受体介导的抗白念珠菌免疫作用及机制

树突细胞相关c型凝集素-1是一种主要的C型凝集素样受体,存在于多种细胞和组织中.它能够特异性识别白念珠菌细胞壁主要成分β-葡聚糖,进而诱导机体产生固有免疫反应和适应性免疫反应.其可单独或与其他模式识别受体相互协同完成上述过程.白念珠菌β-葡聚糖的暴露、树突细胞相关C型凝集素-1表达水平等因素可影响机体免疫效应的强度,对其进行调控将为自念珠菌感染预防和治疗提供一个新的方向.

Abstract：

Dectin-1, a principal C-type lectin pattern-recognition receptor, exists in various types of cells and tissues. It can specifically recognize β-glucans, which are the major cell wall component of Candida albicans, and then trigger the innate and adaptive immune responses in hosts. Dectin-1 can independently, as well as collaboratively with other pattern recognition receptors, induce above mentioned processes. The intensity of immune responses in hosts is modulated by multiple factors such as the exposure of β-glucans on Candida albicans and the expression level of Dectin-1, and this modulation may provide a new direction for the prevention and therapy of Candida albicans infection.     

汉防己甲素对氟康唑抗白念珠菌活性的增效作用

目的 探讨体外汉防己甲素对氟康唑抗白念珠菌活性是否有增效作用.方法 参照微量稀释法确定汉防己甲素对白念珠菌的非细胞毒性剂量,并测定氟康唑单独及联合汉防己甲素时对16株白念珠菌的MIC.结果 在终质量浓度≤40μg/ml汉防己甲素的作用下,白念珠菌的存活率>95%.其中汉防己甲素(30～40μg/mL)可抑制白念珠菌菌丝相形成.氟康唑单独作用时,对16株白念珠菌的MIC值为0.250～64μg/mL;与汉防己甲素(40μg/mL)联合时,氟康唑对受试菌的MIC值降至0.125～16μg/mL,且终点清晰,“拖尾现象”消火.结论 汉防己甲素在体外对氟康唑的抗白念珠菌活性有增效作用.     

汉防己甲素对氟康唑抗白念珠菌增效活性的分子机制探讨

目的 探讨汉防己甲素在体外对氟康唑增效作用的分子机制。方法 分别提取 16 株白念珠菌酵母相总 RNA，采用 RT-PCR 方法比较汉防己甲素作用前及作用24 h后白念珠菌药物外排泵基因 MDR1、FLU1、CDR1、CDR2在氟康唑敏感、剂量依赖性敏感、耐药株中的表达情况。结果 在汉防己甲素作用前，MDR1、FLU1、CDR1、CDR2表达水平在氟康唑敏感、剂量依赖性敏感、耐药株间的差异均有统计学意义（P < 0.05）。汉防己甲素作用24 h后，与作用前相比，MDR1 在氟康唑耐药株中、FLU1在氟康唑剂量依赖性敏感、耐药株中、CDR1和CDR2在氟康唑敏感、剂量依赖性敏感、耐药株中表达水平的差异均有统计学意义（P < 0.05）。结论 汉防己甲素在体外对氟康唑抗白念珠菌活性增效作用的分子机制与抑制药物外排泵基因MDR1、FLU1、CDR1、CDR2的表达均有关。     

白念珠菌对氟康唑耐药的差异性膜蛋白质组学分析

目的 鉴定并分析白念珠菌氟康唑敏感株与耐药株的差异表达膜蛋白质。 方法 以同一亲本来源的白念珠菌氟康唑敏感株CA-3和耐药株CA-16为研究对象,以双向聚丙烯酰胺凝胶电泳（2D-PAGE）技术分析菌株之间膜蛋白图谱的差异,以基质辅助激光解吸电离飞行时间串联质谱（MALDI-TOF/TOF-MS）鉴定表达差异的蛋白质,通过白念珠菌蛋白质数据库进行检索分析。结果 白念珠菌氟康唑耐药株较敏感株有22种差异表达蛋白质点,其中有6种蛋白质上调,为Adh1p、Csp37p、Pgk1p、Pgk1p和2个未命名蛋白质（gi227305312、gi53954641）;16种蛋白质下调,分别为Aco1p、Aco1p、Hsp78p、Gut2p、Sdh12p、Ilv2p、Ndh51p、Ndh51p、Atp1p、Pda1p、Srb1p、Idh1p、Tdh1p、Cyt1p、Cox4p、Cox13p。结论 白念珠菌氟康唑耐药株较敏感株差异膜蛋白主要涉及能量代谢、线粒体功能等。 【关键词】 念珠菌,白色; 氟康唑; 抗药性,真菌; 膜蛋白质类; 蛋白质组学     

SLE患者基因组DNA甲基化水平的检测

Objective To assess the DNA methylation status in peripheral blood mononulclear cells (PBMCs)from patients with systemic lupus erythematosus (SLE) using a constructed methyl-CpG-binding domain (MBD) proteins.Methods A recombinant plasmid MBD-pET30b+ was transformed into E.coli (DH5a)for clonal expansion followed by sequencing.Then,plasmids with correct sequence were transformed into E coli BL21 (DE3) for the expression of MBD proteins under the induction of isopropy-β-D-thiogalactoside (IPTG).The expression products were purified by Ni-NTA chromatography and identified by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE)and Western blotting.Cultured 293T cells and isolated PMBCs from 21 patients with SLE and 15 normal human controls were immunostained with the expressed protein,and analyzed by fluorescence microscopy and flow cytometry.Results The sequence of recombinant plasmid was proved to be consistent with the expectation.SDS-PAGE and Western blotting demonstrated that the molecular weight of expressed tetrameric-protein was 46000,with the presence of N-terminal His-tag and C-terminal HA-tag.As fluorescence microscopy showed,the MBD proteins could specifically bind to intracellular CpG DNA in 293T cells.Patients with SLE had a significantly lower methylation level than the controls did(9.32±1.33 vs 11.66±1.04,t=5.68,P<0.05),and the DNA methylation level negatively correlated、with SLE disease activity index(r= -0.78,P<0.01).Condusions There is a lower DNA methylation in patients with SLE than in normal human controls,and the methylation level negatively correlates with SLE disease activity index.     

寻常性银屑病患者外周血单一核细胞IFN-γ受体和TNF-α受体mRNA表达的研究

Objective To investigate the mRNA expressions of IFN-gamma receptor and TNF-alpha receptor in peripheral blood mononuclear cells (PBMCs) of patients with psoriasis vulgaris and their role in the pathogenesis of psoriasis. Methods Fifty patients with psoriasis vulgaris (37 cases of active psoriasis and 13 cases of stable psoriasis) and 24 healthy human controls were included in this study. PBMCs were isolated from blood samples obtained from all patients and controls. The mRNA expressions of IFN-gamma receptor and TNF-aipha receptor in PBMCs were detected by RT-PCR. The disease severity in patients was evaluated by psoriasis area and severity index (PASI). Results The mRNA expressions of IFN-gamma receptor and TNF-alpha receptor were observed in the PBMCs of all subjects. The mRNA expression levels of IFN-gamma receptor were 0.72 ± 0.17 in healthy controls, 1.11 ± 0.55 in all patients with psoriasis, 1.13 ±0.57 in patients with active psoriasis and 1.03 ± 0.52 in patients with stable psoriasis, respectively. A signifi-cant increase was observed in the expression levels of IFN-gamma receptor mRNA in all psoriatic patients and in patients with active psoriasis compared with those in healthy controls (both P < 0.05), but there was no significant difference between the healthy controls and patients with stable psoriasis (P ＞ 0.05). The expres-sion levels of TNF-alpha receptor mRNA were 2.05 ± 1.34 in healthy controls, 2.70 ± 3.80 in all psoriatic patients, 2.90 ± 4.40 in patients with active psoriasis, 2.14 ± 1.05 in patients with stable psoriasis, respectively;there was no significant difference between psoriatic patients and healthy controls (P ＞ 0.05). However, no correlation was found between the mRNA expression of IFN-gamma receptor, that of TNF-alpha receptor,and disease severity in psoriatic patients. Conclusions The mRNA expression of IFN-gamma receptor in PBMCs is up-regulated in patients with psoriasis vulgaris, which is unrelated to the activity of psoriasis.     

寻常性银屑病患者外周血单一核细胞IFN-γ受体和TNF-α受体mRNA表达的研究

Objective To investigate the mRNA expressions of IFN-gamma receptor and TNF-alpha receptor in peripheral blood mononuclear cells (PBMCs) of patients with psoriasis vulgaris and their role in the pathogenesis of psoriasis. Methods Fifty patients with psoriasis vulgaris (37 cases of active psoriasis and 13 cases of stable psoriasis) and 24 healthy human controls were included in this study. PBMCs were isolated from blood samples obtained from all patients and controls. The mRNA expressions of IFN-gamma receptor and TNF-aipha receptor in PBMCs were detected by RT-PCR. The disease severity in patients was evaluated by psoriasis area and severity index (PASI). Results The mRNA expressions of IFN-gamma receptor and TNF-alpha receptor were observed in the PBMCs of all subjects. The mRNA expression levels of IFN-gamma receptor were 0.72 ± 0.17 in healthy controls, 1.11 ± 0.55 in all patients with psoriasis, 1.13 ±0.57 in patients with active psoriasis and 1.03 ± 0.52 in patients with stable psoriasis, respectively. A signifi-cant increase was observed in the expression levels of IFN-gamma receptor mRNA in all psoriatic patients and in patients with active psoriasis compared with those in healthy controls (both P < 0.05), but there was no significant difference between the healthy controls and patients with stable psoriasis (P ＞ 0.05). The expres-sion levels of TNF-alpha receptor mRNA were 2.05 ± 1.34 in healthy controls, 2.70 ± 3.80 in all psoriatic patients, 2.90 ± 4.40 in patients with active psoriasis, 2.14 ± 1.05 in patients with stable psoriasis, respectively;there was no significant difference between psoriatic patients and healthy controls (P ＞ 0.05). However, no correlation was found between the mRNA expression of IFN-gamma receptor, that of TNF-alpha receptor,and disease severity in psoriatic patients. Conclusions The mRNA expression of IFN-gamma receptor in PBMCs is up-regulated in patients with psoriasis vulgaris, which is unrelated to the activity of psoriasis.     

寻常性银屑病患者外周血单一核细胞IFN-γ受体和TNF-α受体mRNA表达的研究

Objective To investigate the mRNA expressions of IFN-gamma receptor and TNF-alpha receptor in peripheral blood mononuclear cells (PBMCs) of patients with psoriasis vulgaris and their role in the pathogenesis of psoriasis. Methods Fifty patients with psoriasis vulgaris (37 cases of active psoriasis and 13 cases of stable psoriasis) and 24 healthy human controls were included in this study. PBMCs were isolated from blood samples obtained from all patients and controls. The mRNA expressions of IFN-gamma receptor and TNF-aipha receptor in PBMCs were detected by RT-PCR. The disease severity in patients was evaluated by psoriasis area and severity index (PASI). Results The mRNA expressions of IFN-gamma receptor and TNF-alpha receptor were observed in the PBMCs of all subjects. The mRNA expression levels of IFN-gamma receptor were 0.72 ± 0.17 in healthy controls, 1.11 ± 0.55 in all patients with psoriasis, 1.13 ±0.57 in patients with active psoriasis and 1.03 ± 0.52 in patients with stable psoriasis, respectively. A signifi-cant increase was observed in the expression levels of IFN-gamma receptor mRNA in all psoriatic patients and in patients with active psoriasis compared with those in healthy controls (both P < 0.05), but there was no significant difference between the healthy controls and patients with stable psoriasis (P ＞ 0.05). The expres-sion levels of TNF-alpha receptor mRNA were 2.05 ± 1.34 in healthy controls, 2.70 ± 3.80 in all psoriatic patients, 2.90 ± 4.40 in patients with active psoriasis, 2.14 ± 1.05 in patients with stable psoriasis, respectively;there was no significant difference between psoriatic patients and healthy controls (P ＞ 0.05). However, no correlation was found between the mRNA expression of IFN-gamma receptor, that of TNF-alpha receptor,and disease severity in psoriatic patients. Conclusions The mRNA expression of IFN-gamma receptor in PBMCs is up-regulated in patients with psoriasis vulgaris, which is unrelated to the activity of psoriasis.     

内皮素对黑素瘤A375细胞转化生长因子-β1及磷酸化Smad 3蛋白表达的调节

Objective To investigate the effects ofendothelin-1 (ET-1) and endothelin-3 (ET-3) on the expression of transformation growth factor-beta 1 (TGF-β1) and phosphorylation of Smad 3 in malignant melanoma cell line, A375. Methods Cultured A375 cells were classified into 5 groups, i.e. control group (no stimulation), ET-1 group (stimulated with ET-1), ET-1+BQ123 group (treated with ET-1 and BQ123),ET-1 + BQ788 group (treated with ET-1 and BQ788), ET-3 group (stimulated with ET-3), to receive different stimulation. The working concentrations were 0, 0.1, 1, 10, 100 nmol/L for ET-1 and ET-3, 10 μmol/L for BQ123 and BQ788. After another 12- and 24-hour culture, ELISA, RT-PCR and Western blot were used to detect the expression of TGF-β1 protein and mRNA as well as phosphorylated Smad 3 (P-Smad 3). Results The expression of TGF-β1 in A375 cells was up-regulated by ET-1, but down-regulated by ET-3, and both of the effects were in a concentration-dependent manner. Under the stimulation with ET-1 and ET-3 of 100 nmol/L, the level of TGF-β1 reached 1289.38 ± 89.42 ng/L per 105 cells and 85.09 ± 9.37 ng/L per 105 cells, respectively, significantly different from that in unstimulated cells (both P < 0.05). BQ123 signifi-cantly blocked the up-regnlatory effect of ET-1 on the expression TGF-β1 protein(P < 0.05), but BQ788 had no significant influence on the effect, so was the case with TGF-β1 mRNA. Western blot revealed that ET-1significantly elevated the expression of P-Smad 3 in A375 cells (P <0.05), and the elevation was significantly inhibited by BQ123, but not by BQ788. The expression of P-Smad 3 was statistically decreased by ET-3 in A375 cells (P <0.05). Conclusions The expression of TGF-β1 could be enhanced by ET-1, but suppressed by ET-3. It is likely that endothelin receptor A mediates the phosphorylation of Smad 3 induced by ET-1.     

内皮素对黑素瘤A375细胞转化生长因子-β1及磷酸化Smad 3蛋白表达的调节

Objective To investigate the effects ofendothelin-1 (ET-1) and endothelin-3 (ET-3) on the expression of transformation growth factor-beta 1 (TGF-β1) and phosphorylation of Smad 3 in malignant melanoma cell line, A375. Methods Cultured A375 cells were classified into 5 groups, i.e. control group (no stimulation), ET-1 group (stimulated with ET-1), ET-1+BQ123 group (treated with ET-1 and BQ123),ET-1 + BQ788 group (treated with ET-1 and BQ788), ET-3 group (stimulated with ET-3), to receive different stimulation. The working concentrations were 0, 0.1, 1, 10, 100 nmol/L for ET-1 and ET-3, 10 μmol/L for BQ123 and BQ788. After another 12- and 24-hour culture, ELISA, RT-PCR and Western blot were used to detect the expression of TGF-β1 protein and mRNA as well as phosphorylated Smad 3 (P-Smad 3). Results The expression of TGF-β1 in A375 cells was up-regulated by ET-1, but down-regulated by ET-3, and both of the effects were in a concentration-dependent manner. Under the stimulation with ET-1 and ET-3 of 100 nmol/L, the level of TGF-β1 reached 1289.38 ± 89.42 ng/L per 105 cells and 85.09 ± 9.37 ng/L per 105 cells, respectively, significantly different from that in unstimulated cells (both P < 0.05). BQ123 signifi-cantly blocked the up-regnlatory effect of ET-1 on the expression TGF-β1 protein(P < 0.05), but BQ788 had no significant influence on the effect, so was the case with TGF-β1 mRNA. Western blot revealed that ET-1significantly elevated the expression of P-Smad 3 in A375 cells (P <0.05), and the elevation was significantly inhibited by BQ123, but not by BQ788. The expression of P-Smad 3 was statistically decreased by ET-3 in A375 cells (P <0.05). Conclusions The expression of TGF-β1 could be enhanced by ET-1, but suppressed by ET-3. It is likely that endothelin receptor A mediates the phosphorylation of Smad 3 induced by ET-1.     

凝集性生长毛乳头细胞蛋白质组的差异分析

目的分析凝集性生长状态下,毛乳头细胞胞浆蛋白组的表达。方法提取第3代和第10代毛乳头细胞的胞浆总蛋白,采用固相pH梯度双向凝胶电泳技术进行分离,考马斯亮蓝G250染色后经PDQuest软件分析电泳图谱,对差异显著的蛋白点应用MALDI-TOF质谱仪测定其肽质量指纹图谱,在NCBInr数据库鉴定蛋白质。结果获得了较好的双向电泳图谱。图像分析显示:在第3代和第10代毛乳头细胞的样品中分别检测到608±39个和595±31个蛋白质点;对表达显著差异的24个蛋白质点进行肽质量指纹图谱分析,17个得到良好的肽质量指纹图谱,经数据库检索后初步鉴定出15个相匹配的已知蛋白,大多为一些与代谢、信号转导、凋亡和蛋白合成相关的蛋白。结论毛乳头细胞的蛋白组表达模式与其凝集性生长状态密切相关。     

凝集生长毛乳头细胞分泌性蛋白组的初步分析

【摘要】 目的 了解毛乳头细胞在凝集生长状态下分泌性蛋白组的表达。 方法 以凝集和非凝集生长的毛乳头细胞为研究对象,分别制备分泌性蛋白质,以双向凝胶电泳技术和PDQuest软件分析二者之间的蛋白图谱的差异,通过基质辅助激光解吸电离飞行时间串联质谱（MLDI-TOF-MS）鉴定表达差异的蛋白质,通过蛋白质数据库NCBInr检索分析。 结果 建立了重复性好、分辨率高的双向电泳图谱;在凝集生长和非凝集生长的毛乳头细胞分泌性蛋白质中分别检测到（1 134 ± 52）个和（1 078 ± 36）个蛋白点,大多匹配。按照差异量在5倍以上的标准,二者存在差异蛋白质28个,经过MLDI-TOF-MS质谱鉴定出10种差异表达蛋白点,其中8种蛋白质表达上调,分别为Rho GDP分离抑制剂1、细丝蛋白A、胱抑素C、纤维连接蛋白、亲环素A、前胶原C端蛋白酶增强子、组织金属蛋白酶抑制剂、组织金属蛋白酶-2抑制剂。2种蛋白质表达下调,为神经肽h3和基质金属蛋白酶-3金属蛋白酶组织抑制剂-1复合物复合物。 结论 凝集生长和非凝集生长毛乳头细胞差异蛋白主要涉及信号通路、细胞增殖与分化、细胞外基质合成及降解等功能。     

黄苓苷对HaCaT细胞影响的蛋白组学研究

目的 本研究旨在观察黄苓苷对永生化人表皮角质形成细胞HaCAT株差异表达蛋白的影响.方法 培养HaCaT细胞,并加入黄苓苷进行干预.培养24h后收集细胞总蛋白,利用双向电泳方法分离细胞蛋白质,并用ImageMaster图像分析软件比较各组细胞的蛋白质图谱,筛选出的差异蛋白质点应用基质辅助激光解吸离子化-飞行时间质谱(MALDI-TOF-MS)测定其胶内酶解后的肽质量指纹图谱,然后利用Mascot查询软件搜寻Swiss Prot数据库鉴定蛋白质.结果 黄苓苷使HaCaT细胞蛋白质组发生改变,对其中18个差异蛋白质点进行质谱分析,获得15个点的肽质量指纹图,初步鉴定为热休克蛋白beta-1,肌动蛋白、二氢嘧啶酶调节蛋白2等.结论 黄苓苷可通过影响细胞中多种蛋白的表达,增强细胞的稳态和抵抗环境刺激损伤的能力,发挥抗肿瘤活性.     

利用血清蛋白质组学技术筛选银屑病相关蛋白

目的 研究常见亚型银屑病患者血清中差异表达蛋白,筛选银屑病特异性蛋白质标记物。 方法 收集首次发病进展期寻常性银屑病6例、红皮病性银屑病5例及健康人6例的血清,分别将各组标本混合,并去除高丰度白蛋白和免疫球蛋白IgG后,应用双向电泳技术分离血清,比较差异蛋白点。利用基质辅助激光解析电离飞行时间质谱,对候选差异表达蛋白点进行肽质量指纹图谱分析,通过NCBI数据库搜索鉴定蛋白。 结果 获得了3组较好的血清双向电泳图谱。应用胶图分析软件分析,发现寻常性银屑病组、红皮病性银屑病组与健康对照组间差异蛋白点分别为33个和17个,寻常性银屑病组和红皮病性银屑病组之间差异蛋白点26个。共鉴定得到14种蛋白。在银屑病组中表达升高的有补体成分3、白介素16、维生素D结合蛋白、α1抗胰蛋白酶等;红皮病组中补体成分3、补体因子H、α1抗胰蛋白酶、血液结合素、触珠蛋白的表达量高于健康人对照,α1抗胰蛋白酶、补体因子H、补体成分4和触珠蛋白的表达量高于寻常性银屑病组。在红皮病性患者血清中表达下降的有血清淀粉样蛋白P。 结论 寻常性银屑病、红皮病性银屑病与健康人对照间血清蛋白表达谱存在差异。     

皮肤成纤维细胞与角质形成细胞蛋白质组双向电泳图谱的比较

目的建立人皮肤成纤维细胞与角质形成细胞的双向凝胶电泳图谱,并利用计算机图像分析技术初步分析其差异表达的蛋白质。方法采用固相pH梯度双向凝胶电泳分离人皮肤成纤维细胞与角质形成细胞的总蛋白质,凝胶经银染显色后,Im agingM aster 2D图像分析软件进行比较分析,识别差异表达的蛋白质。结果①人皮肤成纤维细胞与角质形成细胞凝胶的平均蛋白质点数分别为685±34和592±27,平均匹配的点数分别为593±29和483±22,匹配率达86.6%和81.7%;②通过比较分析上述两种细胞的双向凝胶电泳图谱,得到平均匹配蛋白点数为315±25。差异表达蛋白点数为281个,其中117个点仅在成纤维细胞中表达,124个点仅在角质形成细胞中表达,126个点在成纤维细胞中为高表达,145个点在成纤维细胞中为低表达。结论本研究建立了分辨率高且重复性较好的人皮肤成纤维细胞与角质形成细胞的双向凝胶电泳图谱,发现两者间存在一些差异表达的蛋白质,为皮肤蛋白质组学的进一步研究奠定基础。     

血清蛋白质组学技术筛选豚鼠银屑病样模型血清蛋白质的实验研究

目的应用血清蛋白质组学技术筛选豚鼠银屑病样模型血清中的差异蛋白质,期望从中发现与银屑病相关的血清蛋白标志物。方法采用双向-差异凝胶电泳(2D-DIGE)和二级质谱分析鉴定豚鼠银屑病样模型血清中的差异蛋白质。结果获得重复性和分辨率较好的血清双向差异凝胶电泳图谱,通过二级质谱分析鉴定得到3个差异的蛋白质。结论豚鼠银屑病样模型血清与正常豚鼠血清比较存在差异表达蛋白质。     

皮肤型红斑狼疮皮损中SASPase的表达和意义

目的 探讨SASPase在皮肤型红斑狼疮皮损中的表达水平及其在发病机制中的意义.方法 无血清培养原代角质形成细胞,将提取的蛋白样品采用固相pH梯度双向凝胶电泳进行分离,应用ImageMaster 2D Platinum 5.0软件对图像进行匹配分析,选取差异表达蛋白质点,经基质辅助激光解吸附飞行时间质谱进行质谱鉴定.并通过免疫印迹验证表达水平.结果 成功培养角质形成细胞,获得重复性较好的双向电泳图谱,匹配点数在1200个左右,匹配率＞80％.鉴定SASPase在CLE皮损角质形成细胞表达升高,免疫印迹验证与双向电泳结果一致.结论 皮肤型红斑狼疮皮损的发生发展可能与SASPase的异常激活和过度表达有关.