SLE患者雌激素受体-α与相关细胞因子的表达及临床相关性研究

目的 探讨SLE患者雌激素受体(ER)对T、B淋巴细胞的作用机制以及T、B淋巴细胞在SLE发病中的协同作用.方法 采用实时荧光定量PCR检测40例SLE患者与40例正常人外周血单核细胞(PBMC)ER-α、IL-10、B淋巴细胞刺激因子(BLyS)mRNA表达水平;分析ER-α、IL-10和BLyS mRNA表达与临床及实验室指标的相关性.结果 SLE组ER-α、IL-10、BLyS mRNA相对表达量显著高于正常人对照组(P＜0.05或P＜0.01),SLE活动期较静止期明显增高(P＜0.05或P＜0.01),且其表达水平与肾损害、蛋白尿、关节炎等临床及实验室指标有相关性.男女SLE组以及男女正常对照组的表达比较差异无统计学意义.结论 IL-10和BLyS与SLE的病情活动性和严重程度相关,ER-α可能在SLE T、B淋巴细胞的作用机制中有重要作用.

Abstract：

Objective To study the mechanism of effects of estrogen receptor (ER) on T and B lymphocytes in patients with SLE and synergistic effect of T and B lymphocytes in the pathogenesis of SLE.Methods Real-time fluorescence quantitative PCR was performed to detect the mRNA expressions of ER-α,interleukin 10 (IL-10) and B lymphocyte stimulator (BLyS) in peripheral blood mononuclear cells (PBMCs)from 40 SLE patients and 40 normal human controls. The clinical and laboratory correlation with the levels of these parameters was analyzed. Results A significant increase was observed in the relative expression levels of ER-α, IL-10 and BLyS mRNA in SLE patients compared with the normal human controls (P ＜ 0.05 or 0.01 ), in active SLE patients compared with inactive SLE patients (P ＜ 0.05 or 0.01 ). Additionally, the mRNA expression levels of the 3 parameters were significantly correlated with the presence of renal damage, proteinuria, arthritis, etc. No statistical difference was observed in the mRNA expression levels of these parameters between female and male patients or between female and male normal controls. Conclusions IL-10 and BLyS appear to be correlated with the disease activity and severity of SLE, and ER-α may play an important role in the action mechanism of T and B lymphocytes in SLE.     

Effects of tacrolimus on the expression of nuclear factor-κB and glucocorticoid receptor by HaCaT cells in vitro

Objective To investigate the effect of tacrolimus on the expression of nuclear factor-κB (NF-κB) in HaCaT cells stimulated by tumor necrosis factor-α(TNF-α),and on the expression of glucocorticoid receptor (GR)α and β in untreated HaCaT cells in vitro.Methods Cultured Ha CaT cells were treated with TNF-α(10μg/L) only,combination of TNF-α(10μg/L) and various concentrations (10-8mol/L, 10-7mol/L,10-6moL/L) of tacrolimus or tacrolimus of different concentrations only.After additional 12-,24-, 36- or 48-hour cnlture, Westem blot and immunofluorescenee-confocal laser scanning microscopy were used to detect the expressions of NF-κB,GRα and GRβ in HaCaT cells.Those untreated HaCaT cells served as the control.Results The relative protein expression level of NF-κB was increased in HaCaT cells after treatment with TNF-α for 24 and 48 hours zompared with untreated ceils (0.73±0.0316 and 0.8925±0.0171 vs 0.4988±0.03506,both P<0.05);however,the increase in NF-κB expression was inhibited by the combination treatment with tacrolimus,and the relative expression level of NF-κB protein was 0.6825±0.0263.0.6200±0.0163 and 0.5575±0.0299 in HaCaT cells treated with TNF-α plus tacrolimus of 10-8mol/L 10-7mol/L and 10-6mol/L,respectively;the difference was significant etween TNF-α-treated cells and those dealt with the combination of NF-α and tacrolimus of 10-7 or 10-6 mol/L (both P<0.05).No significant difference was observed in the expression of NF-κB by HaCaT cells between different time oints treated with tacrolimus of 10-8,10-7 or 10-6 mol/L.Also,there was no zignificant difference in the expression of GRα or GRβ between untreated HaCaT cells and those treated with tacrolimus of 10-8, 10-7 or 10-6 mol/L at any time point.Conclusions Tacrolimus ould inhibit the expression of NF-κB by TNF-α-stimulated HaCaT cells,but does not affect the expression of GRα or GRβ,in untreated HaCaT cells.     

Effects of tacrolimus on the expression of nuclear factor-κB and glucocorticoid receptor by HaCaT cells in vitro

Objective To investigate the effect of tacrolimus on the expression of nuclear factor-κB (NF-κB) in HaCaT cells stimulated by tumor necrosis factor-α(TNF-α),and on the expression of glucocorticoid receptor (GR)α and β in untreated HaCaT cells in vitro.Methods Cultured Ha CaT cells were treated with TNF-α(10μg/L) only,combination of TNF-α(10μg/L) and various concentrations (10-8mol/L, 10-7mol/L,10-6moL/L) of tacrolimus or tacrolimus of different concentrations only.After additional 12-,24-, 36- or 48-hour cnlture, Westem blot and immunofluorescenee-confocal laser scanning microscopy were used to detect the expressions of NF-κB,GRα and GRβ in HaCaT cells.Those untreated HaCaT cells served as the control.Results The relative protein expression level of NF-κB was increased in HaCaT cells after treatment with TNF-α for 24 and 48 hours zompared with untreated ceils (0.73±0.0316 and 0.8925±0.0171 vs 0.4988±0.03506,both P<0.05);however,the increase in NF-κB expression was inhibited by the combination treatment with tacrolimus,and the relative expression level of NF-κB protein was 0.6825±0.0263.0.6200±0.0163 and 0.5575±0.0299 in HaCaT cells treated with TNF-α plus tacrolimus of 10-8mol/L 10-7mol/L and 10-6mol/L,respectively;the difference was significant etween TNF-α-treated cells and those dealt with the combination of NF-α and tacrolimus of 10-7 or 10-6 mol/L (both P<0.05).No significant difference was observed in the expression of NF-κB by HaCaT cells between different time oints treated with tacrolimus of 10-8,10-7 or 10-6 mol/L.Also,there was no zignificant difference in the expression of GRα or GRβ between untreated HaCaT cells and those treated with tacrolimus of 10-8, 10-7 or 10-6 mol/L at any time point.Conclusions Tacrolimus ould inhibit the expression of NF-κB by TNF-α-stimulated HaCaT cells,but does not affect the expression of GRα or GRβ,in untreated HaCaT cells.     

寻常痤疮患者外周血单核细胞TLR2的表达及其与IL-8和TNF-α的相关性研究

目的 探讨Toll样受体2(TLR2)在寻常痤疮发病中的作用及意义.方法 选择轻、中、重度痤疮患者各30例,采用流式细胞仪检测其外周血CD14+单核细胞TLR2的表达,并采用双抗体夹心酶联免疫吸附法(ELISA)检测血清IL-8和TNF-α的水平.另取30例正常人作为对照组.结果 轻、中、重度组痤疮患者外周血单核细胞表面TLR2的表达均显著高于正常人对照组(P＜0.01).各组患者血清中IL-8和TNF-α的水平也显著高于正常人对照组(P＜0.01).直线相关分析显示,各组患者TLR2的表达与IL-8、TNF-α水平均呈正相关性(r分别为0.382、0.517、0.436、0.641、0.725、0.593,P＜0.05).痤疮严重程度与TLR2的表达呈正相关性(r=0.406,P＜0.01).结论 TLR2表达水平及相关细胞因子浓度与痤疮严重程度密切相关.

Abstract：

Objective To explore the role of Toll like receptor 2 (TLR2) in the pathogenesis of acne vulgaris. Methods Venous blood was collected from 30 normal human controls and 90 patients with acne vulgaris. The patients were equally divided into three groups, i.e., mild, moderate and severe groups, according to disease severity. Flow cytometry was performed to detect the expression of TLR2 in PBMCs, and double antibody sandwich ELISA to measure the levels of serum interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α). Results A significant increase was observed in the expression of TLR2 in PBMCs, serum level of IL-8and TNF-α in the three groups of patients with acne vulgaris compared with the normal human controls (all P ＜0.01). The expression of TLR2 was positively correlated with the expression of IL-8 (r=0.382, 0.517,0.436,respectively, all P＜0.05) and TNF-α(r=0.641, 0.725, 0.593, respectively, all P＜0.05) in the patients with mild, moderate and severe acne vulgaris, respectively, and with the severity of acne vulgaris (r = 0.406,P＜0.01 ). Conclusion The expression level of TLR2 and related cytokines seems closely correlated with the severity of acne vulgaris.     

氧化应激所致的人皮肤成纤维细胞衰老中β-连环蛋白的表达研究

目的 观察β-连环蛋白在氧化应激所致的人皮肤成纤维细胞衰老中的变化,探讨其在应激诱导的皮肤衰老中可能的作用.方法 从儿童包皮分离培养原代成纤维细胞,H2O2:处理第2～4代人皮肤成纤维细胞,显微镜观察其形态学改变,β-半乳糖苷酶细胞衰老试剂盒检测β-半乳糖苷酶活性,RT-PCR、Western印迹检测β连环蛋白mRNA和蛋白表达水平.结果 150 μmol/L H2O2:处理2 h后可以诱导人皮肤成纤维细胞衰老.在应激诱导的衰老(SIPS)细胞中β-半乳糖苷酶阳性率达到(37.67±1.53)%.而对照细胞中只有(2.97±0.25)%,两组差异有统计学意义(P<0.01).RT-PCR 结果显示,在对照细胞组β连环蛋白/GAPDH mRNA比值为0.59±0.04,SIPS细胞组为0.29±0.30,对照细胞组明显高于SIPS细胞组,两组差异有统计学意义(t=10.06,P<0.01).Western印迹结果显示,对照细胞组β连环蛋白/GAPDH蛋白比值为0.62±0.03,SIPS细胞组为0.31±0.01,对照细胞组明显高于SIPS细胞组,两组差异有统计学意义(t=14.97,P<0.0).结论 150 μmol/L H2O2:处理2 h可以诱导人皮肤成纤维细胞发生衰老改变,SIPS细胞中β连环蛋白表达水平明显降低,初步推测β连环蛋白可能是调控皮肤衰老的重要靶基因.

Abstract：

Objective To observe the changes of β-catenin expression in human skin fibroblasts (HSFs) after induced by oxidative stress, and to explore its possible roles in oxidative stress-induced premature senescence (SIPS) of HSFs. Methods Fibroblasts were isolated from the foreskin of a child and subjected to a primary culture. The fibroblasts of second to fourth passage were treated with various concentrations of H2O2 for 2 hours to establish an optimized model of stress-induced premature senescence, β-galactosidase assay kit was used to detect the activity of β-galactosidase in H2O2rinduced HSFs, RT-PCR and Western blot to measure the mRNA and protein expressions of β-catenin in control and senescent HSFs. Results Premature senescence of HSFs could be induced by the treatment with H2O2 of 150 μmol/L for 2 hours. The proportion of β-galactosidase-positive cells was (2.97 ± 0.25)% in control HSFs and (37.67 ± 1.53)% in senescent HSFs (P< 0.01). A significant increase was observed in the β-catenin/GAPDH protein ratio and β-catenin/GAPDH mRNA ratio in control HSFs compared with the senescent HSFs (0.62 ± 0.03 vs. 0.31 ± 0.01, t = 14.97, P < 0.01; 0.59 ± 0.04 vs. 0.29 ± 0.30, t = 10.06, P < 0.01). Conclusions The two-hour treatment with H2O2 of 150 μmol/L could induce the premature senescence of HSFs, and there is a notable decrease in the expression of β-catenin in prematurely senescent HSFs induced by oxidative stress, implying that β-catenin is an important target gene for the regulation of skin aging.     

黄芪甲苷对光老化小鼠皮肤中转化生长因子-β信号通路的影响

目的 探讨黄芪甲苷对皮肤光老化的保护机制.方法 BALB/c小鼠分为模型组、模型+基质组、模型+黄芪甲苷组、正常对照组.RT-PCR测定转化生长因子β受体Ⅱ(TGF-βRⅡ)、Smad 7的mRNA表达水平,免疫组化观察TGF-βR Ⅱ、Smad 7在小鼠皮肤组织中的蛋白表达情况.结果 正常对照组中,TGF-βR Ⅱ、Smad 7的灰度值比值分别为0.5688±0.0439、0.5900 ±0.0585,阳性表达率分别为(53.00 ±4.72)%、(47.50±3.81)%;模型组中,TGF-βR Ⅱ、Smad 7的灰度值比值分别为0.2588±0.0283、0.8637±0.0514,阳性表达率分别为(28.20±5.24)%、(82.06±2.18)%;模型+基质组中,TGF-βR Ⅱ、Smad 7的灰度值比值分别为0.2653±0.0456、0.8553±0.0575,阳性表达率分别为(28.74±2.28)%、(82.62±4.02)%;模型+黄芪甲苷组中TGF-βRⅡ、Smad 7的灰度值比值分别为0.3767±0.0374、0.7131±0.0410,阳性表达率分别为:(41.64±2.59)%、(64.36±2.62)%.皮肤组织中TGF-βRⅡ和Smad灰度值比值4组小鼠间比较,F值分别为80.98和736.80,TGF-βRⅡ和Smad 7的阳性表达率4组小鼠间比较,F值分别为45.36和132.25,P值均<0.01.与正常对照组相比,模型组TGF-βR Ⅱ mRNA和蛋白表达明显降低,Smad 7 mRNA和蛋白表达显著升高(P值均<0.01).与模型组和模型+基质组比较,模型+黄芪甲苷组TGF-βRⅡ mRNA和蛋白表达明显升高,Smad 7 mRNA和蛋白表达显著下调(P值均<0.01).模型+基质组TGF-βR Ⅱ、Smad 7的mRNA和蛋白表达与模型组相比差异无统计学意义(P值均>0.01).结论 黄芪甲苷可以通过上调TGF-βRⅡ表达和下调Smad 7表达而改变TGF-β通路的信号转导参与抗光老化.

Abstract：

Objective To study the protective mechanism of astragaloside on skin photoaging. Methods BALB/c mice were randomly divided into four groups: model group irradiated with ultraviolet rays (UV), model plus matrix group pretreated with the matrix before UV irradiation, model plus astragaloside group pretreated with astragaloside 0.08% cream before UV irradiation, normal control group received no irradiation or pretreatment. After 4-week irradiation, the mice were sacrificed, and skin tissues were resected from the back of these mice. Then, reverse transeription PCR (RT-PCR) and immunohistochemistry were performed to detect the mRNA and protein expression of TGF-βR Ⅱ and Smad 7, respectively. Gray scale ratio was used to represent the mRNA levels of TGF-βR Ⅱ and Smad 7. Results There was a significant difference in the mRNA level (F = 80.98, 736.80, respectively, both P < 0.01) and protein positivity rate (F = 45.36,132.25, respectively,both P < 0.01) of TGF-βR Ⅱ and Smad 7 among the 4 groups. The mRNA level and protein positivity rate of TGF-βR Ⅱwere 0.2588±0.0283 and (28.20 ± 5.24)% respectively in the model group, significantly lower than those in the normal control group[0.5688 ± 0.0439, (53.00 ± 4.72)%, both P < 0.01] and model plus astragaloside group [0.3767 ± 0.0374, (41.64 ± 2.59)%, both P< 0.01]; on the contrary, the mRNA level and protein positivity rate of Smad 7 in the model group [0.8637 ± 0.0514, (82.06 ± 2.18)%] were significantly higher than those in the normal control group [0.5900 ± 0.0585, (47.50±3.81)%, both P < 0.01] and model plus astragaloside group [0.7131 ± 0.0410, (64.36 ± 2.62)%, both P< 0.01]. In the model plus astragaloside group, the mRNA level and protein positivity rate of TGF-βR Ⅱ were significantly higher than in the model plus matrix group [0.2653 ± 0.0456, (28.74 ± 2.28)%, both P < 0.01], while those of Smad 7 were statistically lower than in the model plus matrix group [0.8553 ± 0.0575, (82.62 ± 4.02)%, both P < 0.01]. However,no significant difference was observed in the mRNA level or protein positivity rate of TGF-βR Ⅱ or Smad 7 between the model group and model plus matrix group (all P > 0.01). Conclusion Astragaloside can prevent skin photoaging by the alteration of TGF-β pathway via up-regulating TGF-βR Ⅱ expression and down-regulating Smad 7 expression.     

乳房外Paget病皮损中黏蛋白1和黏蛋白2的表达

目的 探讨黏蛋白1(MUC1)和黏蛋白2(MUC2)在乳房外Paget病(EMPD)中的表达情况.方法 生物素蛋白免疫组化法(SP法)检测19例EMPD皮损及19例美容切除术后正常皮肤组织上MUC1与MUC2的表达.结果 19例EMPD皮损常规HE染色显示,3例伴低分化腺癌,6例呈浸润性,10例为上皮内.MUC1在3例伴腺癌Paget病中有2例呈阳性表达,6例浸润性和10例上皮内Paget病均呈阳性表达.MUC2在3例伴腺癌Paget病和6例浸润性Paget病均呈阳性表达,在10例上皮内Paget病中有2例呈阳性表达.MUC1与MUC2在正常皮肤组织呈阴性表达.MUC1在上皮内Paget病中的表达显著高于伴腺癌Paget病和浸润性Paget病(P＜0.05).MUC2在伴腺癌Paget病和浸润性Paget病中的表达显著高于上皮内Paget病(P＜0.05).MUC1和MUC2的表达无明显相关性(r=-0.5,P＞0.05).结论 MUC1在EMPD中呈普遍表达,MUC2在伴有腺癌和浸润性EMPD中呈阳性表达.

Abstract：

Objective To study the expressions of mucin (MUC) 1 and 2 in extramammary Paget's disease(EMPD) lesions. Methods Tissue specimens were obtained from the lesions of 19 patients with EMPD and normal skin of 19 human controls during cosmetic surgery. Streptavidin-perosidase (SP) technique was used to detect the expressions of MUC1 and MUC2 in these specimens. Results As haematoxylin-eosin (HE) staining showed, 3 cases were accompanied by poorly differentiated adenocarcinoma, 6 were invasive Paget's disease and 10 were intraepithelial EMPD. MUC1 was expressed in 2 cases accompanied by poorly differentiated adenocarcinoma, and in all the cases of invasive and intraepithelial EMPD; MUC2 was observed in all the cases of adenocarcinoma-complicated EMPD and invasive EMPD, but only in 2 of 10 cases of intraepithelial EMPD.Neither MUC1 nor MUC2 was observed in normal control specimens. A significant increase was observed in the expression of MUC1 in lesions of intraepithelial EMPD compared with invasive EMPD and adenocarcinoma-complicated EMPD (both P ＜ 0.05), and in the expression of MUC2 in lesions of invasive EMPD and adenocarcinoma-complicated EMPD compared with intraepithelial EMPD (both P ＜ 0.05). The expression of MUC1 was uncorrelated to that of MUC2 (r= -0.5, P＞ 0.05). Conclusions MUC1 is generally expressed in the lesions of EMPD, while MUC2 is expressed in those of adenocarcinoma-complicated EMPD and invasive EMPD.     

银屑病性关节炎患者血清中转化生长因子-β1的检测

目的 探讨银屑病性关节炎患者血清中转化生长因子(TGF)-β1的表达及其意义.方法 分别检测银屑病性关节炎患者与寻常性银屑病患者血清中TGF-β1的水平.结果 38例银屑病性关节炎患者血清中的TGF-β1浓度为375.92±95.88 pg/ml,80例寻常性银屑病患者为510.78±167.97 pg/ml,两组差异有统计学意义(t=4.60,P<0.01).结论 对银屑病性关节炎患者进行血清TGF-β1浓度的动态监测,有助于引起临床医师对银屑病性关节炎骨质疏松的警惕和预防.

Abstract：

Objective To measure the expression of TGF-β1 in the sera of patients with psoriatic arthritis and its significance.Methods The serum level of TGF-β1 was measured in 38 patients with psoriatic arthritis and 80 patients with psoriasis vulgaris.Results A significant difierence was observed between the patients with psoriatic arthritis and those with psoriasis vulgaris(375.92±95.883 pg/ml vs.510.78±167.967 pg/ml,t=4.60,P<0.01).Conclusion Continuous monitoring of serum levels of TGF-β1 may help clinicians to take precautions against osteoporosis in patients with psoriatic arthritis.     

SLE患者外周血T、B淋巴细胞TLR9蛋白表达及与临床指标的关系

Objective To study the expressions of Toll-like receptor 9 protein (TLR9) in peripheral B and T lymphocytes in newly diagnosed, untreated patients with systemic lupus erythematosus (SLE) and their relationship with clinical parameters. Methods Blood samples were obtained from 35 newly diag-nosed, untreated patients with SLE and 16 healthy human controls. B, T lymphocytes and TLR9 protein were labeled with fluorescent antibodies, and the expressions of TLR9 protein were detected by flow cytometry in peripheral B and T lymphocytes. The relationship between TLR9 expression and clinical parameters was assessed. Results The proportions of B and T lymphocytes expressing TLR9 in newly diagnosed, untreated patients were (53.94±17.95)% and (49.33 ± 23.30)%, respectively, compared to (29.40 ± 10.54)% and (29.18 ± 14.78)%, respectively, in healthy controls (t = 6.11,3.73, respectively, both P < 0.01). Additionally,the proportion of B lymphocytes expressing TLR9 correlated negatively with SLE disease activity index (SLEDAI)(r = -0.39, P < 0.05), but positively with the level of serum IgA antibody (r = 0.74, P < 0.01).Condnsions The expression of TLR9 is elevated in peripheral T and B lymphocytes from patients with newly diagnosed, untreated SLE, and the proportion of TLR9-expressing B lymphocytes negatively correlates with SLEDAI, but positively correlates with the serum level of IgA antibody.     

SLE患者外周血T、B淋巴细胞TLR9蛋白表达及与临床指标的关系

Objective To study the expressions of Toll-like receptor 9 protein (TLR9) in peripheral B and T lymphocytes in newly diagnosed, untreated patients with systemic lupus erythematosus (SLE) and their relationship with clinical parameters. Methods Blood samples were obtained from 35 newly diag-nosed, untreated patients with SLE and 16 healthy human controls. B, T lymphocytes and TLR9 protein were labeled with fluorescent antibodies, and the expressions of TLR9 protein were detected by flow cytometry in peripheral B and T lymphocytes. The relationship between TLR9 expression and clinical parameters was assessed. Results The proportions of B and T lymphocytes expressing TLR9 in newly diagnosed, untreated patients were (53.94±17.95)% and (49.33 ± 23.30)%, respectively, compared to (29.40 ± 10.54)% and (29.18 ± 14.78)%, respectively, in healthy controls (t = 6.11,3.73, respectively, both P < 0.01). Additionally,the proportion of B lymphocytes expressing TLR9 correlated negatively with SLE disease activity index (SLEDAI)(r = -0.39, P < 0.05), but positively with the level of serum IgA antibody (r = 0.74, P < 0.01).Condnsions The expression of TLR9 is elevated in peripheral T and B lymphocytes from patients with newly diagnosed, untreated SLE, and the proportion of TLR9-expressing B lymphocytes negatively correlates with SLEDAI, but positively correlates with the serum level of IgA antibody.