fas介导系统性红斑狼疮患者Foxp3+CD4+CD25+Treg细胞凋亡的研究

目的 探讨fas凋亡信号传导途径在系统性红斑狼疮(SLE)患者Foxp3+CD4+CD25+Treg凋亡异常中的作用.方法 选取活动期SLE患者25例、缓解期SLE患者20例及健康对照25名为研究对象,检测所有研究对象外周血Foxp3+CD4+CD25+Treg表面fas的表达,同时分析CD4+CD25+T细胞Foxp3表达.并分别将fas表达率及Foxp3表达率与病情活动性(SLEDAI评分)进行相关分析.结果 (1)外周血Foxp3+CD4+CD25+Treg上fas的表达:活动期SLE组为(23.72±2.35)%,缓解期SLE组为(14.0±2.1)%,对照组为(10.1±1.2)%,在活动期SLE组明显高于缓解期SLE组(P＜0.01)和对照组(P＜0.01),而缓解期SLE组与对照组差异无统计学意义(P＞0.05),fas在Foxp3+CD4+CD25+Treg上的表达与SLEDAI评分呈正相关(r=0.336,P＜0.05).(2)外周血CD4+CD25+T细胞Foxp3的表达:活动期SLE组为(2.83±0.30)%,缓解期SLE组为(5.38±0.63)%,对照组为(8.12-±0.70)%.活动期SLE组外周血 CD4+CD25+T细胞Foxp3表达明显低于缓解期SLE组(P＜0.01)和对照组(P＜0.01);而缓解期SLE组亦低于对照组(P＜0.05).外周血Foxp3表达与SLEDAI评分呈负相关(r=-0.581,P＜0.01).(3)Foxp3与fas的表达呈负相关(r=-0.349,P＜0.01).结论 SLE患者中存在由fas介导的Foxp3+CD4+CD25+Treg的过度凋亡,这可能是导致SLE病情活动的机制之一.

Abstract：

Objective To explore the role of fas apoptosis signal transduction pathway in the abnormal apoptosis of Foxp3 + CD4 + CD25 + Treg in patients with systemic lupus erythematosus ( SLE ).Methods Twenty-five active SLE patients, 20 remission SLE patients and 25 controls were selected. The level of fas expression on peripheral blood Foxp3 + CD4 + CD25 + Treg surface was detected in SLE patients.And analyzed the expression rate of Foxp3 on CD4 + CD25 + T cells was analyzed to explore the relationship between the expression rate and disease activity. Results ( 1 ) The expression rate of fas on Foxp3 + CD4 +CD25 + Treg was (23.72 ± 2. 35 )% , ( 14. 0 ± 2. 1 )% in active and remission SLE groups respectively versus ( 10. 1 ± 1.2)% in control group. The fas expression rate of active SLE group was significantly higher than those of remission SLE group( P ＜ 0. 01 ) and control group ( P ＜ 0. 01 ). And the remission SLE and control groups were not statistically significant ( P ＞0. 05 ). The expression rate of fas on the Foxp3 + CD4 +CD25 + Treg was positively correlated with the SLEDAI ( SLE disease activity index ) score ( r = 0. 336, P ＜0.05). (2) The expression rate of Foxp3 on CD4 +CD25 +T cells was (2.83 ±0.30)%, (5.38 ±0. 63 ) % in active and remission SLE groups respectively versus ( 8. 12 ± 0. 70 ) % in control group. The expression rate of Foxp3 was significantly lower in active SLE group than that in remission SLE group ( P ＜0. 01 )and control group( P ＜0. 01 ). And the Foxp3 expression rate of remission group was also lower than that of control group ( P ＜ 0.05 ). The expression rate of Foxp3 was negatively correlated with the SLEDAI score (r = -0. 581, P ＜ 0. 01 ). (3) The expression rate of Foxp3 was negatively correlated with fas (r=- 0. 349, P ＜ 0. 01 ). Conclusion The abnormal apoptosis of Foxp3 + CD4 + CD25 + Treg mediated by the fas apoptosis signal transduction pathway may be one of the pathogenic mechanisms of disease activity in SLE patients.     

Effect of Chaiqin Chengqi Decoction(柴芩承气汤) on Cholecystokinin Receptor 1-Mediated Signal Transduction of Pancreatic Acinar Cells in Acute Necrotizing Pancreatitis Rats

Objective:To investigate the effect of Chaiqin Chengqi Decoction(柴芩承气汤,CQCQD)on cholecystokinin receptor 1(CCKRI)-mediated signal transduction of pancreatic acinar cell in rats with acute necrotic pancreatitis(ANP).Methods:Twenty-seven Sprague-Dawley rats were randomized into three groups:the control group,the ANP group,and the CQCQD group(9 in each group).ANP rats were induced by two intraperitoneal injections of 8%L-arginine(pH=7.0,4.4 g/kg) over a 2-h period.Rats were treated with 1.5 mL/100 g body weight of CQCQD(CQCQD group) or physiological saline(control and ANP groups) at 2 h interval.And 6 h after induction,pancreatic tissues were collected for histopathological examination.Pancreatic acinar cells were isolated for determination of CCKR1 mRNA and protein expression,phospholipase C(PLC) and inositol-1,4,5-triphosphate(IP3),and determination of fluorescence intensity(Fl)as a measure of intracellular calcium ion concentration[Ca~(2+)]_i.Results:The pancreatic histopathological score(6.2 + 1.1) and the levels of PLC(1,187.2 ±228.2 μg/mL) and IP3(872.2 ±88.4 μg/mL) of acinar cells in the ANP group were higher than those in the control(2.8 ±0.4,682.5 ±121.8 μg/mL,518.4 ±115.8 μg/mL)and the CQCQD(3.8 ±0.8,905.3 ±78.5 μg/mL,611.0 ±42.5 μg/mL) groups(P0.05).[Ca~(2+)]_i Fl for the ANP group(34.8 ±27.0) was higher than that in the control(5.1 ±2.2) and CQCQD(12.6 ±2.5) groups(P0.05).The expression of pancreatic acinar cell CCKR1 mRNA in the ANP group was up-regulated(expression ratio=1.761;P=0.024) compared with the control group.The expression of pancreatic acinar cell CCKR1 mRNA in the CQCQD group was down-regulated(expression ratio=0.311;P=0.035) compared with the ANP group.The ratio of gray values of the CCKR1 and B-actin in the ANP group(1.43 ±0.17) was higher than those in the control(0.70 ±0.15) and CQCQD(0.79 ±0.11) groups(P0.05).Conclusions:Pancreatic acinar cell calcium overload of ANP induced by L-arginine was related to the up-regulated expressions of pancreatic acinar cell CCKR1 mRNA and protein.CQCQD can down-regulate expressions of pancreatic acinar cell CCKR1 mRNA and protein to reduce the PLC and IP3 of pancreatic acinar cells,relieving the calcium overload and reducing the pathological changes in rats with ANP.     

Expression of Toll-like receptor 4 in neonatal cord blood mononuclear cells in patients with preeclampsia

The expression of Toll-like receptor 4 (TLR4) in neonatal cord blood mononuclear cells (MNCs) and serum TNF-α were investigated in order to explore the roles of TLR4 in the pathogenesis of preeclampsia.The study enrolled 27 patients suffering from preeclampsia (experimental group) and 21 normal pregnancy patients (control group).After MNCs were separated, the expression of TLR4 mRNA and protein was detected by using real-time quantitative PCR and Western blotting respectively, and the expression of TNF-α by using ELISA.The results showed the TLR4 mRNA level in cord blood MNCs (2-CT:0.07±0.17), TLR4 protein expression level (absorbance ratio:0.81%±0.15%) and TNF-α level (9.5±1.73 pg/mL) were all increased in experimental group as compared with control group with the differences being statistically significant (P<0.05).There was a positive correlation between the expression of TLR4 mRNA and TNF-α in both experimental group and control group (r=0.54 and 0.53, respectively, P<0.05).It was concluded that TLR4 expression in the experimental group of cord blood MNCs was increased and there was a positive correlation between the expression of TLR4 mRNA and TNF-α in both groups.TLR4-mediated release of inflammatory cytokines may be one of the important reasons leading to preeclampsia.     

Over-expression of VEGF and MMP-9 in residual tumor cells of hepatocellular carcinoma after embolization with lipidol

The expression and implication of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) in residual hepatic tumor cells after lipiodol embolization were investi- gated. Two weeks after transplantation of VX2 tumor cells into the livers of rabbits, a xenograft model of the human hepatic neoplasm was successfully established. Forty rabbits were randomly divided into control group (n=20) and lipiodol group (n=20). For the control group, 1 mL normal saline was injected through the gastroduodenal artery, whereas 0.3 mL/kg lipiodol was applied for the lipiodol group. One week after embolization, the expression level of VEGF in the plasma was measured by using en- zyme-linked immunosorbent assay (ELISA). A three-step immunohistochemical technique (ABC) was employed to detect the protein levels of VEGF and MMP-9 and the quantitative PCR for their mRNA levels was performed in the residual tumor cells. The VEGF in the plasma was significantly higher in the lipiodol group (1.42±0.29 ng/mL) than in the control group (1.12±0.21 ng/mL) (P<0.01). Moreover, the positive rate of VEGF protein in the residual tumor cells was significantly higher in the lipiodol group (62.13%±7.69%) than in the control group (53.16%±9.17%) (P<0.05). Similarly, the MMP-9 expression in the residual tumor cells was higher in the lipiodol group. The mRNA levels of VEGF (2.9313±2.4231) and MMP-9 (3.5721±1.6107) in the lipiodol group were significantly higher than those in the control group (1.5728±0.9453 and 1.7573±1.0641, respectively, P<0.05). Therefore, it was rea- sonable to speculate that the increased expression of VEGF and MMP-9 in residual hepatic tumor cells and tumor angiogenesis post-embolization would be responsible for the increased metastatic potentiality and invasiveness of these cells.     

APP、Fis1、KLRF1和PDCD7基因在急性髓系白血病中的表达及临床意义

目的 探讨淀粉样前体蛋白(APP)、裂殖1同源物(Fis1)、杀伤细胞凝集素样受体亚家族F成员1(KLRF1)和细胞程序性死亡7(PDCD7)mRNA在急性髓系白血病(AML)细胞中的表达及其预后意义.方法 抽取34例AML及10例非恶性血液病患者骨髓.提取总RNA并逆转录成cDNA,SYBR Green I实时定量PCR法检测各基因mRNA表达水 平,采用2-~(△Cr)公式计算基因mRNA相对表达量.结果 AML患者组PDCD7mRNA表达水平均显著高于对照组(Z=-2.213,P=0.027).除M1(1例)、M2a(2例)外的AML各亚型基因表达同对照组比较,APPmRNA在伴有t(8,21)染色体异常的M2表达显著高于对照组(Z=-2.197,P=0.028),而在M4b和M5b中的表达则显著低于对照组(Z=-2.368,P=0.018).APP mRNA在AML亚型间表达有差异,M2显著高于M4和M5(Z=-2.430,P=0.015;Z=-3.175,P=0.001).结论 AML患者高表达PDCD7基因,单核细胞白血病患者低表达APP基因.

Abstract：

Objective To investigate the expression of amyloid precursor protein (APP), Fis1, PDCD7 and KLRF1 mRNA in acute myeloid leukemia (AML) and their prognostic significances. Methods Thirty-four AML patients and 10 patients with nonmalignant hematologic diseases (control group) were recruited in this study. Bone marrows were obtained from these patients to isolate the mononuclear cells, from which total RNA was extracted and reverse transcribed into cDNA. SYBR Green Ⅰ dye-based real-time quantitative PCR method was used to compare the expression of APP, Fis1, PDCD7 and KLRF1 mRNA between two groups. Results The expression level of PDCD7 mRNA in AML patients was significantly higher than that in the control group (Z=-2.213,P=0.027), but APP, Fisl and KLRF1 mRNA expression levels were similar between the two groups (P<0.05). The expression level of APP mRNA in M2 patients with t (8, 21) chromosomal abnormality was significantly higher than that in the control group (Z=-2.197, P=0.028), while APP mRNA expressions in M4b and M5b patients were both lower than those in the control group (Z=-2.368, P-0.018). The expression of APP mRNA differed significantly between the subtypes of AML, higher in M2 than in M4b and M5b (Z=-2.430, P=0.015; Z=-3.175, P=0.001, respectively). Conclusions The expression of PDCD7 mRNA increased significantly in AML patients, and APP mRNA expression is decreased in monocytic leukemia.     

Protective Effect of Norcantharidin on Collagen-Induced Arthritis Rats

Objective: To observe the effect of norcantharidin(NCTD) on collagen-induced arthritis(CIA) rats. Methods: Sixty Sprague-Dawley(SD) rats were randomly divided into 6 groups(n=10): normal group, CIA model group(model group), NCTD low-dose group [1.35 mg/(kg·d)], NCTD middle-dose group [2.7 mg/(kg·d)], NCTD high-dose group [5.4 mg/(kg·d)] and methotrexate(MTX) group [1.8 mg/(kg/w)]. Anesthetized rats were sacrificed by luxation of cervical vertebra after 4 weeks of administration. The arthritis scores were evaluated twice a week. The pathological changes in the ankle joints of rats were observed by hematoxylin-eosin(HE) staining. The serum levels of interleukin(IL) 1β, IL-6, tumor necrosis factor(TNF)-α, vascular endothelial growth factor(VEGF), IL-17 and transform growth factor(TGF) β were detected by enzyme linked immunosorbent assay(ELISA). The mRNA expression of retinoid-related orphan nuclear receptor γ t(ROR γ t) and forkhead box P3(Foxp3) in peripheral blood lymphocytes were confirmed by real-time polymerase chain reaction. Results: MTX and high-dose NCTD not only decreased the arthritis scores but also alleviated the pathological changes in CIA rats' ankle joints compared with the model group(P0.05 or P0.01). All doses of NCTD significantly inhibited the serum levels of IL-6, IL-17 and TNF-α in CIA rats(P0.05). Only middle-and high-dose of NCTD prominently decreased serum IL-1β and TGF-β levels of CIA rats(P0.05). However, NCTD has no effect on vascular endothelial growth factor(VEGF) level in CIA rats. The Foxp3 mRNA expression in all NCTD groups were increased significantly than in the model group(P0.05). The mRNA expression of RORγt in NCTD high-dose group was decreased apparently in comparison with the model group(P0.05). Conclusion: NCTD showed therapeutic effect on CIA rats by inhibition of cytokines and regulation of Th17/Treg cells.     

GMCSF对高氧暴露新生大鼠肺组织RAGE-NF-κB通路的影响

目的 探讨高氧暴露对新生大鼠肺组织高级糖基化终产物受体(RAGE)-核因子κB(NF-κB)信号通路的影响及粒细胞巨噬细胞集落刺激因子(GMCSF)肺损伤保护作用的相关机制.方法 24只3日龄新生大鼠随机平均分为3组:正常对照组(空气环境下7 d)、高氧对照组(95%氧暴露7 d)、高氧干预组(95%氧暴露7 d+GMCSF皮下注射9 μg/kg/次,共3次);光镜观察并盲法进行肺组织病理学损伤评分;RT-PCR法检测肺组织匀浆RAGE mRNA、NF-κB mRNA表达;Western印迹检测肺组织匀浆RAGE、NF-κB蛋白表达;ELISA检测支气管肺泡灌洗液(BALF)及血清中肿瘤坏死因子-α(TNF-α)水平;免疫组化法检测肺组织石蜡切片RAGE表达情况.结果 正常对照组、高氧对照组、高氧干预组肺组织损伤评分分别为0.46±0.20、3.06±0.33、2.31±0.56,差异有统计学意义(P=0.000);3组RAGE mRNA和蛋白表达分别为0.14±0.02、0.34±0.06、0.28±0.04和0.30±0.04、0.76±0.11、0.55±0.08,差异均有统计学意义(均P=0.000);3组NF-κB mRNA和蛋白表达分别为0.41±0.21、0.90±0.36、0.69±0.30和0.41±0.26、0.96±0.43、0.77±0.33,差异均有统计学意义(P=0.000和P=0.017);3组BALF中TNF-α水平分别为76±10、224±42、143±24,差异有统计学意义(P=0.000).以上各指标结果在高氧对照组、高氧干预组均明显高于正常对照组(均P<0.05),高氧干预组低于高氧对照组(P<0.05).3组血清TNF-α水平差异无统计学意义(P>0.05).结论 GMCSF可能通过下调RAGE-NF-κB信号通路对高氧肺损伤发挥保护作用.

Abstract：

Objective To explore the effects of granulocyte-macrophage colony-stimulating factor (GMCSF) on hyperoxia exposure lung injury in newborn rats and elucidate its protective mechanism of operating via the signaling pathway of advanced glycation end products (RAGE)-NF-κB.Methods Twenty-four 3-day-old SD rats from 3 litters were randomly divided into 3 groups. They were hyperoxia exposure plus GMCSF group (group A), hyperoxia exposure group (group B) and air exposure group (group C). The rats from groups A and B were placed in a sealed Plexiglas chamber with a minimal in-and-outflow, providing 6-7 exchanges per hour of chamber volume and maintaining O2 levels above 95%.While the rats in group C only were exposed to air simultaneously.The rats in group A received subcutaneous injections of recombinant murine GMCSF (9 μg/kg) during hyperoxia exposure at 24 h, 72 h and 120 h respectively. And the rats in groups B and C received subcutaneous injections of saline vehicle alone at the same time point. Seven days later, all were sacrificed and immunohistochemistry was employed to assess the expression of RAGE in lung tissue. The levels of tumor necrosis factor-α in bronchoalveolar lavage fluid (BALF) and serum samples were detected by ELISA (enzyme-linked immunosorbent assay). The RAGE mRNA and NF-κB mRNA in tissue homogenates were detected by RT-PCR while RAGE and NF-κB by Western blot. Also the values of lung damage score were calculated with microscopic histology.Results The value of lung damage score in group C, B and A was 0.46±0.20, 3.06±0.33 and 2.31±0.56 respectively, there was significantly difference among three groups (P=0.000). The expression of RAGE mRNA and protein in three groups were 0.14±0.02, 0.34±0.06, 0.28±0.04 and 0.30±0.04, 0.76±0.11, 0.55±0.08 respectively. There were both significantly differences among three groups (P=0.000, P=0.000). The expression of NF-κB mRNA and protein in three groups were 0.41±0.21, 0.90±0.36, 0.69±0.30 and 0.41±0.26, 0.96±0.43, 0.77±0.33 respectively, there were both significantly difference among three groups (P=0.000, P=0.017). The level of TNF-α in BALF was 76±10, 224±42 and 143±24 respectively, there was significantly difference among three groups (P=0.000). All indicators above in group B and group A were significantly more than those in group C (all P<0.05), while these indicators in group A were lower than those in group B. But there was no difference in the level of TNF-α of serum among three groups (P>0.05).Conclusion GMCSF may protect hyperoxia-induced lung injury via down-regulating the signaling pathway of RAGE-NF-κB.     

Expression of Interleukin-17 in Lung and Peripheral Blood of Asthmatic Rats and the Influence of Dexamethasone

The expression of interleukin-17(IL-17) in lung and peripheral blood of asthmatic rats and the influence of dexamethasone,and the role of IL-17 in the pathogenesis of asthma were investigated.Thirty Sprague-Dawley(SD) adult rats were randomly divided into three groups(n=10 in each group):normal group,asthmatic group,and dexamethasone-interfered group.Rat asthmatic model was established by intraperitoneal(i.p.) injection of 10% ovalbumin(OVA) and challenge with 1% OVA via inhalation.Rats in dexamethasone-interfered group were pretreated with dexamethasone(2 mg/kg,i.p.) 30 min before each challenge.The expression of IL-17 protein in serum and bronchoalveolar lavage fluid(BALF) was detected by ELISA.The expression of IL-17 mRNA in peripheral blood mononuclear cells(PBMC) and BALF cells was semi-quantitatively detected by RT-PCR.The expression of IL-17 protein in serum and BALF of asthmatic rats was significantly elevated as compared with normal rats and dexamethsone-interfered rats(P<0.01),and there was significant difference between normal rats and dexamethsone-interfered rats(P<0.05).The expression of IL-17 mRNA in PBMC and BALF cells of asthmatic rats was markedly increased as compared with normal rats and dexamethsone-interfered rats(P<0.01),and significant difference was found between normal rats and dexamethsone-interfered rats(P<0.05).It was concluded that the expression of IL-17 was increased significantly in asthmatic rats and could be inhibited partly by dexamethasone,suggesting that IL-17 might play an important role in the pathogenesis of asthma as an inflammation regulation factor.     

溃疡性结肠炎中Th17/Treg细胞联合检测的临床意义

目的 检测溃疡性结肠炎(UC)患者血液中Th17/Treg细胞的比例变化,探讨其在UC发病中的作用.方法 该院2009年6月～2011年6月消化内科诊治的UC患者42例,另取健康对照42例.流式细胞仪检测Th17和Treg细胞的表达百分比;Trizol提取血液细胞总RNA,Real-time荧光定量PCR检测ROR γt和Foxp3基因的表达;ELISA检测IL-6和TGF-β1的表达水平.结果 UC患者血液中出现了Th17细胞比例的升高和Treg细胞比例的降低,相应的Th17/Treg细胞比值从对照组的(0.54±0.18)升高到UC组的(7.84±2.07)(P ＜0.01).ROR γt基因在UC患者中表达上调,而Foxp3基因则表达下调,与对照组相比二者的比值从(0.767±0.084)升高到(1.351±0.114)(P＜0.01).ELISA检测结果表明UC患者外周血中细胞因子IL-6的表达水平显著高于对照组(P＜0.01),而TGF-β1的表达水平显著低于对照组(P＜0.01).结论 UC患者存在Th17/Treg细胞的比例失衡,Th17/Treg细胞比例的失衡,可能参与LD患者免疫功能的紊乱,在UD患者肠道的损伤中发挥着重要的作用.     

梅毒血清固定患者外周血中Th17/Treg细胞、Foxp3和ROR-γt的表达水平及相关性研究

目的 通过检测梅毒血清固定患者外周血Th 17/Treg细胞比例及其特异性转录因子叉头状转录因子3(Foxp3)和维甲酸相关核孤儿受体(ROR-γt)的mRNA表达水平以及相关的细胞因子水平,探讨其与梅毒血清固定免疫机制的关系.方法 采集60例梅毒血清固定患者(观察组)和60例健康对照者(对照组)的外周血,分离单一核细胞(PBMCs),采用流式细胞仪检测Th17和Treg细胞比例;提取总RNA,采用实时荧光定量PCR(QRT-PCR)法检测Foxp3和ROR-γt的mRNA表达水平;采用ELISA法测定PBMCs上清液中IL-17和转化生长因子-β(TGF-β)水平.结果 观察组外周血中的Treg细胞比例[(9.68±1.54)％]、Foxp3的mRNA[(2.85±0.16)％]和TGF-3[(275.15±20.89) pg/ml]的表达水平明显高于对照组[(5.96±1.27)％、(1.96±0.14)％、(156.33 ±41.49) pg/ml];观察组中的Thl7细胞比例[(1.13±0.31)％]、ROR-γt的mRNA[(0.98±0.16)％]和IL-17的表达水平[(12.21 ±4.73) pg/ml]明显低于对照组[(1.98±0.17)％、(1.85±0.15)％、(18.98±6.89) pg/ml],差异有统计学意义(P＜0.05).结论 外周血中Treg和Th17细胞比例的变化、Foxp3和ROR-γtmRNA表达水平的异常与梅毒血清固定的免疫机制相关,梅毒血清固定患者可能存在Treg/Th17的失衡,且向Treg细胞漂移.     

Treg/Th17细胞、钙卫蛋白在溃疡性结肠炎模型大鼠血清及肠组织中的表达变化

目的 探讨辅助T细胞（Th）、调节性T细胞（Treg）及钙卫蛋白在溃疡性结肠炎(UC)大鼠炎症发生中的作用。方法 30只大鼠随机均分为UC模型组（UC组）、正常对照组(NC组),UC组大鼠采用肠道灌注三硝基苯磺酸/乙醇混合液,复制UC模型;NC组大鼠予生理盐水灌肠。模型制备成功后21 d,观察两组大鼠肠组织形态学改变,采用酶联免疫吸附法检测大鼠血清细胞因子白介素(interleukin, IL)-1β、IL-10、IL-17、IL-23及钙卫蛋白的变化,采用流式细胞术检测大鼠外周血Treg变化,分别采用免疫组化、免疫印迹检测大鼠肠组织IL-17、决定Treg细胞分化及功能的转录调控蛋白（FoxP3）及钙卫蛋白的表达。结果 UC大鼠肠组织炎细胞浸润明显,组织出现损伤;与NC组相比,UC组大鼠血清IL-1β、IL-17、IL-23、钙卫蛋白的表达升高;血清IL-10和外周血Treg表达降低(P<0.01或P<0.05)。UC组大鼠肠组织IL-17、钙卫蛋白的表达较NC组升高(P<0.01或P<0.05),FoxP3的表达较NC组降低(P<0.05)。结论 UC大鼠血清、肠组织中钙卫蛋白、IL-17表达增强,导致Th17/Treg细胞失衡,炎症反应升高,从而促进溃疡性结肠炎的发生和发展。     

终止香烟烟雾暴露后大鼠Th1/Tc1介导气道炎症及调节性T细胞的变化

目的 观察香烟烟雾暴露和终止香烟烟雾暴露后大鼠Th1/Tc1介导气道炎症及支气管肺泡灌洗液(BALF)中调节性T细胞(Treg)的变化.方法 将50只健康清洁级雄性Wistar大鼠随机分为5组:12周正常对照组(简称12周对照组)、24周正常对照组(简称24周对照组)、12周香烟烟雾暴露组(简称12周暴露组)、24周香烟烟雾暴露组(简称24周暴露组)、终止香烟烟雾暴露组(简称终止暴露组).烟熏法复制大鼠气道炎症的动物模型.12周后,12周对照组、12周暴露组处置取材,24周暴露组继续烟熏12周,终止暴露组终止烟雾暴露12周,将后2组及24周对照组处置取材.HE染色观察小气道病理改变,进行气道评分;收集BALF进行细胞学计数和分类计数;酶联免疫吸附法(ELISA)法测BALF中4种细胞因子:Th1/Th2型细胞因子干扰素(IFN)γ、白细胞介素(IL)4及促炎因子IL-8、肿瘤坏死因子(TNF)α的浓度;流式细胞术检测各组大鼠BALF中Treg细胞的比例,RT-PCR检测各组大鼠BALF中Foxp3 mRNA的表达.结果 (1)12周暴露组、24周暴露组、终止暴露组气道炎症评分较12周对照组、24周对照组明显高(均P＜0.01).24周暴露组、终止暴露组气道炎症评分均较12周暴露组高(均P＜0.01).(2)与12周对照组、24周对照组相比,12周暴露组、24周暴露组、终止暴露组BALF中IFN-γ、TNF-α和IL-8高,IL-4低(均P＜0.01).与12周暴露组相比,终止暴露组IFN-γ、IL-4、TNF-α差异均无统计学意义(均P＞0.05),IL-8较高(P＜0.01),24周暴露组IFN-γ、TNF-α和IL-8明显高(均P＜0.01).(3)BALF中Treg细胞比例,12周暴露组(7.4%±0.8%)、24周暴露组(7.8%±1.7%)、终止暴露组(7.0%±1.4%)较12周对照组(4.8%±1.2%)、24周对照组(4.7%±1.2%)高(均P＜0.01),前3组之间BALF中Treg细胞比例差异均无统计学意义(均P＞0.05).(4)12周暴露组(0.22±0.02)、24周暴露组(0.23±0.03)、终止暴露组(0.20±0.04)BALF中Foxp3 mRNA表达较12周暴露组(0.13±0.01)、24周暴露组(0.11±0.02)高(均P＜0.01).前3者之间BALF中Foxp3 mRNA表达差异均无统计学意义(均P＞0.05).结论 香烟暴露致大鼠气道Th1/Tc1介导炎症伴Treg细胞表达增高,终止香烟烟雾暴露后其炎症及Treg细胞高表达仍持续存在,提示该免疫失衡可能是导致终止香烟暴露后Th1/Tc1气道炎症仍持续进展的原因之一.     

类风湿关节炎患者TH17细胞及其相关细胞因子的表达及临床意义

目的 探讨类风湿关节炎(RA)患者TH17细胞及其相关细胞因子的表达及临床意义.方法 收集大连医科大学附属第一医院门诊RA初治活动期患者的外周血标本32例为活动期RA组,健康献血者30例为健康对照组,所有受试者清晨空腹坐位肘静脉采血.应用酶联免疫吸附法(ELISA)及反转录PCR(RT-PCR)法,分别检测外周血血清中IL-17水平和外周血单个核细胞(PBMC)中IL-17mRNA表达水平;将PBMC一组不加刺激,另一组用佛波酯(50 ng/mL)和离子霉素(1μg/mL)刺激4h后,流式细胞仪检测TH17细胞数量.结果 活动期RA患者血清IL-17水平(15.76 ±0.62) pg/mL与健康对照组(8.67±0.12) pg/mL比较,差异有显著性意义(P＜0.05).活动期RA患者PBMC的IL-17mRNA表达水平(1.94±0.46)与健康对照组(0.84±0.16)比较,差异有显著性意义(P＜0.05).RA组血清IL-17水平与mRNA表达水平呈正相关(r=0.70,P＜0.05),且与疾病活动程度(DAS28)评分呈正相关(r=0.99,P＜0.05).活动期RA患者外周血TH17细胞比例(2.50±0.54)％与健康对照组(0.03±0.00)％比较,差异有显著性意义(P＜0.05).但刺激组(3.23±0.05)％与无刺激组(2.50±0.54)％比较,差异无显著性意义(P＞0.05).RA患者组TH17细胞数量与疾病活动程度(DAS28)评分呈正相关(r=0.96,P＜0.05).结论 活动期RA患者TH17细胞及其主要效应性细胞因子IL-17在细胞、蛋白和基因表达水平上均明显升高并与疾病活动程度呈正相关,对临床制定治疗方案、判断疗效有指导意义.     

脂肪间充质干细胞对MRL/lpr小鼠的治疗效果及对脾脏Th17/Treg细胞平衡的影响

目的：探讨移植脂肪间充质干细胞（adipose tissue derived stem cells,ADSCs）对MRL/lpr小鼠的治疗作用及对MRL/lpr小鼠脾脏Th17/Treg细胞平衡的影响。方法：15只12周龄的雌性MRL/lpr小鼠采用随机数字表法分为ADSCs治疗组(ADSCs组)、对照组(control组)和环磷酰胺治疗组（cyclophosphamide,CTX组）,每组5只。ADSCs组经尾静脉注射ADSCs,每次注射的细胞数为1×106/150 μL,每周1次,共注射8次;control组经尾静脉注射等体积磷酸盐缓冲液,每周1次,共注射8次;CTX组经尾静脉注射环磷酰胺,剂量为15 mg/kg（体重）,每周1次,连用2次,休息2周后重复,共注射4次。留取治疗前和治疗后尿液检测24 h尿蛋白浓度。治疗8周后,处死MRL/Ipr小鼠,以流式细胞仪分析脾细胞Th17/Treg细胞比例变化。结果：（1）24 h尿蛋白浓度变化：治疗前,3组小鼠的24 h尿蛋白浓度差异无统计学意义（P>0.05);治疗4周后,ADSCs组和CTX组24 h尿蛋白浓度显著低于control组,差异有统计学意义［(5.02±1.61) g/L vs. (7.10±1.63) g/L、(4.90±0.71) g/L vs. (7.10±1.63) g/L,P<0.05］,而且治疗时间越长,效果越明显,治疗8周后,ADSCs组和CTX组24 h尿蛋白浓度显著低于control组［(2.24±0.73) g/L vs. (10.36±1.64) g/L、(3.80±1.45) g/L vs. (10.36±1.64) g/L,P<0.01］;ADSCs组和CTX组之间24 h尿蛋白浓度差异无统计学意义（P>0.05）。（2）脾脏Treg细胞占CD4+T细胞的百分率：ADSCs组和CTX组脾淋巴细胞中Treg细胞占CD4+T细胞的百分率高于control组,3组分别为13.62%±1.87%、14.14%±1.29%、0.71%±1.23%,但组间差异无统计学意义（P>0.05）。（3）脾脏Th17细胞占CD4+T细胞的百分率：ADSCs组和CTX组脾淋巴细胞中Th17细胞占CD4+T细胞的百分率显著低于control组,3组分别为1.43%±020%、1.63%±0.65%、6.37%±1.64%,差异有统计学意义（P<0.01）。结论：移植ADSCs能够减少MRL/lpr小鼠尿蛋白浓度,治疗时间越长,效果越显著。ADSCs能够降低MRL/lpr小鼠脾脏Th17细胞水平,提高Treg细胞水平,负向调节Th17/Treg细胞平衡,进而发挥抗炎和免疫调节的作用。     

Th17细胞及相关细胞因子在系统性硬化病小鼠模型中的表达及意义

目的:研究T辅助细胞17(Th17)及相关细胞因子在系统性硬化病(systemic sclerosis,SSc)小鼠模型外周血、皮肤、肺部的表达及意义。方法:20只雌性BALB/c小鼠随机分为对照组和博莱霉素注射4周组(SSc组),观察小鼠皮肤/肺部炎症和纤维化(pulmonary fibrosis,PF)的病理切片,流式细胞计数检测外周血、皮肤/肺组织CD4+IL-17+Th17细胞,荧光定量PCR检测小鼠皮肤/肺部维甲酸相关孤独受体(RORγt)、IL-17A和IL-6 mRNA的表达,酶联免疫吸附试验检测血清IL-17、IL-6水平,并分析这些指标的相关性。结果:SSc组比对照组皮肤炎症和肺纤维化评分(Ashcroft评分)明显增加(2.6±0.84 vs.0.4±0.52,2.80±1.81 vs.0.60±0.70),皮肤/肺羟脯氨酸(hydroxyproline,HYP)的含量明显增多[(3.17±1.74)mg/g vs.(1.45±0.40)mg/g,(0.53±0.14)mg/g vs.(0.38±0.16)mg/g],均P<0.05。SSc组外周血、皮肤和肺组织Th17细胞比例较对照组明显增加[(2.07±0.89)%vs.(1.02±0.32)%,(5.80±2.02)%vs.(1.64±0.58)%,(5.24±2.43)%vs.(1.92±0.98)%,P<0.01];与对照组相比,SSc组皮肤/肺组织IL-17A、IL-6 mRNA的表达量,皮肤RORγt mRNA的表达量增高,均P<0.05。SSc组外周血IL-17、IL-6含量明显增高(P<0.01)。外周血Th17细胞、IL-17和IL-6的含量与皮肤/肺部炎症、PF评分、皮肤HYP含量呈密切正相关,皮肤/肺组织Th17细胞分别与皮肤/肺部炎症、皮肤/肺HYP含量密切正相关。结论:Th17细胞在SSc小鼠模型外周血、皮肤及肺组织表达增高,与皮肤/肺部炎症、纤维化病变密切正相关,并通过IL-17、IL-6等细胞因子参与SSc的发病。     

慢性乙型肝炎患者外周血Th17/Treg和Th1免疫失衡研究

目的：探讨Th17、Treg和Th1细胞免疫失衡在慢性乙型肝炎（CHB）发病中的作用。方法：采用荧光定量PCR检测慢性乙型肝炎患者外周血单个核细胞（PBMCs）中Th17、Treg和Th1细胞的特异性转录因子RORC、Foxp3和T-bet的mRNA表达,ELISA分析CHB患者PBMCs培养上清液中白细胞介素（IL）-17、IL-21和血清IL-6、IL-1β的水平,PCR检测外周血HBV DNA;生化分析仪检测谷丙转氨酶（ALT）。结果：与正常对照组相比,CHB患者PBMCs中RORC mRNA、IL-17、IL-21水平明显升高（P〈0.01）;RORC mRNA与ALT呈正相关,与T-bet mRNA呈负相关;T-betmRNA与ALT呈负相关,外周血中IL-6、IL-1β水平也明显增高（P〈0.01）;Foxp3 mRNA与HBV DNA正相关。结论：Th17和Treg、Th1细胞免疫功能失衡与CHB病情密切相关。     

从Th17和CD4+CD25+Foxp3+T细胞功能变化探讨肺结核的耐药机制

目的 分析耐药肺结核患者外周血Th17细胞与CD4+CD25+Foxp3+T细胞比例及细胞因子的变化,从免疫学角度探讨其耐药机制。 方法 分别选取33例耐药肺结核(耐药组)、49例普通肺结核(普通组)及51例健康志愿者(对照组)展开对比研究。检测Th17细胞与CD4+CD25+Foxp3+T细胞比例;采用RT-PCR检测其特异性转录因子RORγt与Foxp3的mRNA表达水平;采用ELISA实验检测IL-17A、IL-17F、TGF-β1及IL-10的血清水平。 结果 普通组Th17细胞比例和RORγt mRNA表达水平低于对照组,耐药组低于普通组;普通组CD4+CD25+Foxp3+T细胞比例和Foxp3 mRNA表达水平高于对照组,耐药组高于普通组;普通组Th17/CD4+CD25+Foxp3+T和RORγt/Foxp3低于对照组,耐药组低于普通组(P＜0.05)。普通组IL-17A、IL-17F水平低于对照组,耐药组低于普通组;普通组TGF-β1、IL-10水平高于对照组,耐药组高于普通组;其组间差异均有统计学意义(P＜0.05)。 结论 Th17/CD4+CD25+Foxp3+T平衡向CD4+CD25+Foxp3+T偏移、免疫系统功能抑制,可能是结核病慢性化和产生耐药的重要机制之一。