Wnt信号通路抑制剂对人神经胶质瘤细胞增殖活力和生长迁移的影响

目的:探讨Wnt信号通路在胶质瘤细胞增殖和生长迁移中的可能作用。方法:建立U251胶质瘤细胞培养体系、并给予不同浓度Wnt信号通路的抑制剂(IWR-1 0,2.5,5.0,10μmol/L)处理后,MTT测定增殖活力、划痕实验测定生长迁移、Western Blot测定Wnt5a、β-catenin蛋白表达水平,分析IWR-1的生物作用与效应途径。结果:MTT表明胶质瘤细胞的IWR-1 5.0、10μmol/L处理24 h、48 h组,细胞增殖活力显著低于对照组(P0.05~0.01)。划痕检测表明IWR-1 5.0、10μmol/L处理48 h组胶质瘤细胞的生长迁移能力低于对照组(P0.05~0.01)。Western Blot表明IWR-1处理48 h组胶质瘤细胞的β-catenin蛋白表达水平低于对照组(P0.05~0.01)。结论:Wnt信号抑制剂IWR-1能够明显抑制人胶质瘤细胞增殖和生长迁移,提示Wnt/β-catenin信号途径可能具有胶质瘤干预治疗靶点的潜在价值。     

芒柄花黄素对体外卵巢癌细胞活力、迁移与侵袭的影响

目的:观察芒柄花黄素对卵巢癌细胞增殖、迁移与侵袭的影响,并初步探讨其机制。方法:体外培养人卵巢浆液性囊腺癌SKOV-3细胞,采用不同浓度(0、25、50和100μmol/L)芒柄花黄素处理细胞48 h,用MTS法检测细胞活力,并筛选出半数抑制浓度。通过划痕迁移实验和Transwell侵袭实验检测芒柄花黄素对SKOV-3细胞迁移与侵袭能力的影响。RT-qPCR法和Western blot法检测细胞中的上皮钙黏素(E-cadherin)和基质金属蛋白酶9(MMP-9)mRNA及蛋白表达的变化。结果:与对照组比较,SKOV-3细胞的活力随着芒柄花黄素浓度增加而下降(P0.01)。50μmol/L芒柄花黄素组细胞迁移距离少于对照组(P0.01)。50μmol/L芒柄花黄素组侵袭穿过Transwell小室底膜的细胞数量显著减少(P0.01)。此外,50μmol/L芒柄花黄素组E-cadherin的mRNA及蛋白水平显著高于对照组(P0.01),而MMP-9的mRNA及蛋白水平显著低于对照组(P0.01)。结论:芒柄花黄素可能通过增加E-cadherin和降低MMP-9的表达来抑制卵巢癌SKOV-3细胞的迁移和侵袭。     

膜联蛋白A5低表达对人宫颈癌HeLa细胞迁移和侵袭的影响

目的探讨膜联蛋白A5(ANXA5)低表达对人宫颈癌HeLa细胞迁移和侵袭的影响。方法将细胞分为干扰组、阴性对照组、转染试剂对照组和空白对照组,采用脂质体转染法将siRNA转染干扰组HeLa细胞,在转染72h后采用RT-PCR和Western blotting检测各组ANXA5的表达以鉴定抑制效率;细胞划痕实验检测各组HeLa细胞的迁移能力;Transwell实验检测各组HeLa细胞的侵袭能力;RT-PCR和Western blotting分别检测ANXA5低表达对E-钙黏蛋白(E-cadherin)和基质金属蛋白酶(MMP)-9 mRNA及蛋白表达的影响。结果干扰组ANXA5蛋白及mRNA的表达均明显低于阴性对照组(P0.05);其表达均被显著抑制,干扰组细胞的迁移及侵袭能力比阴性对照组明显增强(P0.05),E-cadherin mRNA及蛋白的表达显著低于阴性对照组(P0.05);干扰组MMP-9mRNA及蛋白的表达均显著高于阴性对照组(P0.05)。结论靶向ANXA5的siRNA可有效抑制ANXA5的表达,且ANXA5低表达可能通过影响E-cadherin和MMP-9的表达来促进HeLa细胞的迁移和侵袭能力。     

miR-410通过靶向Snail1抑制宫颈癌细胞的迁移和侵袭

目的分析miR-410通过Snail1蛋白抑制宫颈癌细胞的迁移和侵袭的机制。方法通过双荧光素酶报告验证miR-410靶向Snail1。将宫颈癌HeLa细胞系分为4组:对照组、mimic组、mimic+Snail1组和Snail1组。通过质粒转染技术过表达miR-410和/或Snail1。qPCR和Western blot分别用于检测RNA和蛋白的表达水平。分别通过CCK-8法、细胞划痕实验、Transwell实验和细胞共培养检测各组的细胞生长、迁移、侵袭能力和免疫抑制情况。结果 miR-410直接靶向抑制Snail1。Mimic组的Snail1mRNA和蛋白水平显著低于对照组(P0.05),Mimic+Snail1组的Snail1 mRNA和蛋白水平显著高于mimic(P0.05)。mimic组的细胞活力、迁移、侵袭和免疫抑制水平显著低于对照组(P0.05),Snail1组的细胞活力、迁移、侵袭和免疫抑制水平显著高于对照组(P0.05),并且mimic+Snail1组的细胞活力、迁移、侵袭和免疫抑制水平显著高于mimic组(P0.05)。结论 miR-410可通过靶向Snail1蛋白的表达解除免疫抑制并抑制宫颈癌HeLa细胞生长、迁移和侵袭。     

MiR-126 通过MAPK 信号通路抑制oxLDL 处理的血管平滑肌细胞增殖和迁移

目的:探索miR-126 对oxLDL 处理的血管平滑肌细胞(VSMC)生物学功能的影响及其分子机制。方法:采用oxLDL 处理人主动脉血管平滑肌细胞建立模型。实时定量PCR(qRT-PCR)检测miR-126 表达。CCK-8 实验检测细胞增殖。Transwell 实验分析细胞迁移。免疫印迹分析增殖标记蛋白细胞增殖核抗原-67(Ki-67)、增殖细胞核抗原(PCNA)、迁移标记蛋白基质金属蛋白酶9(MMP-9)、血管内皮细胞生长因子(VEGF)促分裂原活化蛋白激酶(MAPK)信号通路相关蛋白ERK1/2、p-ERK1/2、p38、p-p38、JNK 和p-JNK 的表达。结果:模型组的miR-126 表达显著低于对照组(P<0.01)。与模型组相比,miR-126 mimic 组miR-126 表达极显著升高(P<0.001)。模型组和mimic control 组细胞增殖倍数明显高于对照组(P<0.05)。与模型组相比,miR-126 mimic 组细胞增殖倍数明显降低(P<0.05)。与对照组相比,模型组和mimic control 组细胞迁移能力大大提高(P<0.001)。miR-126 mimic 组细胞迁移显著低于模型组(P<0.01)。模型组和mimic control 组细胞增殖标记蛋白Ki-67和PCNA,迁移标记蛋白MMP-9 和VEFG 表达显著高于对照组(P<0.01)。与模型组相比,miR-126 mimic 组增殖标记蛋白Ki-67 和PCNA 及迁移标记蛋白MMP-9 和VEFG 表达明显减弱(P<0.05)。而且,与对照组相比,模型组和mimic control 组中p-ERK1/2/ ERK1/2、p-p38/ p38、p-JNK/ JNK 的相对蛋白表达量的比值显著升高(P<0.01)。miR-126 mimic 组p-ERK1/2/ ERK1/2、p-p38/ p38、p-JNK/ JNK 的相对蛋白表达量的比值则明显低于模型组(P<0.05)。与对照组相比,模型组Ki-67、PCNA、MMP-9、VEGF 表达明显升高(P <0.05)。与模型组相比,miR-126 mimic 组Ki-67、PCNA、MMP-9、VEGF 表达明显降低(P <0.05);Anisomycin 组Ki-67、PCNA、MMP-9、VEGF 表达明显升高(P<0.05)。miR-mimic+ Anisomycin 组Ki-67、PCNA、MMP-9 和VEGF表达与模型组无明显差异。结论:MiR-126 通过MAPK 信号通路抑制oxLDL 处理的血管平滑肌细胞增殖和迁移。     

抑制miR-23a-3p表达对人白血病细胞株HL-60侵袭和迁移的调节作用

目的探讨抑制miR-23a-3p表达对人白血病细胞株HL-60侵袭和迁移的影响及其分子机制。方法转染miR-23a inhibitor到HL-60细胞抑制miR-23a-3p的表达。HL-60细胞分为3组:HL-60组(阴性对照组)、scramble组(转染对照组)和miR-23a inhibitor组。qRT-PCR技术检测MMP-9和VEGF mRNA水平,Transwell实验检测细胞侵袭,划痕实验分析细胞迁移。Western blot法检测MMP-9和VEGF蛋白表达。结果转染scramble后HL-60细胞miR-23a-3p表达无明显变化。转染miR-23a inhibitor后HL-60细胞miR-23a-3p表达显著下降(P0.01)。scramble组与阴性对照组每个视野下的侵袭细胞数目差异无统计学意义。miR-23a inhibitor组每个视野下的侵袭细胞数目显著低于阴性对照组(P0.01)。scramble组与阴性对照组细胞迁移无明显差异。与阴性对照组相比,miR-23a inhibitor组细胞迁移显著降低(P0.01)。转染scramble不会影响MMP-9和VEGF的表达。miR-23a inhibitor组MMP-9和VEGF的mRNA水平显著低于阴性对照组(P0.01)。与阴性对照组相比,miR-23a inhibitor组MMP-9和VEGF的蛋白水平显著下降(P0.001)。结论抑制miR-23a-3p表达可减弱人白血病细胞株HL-60的侵袭和迁移。     

地塞米松抑制哮喘小鼠肺RANTES的表达

目的 研究地塞米松对哮喘小鼠调节激活正常T细胞表达和分泌细胞因子(RANTES)蛋白和mRNA表达的影响.方法 将30只雌性BALB/c小鼠随机分为3组(每组10只):对照组、哮喘组、激素干预组,收集支气管肺泡灌洗液(BALF),行白细胞和嗜酸粒细胞(EOS)计数;酶联免疫吸附法(ELISA)测定BALF和血清中RANTES含量;肺组织切片HE染色,光镜下计数细胞总数和EOS百分比;部分行免疫组化染色检测肺组织RANTES蛋白;用原位杂交法检测肺组织RANTES mRNA表达.结果 哮喘组白细胞总数、EOS百分比、RANTES浓度明显升高(P＜0.01),干预组明显低于哮喘组(P＜0.01);哮喘组肺组织RANTES蛋白及mRNA表达显著高于对照组(P＜0.01),主要表达于上皮细胞;干预组肺组织RANTES蛋白及mRNA表达显著低于哮喘组(P＜0.01).结论 地塞米松可抑制RANTES表达和活性而发挥抗EOS炎症作用,这可能是其控制哮喘发病的重要机制之一.     

阿司匹林对血小板诱导的乳腺癌MCF-7细胞上皮间质转化和迁移侵袭能力的影响

目的　探讨阿司匹林对血小板诱导人乳腺癌MCF-7细胞（MCF-7humanbreast cancercells）上皮间质转化和迁移侵袭能力的影响。 方法　以未经干预的MCF-7细胞为对照组,分别用不同浓度（0.5、2.0 mmol/L）的阿司匹林、花生四烯酸激活的血小板及不同浓度阿司匹林预处理花生四烯酸激活的血小板分别干预MCF-7细胞,采用细胞划痕及Transwell实验检测肿瘤细胞迁移侵袭能力的变化。Western Blot检测肿瘤细胞上皮间质转化标记蛋白E-cadherin及Vimentin的表达。 结果　活化的血小板处理后与对照组相比MCF-7细胞迁移侵袭能力显著增强（P<0.05）,Western Blot显示E-cadherin表达显著减少（P<0.01）,Vimentin的表达显著增加（P<0.01）。阿司匹林预处理血小板活化后干预MCF-7细胞,其迁移侵袭能力较活化血小板组显著降低（P<0.05）,E-cadherin表达显著增高（P<0.01）,Vimentin的表达显著减少（P<0.05）。阿司匹林单独处理后的MCF-7细胞迁移及侵袭能力较对照组无显著变化(P>0.05),E-cadherin及Vimentin的表达亦无显著改变(P>0.05)。 结论　体外实验结果表明,阿司匹林可通过抗血小板活化,抑制血小板诱导的乳腺癌MCF-7细胞上皮间质转化和迁移侵袭能力。     

抑制BCAR1降低肺癌细胞系A549 p-P38表达

目的探讨抑制乳腺癌抗雌激素药物耐药蛋白(BCAR1)表达对肺癌细胞系A549 P38和p-P38表达的影响。方法培养正常A549细胞(对照组)、慢病毒BCAR1基因干扰A549细胞(干扰组)和慢病毒阴性对照A549细胞(阴性对照组),用Western blot检测细胞P38和p-P38表达水平,用克隆形成实验、流式细胞计量术、Transwell实验和划痕实验检测细胞的细胞克隆、细胞周期、细胞侵袭和迁移能力。结果干扰组p-P38表达显著低于其他两组(P0.05);干扰组细胞G1期比例显著高于其他两组(P0.05);干扰组细胞增殖、侵袭和迁移能力低于其他两组(P0.05)。结论抑制BCAR1表达可下调p-P38表达,减弱肺癌A549细胞增殖、迁移和侵袭能力。     

15d-PGJ_2减弱香烟提取物抑制人脐静脉内皮细胞迁移

目的 探讨15-脱氧-△12,14-前列腺素J2(15d-PGJ2)对香烟提取物(CSE)抑制人脐静脉内皮细胞(HUVECs)迁移的保护作用.方法 "划痕法"检测HUVECs的迁移.毛细管样成管实验检测HUVECs的血管新生.westem blot检测occludin蛋白表达.结果 与对照组相比,CSE显著抑制HUVECs迁移,使单个视野划痕内细胞数由629±28降低至364±17(P＜0.01),15d-PGJ2干预可使细胞数回升至546+20(P＜0.01).CSE抑制HUVECs血管新生,15d-PGJ2可逆转CSE对HUVECs血管新生的抑制.与对照组相比,CSE使HUVECs occludin蛋白表达量由0.37±0.04降至0.12±0.03(P＜0.01).15d-PGJ2干预可使其回调至0.28±0.04(P＜0.01),但仍低于对照组(P＜0.05).结论 CASE对HUVEVs迁移的抑制作用可能是通过下调occludin蛋白的表达实现的.15d-PGJ2减弱CSE下调occludin表达而逆转CSE对HUVECs迁移的抑制.     

骨髓间充质干细胞调控尿激酶型纤溶酶原激活物表达及其对大鼠肝星状细胞凋亡的影响**

背景：骨髓间充质干细胞主要通过旁分泌及激活肝细胞生长因子促进肝星状细胞凋亡。 目的：观察骨髓间充质干细胞对尿激酶型纤溶酶原激活物合成的调控及肝细胞生长因子活性的影响,探讨其诱导肝星状细胞凋亡的机制。 方法：实验分为5组：①共培养组：大鼠骨髓间充质干细胞与肝星状细胞建立上下双层细胞共培养体系。②肝星状细胞单独培养组。③纤维原细胞对照组。④UK122干预组：在骨髓间充质干细胞与肝星状细胞共培养6 h前加入尿激酶型纤溶酶原激活物特异性抑制剂UK122。⑤骨髓间充质干细胞空白对照组。 结果与结论：共培养组尿激酶型纤溶酶原激活物mRNA表达较肝星状细胞单独培养明显增加(P < 0.01)。与肝星状细胞单独培养相比,共培养组的肝星状细胞在与骨髓间充质干细胞共培养24 h后表现明显增殖抑制(P < 0.01),且呈现时间依赖性;骨髓间充质干细胞与肝星状细胞共培养后,活性肝细胞生长因子的蛋白表达及肝星状细胞凋亡增加(P < 0.01)。UK122干预后活性肝细胞生长因子表达及肝星状细胞凋亡减少(P < 0.01)。提示骨髓间充质干细胞通过促进分泌尿激酶型纤溶酶原激活物,增加活性肝细胞生长因子表达,促进肝星状细胞凋亡。     

Enhanced tryptophan degradation in systemic lupus erythematosus

In vitro and in vivo, tryptophan degradation was found to be associated with T cell functional loss and tolerance induction. In systemic lupus erythematosus (SLE) besides the Th2-type cytokine interleukin-10, Th1-type cytokines including interferon-gamma (IFN-gamma) are expressed especially during exacerbation of the disease. IFN-gamma stimulates the enzyme indoleamine (2,3)-dioxygenase (IDO) converting tryptophan to the metabolite kynurenine which in macrophages is subsequently degraded to other, partly neurotoxic compounds like quinolinic acid, and finally to nicrotinamides. We measured kynurenine and tryptophan concentrations in the sera of 55 SLE patients. In these patients, the concentrations of tryptophan (median, interquartile range: 53.9, 45.7-64.1 microM) were lower (p < 0.0001), and the kynurenine concentrations (2.45, 1.75-3.40 microM) were increased (p < 0.0005) compared to healthy blood donors (70.0, 63.8-80.6; 1.80, 1.45-2.27 microM, respectively). Also the kynurenine per tryptophan quotients (K/T), which allow to estimate IDO activity, were significantly higher in patients than in normals (0.043, 0.033-0.062 vs. 0.027, 0.021-0.030; p < 0.0001), indicating enhanced IDO-induced tryptophan degradation in SLE. There was no significant relationship between tryptophan, kynurenine and the SLEDAI, and also the correlation of K/T with SLEDAI was rather weak (rs = 0.243, p < 0.05). Higher K/T was found in patients presenting with serositis (p = 0.01), decrease of complement (c3, c4; p < 0.01) and blood count change (anemia, leucopenia, lymphopenia; p = 0.032) than in patients without such disease manifestations. The significant correlation found between K/T and neopterin (rs = 0.808, p < 0.001), a marker of immune activation, points to a role of immune activation to be responsible for tryptophan degradation in SLE patients.     

供体年龄影响融合生长内皮祖细胞对平滑肌细胞表型转换及增殖迁移的作用

目的:探讨老龄和年轻个体来源融合生长状态内皮祖细胞(EPCs)对血管平滑肌细胞(SMCs)表型转换以及增殖和迁移的调节作用。方法:脱臼处死1~2月龄、19~26月龄SD大鼠,应用含15%FBS的DMEM/F12培养基(含内皮细胞生长添加剂100 mg/L、肝素100 mg/L、青霉素、链霉素各1×105U/L)培养EPCs,取1~2月龄大鼠腹主动脉,组织块法培养血管SMCs,应用Di I-Ac-LDL与FITC-UEA-1荧光双染以及α-SM-actin免疫荧光分别对EPCs和SMCs进行鉴定。建立细胞共培养体系,上室为融合生长状态的EPCs,下室为SMCs,实验分4组:(1)第3代SMCs(P3)组;(2)第4代SMCs(P4)组;(3)第4代SMCs与年轻大鼠来源EPCs共培养(P4YE)组;(4)第4代SMCs与老龄大鼠来源EPCs共培养(P4AE)组。Western blotting检测α-SM-actin和osteopontin蛋白的表达;[3H]-TdR掺入法检测SMCs增殖;细胞划痕实验检测SMCs的迁移能力。结果:与P3组相比,P4组的SMCsα-SM-actin表达显著下调,而osteopontin表达显著增强;P4YE组SMCs的α-SM-actin及osteopontin表达与P3组比较未见有显著差别;与P4组相比,年轻和老龄大鼠来源的EPCs均显著促进第4代SMCs的α-SM-actin和下调osteopontin的表达,抑制第4代SMCs的增殖和迁移;与老龄大鼠来源的EPCs相比,年轻大鼠来源的EPCs更能够显著延迟SMCs表型由收缩型向合成型转换,抑制SMCs增殖和迁移。结论:共培养融合生长状态的EPCs使血管SMCs表型转换延迟、抑制SMCs增殖和迁移,年轻大鼠来源的EPCs较老龄大鼠来源的EPC更显著延迟血管SMCs表型由收缩型向合成型转换,并具有更强的抑制血管SMCs增殖和迁移的能力。     

miR-124对C6胶质瘤细胞增殖和迁移能力的影响

目的:研究miR-124对C6胶质瘤细胞增殖和迁移能力的影响.方法:体外培养C6胶质瘤细胞,依据转染不同分为空白对照组、阴性对照组和miR-124拟似物组.实时荧光PCR检测转染效率,绘制生长曲线,MTT实验检测miR-124对C6细胞增殖能力影响,计算抑制率.划痕实验检测miR-124对C6细胞迁移能力影响,计算迁移率.结果:生长曲线显示miR-124拟似物组C6细胞生长能力受到明显抑制,转染后第3天,miR-124拟似物组C6细胞数目显著低于阴性对照组(5.410±0.463Vs.6.917±0.385;P＜0.01);miR-124拟似物组mRNA表达量显著高于阴性对照组和空白对照组(5.92±0.56 vs.0.93±0.13和1.00±0.12;P＜0.01);MTT增殖实验显示miR-124抑制C6细胞增殖能力,miR-124拟似物组抑制率显著高于阴性对照物组(42.90±5.169 vs.10.24±3.351;P＜0.01);划痕实验显示miR-124明显抑制C6细胞迁移能力,阴性对照组迁移率显著高于miR-124拟似物组(98.79±1.210vs.81.72±5.972;P＜0.05).结论:miR-124能够显著抑制C6胶质瘤细胞的增殖和迁移能力.     

Interleukin-8 and leukotriene-B(4), but not formylmethionyl leucylphenylalanine, stimulate CD18-independent migration of neutrophils across human pulmonary endothelial cells in vitro

Although neutrophil migration from the systemic circulation involves the beta2- (or CD18) integrin family, the existence of an alternative, CD18-independent route of neutrophil extravasation to tissues has been demonstrated in animal models. The molecular interactions involved in this alternative migratory route have not yet been characterized. The objective of this study was to assess the CD18-dependency of neutrophil migration across human endothelial cells from an organ known to support CD18-independent migration, the lung, with a view to establishing an in vitro model to facilitate study of CD18-independent migration. Neutrophil migration across human pulmonary artery endothelial cells (HPAECs) in response to three different chemoattractants, formylmethionyl leucylphenyl-alanine (FMLP), interleukin (IL)-8, and leukotriene (LT) B(4), was examined. Results demonstrated that a function-blocking antibody to CD18 decreased FMLP-stimulated migration by 71.7 +/- 4.4% (P < 0.001). In contrast, migration in response to LTB(4) was decreased by only 20.5 +/- 10.2% (P < 0.01), and no significant decrease was observed with migration to IL-8. Neutrophils that migrated to FMLP had 1.7-fold more surface CD11b/CD18 compared with nonmigrated neutrophils (P < 0.01), whereas this integrin complex was not significantly upregulated on neutrophils that had migrated to IL-8 or LTB(4). Further investigation of this migratory route indicated that it did not involve the beta1 integrins (CD29) or the endothelial selectins, E- or P-selectin, nor did it require the activity of either metalloproteinases or neutrophil elastase. These results indicate that neutrophil migration across HPAECs in vitro to IL-8 and LTB(4) is predominantly CD18-independent and provides a much-needed in vitro system for examination of the neutrophil-endothelial interactions involved in this alternative migratory route.     

Induction of indoleamine 2,3-dioxygenase in vivo by IFN-con1.

Indoleamine 2,3-dioxygenase (IDO) activity as determined by increases in serum kynurenine was measured in a group of hepatitis C patients treated with consensus interferon (IFN-con1). Kynurenine levels increased significantly within 2 days of initiation of treatment but returned to normal values by week 4 after treatment. Although IDO is normally induced by IFN-gamma, no such IFN was detected by ELISA or biologic assays. Thus, consensus IFN induces low levels of IDO in vivo without an IFN-gamma intermediate.     

脂肪干细胞来源的外泌体促进肉芽组织来源的成纤维细胞增殖的初步研究

目的研究脂肪干细胞来源的外泌体(ADSC-Exo)能否促进肉芽组织来源的成纤维细胞的增殖。 方法取内蒙古医科大学第三附属医院术后废弃脂肪组织,分离培养脂肪干细胞(ADSC),超滤浓缩离心方法收集ADSC-Exo,电子显微镜下观察其形态,Western Blotting测定表面标志物,Nanosight分析仪检测粒径和浓度;取烧伤创面的肉芽组织,分离培养肉芽组织成纤维细胞,进行波形蛋白免疫组织化学染色。成纤维细胞划痕实验设立空白组和ADSC-Exo组,检测ADSC-Exo在培养24、48 h对成纤维细胞的迁移作用。Transwell共培养实验设立空白对照组、ADSC-Exo共培养组和ADSC共培养组,检测ADSC-Exo与ADSC在培养24、48、72、96 h对成纤维细胞的增殖作用。成纤维细胞划痕实验结果比较采用独立样本t检验;Transwell共培养实验结果采用单因素方差分析。 结果电子显微镜下ADSC-Exo可见囊泡结构,直径在40～100 nm之间,ADSC-Exo标志物CD81、CD63的蛋白表达阳性,Nanosight检测显示其直径峰值聚集于55 nm。成纤维细胞镜下外形呈多角形或长梭形,波形蛋白免疫组织化学染色示细胞质呈棕黄色。培养24 h,ADSC-Exo组划痕迁移面积(46.2±9.8)%与空白组(31.7±8.6)%比较,差异有统计学意义(t＝2.72, P<0.05);培养48 h,ADSC-Exo组迁移面积(85.5±5.3)%与空白组(71.2±8.9)%比较,差异有统计学意义(t＝3.37, P<0.01)。Transwell共培养结果示培养48 h ADSC共培养组吸光度值(0.37±0.05)与空白对照组(0.29±0.06)比较差异有统计学意义(P<0.05);培养72 h,ADSC-Exo共培养组吸光度值(0.51±0.05)和ADSC共培养组吸光度值(0.53±0.08)显著高于空白对照组(0.38±0.06),培养96 h,ADSC-Exo共培养组吸光度值(0.68±0.07)和ADSC共培养组吸光度值(0.72±0.11)显著高于空白对照组(0.54±0.07),差异均有统计学意义(P值均小于0.05)。 结论超滤浓缩离心方法能够分离ADSC-Exo;ADSC-Exo可显著促进肉芽组织来源成纤维细胞的增殖和迁移能力。     

The IDO genetic polymorphisms and postpartum depressive symptoms: an association study in Chinese parturients who underwent cesarean section

Postpartum depressive symptoms (PDS) are not an uncommon mood disorder in postpartum women. Our previous research indicated a role for increased tryptophan (TRP) metabolism along the kynurenine pathway (KP) in the pathogenesis of PDS. Accordingly, this study was going to investigate the association of indoleamine-2,3-dioxygenase (IDO, a key enzyme of KP) genetic polymorphisms with PDS. Seven hundred twenty-five women receiving cesarean section were enrolled in this study. PDS was determined by an Edinburgh Postnatal Depression Scale (EPDS) score ≥ 13. Subsequently, 48 parturients with PDS and 48 parturients without PDS were selected for investigation of perinatal serum concentrations of TRP, kynurenine (KYN), and KYN/TRP ratio, the latter is the representative of IDO activity. In addition, seven single nucleotide polymorphisms of the IDO gene were examined. Following this genotyping, 50 parturients carrying the IDO rs10108662 AA genotype and 50 parturients carrying the IDO rs10108662 AC + CC genotype were selected for comparisons of TRP, KYN, and KYN/TRP ratio levels. This study showed the PDS incidence of 6.9% in the Chinese population, with PDS characterized by increased IDO activity (p < 0.05), versus women without PDS. We also found that the variations of IDO1 gene rs10108662 were significantly related to PDS incidence (p < 0.05). Furthermore, there was a significant difference in IDO activity between the IDO rs10108662 CA + AA, versus CC, genotypes. Our findings indicate a role of the kynurenine pathway in the development of PDS, rs10108662 genetic polymorphism resulting in changes of IDO activity might contribute to PDS pathogenesis.

     

沉默NLRP3炎性小体表达降低香烟烟雾提取物导致的人肺动脉内皮细胞凋亡

目的探讨NLPR3炎性小体(含NLR家族pyrin结构域蛋白3)对香烟烟雾提取物(CSE)诱导人肺动脉内皮细胞(HPAECs)凋亡的影响。方法用NLRP3 siRNA和NC siRNA(阴性对照)分别转染HPAECs后(分别为siNLRP3组、siNC组),再以10%CSE刺激12 h,应用annexin V/PI流式法检测这两组细胞在CSE刺激前后的凋亡水平。再将HPAECs分为对照(control)组、CSE组(加入10%CSE液)、NAC(乙酰半胱氨酸,N-acetylcysteine)+CSE组(以10 mmol/L NAC预处理1h后再以10%CSE培养液处理)及NAC组(以10 mmol/L NAC单独处理),均培养12 h后以DCFH-DA光探针标记法观察细胞内活性氧(reactive oxygen species,ROS),Western blot法测定NLRP3、caspase-1蛋白表达。结果siNC组在CSE刺激后,HPAECs存活率下降,凋亡率较CSE刺激前明显增加(P<0.05),SiNLRP3组在CSE刺激后,细胞凋亡率较siNC组同等CSE刺激后有所减少(P<0.05)。分组处理HPAECs后,CSE组细胞胞内ROS水平较对照组升高(P<0.05),NAC+CSE组细胞胞内ROS水平较CSE组有所下降(P<0.05)。CSE组NLRP3、caspase-1蛋白表达较对照组显著升高(P<0.05),而NAC+CSE组细胞NLRP3、caspase-1表达较CSE组下降(P<0.05)。结论CSE能诱导HPAECs发生凋亡及胞内ROS增加,伴有NLRP3炎性小体表达增加。NLRP3蛋白表达沉默能抑制CSE对HPAEC的促凋亡作用。     

Indoleamine 2,3 dioxygenase and human leucocyte antigen-G inhibit the T-cell alloproliferative response through two independent pathways

Both human leucocyte antigen (HLA)-G and indoleamine 2,3 dioxygenase (IDO) are key molecules involved in immune tolerance. HLA-G is a non-classical HLA class I molecule that can be expressed in both membrane-bound (HLA-G1) and soluble (HLA-G5) forms, both of which exhibit tolerogenic properties via interaction with inhibitory receptors present on natural killer (NK) cells, T cells and antigen-presenting cells (APC). IDO is an enzyme that acts by depleting the surrounding microenvironment of the essential amino acid, tryptophan, thereby inhibiting T-cell proliferation. Our present study was aimed at analysing the potential link that may exist between IDO and HLA-G. Our results showed that during allogeneic reactions, soluble HLA-G expression was not regulated by the addition of IDO substrate (i.e. tryptophan), metabolite (i.e. kynurenine) or inhibitor (i.e. 1-methyl-tryptophan), that IDO activity was not altered by HLA-G5 treatment, and that HLA-G5-mediated inhibition of the T-cell alloproliferative response was neither affected by the presence of tryptophan and kynurenine nor reversed after IDO activity blockage, demonstrating that HLA-G5 can exert its function in the absence of functional IDO. Similarly, inhibition of the T-cell alloresponse, induced by HLA-G1-expressing antigen-presenting cells, was not altered by IDO metabolites or inhibitor. Taken together, these findings show that the function and expression of IDO and HLA-G5 are not mutually influenced, but rather inhibit the T-cell alloproliferative response through two independent pathways. IDO and HLA-G are thus complementary for inducing and maintaining immune tolerance in physiological (pregnancy) and pathological (tumour and allograft) situations.