TRPC1/STIM1复合体调节人脐静脉内皮细胞钙池操纵性钙通道和受体操纵性钙通道介导的钙内流和NO生成

目的研究瞬时受体电位通道1(TRPC1)/基质交联分子1(STIM1)复合体在人脐静脉内皮细胞(HUVEC)钙池操纵性钙通道(SOCC)和受体操纵性钙通道(ROCC)介导的钙内流和NO生成中的作用。方法取2~3代HUVEC,将构建的TRPC1和STIM1干扰质粒分别转染HUVEC,观察细胞转染效果的同时采用real-time PCR和Western blot检测TRPC1、STIM1 mRNA和蛋白的表达。将细胞分别与钙敏感受体(CaR)激动剂精胺、ROCC模拟剂(TPA)+CaR负性变构调节剂Calhex231、蛋白激酶C(PKC)抑制剂Ro31-8220及经典型PKCs和PKCμ抑制剂Go6967孵育后,用荧光探针Fura-2/AM及DAF-FM负载方法同步检测[Ca2+]i和NO生成的变化;随后用TRPC1和STIM1干扰质粒同时转染HUVEC,与精胺孵育后检测[Ca2+]i和NO生成,免疫共沉淀法检测TRPC1和STIM1的相互作用。结果与Control组相比,TRPC1转染组和STIM1转染组TRPC1、STIM1 mRNA和蛋白表达均明显降低(P0.05);在四种不同处理作用下,TRPC1转染组和STIM1转染组[Ca2+]i△ratio值和NO净荧光强度值均明显降低(P0.05);与Control组、TRPC1转染组及STIM1转染组相比,TRPC1和STIM1共转染组[Ca2+]i△ratio值和NO净荧光强度值均明显降低(P0.05);TRPC1与STIM1相互作用形成复合体,且在CaR激动剂的剌激下作用增强。结论 TRPC1/STIM1复合体共同调节CaR经SOCC和ROCC激活介导的钙内流和NO生成。     

经典瞬时受体电位通道3在染料木黄酮抑制非小细胞肺癌H1299细胞增殖中的作用

目的旨在探讨经典瞬时受体电位(TRPC)通道(TRPC)3在染料木黄酮(Gen)抑制非小细胞肺癌细胞增殖中的作用。方法用MTT法检测癌细胞增殖活力,用荧光定量RT-PCR和蛋白免疫印迹法检测TRPC通道表达水平,用1-油酰基-2-乙酰基-sn-丙三醇(OAG)诱导的钙内流实验检测TRPC3功能。结果在所有Gen给药浓度,H1299细胞增殖率均低于A549细胞(P0.01)。H1299细胞的TRPC3 m RNA和蛋白表达都显著高于A549细胞(P0.01)。用sh NC和sh C3i质粒转染H1299细胞,在Gen处理24、48、72 h三个时间点,sh C3i及Gen处理两个转染细胞的增殖率都低于sh NC细胞(P0.01)。在24、48 h,sh C3i,sh NC+Gen和sh C3i+Gen三组间无差异,在72 h,sh C3i+Gen组低于sh C3i和sh NC+Gen组(P0.05)。Gen对sh C3i细胞的增殖抑制率(9.9%)低于其对sh NC细胞的增殖抑制率(56%)。Gen处理不影响H1299细胞TRPC3蛋白表达,但在OAG诱导的钙内流检测,sh C3i细胞,Gen处理的sh NC及sh C3i细胞的瞬时钙内流均低于sh NC细胞,Gen对sh C3i细胞钙内流抑制作用低于其对sh NC细胞钙内流抑制作用。结论 Gen抑制TRPC3通道功能可能是其抑制H1299细胞增殖的作用机制。     

钙池操纵性钙通道的激活信号分子与肺动脉高压

钙池操纵性钙通道是非兴奋细胞上Ca2+内流的主要途径,以钙释放激活钙离子流最为典型。目前发现,由基质交感分子、Orai和经典瞬时感受器电位亚家族等介导的钙池操纵性钙内流参与了多种生理及病理过程。文章通过介绍钙池操纵性钙内流复合体的分子组成及其相互作用模式,以及钙池操纵性钙内流在肺动脉高压发病过程中的作用,旨在为肺动脉高压的防治提供新的研究方向。     

血小板钙库操纵性钙通道蛋白与冠状动脉斑块易损性的相关性研究

目的:研究冠心病患者血小板钙库操纵性钙通道蛋白(SOCC)包括基质交联分子1(STIM1)、钙释放激活钙通道蛋白Orai1及瞬时受体电位通道1(TRPC1)水平的变化与冠状动脉斑块易损性之间的关系。方法:入选2015年1月1日至2016年8月31日,在北京安贞医院住院行冠状动脉造影确诊为单支单处冠状动脉临界病变的患者118例,采集血液标本并应用流式细胞学检测法测定血小板钙离子信号蛋白STIM1、Orai1、TRPC1水平。根据冠状动脉血管内超声(IVUS)分为稳定斑块(SP)组76例,易损斑块(VP)组42例,VP组行介入治疗,SP组按照冠心病二级预防用药保守治疗,1年后对SP组患者复查IVUS,根据斑块性质变化分别分为进展为易损斑块组(SVP组)和未进展为易损斑块组(SSP组),并复测血小板钙离子信号蛋白水平。结果:VP组血小板STIM1、Orai1、TRPC1水平明显高于SP组(P<0.05)。1年后对SP组患者复查IVUS,其中16例进展为VP(SVP组),剩余60例仍为SP(SSP组),SVP组血小板STIM1、Orai1、TRPC1水平明显高于SSP组(P<0.05),SSP组SOCC水平较治疗前有下降趋势,但无统计学意义(P>0.05)。结论:血小板钙库操纵性钙通道蛋白与冠状动脉粥样硬化斑块的易损性密切相关,有望成为预测冠状动脉高危事件的新型临床检测指标。     

SOCE通道在消化系肿瘤中的研究进展

钙库操纵性钙离子内流(store-operated calcium entry,SOCE)是介导胞外Ca2+进入细胞内的重要通道之一.其核心蛋白是位于内质网上的基质相互作用分子1(stromal interaction molecule 1,STIM1)及位于细胞膜上的钙释放激活钙通道蛋白1(calcium release-activated calcium channel protein 1,CRCM1/Orai1).随着研究的不断深入,SOCE通道在肿瘤中的作用机制逐渐阐明并加以完善,同样在消化系肿瘤中SOCE通道也发挥了重要作用.本文重点对SOCE通道基本信息、及其在消化系肿瘤中研究概况进行综述,并初步分析其作用机制.     

瞬时受体电位通道C亚族在心血管疾病中的研究进展

瞬时受体电位通道C亚族（canonical transient receptorpotential,TRPC）是位于细胞膜上的一种非选择性阳离子通道蛋白,分为7种亚型（TRPC1-7）,其中TRPC2在人类是假基因。近年来研究发现,TRPC通道的激活与高血压、肺动脉高压、心室重构、扩张性心肌病及动脉粥样硬化等心血管疾病发生、发展关系密切。血管紧张素Ⅱ、三磷酸腺苷、内皮素、去甲肾上腺素、加压素、乙酰胆碱、多种生长因子、钙池的衰竭和膜牵张等诸多因素都能够激活TRPC通道。根据激活机制的不同,TRPC通道可分为受体操纵性通道（receptor-operated channels,ROC）和储存操纵性通道（store-operated channels,SOC）。两个途径的激活均可改变阳离子(如Ca²⁺和Na⁺)内流,从而在调节心血管系统的功能方面发挥重要作用。本综述集中阐述TRPC通道C亚族在心血管疾病中的最新研究进展。     

钌红、丁卡因对肺动脉高压模型大鼠肺动脉平滑肌细胞[Ca2+]i释放功能的影响

目的:分析肺动脉高压时肺动脉平滑肌细胞雷诺定(Ryanodine)受体[Ca2+]i释放功能的改变及钌红(Rutheniumred)、丁卡因对肺动脉高压模型大鼠肺动脉平滑肌细胞[Ca2+]i释放功能的影响.方法:清洁级Wister大白鼠25只,腹腔注射野百合碱,建立大鼠肺动脉高压模型,死亡2只,最终23只;原代培养肺动脉平滑肌细胞;Fura-2/AM(钙离于荧光指示剂)负载培养细胞;荧光测钙技术比较雷诺定诱导的野百合碱组(n=15)及对照组(n=8)[Ca2+]i数值,观测钌红、丁卡因对雷诺定受体激动剂雷诺定诱导的[Ca2+]i变化的影响.结果:10 nmol/L雷诺定使对照组[Ca2+]i平均增加(93.31±12.41)nmoL/L,使野百合碱组[Ca2+]i平均增加(141.71±13,59) nmol/L;两组样本[Ca2+]i增加的数值比较差异有统计学意义(P＜0.01).钌红浓度为o.5～ 10 μmol/L、丁卡因浓度为2～40 μnoL/L时,可使雷诺定受体激动剂雷诺定诱导的[Ca2+]i值最大下降(115.32±7.47) nmol/L和(107.97±7.11) nmol/L.结论:肺动脉高压大鼠肺动脉平滑肌细胞对雷诺定受体激动剂雷诺定的敏感性增强;钌红、丁卡因对雷诺定诱导的肺动脉高压大鼠肺动脉平滑肌细胞[Ca2+]i上升有明显的抑制作用.     

钙库操纵性钙通道特性及其与低氧性肺血管收缩、重构的关系

肺动脉平滑肌细胞内Ca2+浓度增加是导致低氧性肺血管收缩与重构的重要分子基础.由瞬时受体电位蛋白构成的钙库操纵性钙通道(store-operated channels,SOC)是调节细胞内Ca2+浓度的重要机制,并参与了肺动脉高压时血管收缩和重构过程.充分了解SOC通道的特性,对深入认识肺动脉高压发病的病理生理学机制、指导临床治疗策略具有重要意义.     

基质相互作用分子1在炎性免疫细胞中的作用

近年来基质相互作用分子(stromal interaction molecule,STIM)成为钙库调控的钙内流(store operated calcium entry,SOCE)研究中的一个热点,有STIM1和STIM2两种亚型,STIM1与钙库调控的钙通道(store operated calcium channel,SOC)激活高度相关,这在Roos等[1]及Liou等[2]的实验中得到证实,而STIM2在HEK293,A7rR5细胞研究中发现对SOC有抑制作用.SOC广泛分布于细胞膜上,以非兴奋性细胞为主,是胞外Ca2+内流的主要通道之一,也称为Ca2+释放激活的钙通道(Ca2+release-activated Ca2+channels,CRAC).     

甲状旁腺钙受体与肾性甲旁亢关系的研究进展

甲状旁腺钙受体 (CaR)是与G蛋白相偶联的膜受体。胞外Ca2 + 通过激活CaR ,可快速、独立地影响甲状旁腺激素 (PTH)的分泌。慢性肾功能衰竭 (肾衰 )继发甲状旁腺功能亢进 (甲旁亢 )时 ,Ca调定点升高 ,PTH过度分泌。多数学者认为这与甲状旁腺CaR的mRNA表达下降及受体蛋白合成减少有关 ,而与CaR的基因突变无关。CaR的激动剂能模仿甚至增强胞外Ca2 + 对甲状旁腺细胞的效应 ,初步证实它能快速、安全、有效地抑制PTH分泌 ,延缓肾性骨病的发展。     

Cytochrome 1A1 and 1B1 gene diversity in the Zanzibar islands

Amodiaquine (AQ) is a 4‐aminoquinoline widely used in the treatment of malaria as part of the artemisinin combination therapy (ACT). AQ is metabolised towards its main metabolite desethylamodiaquine mainly by cytochrome P450 2C8 (CYP2C8). CYP1A1 and CYP1B1 play a minor role in the metabolism but they seem to be significantly involved in the formation of the short‐lived quinine‐imine. To complete the genetic variation picture of the main genes involved in AQ metabolism in the Zanzibar population, previously characterised for CYP2C8, we analysed in this study CYP1A1 and CYP1B1 main genetic polymorphisms. The results obtained show a low frequency of the CYP1A1*2B/C allele (2.4%) and a high frequency of CYP1B1*6 (approximately 42%) followed by CYP1B1*2 (approximately 27%) in Zanzibar islands. Genotype data for CYP1A1 and CYP1B1 show a low incidence of fast metabolisers, revealing a relatively safe genetic background in Zanzibar’s population regarding the appearance of adverse effects.     

CYP1A1 , GSTM1 , GSTT1 and NQO1 polymorphisms and colorectal adenomas in Japanese men

AIM: To investigate the role of functional genetic poly-morphisms of metabolic enzymes of tobacco carcinogens in the development of colorectal adenomas. METHODS: The study subjects were 455 patients with colorectal adenomas and 1052 controls with no polyps who underwent total colonoscopy in a preretirement health examination at two Self Defense Forces hospitals. The genetic polymorphisms studied wereCYP1A1*2A (rs 4646903), CYP1A1*2C (rs 1048943), GSTM1 (null or non-null genotype), GSTT1 (null or non-null genotype) and NQO1 C609T (rs 1800566). Genotypes were determined by the polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR method using genomic DNA extracted from the buffy coat. Cigarette smoking and other life-style factors were ascertained by a self-administered questionnaire. The associations of the polymorphisms with colorectal adenomas were examined by means of OR and 95%CI, which were derived from logistic regression analysis. Statistical adjustment was made for smoking, alcohol use, body mass index and other factors. The gene-gene interaction and effect modification of smoking were evaluated by the likelihood ratio test. RESULTS: None of the five polymorphisms showed a significant association with colorectal adenomas, nor was the combination of GSTM1 and GSTT1 . A borderline significant interaction was observed for the combination of CYP1A1*2C and NQO1 (P = 0.051). The OR associated with CYP1A1*2C was significantly lower than unity among individuals with the NQO1 609CC genotype. The adjusted OR for the combination of the CYP1A1*2C allele and NQO1 609CC genotype was 0.61 (95%CI: 0.42-0.91). Although the interaction was not statistically significant (P = 0.24), the OR for individuals carrying the CYP1A1*2C allele and GSTT1 null genotype decreased significantly compared with those who had neither CYP1A1*2C allele nor GSTT1 null genotype (adjusted OR: 0.69, 95%CI: 0.49-0.97). Smoking did not modify the associations of the individual polymorphisms with colorectal adenomas. There w     

Trib1 and Evi1 cooperate with Hoxa and Meis1 in myeloid leukemogenesis

Cooperative activation of Meis1 and Hoxa9 perturbs myeloid differentiation and eventually leads myeloid progenitors to leukemia, yet it remains to be clarified what kinds of subsequent molecular processes are required for development of overt leukemia. To understand the molecular pathway in Hoxa9/Meis1-induced leukemogenesis, retroviral insertional mutagenesis was applied using retrovirus-mediated gene transfer. The mice that received Hoxa9/Meis1-transduced bone marrow cells developed acute myeloid leukemia (AML), and Trib1, Evi1, Ahi1, Raralpha, Pitpnb, and AK039950 were identified as candidate cooperative genes located near common retroviral integration sites. Trib1 and Evi1 were up-regulated due to retroviral insertions, and coexpression of these genes significantly accelerated the onset of Hoxa9/Meis1-induced AML, suggesting that Trib1 and Evi1 are the key collaborators. Furthermore, Trib1 by itself is a novel myeloid oncogene, enhancing phosphorylation of ERK, resulting in inhibition of apoptosis. These results demonstrate the importance of specific oncogene interaction in myeloid leukemogenesis.     

CYP1A1, CYP2E1 and EPHX1 polymorphisms in sporadic colorectal neoplasms

AIM To investigate the contribution of polymorphisms in the CYP1A1, CYP2E1 and EPHX1 genes on sporadic colorectal cancer(SCRC) risk. METHODS Six hundred forty-one individuals(227 patients with SCRC and 400 controls) were enrolled in the study. The variables analyzed were age, gender, tobacco and alcohol consumption, and clinical and histopathological tumor parameters. The CYP1A1 *2A, CYP1A1 *2C CYP2E1 *5B and CYP2E1 *6 polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP). The EPHX1 Tyr113 His, EPHX1 His139 Arg and CYP1A1 *2C polymorphisms were detected by real-time PCR. Chisquared test and binary logistic regression were used in the statistical analysis. Haplotype analysis was conducted using the Haploview program, version 2.05.RESULTS Age over 6 2 years was a risk factor for SCRC development(OR = 7.54, 95%CI: 4.94-11.50, P 0.01). Male individuals were less susceptible to SCRC(OR = 0.55, 95%CI: 0.35-0.85, P 0.01). The CYP2E1*5B polymorphism was associated with SCRC in the codominant(heterozygous genotype: OR = 2.66, 95%CI: 1.64-4.32, P 0.01), dominant(OR = 2.82, 95%CI: 1.74-4.55, P 0.01), overdominant(OR = 2.58, 95%CI: 1.59-4.19, P 0.01), and log-additive models(OR = 2.84, 95%CI: 1.78-4.52, P 0.01). The CYP2E1*6 polymorphism was associated with an increased SCRC risk in codominant(heterozygous genotype: OR = 2.81, 95%CI: 1.84-4.28, P 0.01; homozygous polymorphic : OR = 7. 3 2, 9 5 % C I : 1.85-28.96, P 0.01), dominant(OR = 2.97, 95%CI: 1.97-4.50, P 0.01), recessive(OR = 5.26, 95%CI: 1.35-20.50, P = 0.016), overdominant(OR = 2.64, 95%CI: 1.74-4.01, P 0.01), and log-additive models(OR = 2.78, 95%CI: 1.91-4.06, P 0.01). The haplotype formed by the minor alleles of the CYP2E1*5B(C) and CYP2E1*6(A) polymorphisms was associated with SCRC(P = 0.002). However, the CYP1A1 *2A, CYP1A1 *2C, EPHX1 Tyr113 His and EPHX1 His139 Arg polymorphisms were not associated with SCRC.CONCLUSION In conclusion, the results demonstrated that CYP2E1*5B and CYP2E1*6 minor alleles play a role in the development of SCRC.     

8-溴-环磷酸腺苷对单核细胞株THP1中ABCA1及细胞间黏附分子的影响

目的 观察8-Br-cAMP刺激下,单核细胞株THP1中ABCA1、细胞间黏附分子-1(ICAM-1)及 白介素-1β(IL-1β)mRNA和蛋白质表达的变化,阐明ABCA1基因在动脉粥样硬化(AS)形成中的可能机制.方法 复苏培养THP1单核细胞,加入8-Br-cAMP(0.5 mmol/L)刺激3、6、12、24 h,设未加刺激同时孵育24 h的细胞为阴性对照组;以荧光定量RT-PCR和Western印迹法及ELISA法检测ABCA1、ICAM-1及IL-1β mRNA和蛋白质表达量;用ABCA1的反义寡核苷酸(100 nmol/L)转染THP1细胞,给予8-Br-cAMP刺激,同样的方法观察上述指标的改变.结果 予8-Br-cAMP刺激后,THP1细胞中ABCA1、ICAM-1 mRNA和蛋白质及IL-1β蛋白质表达均增高,给予反义寡核苷酸转染后氧化低密度脂蛋白(Ox-LDL)刺激3、6 h上述指标的mRNA表达降低 (P<0.01),12、24 h蛋白质表达降低 (P<0.01).结论 THP1单核细胞中,8-Br-cAMP激活ABCA1不仅可增加细胞内胆固醇外运,还具有增加炎症因子表达的作用,在AS的发生中发挥双重作用.     

牛磺酸下调单核-巨噬细胞ACAT-1表达

目的:观察牛磺酸对THP-1及THP-1源性巨噬细胞酰基辅酶A:胆固醇酰基转移酶1(ACAT-1)表达及活性的影响。方法:将THP-1细胞与PMA孵育48h使之分化为巨噬细胞,在有或无干扰素-γ(IFN-γ)的条件下,用不同浓度的牛磺酸与THP-1持续作用不同的时间,RTPCR检测ACAT-1mRNA水平,WesternBlot检测其蛋白表达,并测定其酶活性。结果:牛磺酸显著下调THP1及THP1源性巨噬细胞的ACAT1mRNA和蛋白表达水平以及ACAT-1活性(P<0.05～0.01,n=3);IFNγ促进ACAT1mRNA和蛋白表达、增强ACAT-1活性(P<0.05,n=3)的作用被牛磺酸部分抑制(P<0.05,n=3)。结论:牛磺酸不但能降低单核巨噬细胞ACAT-1的基础表达和活性,而且能抑制由IFN-γ诱导的ACAT-1表达和活性,这可能是其抗动脉粥样硬化的机制之一。     

NRG-1及其受体在心血管系统中的研究进展

近年来,人们研究发现表皮生长因子家族中的神经调节蛋白I（Neuregulin-1,NRG-1）及其受体ErbB在心血管系统的发育形成及疾病转归中起到了重要的作用。本文将综述NRG-1及其受体在心脏发育形成及其在心血管相关疾病中起到的作用,为临床应用提供依据。