细粒棘球蚴重组抗原B 8-kDa亚单位1对囊型包虫病的血清学诊断价值

目的通过基因工程技术获得细粒棘球蚴抗原B8-kDa亚单位1重组蛋白(rEgAgB8/1),探讨其对囊型包虫病(CE)的血清学诊断价值。方法将构建的rEgAgB8/1原核表达质粒(pET32b-rEgAgB8/1)转化至E.coli BL-21(DE3)中,用IPTG诱导表达,经亲和层析纯化获得高纯度rEgAgB8/1,以rEgAgB8/1为抗原,应用ELISA和Immuno blotting方法对31例手术确诊的囊型包虫病病人血清进行了回顾性检测与分析。结果ELISA和Immuno blotting方法检测CE病人血清阳性率均为90.3%(28/31),3例血清学检测阴性的CE病人均为初次诊断为CE及单纯性肝脏单发感染的病人;血清抗体水平随着病人棘球蚴囊数目增加而有所增加,棘球蚴囊的数目与血清抗体水平的比较用单因素方差分析有显著性差异(F=5.06,P=0.0142),1个囊与2个囊/3个囊组间血清抗体水平有显著差异,2个囊与3个囊组间差异无统计学意义。结论rEgAgB8/1重组蛋白抗原对囊型包虫病有较高的血清学诊断价值,多囊型包虫病人血清抗体水平高于单囊型包虫病人。     

原发性肝癌患者血清TGF-β1、IL-8及T细胞亚群水平的变化

转化生长因子(TGF)包括两类多肽类生长因子,TGF-α与TGF-β[1].TGF-β属于多功能蛋白质,能够影响人体内多种细胞的生长、分化及凋亡,并具有调节免疫的功能.TGF-β包括三个亚型:TGF-β1,TGF-β2和TGF-β3.TGF-β3主要存在于人体内.有研究表明,TGF-β3对人体内肿瘤细胞的生长、进展及凋亡起着一定的作用[2].本文探讨原发性肝癌患者血清TGF-β1表达水平及其临床意义.     

肝脏表达Niemann-Pick C1-Like 1调节肝X受体诱导的小鼠胆固醇分泌

目的:探讨小鼠肝脏过表达人NPC1L1对LXR诱导的小鼠胆固醇分泌的影响。方法:给予野生型小鼠(WT)和肝脏过表达人NPC1L1的转基因小鼠(L1Tg)胃饲LXR激动剂(T0901317)7 d后,抽提小鼠粪便总脂质并检测核心甾醇的含量,检测小鼠的血浆脂质水平,分析肝脏ABCG5和ABCG8的mRNA表达水平。结果:L1Tg小鼠与WT小鼠相比,除血浆游离胆固醇含量显著升高外,粪便核心甾醇含量及肝脏ABCG5和G8的mRNA水平差异无统计学意义;在胃饲T0901317 1 w后,WT小鼠的粪便核心甾醇含量由[(3.22±0.44)升高到(28.68±1.05)μmol.d-1.100 g-1],血浆总胆固醇、游离胆固醇、胆固醇酯和磷脂的含量均显著升高,同时肝脏的ABCG5和G8的mRNA水平也分别上调了5倍和2倍;然而,L1Tg小鼠经T0901317处理后,与T0901317处理的WT小鼠相比,粪便核心甾醇的分泌减少了56%,血浆游离胆固醇含量升高了40%,肝脏的ABCG5和G8的mRNA水平分别降低了52.4%和40.6%。结论:肝脏特异表达人NPC1L1降低了LXR诱导的小鼠粪便核心甾醇的分泌,并升高了血浆游离胆固醇水平,可能与下调LXR诱导的肝脏ABCG5和G8 mRNA表述水平有一定关系。     

人肌纤生成调节因子1真核表达载体的构建及其在乳鼠心肌细胞中的表达

目的构建人肌纤生成调节因子1全长的真核表达质粒,并观察其在HEK293T细胞系及Sprague-Daw-ley乳鼠心肌细胞中的表达。方法从NCBI GenBank数据库中克隆得到人肌纤生成调节因子1基因(AF417001)全长序列,与真核表达载体质粒pcDNA3.1/Myc-His(-)B连接并转化大肠杆菌XL1-Blue,筛选阳性克隆,T7引物测序,转染细胞后以逆转录聚合酶链反应、Western Blotting方法检测人肌纤生成调节因子1的表达。结果pcDNA3.1/Myc-His(-)B-hMR-1质粒经测序证实目的基因序列正确,无碱基突变。该质粒转染到HEK293T细胞系和乳鼠心肌细胞后人肌纤生成调节因子1的转录水平及表达水平明显增高。结论成功构建了人肌纤生成调节因子1全长的真核表达载体并确定了简便有效的乳鼠心肌细胞瞬时转染方法。     

游泳运动对β淀粉样前体蛋白/早老素1小鼠海马紧密连接蛋白表达和学习记忆的影响

目的 探讨游泳运动对β淀粉样前体蛋白(APP)/早老素1(PS1)双转基因小鼠闭锁小带蛋白1(ZO-1)、闭合蛋白(occludin)表达和学习记忆的影响。方法 将20只APP/PS1小鼠随机分为模型组和游泳组每组10只,对照组选取同窝阴性小鼠10只。游泳组采用游泳运动干预,用新物体识别实验、免疫蛋白印迹法以及免疫荧光检测小鼠学习记忆能力、海马紧密连接蛋白ZO-1、occludin表达和海马CA1区神经元表达情况;硫黄素S染色法检测海马β淀粉样蛋白(Aβ)斑块沉积情况。结果 与对照组比较,模型组辨别指数(RI)明显降低(P<0.01);与模型组比较,游泳组RI明显升高[(63.63±13.35)%vs(50.38±9.83)%,P<0.01]。与对照组比较,模型组ZO-1、occludin明显降低(P<0.05,P<0.01);与模型组比较,游泳组ZO-1、occludin明显增加(0.81±0.02 vs 0.38±0.08,1.07±0.03 vs 0.68±0.12,P<0.05)。与对照组比较,模型组神经元阳性表达明显减少(P<0.01);与...     

胰岛素样生长因子1受体α亚单位基因重组真核表达质粒的构建

目的 构建编码胰岛素样生长因子1受体(IGF-1R)α亚单位基因的重组真核表达质粒.方法 根据GenBank中人IGF-1Rα亚单位基因编码序列设计并合成引物,用TRIzol一步法从Graves病患者甲状腺组织中提取总RNA,逆转录成cDNA并将其作为模板,PCR扩增获得IGF-1Rα亚单位编码基因片段.胶回收纯化后再...     

IL-6对肝细胞癌患者CD8+T淋巴细胞程序性死亡受体1表达和功能的影响

目的观察血浆IL-6及程序性死亡受体(PD-1)在肝细胞癌(HCC)患者外周血CD8~+T淋巴细胞中的表达,评估IL-6对HCC患者CD8~+T淋巴细胞中PD-1表达和功能的影响。方法纳入2019年1月—2019年9月期间在陕西省人民医院或空军军医大学第二附属医院(第四军医大学唐都医院)就诊的HCC患者44例(HCC组),同时纳入年龄和性别匹配的健康对照者19例(HC组),采集外周血,分离血浆和外周血单个核细胞,分选CD8~+T淋巴细胞,ELISA法检测血浆IL-6水平,流式细胞术检测PD-1在CD8~+T淋巴细胞中的表达水平。使用IL-6中和抗体刺激分选的CD8~+T淋巴细胞24 h,CCK-8法检测细胞增殖,ELISA法检测培养上清IFNγ和TNFα水平,实时定量PCR法检测穿孔素、颗粒酶B和颗粒溶素mRNA相对表达量,Western blot法检测STAT3和Src磷酸化水平。计量资料两组间比较采用t检验或配对t检验;计数资料组间比较采用χ~2检验。结果 HCC组患者血浆IL-6水平较HC组显著升高~([(99.67±20.92) pg/m L vs (81.05±16.76) pg/m L,t=3.427,P=0.001 1])。虽然CD3~+CD8~+T淋巴细胞比例在HCC组和HC组患者之间的差异无统计学意义(P 0.05),但PD-1~+CD8~+细胞比例在HCC组患者中显著升高(3.79%±1.36%vs 2.20%±0.47%,t=5.335,P 0.000 1)。使用IL-6中和抗体抑制HCC组患者CD8~+T淋巴细胞的IL-6虽不影响细胞增殖,但可降低PD-1表达(2.67%±0.91%vs 3.33%±1.12%,t=2.177,P=0.035),增加IFNγ分泌~([(13.50±3.82) pg/m L vs (10.82±1.37) pg/m L,t=3.170,P=0.002 8]),穿孔素和颗粒酶B mRNA相对表达量亦显著升高(t值分别为6.161、14.140,P值均0.000 1),同时伴有磷酸化STAT3水平降低(P 0.000 1)。结论抗人IL-6中和抗体可能通过增加穿孔素和颗粒酶B水平、增强细胞因子分泌以及抑制PD-1表达增强HCC患者CD8~+T淋巴细胞的功能。     

神经调节蛋白-1β对糖尿病心肌病大鼠血管内皮生成因子/磷酸化血管内皮生长因子受体-1信号通路的影响

目的运用神经调节蛋白-1β(beuregulin-1β,NRG-1β)干预糖尿病心肌病大鼠,并了解其对血管内皮生成因子(vascular endothelial growth factor,VEGF)/磷酸化血管内皮生长因子受体-1(vascular endothelial growth factor receptor-1,FLK-1)信号通路的影响。方法腹腔注射链脲霉素(strzptozotocin,STZ)(55 mg/kg)诱导糖尿病心肌病大鼠模型,干预组大鼠在建模成功后12周给予重组人NRG-1β10μg/(kg·d)鼠尾静脉注射,共10 d。运用MPA心脏功能分析系统评估大鼠心脏功能,运用同位素标记的微球评估大鼠心肌血流量;通过CD31标记免疫组化测定大鼠毛细血管密度;运用Western blot测定蛋白表达。结果与正常组比较,糖尿病心肌病组大鼠左心室功能显著下降,心肌毛细血管密度及心肌血流量显著减少,VEGF及磷酸化FLK-1、ErbB2、ErbB3浓度显著下降;与糖尿病心肌病组大鼠比较,NRG-1β干预组大鼠左心室功能、毛细血管密度显著增加;VEGF及磷酸化FLK-1、ErbB2、ErbB3浓度显著增加,差异有统计学意义(P0.05)。结论 NRG-1β能增强ErbB2、ErbB3磷酸化受体表达,改善糖尿病心肌病大鼠心脏功能,其可能机制是通过增强VEGF/FLK-1信号传导,促进糖尿病心肌血管生成来实现。     

柔红霉素对人急性早幼粒细胞白血病细胞株HL-60细胞及干扰素调节因子1、8、9表达的影响

目的研究柔红霉素(Daunorubicin,DNR)对人急性早幼粒细胞白血病(APL)细胞株HL-60细胞中干扰素调节因子1(IRF1)-mRNA、干扰素调节因子8(IRF8)-mRNA、干扰素调节因子9(IRF9)-mRNA表达的影响。方法将HL-60细胞株设为HL-60组、HL-60+DNR组,同时取3例正常人外周血白细胞为NC组。HL-60组为未加药处理组,HL-60+DNR组为小剂量DNR持续作用HL-60细胞10 d。采用实时荧光定量聚合酶链反应(RT-PCR)法检测IRF1-mRNA、IRF8-mRNA、IRF9-mRNA转录水平,每组实验重复3次。结果与NC组相比,IRF1-mRNA、IRF8-mRNA转录水平在HL-60组显著下调(P0.05),而HL-60+DNR组显著上调(P0.05);与NC组相比,IRF9-mRNA转录水平在HL-60+DNR显著上调(P0.05),在HL-60组则表现为下调,但无统计学差异(P0.05)。结论 DNR可上调HL-60细胞中的IRF1、IRF8、IRF9表达水平。APL经DNR治疗后,可能通过上调IRF1、IRF8、IRF9的表达诱导白血病细胞的凋亡,促进其成熟,促进病情的缓解。     

Stabilization of Ca2+-permeable AMPA receptors at perisynaptic sites by GluR1-S845 phosphorylation

AMPA receptor (AMPAR) channel properties and function are regulated by its subunit composition and phosphorylation. Certain types of neural activity can recruit Ca²⁺-permeable (CP) AMPARs, such as GluR1 homomers, to synapses likely via lateral diffusion from extrasynaptic sites. Here we show that GluR1-S845 phosphorylation can alter the subunit composition of perisynaptic AMPARs by providing stability to GluR1 homomers. Using mice specifically lacking phosphorylation of the GluR1-S845 site (GluR1-S845A mutants), we demonstrate that this site is necessary for maintaining CP-AMPARs. Specifically, in the GluR1-S845A mutants, CP-AMPARs were absent from perisynaptic locations mainly due to lysosomal degradation. This regulation was mimicked by acute desphosphorylation of the GluR1-S845 site in wild-type mice by NMDA application. Furthermore, long-term depression (LTD) was associated with a reduction in perisynaptic CP-AMPAR levels. Our findings suggest that GluR1-S845 is necessary for maintaining CP-AMPARs on the surface, especially at perisynaptic sites, and suggest that the regulation of these receptors is involved in synaptic plasticity.     

Expression of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) GluR2/3 receptors in the developing rat pineal gland

The expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type glutamate (GluR2/3) receptors and N-methyl-D-aspartate receptor subtype 1 (NMDAR1) was carried out by immunohistochemistry, double immunofluorescence and real-time RT-PCR analysis in the pineal glands of 1-day to 6-wk-old rats in the present study. GluR2/3 immunopositive cells were distributed throughout the pineal gland and showed branching processes in all age groups. The NMDAR1 immunoreactivity, however, was observed in fewer branched cells. A constitutive mRNA expression of NMDAR1, GluR2 and GluR3 was detected in the pineal glands of various ages and showed no significant difference between the age groups studied. Immunohistochemical and double immunofluorescence results showed that the GluR2/3 were mainly expressed and co-localized with OX-42-positive microglia/macrophages and the glial fibrillary acidic protein (GFAP)-positive astrocytes. Co-localization of NMDAR1 with OX-42- and GFAP-positive cells was much less. The expression of these receptors on the glial cells suggests that they may be involved in the development and growth of the pineal gland in the early postnatal period (1 day to 3 wk) and subsequently in the regulation of melatonin synthesis.     

Enhanced synaptic plasticity in mice with phosphomimetic mutation of the GluA1 AMPA receptor

Phosphorylation of the GluA1 subunit of AMPA receptors has been proposed to regulate receptor trafficking and synaptic transmission and plasticity. However, it remains unclear whether GluA1 phosphorylation is permissive or sufficient for enacting these functional changes. Here we investigate the role of GluA1 phosphorylation at S831 and S845 residues in the hippocampus through the analyses of GluA1 S831D/S845D phosphomimetic knock-in mice. S831D/S845D mice showed normal total and surface expression and subcellular localization of GluA1 as well as intact basal synaptic transmission. In addition, theta-burst stimulation, a protocol that was sufficient to induce robust long-term potentiation (LTP) in WT mice, resulted in LTP of similar magnitude in S831D/S845D mice. However, S831D/S845D mice showed LTP induced with 10-Hz stimulation, a protocol that is weaker than theta-burst stimulation and was not sufficient to induce LTP in WT mice. Moreover, S831D/S845D mice exhibited LTP induced with spike-timing-dependent plasticity (STDP) protocol at a long pre-post interval that was subthreshold for WT mice, although a suprathreshold STDP protocol at a short pre-post interval resulted in similarly robust LTP for WT and S831D/S845D mice. These results indicate that phosphorylation of GluA1 at S831 and S845 is sufficient to lower the threshold for LTP induction, increasing the probability of synaptic plasticity.     

纹状体神经元GluR1Ser831磷酸化对帕金森病异动症发病机制的影响

目的 从纹状体神经元谷氨酸受体1的831位丝氨酸（GluRlSer831）磷酸化角度，探讨长期左旋多巴治疗帕金森病（PD）的异动症发病机制。方法 通过6羟基多巴胺立体定向注射至大鼠前脑内侧束建立PD动物模型，然后左旋多巴甲酯腹腔注射治疗（25mg·kg^-1·d^-1，每天2次）22d，评估左旋多巴处理剂峰旋转行为情况；运用免疫荧光与免疫印迹法（Western blot）观察和检测纹状体区谷氨酸受体GluR1亚细胞分布及GluR1Ser831磷酸化的表达情况。结果 PD大鼠应用左旋多巴长期处理后出现明显的剂峰旋转行为增强，与PD患者异动症具有相似特征。PD大鼠损伤侧纹状体细胞膜上GluR1和GluR1Ser831磷酸化的数量分别减少至（73．0±4．8）％和（51．4±4．3）％；长期左旋多巴处理又使损伤侧纹状体细胞膜上GluR1和GluR1Ser831磷酸化的数量分别增加至（104．2±5．5）％和（93．7±3．1）％；而损伤侧纹状体总蛋白GluR1的数量未发生明显变化。GluR1和GluR1Ser831磷酸化的这些改变独特发生在纹状体小清蛋白阳性中间神经元上。结论 纹状体小清蛋白阳性中间神经元上GluR1及GluR1Ser831磷酸化的改变，可能与PD异动症的发生有关。     

Expression of Erk kinase,AMPA receptor subunits GluR1 and GluR2,and protection of chondroitin sulfate in brain of rats with chronic fluorosis

<正>Objective To study the mechanism of central nervous system (CNS) injury in chronic fluorosis and the neuroprotective effect of chondroitin sulfate (CS).Methods Forty-eight female Sprague-Dawley rats weigh-     

Hypocholesterolemia, foam cell accumulation, but no atherosclerosis in mice lacking ABC-transporter A1 and scavenger receptor BI

High-density lipoprotein (HDL) mediated reverse cholesterol transport (RCT) is regarded to be crucial for prevention of foam cell formation and atherosclerosis. ABC-transporter A1 (ABCA1) and scavenger receptor BI (SR-BI) are involved in the biogenesis of HDL and the selective delivery of HDL cholesterol to the liver, respectively. In the present study, we phenotypically characterized mice lacking these two proteins essential for HDL metabolism. ABCA1 × SR-BI double knockout (dKO) mice showed severe hypocholesterolemia mainly due to HDL loss, despite a 90% reduction of HDL cholesterol uptake by liver. VLDL production was increased in dKO mice. However, non-HDL cholesterol levels were reduced, probably due to enhanced clearance via LRP1. Hepatobiliary cholesterol transport and fecal sterol excretion were not impaired in dKO mice. In contrast, the macrophage RCT in dKO mice was markedly impaired as compared to WT mice, associated with the accumulation of macrophage foam cells in the lung and Peyer's patches. Strikingly, no atherosclerotic lesion formation was observed in dKO mice. In conclusion, both ABCA1 and SR-BI are essential for maintaining a properly functioning HDL-mediated macrophage RCT, while the potential anti-atherosclerotic functions of ABCA1 and SR-BI are not evident in dKO mice due to the absence of pro-atherogenic lipoproteins.     

游泳对高脂饮食老年大鼠高密度脂蛋白及载脂蛋白A1基因表达的影响

目的探讨运动对高脂饮食老年大鼠血脂代谢的影响. 方法建立饮食性高脂血症大鼠模型,采用不同时间(45 min和90 min)的游泳运动,测定各组大鼠血清胆固醇(TC)、血清高密度脂蛋白胆固醇(HDL-C)和载脂蛋白A1(apoA1);并用逆转录聚合酶链式反应(RT-PCR)检测各组大鼠肝脏apoA1 mRNA水平. 结果参加运动的高脂饮食大鼠血清HDL-C分别为(1.48±0.24)mmol/L和(1.49±0.25)mmol/L,apoA1分别为(0.13±0.07)mmol/L和(0.17±0.03)mmol/L,均较对照组和单纯高脂组升高(P＜0.05),高脂饮食的老年大鼠肝脏apoA1mRNA表达明显减少为0.651±0.24(P＜0.01),而游泳运动则使其显著恢复,两运动组apoA1 mRNA表达量分别为1.623±0.61和1.577±0.49,差异无显著性(P＜0.05). 结论游泳运动通过调节大鼠肝脏apoA1 mRNA的表达,影响高脂饮食大鼠血浆高密度脂蛋白(HDL)的变化.     

The antidepressant-like effect of the melatonin receptor ligand luzindole in mice during forced swimming requires expression of MT2 but not MT1 melatonin receptors

We previously reported an antidepressant-like effect in C3H/HeN mice during the forced swimming test (FST) following treatment with the MT1/MT2 melatonin receptor ligand, luzindole. This study investigated the role melatonin receptors (MT1 and/or MT2) may play in the effect of luzindole in the FST using C3H/HeN mice with a genetic deletion of either MT1 (MT1KO) or MT2 (MT2KO) melatonin receptors. In the light phase (ZT 9-11), luzindole (30 mg/kg, i.p.) significantly decreased immobility during swimming in both wild type (WT) (135.6 +/- 25.3 s, n = 7) and MT(1)KO (132.6 +/- 13.3 s, n = 8) as compared with vehicle-treated mice (WT: 207.1 +/- 6.0 s, n = 7; MT1KO: 209.5 +/- 6.2 s, n = 8) (P < 0.001). In the dark phase (ZT 20-22), luzindole also decreased time of immobility in both WT (89.5 +/- 13.9 s, n = 8) and MT1KO (66.5 +/- 6.4 s, n = 8) mice as compared with the vehicle treated (WT: 193.8 +/- 3.5, n = 6; MT1KO: 176.6 +/- 6.2 s, n = 8) (P < 0.001). Genetic disruption of the MT1 gene did not alter the diurnal rhythm of serum melatonin in MT1KO mice (ZT 9-11: 1.3 +/- 0.6 pg/mL, n = 7; ZT 20-22: 10.3 +/- 1.1 pg/mL, n = 8) as compared with WT (ZT 9-11: 1.4 +/- 0.7 pg/mL; ZT 20-22: 10.6 pg/mL). Swimming did not alter the serum melatonin diurnal rhythm in WT and MT1KO mice. Decreases in immobility of WT and MT1KO mice by luzindole treatment were not affected by gender or age (3 months versus 8 months). In contrast, luzindole did not decrease immobility during the FST in MT2KO mice. We conclude that the antidepressant-like effect of luzindole may be mediated through blockade of MT2 rather than MT1 melatonin receptors.     

H1 histone subtype constitution and phosphorylation state of the ageing cell system of human peripheral blood lymphocytes

Until a few years ago, the H1 histones were exclusively considered to be the architectural proteins of chromatin involved in chromatin condensation. However there is now increasing data to support the hypothesis that the H1 subtypes are involved in genomic integrity and that they may have unexpected functional roles in various biological processes such as in differentiation and DNA repair, apoptosis and lifespan. Moreover, the H1 histones are phosphorylated to a great extent. Recent work has implicated phosphorylation of H1 in the regulation of chromatin remodeling. In light of the fact that chromatin reorganization and heterochromatin formation has been shown to take place during ageing and senescence, in the present investigation, we have analyzed the changes that take place in the somatic H1 linker histone subtype profile and their phosphorylation states in human peripheral blood lymphocytes as a function of donor age. Results from this work show that there is a significant age-related dephosphorylation of H1.4 and H1.5 and an increase in the heterochromatin protein HP1alpha as a function of donor age. These results indicate that dephosphorylation of H1 histones may be related to an increase in senescence-associated heterochromatin formation during the in vivo ageing of human peripheral blood lymphocytes.