败血症休克大鼠血管L-精氨酸/一氧化氮途径的变化

目的:观察败血症休克大鼠主动脉内膜、中膜和外膜一氧化氮合成途径的改变。方法:雄性Wistar大鼠盲肠结扎并穿孔复制败血症休克模型,分别测定假手术组、早期休克组和晚期休克组大鼠主动脉内膜、中膜和外膜的亚硝酸盐(NO^-₂)含量、一氧化氮合酶(NOS)活性及L-精氨酸(L-Arg)转运;免疫组化染色检测诱导型一氧化氮合酶(iNOS)在主动脉各层的分布。结果:早期及晚期败血症休克大鼠主动脉内膜产生的NO^-₂含量、NOS活性及L-Arg转运速率均低于假手术组,而中膜和外膜的NO^-₂、NOS活性及L-Arg转运速率则显著高于假手术组,外膜增加的程度尤为显著。免疫组织化学染色显示,败血症休克时血管中膜和外膜尤其是外膜iNOS阳性染色明显较强。结论:败血症休克时血管内膜NO合成受到抑制,而中膜和外膜NO合成显著增强,这一改变与休克状态下血管中L-Arg转运、iNOS表达及其活性的变化有关。     

一氧化氮诱导大鼠肺动脉平滑肌细胞凋亡机制研究

目的:研究一氧化氮(NO)诱导大鼠肺动脉平滑肌细胞(PASMC)凋亡的作用机制。方法:体外培养Wistar大鼠PASMC,加入NO供体硝普钠(SNP)于常氧和低氧条件下孵育12h或24h,通过流式细胞术碘化丙啶染色法观察PASMC细胞周期时段及凋亡亚二倍体峰变化,应用细胞免疫化学法检测PASMCcaspase-3和NF-κB的表达,同时行DNA断裂琼脂糖凝胶电泳。结果:SNP作用后PASMC出现剂量-依赖性促凋亡作用(P＜0.01),表现为凋亡指数与caspase-3表达的不同程度的增强。随凋亡的进展,出现PASMC凋亡核小体DNAladder,同时PASMCNF-κB阳性核表达较对照组少(P＜0.01)。结论:外源性NO诱导大鼠PASMC凋亡,caspase-3与NF-κB可能涉及PASMC凋亡的调控信号途径。     

心血管组织钙化时内源性硫化氢系统下调

目的：观察心血管组织钙化时内源性硫化氢生成系统(CSE/H2S)的变化，以探讨内源性硫化氢在心血管组织钙化中的作用及血管钙化的细胞分子机制。方法：在维生素D3 (Vit D3)和尼古丁(nicotine)诱导大鼠血管钙化模型上，测定钙含量、［45Ca2+］沉积及碱性磷酸酶（ALP）活性判断心血管钙化程度,采用生化法测定血浆、心肌组织和主动脉H2S含量及CSE活性，半定量RT-PCR方法测定心血管组织CSE mRNA水平。结果：钙化组大鼠心肌组织钙含量较对照组高3.8倍,主动脉钙含量、［45Ca2+］ 沉积及ALP 活性分别较对照组高6.8倍、1.4倍和 1.9倍（P＜0.01）；钙化组大鼠血浆H2S含量较对照组低39%（P<0.01），心肌和主动脉组织的 H2S含量也分别较对照组低39%和31%，CSE mRNA表达也分别低28％和36％（P<0.01），CSE活性分别低56%和53%(P<0.01)。结论：钙化心血管组织CSE/H2S通路受抑制，内源性H2S生成减少。     

牛磺酸拮抗同型半胱氨酸诱导的线粒体呼吸链自由基生成和同型半胱氨酸抑制线粒体牛磺酸转运体

目的:观察同型半胱氨酸(Hcy)对心肌线粒体电子漏及自由基生成的影响,以及观察牛磺酸的拮抗效应。方法: 分离大鼠心肌线粒体,超声波破碎线粒体制备亚线粒体,纯化制备猪心肌线粒体琥珀酸细胞色素C还原酶(SCR)重组体。分别用Hcy和/或牛磺酸共同孵育心肌线粒体、亚线粒体、SCR;用化学发光法测定H₂O₂^- 及O₂^- 生成;并用滤膜抽滤法观察线粒体膜牛磺酸转运体的性质及Hcy对牛磺酸转运功能的影响。结果: Hcy呈浓度依赖性地刺激大鼠心肌线粒体、亚线粒体氧自由基生成及SCR电子漏增加,牛磺酸自身不影响心肌线粒体、亚线粒体及SCR氧自由基生成,但呈浓度依赖性地抑制Hcy诱导的线粒体、亚线粒体及呼吸链重组体氧自由基生成。线粒体膜上存在Na+依赖性的牛磺酸转运体,Hcy呈浓度依赖性地抑制牛磺酸转运体对牛磺酸的转运功能。结论: 牛磺酸抑制Hcy刺激的线粒体呼吸链电子漏增加及氧自由基生成。线粒体膜上存在Na+依赖性的牛磺酸转运体,Hcy抑制线粒体膜上牛磺酸转运体对牛磺酸的转运功能。     

Early-life epileptiform discharges exert both rapid and long-lasting effects on AMPAR subunit composition and distribution in developing neurons

The perinatal period of brain is characterized by dynamic changes in structure and high propensity for epilepsy. Animal models have shown that alterations of AMPA receptor (AMPAR) assembly or function may be related to seizure-induced cell damage, long-lasting impairments in brain development and seizure threshold. However, effects of earlier epileptiform discharges on AMPAR composition and sub-cellular distribution remain understudied. In this study, we analyzed age-dependent variation of relative GluR1 and GluR2 protein levels in primary cultured rat cortical neurons at 7 DIV, 12 DIV, 17 DIV and 21 DIV. By inducing a single event of epileptiform activity at 6 DIV, we tested the effects of early-life seizure-like insults on AMPAR subunit distribution. We found a significant increase in synaptosomal membrane GluR1 expression in magnesium-free (MGF) medium-treated neurons at each time point detected (p < 0.05), while GluR2 expression increased at 7 DIV, and declined at 17 DIV and 21 DIV respectively (p < 0.05). That is, a trend of high GluR1 with much lower GluR2 expression on the surface membrane of epileptiform discharges experienced neurons over time in culture was presented. These findings in an in vitro model of early-life seizure may inform rodent models of epilepsy, as well as the cellular mechanism involved in epilepsy-associated brain dysfunction.     

钙调神经磷酸酶在大鼠不同组织中的分布及活性

目的:观察钙调神经磷酸酶(CaN)在大鼠不同器官组织中的活性和蛋白分布。方法:取正常大鼠不同器官组织,应用Westernblot及免疫组织化学方法测定CaN催化亚单位α亚型(CnAα)在各组织中的蛋白含量及分布,并用[³²P]标记的底物肽测定各组织的CaN活性。结果:①Westernblot结果显示,CnAα在大鼠脑组织中有丰富表达,心脏、骨骼肌和肺中亦有表达,但肾脏及主动脉未检测出有CnAα表达。②免疫组织化学显示,大鼠脑组织、心肌细胞胞浆内、肺内巨噬细胞及细支气管周围的结缔组织、主动脉外膜、肾小血管周围结缔组织及肾远曲小管及集合管的外壁中存在免疫反应阳性产物。③CaN活性在脑组织最高,其次为骨骼肌、心肌和肺组织,主动脉和肾脏最低。结论:钙调神经磷酸酶在体内具有广泛分布,可能参与多种器官组织的功能调节。     

Anti-myeloperoxidase IgG subclass distribution and avidity in sera from patients with propylthiouracil-induced antineutrophil cytoplasmic antibodies associated vasculitis

OBJECTIVE: Propylthiouracil (PTU) could induce MPO-ANCA-positive vasculitis. The aim of this study was to compare the IgG subclass distribution and avidity of MPO-ANCA in sera from patients with primary ANCA-associated vasculitis (AASV) and PTU-induced vasculitis. METHODS: Nineteen patients with primary AASV with MPO-ANCA and thirteen patients with PTU-induced vasculitis were enrolled in the current study. Sera in both active phase and remission were collected. Anti-MPO IgG subclasses were detected by antigen specific ELISAs using specific monoclonal antibodies as second antibodies, and MPO-ANCA avidity was assessed by antigen-inhibition ELISAs. RESULTS: In primary AASV, all four anti-MPO IgG subclasses could be detected in active phase with IgG1 (100%), IgG2 (73.7%), IgG3 (63.2%) and IgG4 (94.7%), and in remission, IgG1 and IgG4 subclasses in most patients remained positive. However, in PTU-induced vasculitis, anti-MPO IgG3 subclass could not be detected, the anti-MPO IgG subclasses in active phase were IgG1 (100%), IgG2 (61.5%) and IgG4 (46.2%). Furthermore, five out of the six patients (88.8%) with PTU-induced vasculitis with positive IgG4 subclass in active phase turned to negative in remission, however, only eight out of the fourteen patients (57.1%) with primary AASV turned to negative. The median avidity constant of MPO-ANCA was 56 (8.96 to >140) x 10(7) mol/l for patients with primary AASV and 0.7 (<0.28 to >140) x 10(7) mol/l for patients with PTU-induced vasculitis respectively. Furthermore, the relative levels of MPO-ANCA avidity were associated with elevation of ESR in primary AASV and were associated with BVAS scores in patients with PTU-induced vasculitis, respectively. CONCLUSION: MPO-ANCA IgG subclass distribution and avidity were different between patients with primary AASV and PTU-induced vasculitis. It was suggested that the mechanism of ANCA production in PTU-induced vasculitis was different from that in primary AASV, and the avidity of MPO-ANCA might be associated with disease activity.     

在光纤芯片上进行高速个体化全基因组测序

新近发展的在光纤芯片上做高通量测序的技术，可以将人类全基因组序列的测定时间由目前所用的10年时间缩短到100d。光纤芯片上的基因组测序结合了磁珠、乳液PCR和焦磷酸测序法等技术，利用光纤优良的表面性质和有效的加工工艺，将光纤制作成一种基因芯片。在芯片上用磁珠来耦联DNA、RNA等微量反应物。乳液PCR可以获得大量的不同的基因组DNA片段。乳液PCR和光纤芯片都是由于有了磁珠作为微量反应物的载体，从而可以将反应一再小型化。而这些技术的结合，工作效率是目前常规的测序方法的100多倍。成功实现了基因组测序反应小型化、高通量化的思想，大大降低了成本，减少了完成全基因组测序的时间。     

北京地区人乳头瘤病毒16型感染及其E6/E7基因变异与宫颈病变的相关性研究

目的探讨HPV16感染及其E6/E7基因变异与宫颈病变的相关性。方法采用导流杂交技术进行HPV感染分型检测,PCR扩增出80份HPV16阳性宫颈病变的E6/E7基因、克隆入pMD18-T载体,双向测序分析基因变异与宫颈病变相关性。结果HPV16在宫颈病变患者中的检出率最高为33.3%(154/463),与病变程度相关(P<0.05)。E6/E7基因72份测序成功,DNA序列变异发生率为88.9%(64/72)。氨基酸序列E6-D32E(T96G)和E7-N29S(A86G)位点突变同时伴随存在,D32E/N29S的检出率为38.9%(28/72),与宫颈病变程度相关(P<0.05)。结论HPV16是北京地区来源的宫颈病变中最常见的致病型,其D32E/N29S变异与病变程度相关。     

趋化因子受体CCR5反义RNA在真核细胞中的表达

目的 构建趋化因子受体CCR5反义RNA真核表达载体并获取重组假病毒颗粒以用于抗HIV-1研究，方法 用RT-PCR法从健康人外周血单个核细胞（PBMCs)中获得趋化因子受体CCR5翻译起始区的基因片段，并以正、反两个方面定向插入到真核表达载体pLXSN上，重组载体用脂质体转染剂（lipofectAMINE)转染PA317包装细胞，抗-G418克隆的细胞上清经逆转录后用荧光定量PCR（FQ-PCR）测定假病毒滴度，进一步感染NIH/3T3细胞。结果 CCR5正、反义RNA的真核表达载体。经PA317细胞包装形成的假病毒颗粒已成功地感染NIH/3T3细胞，目的基因在该细胞中得到整合与表达。结论 从PBMCs中获得的趋化因子受体CCR5基因片段通过逆转录病毒载体可转移至真核细胞中并得到表达，为进一步研究CCR5反义RNA的抗HIV-1作用奠定了基础。