RNA-binding protein is involved in aggregation of light neurofilament protein and is implicated in the pathogenesis of motor neuron degeneration

Abnormal protein aggregation is emerging as a common theme in the pathogenesis of neurodegenerative disease. Our previous studies have shown that overexpression of untranslated light neurofilament (NF-L) RNA causes motor neuron degeneration in transgenic mice, leads to accumulation of ubiquitinated aggregates in degenerating cultured motor neurons and triggers aggregation of NF-L protein and co-aggregation of mutant SOD1 protein in neuronal cells. Here, we report that p190RhoGEF, an RNA-binding protein that binds to a destabilizing element in NF-L mRNA, is involved in aggregation of NF-L protein and is implicated in the pathogenesis of motor neuron degeneration. We show that p190RhoGEF co-aggregates with unassembled NF-L protein and that co-aggregation is associated with down-regulation of parent NF-L mRNA in neuronal cells. Co-expression of NF-M increases NF assembly and reduces RNA-triggered aggregation as well as loss of solubility of NF-L protein. siRNA-induced down-regulation of p190RhoGEF not only reduces aggregation and promotes assembly of NF-L and NF-M, but also causes reversal of aggregation and recovery of NF assembly in transfected cells. Examination of transgenic models of motor neuron disease shows that prominent aggregates of p190RhoGEF and NF-L and down-regulation of NF-L expression occur in degenerating motor neurons of mice expressing untranslated NF-L RNA or a G93A mutant SOD1 transgene. Moreover, aggregates of p190RhoGEF and NF-L appear as early pathological changes in presymptomatic G93A mutant SOD1 transgenic mice. Together, the findings indicate that p190RhoGEF is involved in aggregation of NF-L protein and support a working hypothesis that aggregation of p190RhoGEF and NF-L is an upstream event triggering neurotoxicity in motor neuron disease.     

抗肝癌细胞噬菌体单链抗体库的构建及筛选

构建抗人肝癌细胞单链抗体库 ,从中筛选与肝癌细胞特异结合的高亲和力单链抗体。从HepG2细胞免疫的BALB/c小鼠脾脏提取总RNA ,RT PCR扩增小鼠抗体重、轻链可变区基因 ,用 (Gly4Ser) 3 连接肽基因 ,经重叠延伸反应 ,在体外将VH 和VL 连接成单链抗体 (scFv)基因 ,并克隆入噬菌粒载体pCANTAB5E中 ,构建噬菌体单链抗体库。以HepG2细胞为抗原对抗体库进行淘选 ,ELISA法鉴定各单克隆与肝癌细胞的结合活性 ,并对阳性克隆进行表达。成功构建了库容为 1 1× 10 6抗肝癌细胞的噬菌体单链抗体库 ,经筛选得到了与HepG2细胞具有较强结合能力的单链抗体 ,实现了scFv在大肠杆菌中的可溶性表达。序列测定结果表明 ,VH 和VL 基因符合小鼠抗体可变区特征 ,scFv基因拼接正确     

慢性乙型肝炎患者血清HBV DNA水平与肝纤维化标志物的关系

目的研究慢性乙型肝炎患者血清HBV复制水平和肝纤维化血清学标志物的关系。方法入选临床确诊为慢性乙型肝炎的157例患者，其中49例为早期肝硬化，采用荧光定量PCR检测血清HBVDNA水平，放射免疫法和酶免疫法检测肝纤维化血清标志物：透明质酸、层黏蛋白、Ⅲ型前胶原N端肽和Ⅳ型胶原，对血清HBVDNA水平和肝纤维化标志物的关系进行研究分析，并对49例早期肝硬化患者和108例无肝硬化患者的血清HBVDNA及肝纤维化血清标志物水平进行比较。结果慢性乙型肝炎患者的血清HBVDNA水平和肝纤维化标志物水平无显著相关性（P〉0．05），早期肝硬化患者的血清肝纤维化标志物水平显著高于无肝硬化的患者，而HBVDNA水平却低于无肝硬化的患者（P〈0．05）。结论慢性乙型肝炎患者的血清HBVDNA水平和肝纤维化标志物水平无显著相关性。     

ctB和ureⅠ双基因融合表达及其免疫特性分析

目的：构建霍乱毒素B亚单位（CtB）和幽门螺杆菌尿素膜通道蛋白（UreⅠ）融合的原核表达质粒pET32a（＋）ctB/ure Ⅰ,并初步研究融合蛋白Ct B/Ure Ⅰ的表达特性和免疫特性.方法：PCR从pUC18 ctB中克隆ctB基因,定向在pET32a（＋）/ureⅠ的ureⅠ基因5＇端插入ctB基因,构建ctB和ure Ⅰ双基因原核表达质粒pET32a（＋）ctB/ure Ⅰ,转该质粒于E.coli BL-21（DE3）,经酶切和序列分析鉴定工程菌.IPTG诱导表达,HP-His亲和层析纯化,SDS-PAGE和Gel-Pro Analizer4分析,重组蛋白免疫BALB/c小鼠.用Western blot和ELISA分析重组蛋白的免疫特性.结果：工程菌含完整的ctB和ure Ⅰ基因,与相对应基因的序列同源性分别为100%.在22℃,1 mmol/L IPTG诱导4 h后,重组蛋白的表达占菌体总蛋白12%,亲和层析纯化后蛋白纯度为94.3%.Western blot表明重组蛋白分别能与相应的抗体反应,该蛋白免疫小鼠后能产生相应的IgG抗体.结论：成功构建了能表达CtB/Ure Ⅰ蛋白的大肠杆菌表达菌株.对融合蛋白表达和纯化后,初步证明了该重组蛋白有CtB和Ure Ⅰ的双特异反应原性和免疫原性,为研究新型幽门螺杆菌疫苗奠定了坚实的基础.     

HIV-1高暴露持续血清阴性者对Nef、Gag肽段库的特异性CTL应答的研究

目的 研究中国HIV-1高暴露持续血清阴性（highly exposed persistently seronegative,HEPS）者的Nef、Gag特异性细胞毒性T淋巴细胞（cytotoxic T lymphocyte,CTL）应答特点,探讨HIV-1特异性CTL应答在这类特殊人群中抵抗感染的作用机制.方法 选取10例HIV-1高暴露持续血清阴性者,11例经性接触感染且从未接受抗病毒治疗的HIV/AIDS患者及4例未经暴露的健康志愿者.以覆盖HIV-1 gag全长和部分nef的14个肽段库为刺激原,应用IFN-γ ELISPOT法测定3组人群的特异性CTL应答,并对3组的应答强度、宽度以及对肽段库识别比例进行比较.结果 50%（5/10）的HEPS,100%（11/11）的HIV/AIDS患者均存在Nef及Gag特异性CTL应答,而4例健康对照均为阴性.存在应答的HEPS者对14个肽段库的平均应答强度和宽度分别是HIV/AIDS患者的4.3%和37.7%.在HEPS者中主要识别的肽段库均为HIV/AIDS患者中识别比例相对较低的肽段库.结论 与HIV/AIDS患者相比,HEPS者中的HIV-1特异性CTL应答存在着不同的特点和规律,可能在保护机体免于HIV-1感染中发挥着重要作用.     

Effects of magnesium on prostacyclin synthesis and intracellular free calcium concentration in vascular cells.

This study investigated the effects of extracellular magnesium concentration ([Mg2+]e; 0.3-3 mM) on intracellular free calcium concentration ([Ca2+]i) and prostacyclin (PGI2) production in cultured human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells from rats (VSMC) under basal and agonist-stimulated conditions. We used histamine as agonist which increases [Ca2+]i and PGI2 production in HUVEC, norepinephrine in VSMC. [Mg2+]e dose-dependently increased basal and agonist-stimulated PGI2 production in both cells. [Mg2+]e dose-dependently reduced basal [Ca2+]i in VSMC, but did not influence in HUVEC. In both cells, increasing [Mg2+]e reduced agonist-stimulated [Ca2+]i responses. Furthermore, [Mg2+]e dose-dependently reduced agonist-stimulated [Ca2+]i in Ca(2+)-free buffer, indicating intracellular Ca2+ release. In VSMC, 10(-6) M diltiazem and 10(-7) M nifedipine, Ca2+ channel blockers, reduced agonist-stimulated [Ca2+]i as well as 3 mM Mg2+, but did not affect PGI2 production. [Mg2+]e amplified dose-dependently arachidonic acid-induced PGI2 production in both cells, suggesting the activation of cyclooxygenase and/or PGI2 synthetase. Our results suggest that [Mg2+]e influences intracellular Ca2+ mobilization of not only vascular smooth muscle cells but also endothelial cells by inhibiting both Ca2+ influx and intracellular Ca2+ release. [Mg2+]e enhances PGI2 production in both types of cells, although the mechanism is likely to be independent from Ca2+ mobilization.     

apoH基因多态性与脑卒中的相关性研究及其对血脂的影响

目的：探讨载脂蛋白H（apolipoprotein H,apoH）基因第8外显子G1025C（Try316Ser）多态性与长沙地区汉族人脑卒中的关系及其对血 脂的影响。方法：采用PCR－单链构象多态技术和DNA直接测序法检测长沙地区汉族260例脑卒中患者、20个脑卒中家系成员和100名健康对照者的apoH基因第8外显子G1025C（Try316Ser）多态性；酶联免疫吸附法检测抗磷脂抗体水平；酶法测定总胆固醇（TC）、甘油三酯（TG）、高密度脂蛋白胆固醇（HDL－C）、低密度脂蛋白胆固醇（LDL－C），免疫比浊法测定apoA-I及apo B100，酶联免疫吸附法测定脂蛋白a血清水平。结果：长沙地区汉族人apoH基因G1025C（Try316Ser）多态存在GG、GC两种基因型，脑卒中组及其各亚组G1025C（Try316Ser）多态基因频率分布与对照组比较差异无显著性（P＞0．05）；分析不同基因型对血脂、脂蛋白水平的影响，发现脑梗塞组和对照组GC基因型血清TG水平均明显高于GG基因型（P＜0．05）。结论：长沙地区汉族人apoH基因G1025C（Try316Ser）多态性可能与脑卒中的易患性无关，但与血脂代谢有一定关联。     

仰俯卧位对早产儿肺炎患儿肺功能的影响

目的探讨仰俯卧位对早产儿肺炎患儿肺功能的影响.方法应用美国Bicore CP-100新生儿肺功能仪分别对30名不吸氧组(SaO2≥90%)和18名吸氧组(SaO2<90%)早产儿肺炎患儿的肺功能值进行检测.结果不吸氧组患儿俯卧位呼吸频率明显低于仰卧位(43.4±10.2 v 49.8±11.7);俯卧位潮气量明显高于仰卧位(3.11±0.92 v 2.48±0.77 ml/kg);俯卧位气道阻力明显低于仰卧位(98.6±50.1 v 117.4±41.5cm H2O/L/s,);俯卧位动态肺顺应性明显高于仰卧位(1.115±0.401 v 0.914±0.48ml/cm H2O/kg);俯卧位呼吸功明显高于仰卧位(8.0±3.8 v 5.9±2.7gm cm/kg).吸氧组患儿俯卧位呼吸频率明显低于仰卧位(52.7±10.2 v 56.1±12.4);潮气量明显高于仰卧位(2.13±0.42 v 1.79±0.66 ml/kg);俯卧位气道阻力明显低于仰卧位(290.4±107.0 v 319.1±104.1cm H2O/L/s);俯卧位动态肺顺应性明显高于仰卧位(0.562±0.192 v 0.488±0.217ml/cm H2O/kg);俯卧位呼吸功明显高于仰卧位(10.1±4.7 v 7.3±3.4gm cm/kg).结论无论吸氧与否俯卧位均可提高患儿的潮气量、动态肺顺应性和呼吸功,降低呼吸频率和气道阻力,俯卧位为早产儿肺炎患儿的适宜体位.