一个新的胆固醇酯转运蛋白基因移码突变的鉴定

收集在脂质专科门诊就诊者中血清高密度脂蛋白－胆固醇（ＨＤＬ－Ｃ）高于１．８０ｍｍｏｌ／Ｌ的全血标本，通过聚合酶链反应（ＰＣＲ）技术扩增胆固醇酯转运蛋白（ＣＥＴＰ）基因，继而采用荧光直读法进行ＣＥＴＰ基因组ＤＮＡ序列分析，发现一女性先驱者为ＣＥＴＰ基因新的移码突变杂合子，即ＣＥＴＰ第３８位密码子中胞嘧啶碱基的缺失引起阅读框架的改变，致使第４８位密码子变为提前的转录终止信号。从该基因变异的性质和该先驱者的血脂变化（ＨＤＬ－Ｃ２．５１ｍｍｏｌ／Ｌ）提示该基因突变为一ＣＥＴＰ合成缺陷的无义突变。     

膜铁转运蛋白1突变斑马鱼骨形态及骨钙素的变化

目的观察膜铁转运蛋白1(ferroportin1,FPN1)突变斑马鱼(铁过载动物模型)骨发育及骨量变化,初步了解铁过载状况对骨代谢的影响。方法分别对受精后36和48h的突变体(weh)和野生型(WT)斑马鱼进行邻联茴香胺血液染色,观察weh及WT血红蛋白量的变化,比较铁过载状况;对受精后第14天的weh和WT进行阿尔辛蓝/茜素红染色,比较骨形态差异;分别提取受精后第5天和第7天的weh和WT总RNA,采用实时定量PCR方法检测骨钙素(BGP)的基因表达,比较骨代谢差异。结果邻联茴香胺血液染色后,与WT相比,weh在受精后36h未能着色,48h后轻微着色;对受精后第14天斑马鱼进行阿尔辛蓝/茜素红染色并与WT比较结果显示,weh茜素红着色较浅且肋骨及尾鳍未发育;与WT相比,受精后第5天和第7天weh骨钙素表达量较低(P0.05)。结论 FPN1突变斑马鱼体内铁代谢异常,同时骨量减低,骨发育迟缓。     

123株人类免疫缺陷病毒-1重组亚型主要耐药性突变的基因屏障

目的 比较HIV-1 CRF01_AE、CRF07_BC和CRF08_BC毒株发生蛋白酶抑制剂(PI)、核苷类反转录酶抑制剂(NRTI)和非核苷类反转录酶抑制剂(NNRTI)主要耐药性突变的基因屏障,分析不同亚型耐药模式的差异.方法 纳入在广西壮族自治区南宁、柳州两市未经治疗的HIV感染者190例,采集外周静脉血,从血浆中提取HIV-1 RNA,扩增pol区并测序,用系统进化树分析序列的亚型,统计各序列每个主要突变位点由野生型密码子突变为耐药密码子所需的碱基转换和颠换的数量,根据转换与颠换发生概率约2.5:1的规律,将每个转换赋值设为1,每个颠换赋值设为2.5,计算各突变赋值之和,即为基因屏障值,采用Kruskal-Wallis检验法和Nemenyi法比较不同亚型的基因屏障差异.结果 共获得CRF01_AE、CRF07_BC和CRF08_BC毒株123株.CRF08_BC发生T/S69D的基因屏障低于CRF01_AE和CRF07_BC(χ2=107.501,P＜0.01),CRF01_AE和CRF07_BC发生V118I和L210W的基因屏障低于CRF08_BC,CRF07_BC发生V106M的基因屏障低于CRF01_AE和CRF08_BC.结论 在相同的选择压力下,CRF01_AE和CRF07_BC比CRF08_BC易发生V118I和L210W,CRF08_BC比CRF01_AE和CRF07_BC易发生T/S69D,CRF07_BC比CRF08_BC和CRF01_AE易发生V106M.     

糖尿病大鼠脑葡萄糖转运子1和3蛋白表达随血糖浓度而变化

以Wistar大鼠建立链脲佐菌素糖尿病模型，用胰岛素使其血糖控制在〈10、10～14和〉16．7mmol／L3个不同水平，用免疫组化法检测对照组及3个糖尿病组大鼠大脑中葡萄糖转运蛋白（GLUT）1和3蛋白表达变化。结果发现慢性高血糖可引起脑内GLUT1和GLUT3蛋白表达降调节。     

突变乙型肝炎病毒核心蛋白的克隆及表达

目的 克隆并表达发生长片段缺失的HBV核心蛋白(HBV-C)基因,并对其进行DNA序列、蛋白质结构和抗原性分析.方法 采用PCR方法从1株野生型HBV基因组中扩增得到发生长片段缺失的HBV-C基因,克隆至pUCm-T质粒,进行测序、同源性比较和蛋白质结构分析;再将基因编码区克隆至原核表达载体pET-28a,构建含HBV-C基因的重组表达质粒,转化至大肠埃希菌BL21中进行诱导表达并检测其抗原性.结果 PCR扩增出的HBV-C基因长度经序列分析表明,其核苷酸序列缺失了220 bp至317 bp之间的98个碱基,造成从第74个氨基酸起发生移码突变并失去了抗原性.结论 成功克隆和表达了发生长片段缺失的HBV-C基因.     

尿酸转运蛋白及其相互作用蛋白在尿酸代谢中的作用研究进展

尿酸的重吸收和排泄依赖尿酸转运蛋白的作用,人体内尿酸转运蛋白数目众多,如葡萄糖转运蛋白9(GLUT9)、有机阴离子转运体1(OAT1)、尿酸盐转运蛋白1(URAT1)等,但单一尿酸转运蛋白的错义翻译很少直接导致高尿酸血症的发生,尿酸转运蛋白的相互作用蛋白,如跨膜整合蛋白2B(ITM2B)、乙醛脱氢酶16家族A1(ALD...     

长QT综合征相关基因新突变G52R-KCNE1的功能研究

目的 了解长QT综合征相关基因新突变G52R-KCNE1的功能。方法 采用交叠PCR方法体外制备突变体G52R-KCNE1,并克隆到原核细胞表达载体中;体外合成RNA、显微注射入卵母细胞,在卵母细胞中共表达野生型KCNQ1(WT-KCNQ1)、野生型KCNE1(WT-KCNE1)和突变体G52R-KCNE1,采用标准双电极电压钳了解突变体的功能。结果 突变体G52R-KCNE1不能放大WT-KCNQ1通道的电流强度;当WT-KCNE1和G52R-KCNE1等量注射时,G52R-KCNE1可降低WT-KCNQ1／WT-KCNE1通道约50％的电流强度,而不影响该通道的激活动力学。结论 位于跨膜区第52位的甘氨酸对维持KCNE1的功能非常重要;突变体G52R-KCNE1明显抑制WT-KCNE1通道的功能,这可能是突变基因携带者心肌细胞复极化延缓,QT间期延长的分子电生理机制。     

包膜蛋白突变对乙型肝炎病毒包装的影响

目的　探讨包膜蛋白突变对HBV包装的影响。方法　构建包膜蛋白突变的全长HBV基因组表达载体:前S1突变HBV表达载体pHBV mS1、Sloop突变HBV表达载体pHBV mS和前S1、S共突变HBV表达载体pHBV mS1S。分别转染HepG2细胞,以野生型HBV质粒 (adwR9 )转染HepG2细胞为对照;pHBV mS1S和pcDNA3分别与adwR9共转染HepG2细胞。ELISA方法检测上清液中和胞内S抗原;荧光定量PCR检测胞内和上清液中病毒量。结果　突变体pHBV mS1、pHBV mS和pHBV mS1S与对照组adwR9中S蛋白表达量和分泌量无明显差别;单独转染的突变体胞内病毒量较adwR9高,尤其以pHBV mS1S明显,而突变体上清中病毒量较对照组adwR9低,尤其以pHBV mS1S明显;pHBV mS1S与adwR9共转染组上清液中病毒量较pcDNA3与adwR9共转染组低。结论包膜蛋白突变对S蛋白表达量和分泌量无影响,但影响病毒包装,使病毒分泌量下降。     

食管癌P21WAF1表达与P53突变的关系

目的探讨P21WAF1和P53基因蛋白表达在食管癌发生发展的作用机制以及它们与食管癌临床病理等特征的关系.方法应用S-P免疫组化方法对144例食管癌病变组织进行了P21WAF1和P53蛋白的定位观察.结果食管癌P21WAF1基因蛋白阳性率仅为39%,且与肿瘤细胞分化及淋巴转移有关;P53蛋白阳性率为65%,也与肿瘤细胞分化及淋巴节转移相关.另外,P21WAF1和P53蛋白的表达在不典型增生组存在明显的相关性.结论p21WAF1和p53基因在食管磷癌发生发展机制中起着重要作用,并且,在食管癌癌前病变之中,p21WAF1基因依赖与p53基因通路.     

老年高血压发病机制研究新进展

老年高血压除具有原发性高血压的共同特点外,尚有其独特决定因素及特点。老年高血压常以收缩期高血压为主要特征。其机制为大动脉壁的结构和功能的改变。随着年龄增加,动脉变硬,顺应性下降,引起血流量改变,脉波速度加快,使收缩期血流回冲变早,形成向后反射波,导致收缩晚期主动脉、左室高压,减少了舒张甲．期局部血管床的灌注。     

SGLT2抑制剂治疗心力衰竭潜在机制的新认识

钠-葡萄糖协同转运蛋白2 (SGLT2)抑制剂已成为治疗心力衰竭(心衰)的新兴药物,但其作用机制尚不清楚.健康人心肌细胞仅表达SGLT1,SGLT2定位在肾脏近曲小管和心外膜脂肪组织(EAT),在疾病状态下高表达.舒张性心衰患者发生EAT堆积,EAT高表达SGLT2和分泌脂肪细胞因子,介导心肌纤维化和心肌肥厚;心肌细胞...     

先天性心脏病相关HAND1基因新突变研究

目的:研究先天性心脏病(congenital heart disease,CHD)相关HAND1基因新突变。方法:入选CHD患儿136例及健康对照儿童200名,抽提基因组DNA,通过聚合酶链反应测序筛查HAND1基因突变。应用计算机软件评估突变氨基酸的保守性并预测突变的致病性,应用双荧光素酶报告基因分析系统分析突变对HAND1功能的影响。结果:在1例法洛四联征患儿发现1种新的HAND1基因杂合突变,其编码核苷酸序列第389位的胸腺嘧啶突变为鸟嘌呤(c.389TG),相应氨基酸序列的第130位亮氨酸变为精氨酸(p.L130R)。该突变改变了进化上保守的氨基酸序列并被预测为有致病性,功能研究揭示突变型HAND1的转录激活功能显著降低。结论:该HAND1基因功能缺失性新突变可能是CHD的少见分子病因。     

Cellular targets and mechanisms of nitros(yl)ation: an insight into their nature and kinetics in vivo

There is mounting evidence that the established paradigm of nitric oxide (NO) biochemistry, from formation through NO synthases, over interaction with soluble guanylyl cyclase, to eventual disposal as nitrite/nitrate, represents only part of a richer chemistry through which NO elicits biological signaling. Additional pathways have been suggested that include interaction of NO-derived metabolites with thiols and metals to form S-nitrosothiols (RSNOs) and metal nitrosyls. Despite the overwhelming attention paid in this regard to RSNOs, little is known about the stability of these species, their significance outside the circulation, and whether other nitros(yl)ation products are of equal importance. We here show that N-nitrosation and heme-nitrosylation are indeed as ubiquitous as S-nitrosation in vivo and that the products of these reactions are constitutively present throughout the organ system. Our study further reveals that all NO-derived products are highly dynamic, have fairly short lifetimes, and are linked to tissue oxygenation and redox state. Experimental evidence further suggests that nitroso formation occurs substantially by means of oxidative nitrosylation rather than NO autoxidation, explaining why S-nitrosation can compete effectively with nitrosylation. Moreover, tissue nitrite can serve as a significant extravascular pool of NO during brief periods of hypoxia, and tissue nitrate/nitrite ratios can serve as indicators of the balance between local oxidative and nitrosative stress. These findings vastly expand our understanding of the fate of NO in vivo and provide a framework for further exploration of the significance of nitrosative events in redox sensing and signaling. The findings also raise the intriguing possibility that N-nitrosation is directly involved in the modulation of protein function.     

Cellular localization of fibronectin gene expression in the seminiferous tubule

The cellular location of fibronectin expression within the seminiferous tubule was investigated in order to better understand testicular cell functions and cell-cell interactions. Peritubular cells were shown to actively synthesize and secrete fibronectin in culture by the detection of a radiolabeled 220 kDa secreted protein that is immunologically similar to fibronectin and by the quantitation of fibronectin in peritubular cell conditioned medium with a fibronectin enzyme-linked immunosorbent assay. Sertoli cells did not produce detectable levels of fibronectin when assayed by either of these procedures. A 6.5 kb fibronectin messenger RNA was detected in freshly isolated or cultured peritubular cells, but no fibronectin gene expression was detected in Sertoli cells or developing germinal cells. Combined results imply that the peritubular cells are the only apparent site of fibronectin expression within the seminiferous tubule. During the development of the testis the levels of fibronectin expression increased to a maximum at early puberty (15-day-old rats) and then slowly declined. The results demonstrate that fibronectin can be utilized as a unique functional and biochemical marker for peritubular cells when compared to other cell types in the seminiferous tubule. Production of fibronectin by peritubular cells provides an example of the ability of peritubular cells and Sertoli cells to cooperate in the production of individual components of the basement membrane of the seminiferous tubule. This cellular interaction is an example of a mesenchymal/stromal-epithelial interaction which is postulated to be important for the physiology of many tissues.     

New insights into the pathophysiology of oedema in nephrotic syndrome

Oedema is a common clinical manifestation of nephrotic syndrome. However, the pathophysiological mechanism of sodium retention in nephrotic syndrome has been intensely debated for decades. Several clinical and experimental observations argue against the classic or "underfill" hypothesis of oedema formation in nephrotic syndrome. In many patients, oedema formation in nephrotic syndrome is due to the kidney being intrinsically unable to excrete salt and is unrelated to systemic factors (i.e. hypoalbuminaemia, decreased “effective” arterial blood volume, and secondary hyperaldosteronism). The cortical collecting duct is the nephron site of sodium retention in nephrotic syndrome. Activation of the epithelial sodium channel in the cortical collecting duct is responsible for sodium retention in nephrotic syndrome. In nephrotic syndrome, a defective glomerular filtration barrier allows the passage of proteolytic enzymes or their precursors, which have the ability to activate the epithelial sodium channel, thereby causing the the subsequent sodium retention and oedema.