重组腺相关病毒(rAAV)介导的人凝血因子IX高活性突变衍生物在血友病B基因治疗研究中的应用

采用定点诱变技术,将R338A点突变引入人凝血因子Ⅸ基因,并构建于AAV载体上,以rHSV/AAV杂合辅助病毒系统介导制备rAAV-hFIX重组病毒,然后,经肌肉注射对血友病B小鼠进行治疗实验,观察该突变基因在小鼠体内的表达、活性以及机体对该突变衍生物的免疫反应与治疗效果.结果显示:(i)治疗小鼠体内可检测到hFIX-R338A突变衍生物的存在,并持续15周以上;(ii)突变衍生物hFIX-R338A在小鼠血浆中的凝血活性达(34.2±5.23)%,显著高于野生型Ⅸ因子凝血活性((14.27±3.4)%);(iii)治疗小鼠体内未检测到抗Ⅸ因子突变衍生物抗体的存在;(iv)未发现与治疗相关的局部及全身性毒副作用.提示:以AAV介导人凝血因子Ⅸ高活性突变衍生物hFLX-R338A基因治疗可能成为替代野生型Ⅸ因子进行血友病B基因治疗的一个更为有效的途径.     

Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associ-ated viral vector containing hFIXR338A, prepared by rHSV/AAV hybrid helper virus system, was directly introduced to the hind leg muscle of factor IX knock out mice. The expression and the biological activity of human factor IX mutant, hFIXR338A, and the immune response against it in the treated mice were assayed and detected. The results showed that (i) the high-level expression of human factor IX mutant protein, hFIXR338A, has been detected in rAAV-hFIXR338A treated hemophilia B mice and lasted more than 15 weeks; (ii) the clotting activity of hFIXR338A in plasma is 34.2%± 5.23%, which is remarkably higher than that of (14.27%±3.4%) of wild type hFIX treated mice in the activated partial thromboplastin assay; (iii) immune response against factor IX R338A was absent, with no factor IX mutant protein (hFIXR338A) inhibitors deve     

耐钙心肌细胞的分离和电生理特性观察

用快速、恒压的无钙和胶原酶Ｔｙｒｏｄｅ液相继灌流豚鼠心脏冠脉系统后,再经无钙液室温浸泡心脏和用改变的Ｋ－Ｂ液帮助分离细胞的恢复,可获得耐钙的游离心肌细胞。全细胞电流记录：静息电位为－７２±９ｍＶ（ｎ＝１２）,并显示出快内向电流（ＩＮａ）,可被异搏定阻断的慢钙离子流和时间依赖性外向钾流（Ｉｋ）;单通道记录分别显示了Ｎａ＋Ｃａ２＋和Ｋ＋通道的电压依赖性等特征。结果表明了用此法分离的细胞具有耐钙性和正常电生理特性。     

海葵毒素对心肌钠通道开放模式、动作电位及心电图QT间期的影响

应用膜片箝技术记录游离豚鼠心肌细胞钠通道电流, 细胞内微电极技术记录心室乳头肌的动作电位和心电图机记录豚鼠的心电图。使用与心肌细胞钠通道有高度亲和力的海葵毒素(sea anemone toxin, ATXⅡ)改变钠通道开放的动力过程, 从三个水平来研究钠通道、动作电位、心电图变化的关系, 并试图探讨长QT综合征(long QT syndrome, LQTs)的发病机制。结果显示: ATXⅡ使钠通道的开放频率增加, 钠通道中“长时间开放模式”的开放时间常数增大, 动作电位的持续时间APD50和APD90也分别增加了23%和27%。 ATXⅡ使动物心电图QT间期延长18.6%, QTc (校正的QT间期)增大18.9%。这些结果提示, 钠通道动力过程的变化对动作电位和心电图QT间期有重要影响, 钠通道功能或结构的变异可能是临床上部分长QT综合征产生的原因。     

Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associ- ated viral vector containing hFIXR338A, prepared by rHSV/AAV hybrid helper virus system, was directly introduced to the hind leg muscle of factor IX knock out mice. The expression and the biological activity of human factor IX mutant, hFIXR338A, and the immune response against it in the treated mice were assayed and detected. The results showed that (i) the high-level expression of human factor IX mutant protein, hFIXR338A, has been detected in rAAV-hFIXR338A treated hemophilia B mice and lasted more than 15 weeks; (ii) the clotting activity of hFIXR338A in plasma is 34.2%± 5.23%, which is remarkably higher than that of (14.27% ± 3.4%) of wild type hFIX treated mice in the activated partial thromboplastin assay; (iii) immune response against factor IX R338A was absent, with no factor IX mutant protein (hFIXR338A) inhibitors development in the treated mice; and (iv) no local or systemic side-effects and toxicity associated with the gene transfer were found. It demonstrated the potential use of treating hemophilia B by recombinant adeno-associated viral vectors with mutant hFIXR338A gene, an alternative strategy for hemophilia B gene therapy to wild-type human factor IX.     

银杉花粉生命力及其变异

中国特有植物银杉(Cathay argyrophylla)是最濒危的松杉类植物之一。研究表明,受精前胚珠的高败育率是银杉生殖效率低的重要原因之一,但迄今对银杉花粉活力及其变异仍不得而知。由于花粉活力对授粉、受精、种子产量和质量以及后代适合度都有直接影响,该研究采用TTC染色和体外萌发法测定了来自大瑶山(Dayaoshan, DYS)和花坪(Huaping, HP)两个地区7个种群16个个体52份银杉花粉样品的生活力。结果表明银杉花粉的活力水平(93.3%)与其它裸子植物相当,干燥低温条件下银杉的花粉活力比较稳定,体外萌发的适宜蔗糖浓度在13%左右。ANOVA分析揭示种群内个体间的花粉活力差异不大(p>0.05),而地区内的种群间及地区间的花粉活力有显著差异(p<0.05)。银杉花粉活力与其生殖成功没有相关性。在上述基础上,从控制授粉的角度讨论了银杉的保护策略和复壮的可能性。     

珍稀植物太行花的遗传多态性及其分化

利用ISSR引物检测太行花的遗传多态性,以揭示太行花原变种和缘毛太行花变种的遗传分化和相互关系.结果表明,太行花拥有较高的遗传多态性(PPL=70.65,He=0.249 0,I=0.374 3),两个变种具有近似的遗传多态性值,遗传一致性高达0.940 6.ISSR标记监测到两变种间存在着广泛的基因交流,Nm=4.907 1.根据遗传相似系数,两变种的绝大部分个体分别被聚到各自的类群,这一结果从核酸水平上支持了变种的划分.太行花种内高度的遗传变异,为其驯化和新品种的培育提供了丰富的遗传基础.     

简便的窦房结细胞分离和穿孔膜片箝

用含胶原酶、弹性蛋白酶的无钙溶液和改良的回复式液体运动来酶解和机械分离兔心窦房结自律细胞。为防止“衰减”现象的出现（ｒｕｎｄｏｗｎ）,运用含ＡｍｐｈｏｔｅｒｉｃｉｎＢ的电极液作穿孔膜片箝。通过穿孔膜片箝的形成过程,记录分离细胞的自律性动作电位、慢内向离子流、外向钾流和起搏离子流Ｉｆ,表明本分离方法和穿孔膜片箝的优越性和简便性     

不同部位脑卒中患者认知功能损害的特点分析

目的：探讨不同部位缺血性脑卒中急性期患者认知功能损害的特点。方法：收集230例脑梗死急性期（1-14天）患者,包括额叶31例,颞叶27例,顶叶26例,枕叶21例,基底节47例,丘脑35例,小脑23例,脑干20例;采用中文版蒙特利尔认知评估量表（montreal cognitive assessment,MoCA）对受试者进行认知功能测评。结果：（1）各病变部位认知障碍的发生率存在显著性差异（P〈0.05）,额叶组及丘脑组认知障碍发生率最高,达90%以上,其次为颞叶组,达到80%以上,小脑组及脑干组最低,约30%左右。（2）额叶组在视空间与执行功能、注意认知域分值低于其他各组（P〈0.05）;颞叶组在命名、延迟回忆认知域分值低于其他各组;顶叶组及枕叶组MoCA总分分值低于基底节、小脑、脑干组（P〈0.05）;丘脑组不仅在视空间与执行功能、注意认知域得分低于顶叶、枕叶、基底节、小脑和脑干组,且其语言及定向认知域分值低于其他各组（P〈0.05）。结论：不同部位脑梗死患者认知障碍的发生率及认知功能损害的特点不同。