凝血因子IX基因剔除小鼠的遗传及血液学指标检测

目的　鉴定凝血因子IX基因剔除小鼠。方法　采用PCR扩增检测小鼠的DNA样品以及采用一期法检定小鼠血浆FIX活性和血浆凝血酶原时间 (plasmaprothrombintime ,PT)、白陶土部分凝血活酶时间 (Kaolinpartialthromboplastintime ,KPTT)值。结果　小鼠PCR检测为阳性 ,FIX活性 <5 %。结论　凝血因子IX基因剔除小鼠能稳定遗传 ,鉴定结果提示该小鼠符合人血友病B相应临床症状。     

基于凝血因子IX基因剔除小鼠建立血友病乙转基因动物模型

为在凝血基因IX基因剔除小鼠基础上建立基因组中整合有含特定点突变的人凝血因子IX基因表达载体原转基因小鼠家系,为血友病乙的研究提供更接近临床实际的动物模型。利用体外定点突变技术,构建含有特定点突变的人凝因IX基因表达载体,该载体包括由人凝血因子IX编码区及第一内含子构成的人凝血因子IX基因（hFIXml）、4个拷贝的MCK增强子（MCKe）、鸡β－肌动蛋白启动子（bA）及PolyA,命名为pMe4bAIXml质粒。将其线性化后,用显微注射法注射入817只凝血因子IX基因剔除小鼠受精卵雄原核,再将它们分别回输45只假孕受体母鼠的输卵管中,共产仔69只,存活63只。采用PCR与基因组Southern杂交筛选法鉴定小鼠,证实6只小鼠基因组中整合有含特定点突变的pMe4bAIXml质粒,并对1只小鼠的PCR产物进行测序,证实转基因结构特征符合设计要求。     

人凝血因子IX突变型研究现状

血友病B是一种性连锁隐性遗传病,其发病机制是位于X染色体上的人凝血因子IX(hFIX)基因发生了突变,导致血浆中hFIX含量或活性大幅下降,从而使得内源性凝血途径受到阻碍,无法进行正常的凝血。文章综述了hFIX基因及其编码蛋白质的结构和功能,并分类详细论述了血友病B中发现的几种主要突变类型。其中包括奠基者效应造成的突变、调控区的突变、编码区的突变、内含子剪切位点的突变及另外两种较为特殊的突变,同时介绍了这些突变所造成的生物学效应。最后还简要介绍了一种能提高hFIX蛋白凝血活性的突变类型(第338位Arg→Ala),并对其应用作了展望。     

人凝血因子IX突变型研究现状

血友病B是一种性连锁隐性遗传病,其发病机制是位于X染色体上的人凝血因子IX（hFIX）基因发生了突变,导致血浆中hFIX含量或活性大幅下降,从而使得内源性凝血途径受到阻碍,无法进行正常的凝血。本文综述了hFIX基因及其编码蛋白质的结构和功能,并分类详细论述了血友病B中发现的几种主要突变类型。其中包括奠基者效应造成的突变、调控区的突变、编码区的突变、内含子剪切位点的突变及另外两种较为特殊的突变,同时介绍了这些突变所造成的生物学效应。最后还简要介绍了一种能提高hFIX蛋白凝血活性的突变类型（第338位Arg→Ala）,并对其应用作了展望。     

凝血因子IX基因剔除小鼠的培育历史与建系方法

目的　培育凝血因子IX基因剔除小鼠近交系。方法　采用全同胞兄妹对交配 ,每代选用第一胎小鼠留种 ,记录繁殖性能 ;第 8代开始筛选纯合子。结果　凝血因子IX基因剔除小鼠已近交培育到第 12代 ,交配年龄 70 - 90d ,平均每窝产仔数 5只以上 ,离乳数 3只以上。结论　纯合子FIX剔除基因小鼠在子代鼠中能稳定遗传     

血友病A基因治疗载体研究现状

血友病A是X染色体隐性遗传出血性疾病。其发病原因是患者血液中先天缺乏凝血因子FⅧ。用于血友病A基因治疗研究的载体有病毒载体和非病毒载体,目前研究较多的是病毒载体,主要有逆转录病毒载体和慢病毒载体,腺病毒载体及腺相关病毒载体等。非病毒载体主要有质粒、脂质体、转座子等。文章拟对血友病A基因治疗各载体的特点和研究进展作一综述。     

人羊水祖细胞的分离和基因修饰

探讨从孕中期羊水中分离出人羊水祖细胞的有效方法和FIX基因修饰后的效果,为血友病B的产前治疗提供可行的基础。从镜下分离出呈致密克隆生长的梭形细胞集落,经培养传代后,通过第3代慢病毒载体将hFIX导入该细胞,经酶联免疫反应(ELISA)等方法检测hFIX的表达并检测凝血活性。用这种方法得到的羊水祖细胞呈成纤维细胞样,倍增时间为39.05 h,该细胞在不仅在蛋白水平表达干细胞表面分子SSEA4,TRA1-60,在基因水平还可检测到NANOG,OCT4,SOX2mRNA的表达。羊水祖细胞导入hFIX cDNA后,能合成并分泌hFIX蛋白,传代后48 h在上清液中的浓度为20.37%±2.77%,凝血活性16.42%±1.78%。上清液中的浓度在第4天达到平台期,为50.35%±5.42%,凝血活性可达45.34%±4.67%。ELISA检测显示转染后的羊水细胞表达的hFIX蛋白的水平呈现基本稳定趋势,波动幅度较小;同时检测FIX凝血活性也与蛋白浓度呈正相关。从羊水中可以分离得到具有多能性祖细胞,转染了hFIX的羊水祖细胞在体外能持续合成有凝血功能的hFIX蛋白,为血友病B产前治疗的新方法提供了实验依据。     

凝血因子药物市场分析

凝血因子是一类特殊的药物,是血友病等血液疾病的治疗药物,目前已经成为血液制品的重要组成部分。国外已经有二十多种重组凝血因子药物上市,2015年全球重组凝血因子药物的市场规模已经达到78.54亿美元,未来还将持续增长,Baxalta公司的重组凝血因子产品销售额位居全球首位,达到28.40亿美元。国外有多种重组凝血因子处于研发阶段,其中长效重组凝血因子将成为新的市场增长点。国内各类凝血因子药物的批签发状况良好,且随着国家发展和改革委员会取消血液制品最高零售价,各类产品价格均有不同幅度增长,其中人纤原蛋白原增长幅度最高,达到189%。国产凝血因子市场空间巨大,但存在产品供给和研发力度不足等问题,发展受到限制,必须改革行业管理制度、提高血浆分离技术、加强重组产品研发。     

利用CRISPR/Cas系统对血友病B小鼠进行基因纠正的初步探索

血友病B是人类常见的X染色体遗传的隐性遗传疾病,目前对于遗传疾病的治疗还没有很好的办法,通过基因治疗可以实现对遗传病的彻底治愈。本研究利用高效基因编辑技术CRISPR/Cas系统,对血友病B小鼠尝试基因修复进行初步的探索,将突变的凝血因子IX(Factor IX,FIX)基因进行同源替换,修复为正常序列。本研究中将同一种纯合突变类型的小鼠交配之后得到的受精卵,进行细胞核显微注射。对收集到的8枚囊胚的基因组中的靶位点,进行巢氏PCR扩增,直接进行测序检测基因型序列。检测结果为4枚囊胚的基因被成功编辑,其中3枚囊胚为靶序列的基因敲除突变,而有1枚囊胚成功进行同源重组,实现基因纠正。     

人凝血酶原复合物分离纯化工艺的研究进展

人凝血酶原复合物(PCC)为人血浆中微量蛋白组份,其浓缩制剂由凝血因子II、VII、IX、X组成,在临床上具有治疗乙型血友病等疾病的确切疗效。本文综述了近来PCC及FIX浓缩制剂分离纯化工艺的研究进展及结合蛋白新型分离纯化技术的发展情况,讨论了该产品制备新工艺的开发方向。     

Ca(2+) -induced binding of anticoagulation factor II from the venom of Agkistrodon acutus with factor IX

Anticoagulation factor II (ACF II), a coagulation factor X- binding protein from the venom of Agkistrodon acutus has both anticoagulant and hypotensive activities. Previous studies show that ACF II binds specifically with activated factor X (FXa) in a Ca(2+) -dependent manner and inhibits intrinsic coagulation pathway. In this study, the inhibition of extrinsic coagulation pathway by ACF II was measured in vivo by prothrombin time assay and the binding of ACF II to factor IX (FIX) was investigated by native polyacrylamide gel electrophoresis and surface plasmon resonance (SPR). The results indicate that ACF II also inhibits extrinsic coagulation pathway, but does not inhibit thrombin activity. ACF II also binds with FIX with high binding affinity in a Ca(2+) -dependent manner and their maximal binding occurs at about 0.1 mM Ca(2+) . ACF II has similar binding affinity to FIX and FX as determined by SPR. Ca(2+) has a slight effect on the secondary structure of FIX as determined by circular dichroism spectroscopy. Ca(2+) ions are required to maintain in vivo function of FIX Gla domain for its recognition of ACF II. However, Ca(2+) at high concentrations (>0.1 mM) inhibits the binding of ACF II to FIX. Ca(2+) functions as a switch for the binding between ACF II and FIX. ACF II extends activated partial thromboplastin time more strongly than prothrombin time, suggesting that the binding of ACF II with FIX may play a dominant role in the anticoagulation of ACF II in vivo.     

Random analysis of amino acid pairs sensitive to variants in human coagulation factor IX precursor

In this study, we used a random approach to determine which amino acid pairs in human coagulation factor IX precursor are more sensitive to its 99 variants. The results show that the randomly unpredictable amino acid pairs are more sensitive to variants.     

利用烟草表达人凝血IX因子(hFIX)的研究

血友病B是凝血IX因子(hFIX)缺乏所导致的一种出血性疾病,通过输血和hFIX浓缩剂进行治疗疗效显著,但存在治疗费用高和安全隐患,因而获得安全、廉价的人凝血IX因子对血友病B治疗具有重要意义。植物系统表达外源蛋白在生产成本和安全性方面具有优势。为此,构建含人凝血IX因子基因(hFIX,2.8kb)植物双元表达载体p35s2300∷gus∷noster,用农杆菌介导法转化烟草“百日红”,通过PCR和Southernblot分析证实获得4株独立转基因植株,hFIX在转基因烟草基因组中的拷贝数为1～4个;RTPCR和ELISA检测结果表明,hFIX在转录和翻译水平已成功表达,hFIX在转基因烟草叶片中的表达量为2.5～8.8ng/g·FW,并具有免疫活性。为利用植物系统表达hFIX的后续研究作了必要准备,也为利用植物系统表达其他药用蛋白研究提供了一些理论和实验参考。     

Expression of functional recombinant human factor IX in milk of mice

Human factor IX is synthesized in the liver and secreted in the blood, where it participates in a group of reactions involving coagulation factors and proteins that permit sanguinary coagulation. In this work two lines of transgenic mice were developed to express the FIX gene in the mammalian glands under control of milk β-casein promoter. The founding females secreted the FIX in their milk (3% total soluble protein). The stable integration of transgene was confirmed by southern blot analysis. The presence of the FIX recombinant protein in the milk of transgenic females was confirmed by western blot and the clotting activity was revealed in blood-clotting assays. The coagulation activity in human blood treated with recombinant FIX increased while the time of coagulation decreased. Our results confirm the production of a large amount of recombinant biologically active FIX in the mammary gland of transgenic mice.     

利用烟草表达人凝血IX因子 (hFIX)的研究

血友病B是凝血IX因子（hFIX）缺乏所导致的一种出血性疾病,通过输血和hFIX浓缩剂进行治疗疗效显著,但存在治疗费用高和安全隐患,因而获得安全、廉价的人凝血IX因子对血友病B治疗具有重要意义。植物系统表达外源蛋白在生产成本和安全性方面具有优势。为此,本研究构建含人凝血IX因子基因（hFIX,2.8kb）植物双元表达载体p35s-2300：：gus：：noster,用农杆菌介导法转化烟草 "百日红",通过PCR和Southern blot分析证实获得4株独立转基因植株, hFIX在转基因烟草基因组中的拷贝数为1-4个;RT-PCR和ELISA检测结果表明,hFIX在转录和翻译水平已成功表达,hFIX在转基因烟草叶片中的表达量为2.5~8.8ng/g·FW,并具有免疫活性。本研究为利用植物系统表达hFIX的后续研究作了必要准备,也为利用植物系统表达其他药用蛋白研究提供了一些理论和实验参考。     

Transgenic Mice Can Express Mutant Human Coagulation Factor IX with Higher Level of Clotting Activity

To improve the available values of transgenic animals, we produced a mutant human coagulation factor IX minigene (including cDNA and intron I) with arginine at 338 changed to alanine (R338A-hFIX) by using a direct mutation technique. The R338A-hFIX minigene was then cloned into a plasmid carrying the goat β-casein promoter to get a mammary gland-specific expression vector. The clotting activity in the supernatant of the transfected HC-11 cells increased to approximately three times more than that of wild-type hFIX. Nine transgenic mice (three females and six males) were produced, and the copy number of the foreign gene was very different, ranging from 1 to 43 in different lines. ELISA, Western blot, and clotting assay experiments showed that the transgenic mice could express R338A-hFIX, showing higher average levels of clotting activity than wild-type hFIX in the milk (103.76% vs. 49.95%). The highest concentration and clotting activity of hFIX reached 26 μg/mL and 1287% in one founder (F₀-7), which was over 10 times higher than that in human plasma. Furthermore, RT-PCR, APTT assay, and histological analysis indicated that hFIX was expressed specifically in the mammary gland without affecting the intrinsic coagulation pathway and physiologic performance of the local tissue.     

Constitutive expression of human coagulating factor IX in HeLa cells by homologous recombination of the promoter

Constitutive expression of hFIX protein in nonhepatocytes was studied. The gene targeting vector was constructed and transferred into HeLa cells. With the detection system of PCR, we demonstrated that the endogenoushFIX promoter was replaced with anhCMV promoter when targeted insertion of the constructor was directed by the sequence homology. The expression ofhFIX in the modified HeLa cells, 11.2 ng/10⁶ cell/24 h, strongly suggested thathFIX gene could be activated by a powerful promoter in nonhepatocytes. The results would make it possible to examine the feasibility of re-regulate gene expression by promoter replacement.     

罕见的异常核型6例报告

ReportofSixRareCasesofAbnormalKaryotypesLiGuixinSongGuangjiangSongXingjun(BiologyDepartment,TaishanMedicalCollege,Taiah,Shandong271000)我定在对外遗传咨询并进行染色体检查时,发现6例异常核型,经医学细胞遗传学国家培训中心夏家辉等专家鉴定,为世界首报枝型．现报告如下：例1女,26岁,表型正常．妊娠4次,均于3个月自然流产．无任何诱因．常规染色体检查,核型为46,XX但有两条异常染色体．经G显带分析,异常核型为46,XX,t（1;8）（lqter-1p32：：8q22-8qter;8pier-8q22：：1p32－1pter）（图1）．例2女,25岁…     

High expression of human clotting factor IX cDNA in myoblasts C2C12 cells and C3H mice

Mouse myoblast C2C12 cell was used as target cell for gene transfer study of human clotting factor IX (hFIX) cDNA. In addition to the previously constructed retroviral vectors XLIX, LNCIX and GINaCIX, GlNaMCIX with hFIX driven by muscle creatine kinase (MCK) enhancer and human cytomegalovirus (CMV) was constructed, based on the retroviral vector GINa. These four retroviral vectors were used to transduce mouse my-oblasts C2C12. With ELISA assays, it has been found that the expression levels of human clotting factor IX detected in those transduced C2C12 cells are GlNaMCIX>GlNaCIX> LNCIX>XLIX. Mixed colonal cells transduced with GlNaMCIX expressed hFIX protein at the level of 640 ng/106 cell every 24 h. The modified C2C12 cells transduced with GlNaMCIX were implanted into skeletal muscle of the hindlegs of C3H mice; a stable expression of hFIX was detected and lasted for 35 d, with a maximum level of 206 ng/mL plasma. The regulation of hFIX cDNA expression in myoblasts was discussed and it was strongly sug     

Utility of intraperitoneal administration as a route of AAV serotype 5 vector-mediated neonatal gene transfer

BACKGROUND: Gene transfer into a fetus or neonate can be a fundamental approach for treating genetic diseases, particularly disorders that have irreversible manifestations in adulthood. Although the potential utility of this technique has been suggested, the advantages of neonatal gene transfer have not been widely investigated. Here, we tested the usefulness of neonatal gene transfer using adeno-associated virus (AAV) vectors by comparing the administration routes and vector doses. METHODS: To determine the optimal administration route, neonates were subjected to intravenous (i.v.) or intraperitoneal (i.p.) injections of AAV5-based vectors encoding the human coagulation factor IX (hfIX) gene, and the dose response was examined. To determine the distribution of transgene expression, vectors encoding lacZ or luciferase (luc) genes were used and assessed by X-gal staining and in vivo imaging, respectively. After the observation period, the vector distribution across tissues was quantified. RESULTS: The factor IX concentration was higher in i.p.-injected mice than in i.v.-injected mice. All transgenes administered by i.p. injection were more efficiently expressed in neonates than in adults. The expression was confined to the peritoneal tissue. Interestingly, a sex-related difference was observed in transgene expression in adults, whereas this difference was not apparent in neonates. CONCLUSIONS: AAV vector administration to neonates using the i.p. route was clearly advantageous in obtaining robust transgene expression. Vector genomes and transgene expression were observed mainly in the peritoneal tissue. These findings indicate the advantages of neonatal gene therapy and would help in designing strategies for gene therapy using AAV vectors.