中华医学遗传学杂志
中華醫學遺傳學雜誌
중화의학유전학잡지
CHINESE JOURNAL OF MEDICAL GENETICS
2010年
1期
1-6
,共6页
匡颖%王津津%卢希彬%陆顺元%张良梁%沈春玲%费俭%王铸钢
劻穎%王津津%盧希彬%陸順元%張良樑%瀋春玲%費儉%王鑄鋼
광영%왕진진%로희빈%륙순원%장량량%침춘령%비검%왕주강
血友病A%基因剔除%凝血因子Ⅷ%四倍体囊胚补偿技术
血友病A%基因剔除%凝血因子Ⅷ%四倍體囊胚補償技術
혈우병A%기인척제%응혈인자Ⅷ%사배체낭배보상기술
hemophilia A%gene knockout%coagulation factor Ⅷ%tetraploid embryo complementation
目的 建立凝血因子Ⅷ(factor Ⅷ,FⅧ)基因剔除小鼠模型,为研究治疗血友病A提供理想的动物模型.方法 应用ET克隆、胚胎干细胞同源重组和四倍体囊胚补偿技术,将小鼠FⅧ基因第16～19外显子靶向剔除,并应用PCR、逆转录PCR(reverse transcriptase-PCR,RT-PCR)及免疫组织化学染色技术,从DNA、RNA、蛋白水平检测FⅧ基因的转录及表达情况.通过检测激活的部分凝血活酶时间(activated partial thromboplastin time,APTT)和FⅧ的促凝活性(FⅧ∶C)进行FⅧ基因剔除小鼠表型鉴定.结果 PCR检测在剔除小鼠中未扩增出FⅧ基因的条带;RT-PCR分析显示剔除区域该基因mRNA转录本缺失;免疫组织化学染色显示在雄性半合子(-/y)和雌性纯合子(-/-)小鼠的肝脏中凝血因子Ⅷ表达缺失;FⅧ∶C和APTT检测结果显示雌性杂合子(+/-)FⅧ活性为野生型(wt)的80%,其APTT值较wt略有延长,而-/y和-/-小鼠的FⅧ的活性仅为wt小鼠的8%和10%,且APTT值均大于120s.结论 FⅧ基因剔除小鼠具有与人类血友病A患者相似的表型,它的建立将促进我国血友病A治疗新技术的发展.
目的 建立凝血因子Ⅷ(factor Ⅷ,FⅧ)基因剔除小鼠模型,為研究治療血友病A提供理想的動物模型.方法 應用ET剋隆、胚胎榦細胞同源重組和四倍體囊胚補償技術,將小鼠FⅧ基因第16～19外顯子靶嚮剔除,併應用PCR、逆轉錄PCR(reverse transcriptase-PCR,RT-PCR)及免疫組織化學染色技術,從DNA、RNA、蛋白水平檢測FⅧ基因的轉錄及錶達情況.通過檢測激活的部分凝血活酶時間(activated partial thromboplastin time,APTT)和FⅧ的促凝活性(FⅧ∶C)進行FⅧ基因剔除小鼠錶型鑒定.結果 PCR檢測在剔除小鼠中未擴增齣FⅧ基因的條帶;RT-PCR分析顯示剔除區域該基因mRNA轉錄本缺失;免疫組織化學染色顯示在雄性半閤子(-/y)和雌性純閤子(-/-)小鼠的肝髒中凝血因子Ⅷ錶達缺失;FⅧ∶C和APTT檢測結果顯示雌性雜閤子(+/-)FⅧ活性為野生型(wt)的80%,其APTT值較wt略有延長,而-/y和-/-小鼠的FⅧ的活性僅為wt小鼠的8%和10%,且APTT值均大于120s.結論 FⅧ基因剔除小鼠具有與人類血友病A患者相似的錶型,它的建立將促進我國血友病A治療新技術的髮展.
목적 건립응혈인자Ⅷ(factor Ⅷ,FⅧ)기인척제소서모형,위연구치료혈우병A제공이상적동물모형.방법 응용ET극륭、배태간세포동원중조화사배체낭배보상기술,장소서FⅧ기인제16～19외현자파향척제,병응용PCR、역전록PCR(reverse transcriptase-PCR,RT-PCR)급면역조직화학염색기술,종DNA、RNA、단백수평검측FⅧ기인적전록급표체정황.통과검측격활적부분응혈활매시간(activated partial thromboplastin time,APTT)화FⅧ적촉응활성(FⅧ∶C)진행FⅧ기인척제소서표형감정.결과 PCR검측재척제소서중미확증출FⅧ기인적조대;RT-PCR분석현시척제구역해기인mRNA전록본결실;면역조직화학염색현시재웅성반합자(-/y)화자성순합자(-/-)소서적간장중응혈인자Ⅷ표체결실;FⅧ∶C화APTT검측결과현시자성잡합자(+/-)FⅧ활성위야생형(wt)적80%,기APTT치교wt략유연장,이-/y화-/-소서적FⅧ적활성부위wt소서적8%화10%,차APTT치균대우120s.결론 FⅧ기인척제소서구유여인류혈우병A환자상사적표형,타적건립장촉진아국혈우병A치료신기술적발전.
Objective Factor Ⅷ (FⅧ) gene knockout mouse model was established for further study on the treatment of hemophilia A. Methods Exons 16-19 of the mouse FⅧ gene were knocked out by ET clone, ES homologous recombination and tetraploid embryo compensation technology. PCR, reverse transcriptase-PCR (RT-PCR) and immunohistochemistry were used to detect the transcription and translation pattern of F Ⅷ. The phenotype of the knockout mice was analyzed by examining the activated immunohistochemistry confirmed that FⅧ was deficient in the FⅧ gene knockout mouse. The APTT results showed that FⅧ-deficient mouse plasma had a prolonged clotting time compared to normal mouse plasma.mice, respectively. Conclusion The phenotype of the FⅧ gene knockout mouse appears grossly similar to that of human with hemophilia A. Establishment of this model may promote the development of new technologies of treatment to hemophilia A.
