CAJ | 학술논문

目的通过高压尾静脉注射将携带attB和人凝血因子Ⅸ(hFⅨ)的质粒与表达phiC31的质粒共同导入血友病B小鼠肝脏细胞,检测目的质粒能否整合入小鼠基因组并持续表达.方法 ①构建表达hFⅨ并携带attB核心序列的真核表达载体attB-hFⅨ-pIRES2-EGFP,并在体外验证该载体能否表达目的基因.②用高压尾静脉注射法将该载体与表达phiC31的质粒CMV-int共注入血友病B小鼠,attB-hFⅨ-pIRES2-EGFP单独注射为对照.③ELISA的方法检测hFⅨ在其体内的表达;用出血时间评价血友病小鼠出血症状是否改善;用巢式PCR检测attB-hFⅨ-pIRES2-EGFP是否整合到基因组的整合热点mpsL1(mouse pseudo-site from liver 1)位点.结果经鉴定,attB-hFⅨ-pIRES2-EGFP载体构建成功,并在体外表达hFⅨ.高压尾静脉注入血友病B小鼠24 h后,hFⅨ血清水平达到最高值为(1533±239)ng/ml,此时小鼠的出血症状明显改善.但是不论是否与CMV-int共注射,此后hFⅨ水平迅速下降,在注射后10 d内降到本底水平.巢式PCR的结果证实,attB-hFⅨ-pIRES2-EGFP整合到小鼠肝脏基因组的mpsL1位点.结论 phiC31可以将34 bp的attB最短序列整合到小鼠基因组的整合热点mpsL1;由CMV启动hFⅨ的能够瞬间高表达并有效改善血友病小鼠的出血症状;但是外来DNA进入细胞后,无论是否整合到基因组均被迅速沉默,说明肺脏和肝脏对整合入基因组的CMV启动子表达调控的机制不尽相同,因此对用于基因治疗的裸DNA进行改进使其适合在靶器官表达是十分必要的.
목적 통과고압미정맥주사장휴대attB화인응혈인자Ⅸ(hFⅨ)적질립여표체phiC31적질립공동도입혈우병B소서간장세포,검측목적 질립능부정합입소서기인조병지속표체.방법 ①구건표체hFⅨ병휴대attB핵심서렬적진핵표체재체attB-hFⅨ-pIRES2-EGFP,병재체외험증해재체능부표체목적 기인.②용고압미정맥주사법장해재체여표체phiC31적질립CMV-int공주입혈우병B소서,attB-hFⅨ-pIRES2-EGFP단독주사위대조.③ELISA적방법검측hFⅨ재기체내적표체;용출혈시간평개혈우병소서출혈증상시부개선;용소식PCR검측attB-hFⅨ-pIRES2-EGFP시부정합도기인조적정합열점mpsL1(mouse pseudo-site from liver 1)위점.결과 경감정,attB-hFⅨ-pIRES2-EGFP재체구건성공,병재체외표체hFⅨ.고압미정맥주입혈우병B소서24 h후,hFⅨ혈청수평체도최고치위(1533±239)ng/ml,차시소서적출혈증상명현개선.단시불론시부여CMV-int공주사,차후hFⅨ수평신속하강,재주사후10 d내강도본저수평.소식PCR적결과증실,attB-hFⅨ-pIRES2-EGFP정합도소서간장기인조적mpsL1위점.결론 phiC31가이장34 bp적attB최단서렬정합도소서기인조적정합열점mpsL1;유CMV계동hFⅨ적능구순간고표체병유효개선혈우병소서적출혈증상;단시외래DNA진입세포후,무론시부정합도기인조균피신속침묵,설명폐장화간장대정합입기인조적CMV계동자표체조공적궤제불진상동,인차대용우기인치료적라DNA진행개진사기괄합재파기관표체시십분필요적.
Objective To investigate whether the plasmid bearing attB and huamn coagulation factor Ⅸ (hFⅨ) coding sequence could insert into hemophilia B mice genome and persistently express hFⅨ with co-injected integrase. Methods The plasmid attB-hF Ⅸ-pIRES2-EGFP was constructed, which bore attB site and hFⅨ coding sequence and was proved in vitro to express hFⅨ . The plasmid and CMV-int expressing integrase was co-infused rapidly in a large-volume solution through tail vein of hemophilia B mice. Mice infused with the plasmid alone served as controls. ELISA was performed to determine serum hFⅨ level. Correction of coagulation defect in vivo by plasmid infusion was assessed by bleeding time. Genomic integration of the plasmid was determined by nested PCR. Results The plasmid attB-hFⅨ-pIRES2-EGFP was successfully constructed. The hemophilia B mice produced ( 1533 ±239) ng/ml hFⅨ at 24 hour after infusion of the hF Ⅸ encoding plasmid and the bleeding diathesis of the hemophilia B mice was significantly corrected as measured by clotting assays. However, whether or not co-injected with CMV-int, the serum hFⅨ level decreased to background level in 10 days after infusion. Nested-PCR results indicated that the integrase phiC31 resulted in the integration of the plasmid in the mouse liver chromosomes. Conclusion Integrase phiC31 can catalyze recombination of 34 bp attB and pseudo-attP. Human FⅨ driven by CMV promoter can be transiently and highly expressed after infusion, but rapidly silenced in vivo.