中华血液学杂志
中華血液學雜誌
중화혈액학잡지
Chinese Journal of Hematology
2009年
3期
166-170
,共5页
程海%徐开林%孙海英%鹿群先%何徐彭%潘秀英
程海%徐開林%孫海英%鹿群先%何徐彭%潘秀英
정해%서개림%손해영%록군선%하서팽%반수영
慢病毒属%因子Ⅷ%血友病A%体外表达%基因治疗
慢病毒屬%因子Ⅷ%血友病A%體外錶達%基因治療
만병독속%인자Ⅷ%혈우병A%체외표체%기인치료
Lentiviral vector%Human coagulation factorⅧ%Hemophilia A%In vitro%Gene therapy
目的 构建含有人凝血因子Ⅷ(FⅧ)基因的慢病毒载体pXZ208-BDDhFⅧ,观察其在体外培养细胞中的表达情况.方法 用限制性内切酶酶切法获得B区缺失的人FⅧ基因(BDDhFⅧ cDNA)片段,将其克隆至慢病毒载体pXZ208的多克隆位点,构建慢病毒表达载体pXZ208-BDDhFⅧ,限制性内切酶酶切法鉴定载体的连接方向.采用三质粒共转染293T细胞制备病毒颗粒,包装后感染人肺、肾HLF细胞、张氏肝(Chang-Liver)细胞和成人骨髓基质细胞(MSC),并以pXZ171作为对照.流式细胞术(FCM)检测载体的感染效率,一期法检测细胞培养上清中FⅧ的活性,PCR检测BDDhFⅧ基因的整合.结果 成功构建了慢病毒载体pXZ208-BDDhFⅧ,包装后能有效感染多种靶细胞,感染效率分别为:HLF细胞(74.52±7.57)%、MSC(42.34±5.84)%和Chang-Live细胞(27.24±6.53)%.感染后48 h检测到HLF细胞、Chang-Liver细胞和MSC培养上清中有FⅧ的表达,FⅧ活性分别为(54.1±5.6)%、(22.5±2.9)%和(12.5±2.7)%.PCR检测到目的 基因的整合.结论 成功构建了慢病毒表达载体pXZ208-BDDhFⅧ,在体外可以有效感染多种靶细胞并表达有活性的FⅧ.
目的 構建含有人凝血因子Ⅷ(FⅧ)基因的慢病毒載體pXZ208-BDDhFⅧ,觀察其在體外培養細胞中的錶達情況.方法 用限製性內切酶酶切法穫得B區缺失的人FⅧ基因(BDDhFⅧ cDNA)片段,將其剋隆至慢病毒載體pXZ208的多剋隆位點,構建慢病毒錶達載體pXZ208-BDDhFⅧ,限製性內切酶酶切法鑒定載體的連接方嚮.採用三質粒共轉染293T細胞製備病毒顆粒,包裝後感染人肺、腎HLF細胞、張氏肝(Chang-Liver)細胞和成人骨髓基質細胞(MSC),併以pXZ171作為對照.流式細胞術(FCM)檢測載體的感染效率,一期法檢測細胞培養上清中FⅧ的活性,PCR檢測BDDhFⅧ基因的整閤.結果 成功構建瞭慢病毒載體pXZ208-BDDhFⅧ,包裝後能有效感染多種靶細胞,感染效率分彆為:HLF細胞(74.52±7.57)%、MSC(42.34±5.84)%和Chang-Live細胞(27.24±6.53)%.感染後48 h檢測到HLF細胞、Chang-Liver細胞和MSC培養上清中有FⅧ的錶達,FⅧ活性分彆為(54.1±5.6)%、(22.5±2.9)%和(12.5±2.7)%.PCR檢測到目的 基因的整閤.結論 成功構建瞭慢病毒錶達載體pXZ208-BDDhFⅧ,在體外可以有效感染多種靶細胞併錶達有活性的FⅧ.
목적 구건함유인응혈인자Ⅷ(FⅧ)기인적만병독재체pXZ208-BDDhFⅧ,관찰기재체외배양세포중적표체정황.방법 용한제성내절매매절법획득B구결실적인FⅧ기인(BDDhFⅧ cDNA)편단,장기극륭지만병독재체pXZ208적다극륭위점,구건만병독표체재체pXZ208-BDDhFⅧ,한제성내절매매절법감정재체적련접방향.채용삼질립공전염293T세포제비병독과립,포장후감염인폐、신HLF세포、장씨간(Chang-Liver)세포화성인골수기질세포(MSC),병이pXZ171작위대조.류식세포술(FCM)검측재체적감염효솔,일기법검측세포배양상청중FⅧ적활성,PCR검측BDDhFⅧ기인적정합.결과 성공구건료만병독재체pXZ208-BDDhFⅧ,포장후능유효감염다충파세포,감염효솔분별위:HLF세포(74.52±7.57)%、MSC(42.34±5.84)%화Chang-Live세포(27.24±6.53)%.감염후48 h검측도HLF세포、Chang-Liver세포화MSC배양상청중유FⅧ적표체,FⅧ활성분별위(54.1±5.6)%、(22.5±2.9)%화(12.5±2.7)%.PCR검측도목적 기인적정합.결론 성공구건료만병독표체재체pXZ208-BDDhFⅧ,재체외가이유효감염다충파세포병표체유활성적FⅧ.
Objective To construct a recombinant lentiviral vector(pXZ208-BDDhFⅧ)mediating B-domain-deleted human coagulation factorⅧ(BDDhFⅧ)gene and investigate its expression in HLF,ChangLiver and MSC cells.Methods BDDhFⅧ gene fragment wag separated by endonueleage digestion and wag cloned into the multiple cloning sites of pXZ208 to construct a recombinant lentiviral vector pXZ208-BDDhFⅧ.Viral particles were prepared by meang of three-plasmid cotransfection of 293T package cells by calcium phosphate precipitation.After infection,the coagulant activity of human FⅧ in the culture medium of 293T,HLF,Chang-Liver and MSC cells wag assayed by one-stage method.The gene transduetion efficiency was assayed by flow cytometry(FCM).Furthermore,PCR wag performed to test the integration of BDDhFⅧ.Resuits The infection rates of HLF,Chang-Liver and MSC were(74.52±7.57)%,(27.24±6.53)%and (42.34±5.84)%respectively.The activities of FⅧ in Sllperuatants of HLF,Chang-Liver and MSC were (54.1±5.6)%,(22.5±2.9)%and(12.5±2.7)%respectively.BDDhFⅧ gene integration was detected in all the infected cells.Conclusion The recombinant lentiviral vector pXZ208-BDDhFⅧ was sneakingsfully constructed and efficiently integrated into target cells to express human FⅧ activity in vitro.
