部分切肝对大鼠肝细胞门静脉途径转基因效率的影响

IMPACT OF PARTIAL HEPATECLOMY ON TRANSFECTION EMCIENCY OF RAT HEPATOCYTES VIA PORTAL VEIN

目的 :探讨 70 %切肝对大鼠肝细胞门静脉途径转基因效率的影响。方法 :Wistar大鼠 70 %切肝 [假手术组 (shamoperation ,S)仅行开腹术 ]建立肝再生模型 ,术后 2 4h肝隔离门静脉注射脂质体 -质粒DNA复合物(lipofectamine - pEGFP -N1DNAcomplexes,lipoplexes)转绿色荧光蛋白 (greenfluorescentprotein ,GFP)基因至肝细胞 ,对照组注射生理盐水 (NS)。转染后 4d(分 4组 ,每组 5只 :切肝并于门静脉注射lipoplexes组 ,He4;切肝并于门静脉注射NS组 ,Hc4;假手术并于门静脉注射lipoplexes组 ,Se4;假手术并于门静脉注射NS组 ,Sc4)、转染后 15d(与转染后 4d的对应分组为 :He15 ;Hc15 ;Se15 ;Sc15 )均以胶原酶消化、Percoll液梯离心法获取纯化肝细胞悬液 ;以NS组为对照并以GFP为荧光标记物 ,用流式细胞仪 (flowcytometry ,FCM )分析各实验组肝细胞的转染率。结果 :FCM分析He4组、Se4组、He15组、Se15组的平均转染率 (% )分别为 :2 7.5 0±18.15、1.0 6± 0 .87、3.40± 2 .0 9、0 .32± 0 .19;He4明显高于Se4(P <0 .0 1) ,He15明显高于Se15 (P <0 .0 5 )。结论

Objective:To analyze the impact of partial hepatectomy(PH) on transfection efficiency(TE) of rat hepatocytes via portal vein(PV).Methods:Regenerating liver models (RLM) of Wistar rats (280±10g) were established by PH(70%),while group of sham operation(S) just experienced laparotomy,and lipofectamine-pEGFP-N 1DNA complexes(lipoplexes:control groups using normal saline,NS) were injected into PV to transfer green fluorescent protein(GFP)gene to hepatocytes with hepatic isolation 24 hours post-operation.Purified hepatocytes were obtained via collagenase degesting followed by percoll gradient centrifugation 4 days (there were 4 subgroups with 5 rats in each one:hepatectomy with lipoplexes injected into PV,He4;hepatectomy with NS injected into PV,Hn4;sham operation with lipoplexes injected into PV,Se4;sham operation with NS injected into PV,Sn4),and 15 days(similar subgroups:He15;Hn15;Se15;Sn15)post-transfection respectively.TE of rat hepatocytes were analyzed by flow cytometry (FCM) using GFP as fluorescent marker.Results:The mean TE(%) analyzed by FCM in He4,Se4;He15 and Se15 were 27.50±18.15、1.06±0.87、3.40±2.09、0.32±0.19;The efficiency of He4 and He15 were evidently higher than that of Se4 and Se15 respectively (P<0.01;P<0.05).Conclusions:PH could notably improve both transient and stable TE of rat hepatocytes via PV.