电穿孔对原代大鼠肝细胞生物学性状影响

Influence of electroporation on the biological activities of primary rat hepatocytes

目的探讨不同电穿孔条件对原代大鼠肝细胞生物学性状的影响，优化外源基因转染原代肝细胞的条件。方法以低电压 （２２０～４００ Ｖ）、高电容（５００～ ９５０μＦ）单脉冲电击原代大鼠肝细胞后接种培养，分别于 ３６ ｈ～９ｄ不同时点采用台盼蓝拒染法与 ＭＴＴ法观测肝细胞活力，并检测肝细胞培养上清液中白蛋白（ＡＬＢ）、ＡＬＴ与乳酸脱氢酶 （ＬＤＨ）含量。以未电穿孔肝细胞为对照组。结果培养 ３６ ｈ～９ ｄ各组肝细胞存活率均高于９０％，贴壁率高于９５％。培养３６ ｈ肝细胞上清液中ＡＬＢ、ＡＬＴ与ＬＤＨ值，电穿孔Ｂ组（２２０ Ｖ，９５０μＦ）、Ｃ组（４００ Ｖ， ９５０μＦ）明显高于对照组；但 ＭＴＴ法检测各电穿孔组肝细胞活力与对照组比较差异无显著性。结论该电穿孔条件不影响肝细胞的存活能力，还能促进肝细胞ＡＬＢ等蛋白的合成与释放，可作为外源基因转染原代肝细胞的优选方法之一。

Objective To investigate the influence of electroporation on the biological activities of primary rat hepatocyte and to optimize the electroporation conditions introducing foreign genes into hepatocytes. Methods A singlepulse procedure was performed at low voltage (220~400 V) but high capacitance (500~950 μF). Its influence on hepatocyte activities was detected by Trypan blue exclusion (TBE) and Mrs analysis. Besides, ALB, ALT and LDH in the supernatants of hepatocytes were tested by biochemical assay. Results Little hepatocyte damage and high survival rate (>90%) was found from 36 hours till 9th day of culture. At 36th hour after electroporation, ALB, ALT and LDH in the supernatants of Group B(220V, 950 μF) and C(400V, 950 μF) were higher than those of control group. Whereas TBE and MTT analysis failed to indicate the significant difference of cell viability between electroporation groups and control group. Conclusions This electroporation procedure is one of the optimal choices to introduce foreign genes into primary rat hepatocytes.