Yao Yun-Qing, Zhang Ding-Feng, Huang Ai-Long, Luo Yun, Zhang Da-Zhi, Wang Bo, Zhou Wei-Ping, Ren Hong, Guo Shu-Hua
Department of Infectious Diseases of the First Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 400016, China.
World J Gastroenterol. 2002 Oct;8(5):893-6. doi: 10.3748/wjg.v8.i5.893.
To investigate the effects of electroporation on primary rat hepatocyte and to optimize the electroporation conditions introducing foreign genes into primary hepatocytes.
A single-pulse procedure was performed at low voltage (220-400 V) but with high capacitance (500-950 microF). Hepatocytes were divided into 4 groups according to the electroporation conditions: group I, 220 V and 500 microF; group II, 220 V and 950 microF; group III, 400 V and 950 microF,and group IV. The control group was freshly isolated hepatocytes and directly cultured under the same conditions as those of electroporation groups. The effects of electroporation on primary rat hepatocytes were detected by trypan blue exclusion (TBE) and MTT analysis. Besides, albumin (Alb), alanine transaminase (ALT) and lactate dehydrogenase (LDH) in the supernatants of cultured hepatocytes were measured by biochemical assay.
Between day 1 and day 15 after incubation, primary rat hepatocytes of each electroporation group appeared normal, being the same with those of control group. TBE staining showed that slight hepatocyte damage and high survival rate were found in the electroporation groups and the control group. Cultured for 3, 7, 11 and 15 days, hepatocyte viability was approximately 92.6+/-2.5 %, 89.5+/-3.3 %, 82.0+/-3.5 % and 74.3+/-1.2 %, respectively. MTT analysis indicated that the viabilities of hepatocytes had no significant difference between each electroporation group, and those were similar to that of control group. At the 36th hour after electroporation, Alb, ALT and LDH in the supernatants of control group were 5.3+/-0.1 g x L(-1), 183.7+/-8.4 nkat x L(-1) and 896.8+/-58.5 nkat x L(-1); those of group II were 5.7+/-0.1 g x L(-1), 215.4+/-16.7 nkat x L(-1) and 1063.8+/-51.8 nkat x L(-1); and those of group III were 5.8+/-0.2 g x L(-1), 217.1+/-8.4 nkat x L(-1) and 1063.8+/-10.0 nkat x L(-1). Statistically, the proteins of group II and group III were significantly higher than those of control group (P<0.05), whereas the protein production of group I, Alb, ALT and LDH were 5.3+/-0.2 g x L(-1), 205.4+/-3.3 nkat x L(-1) and 1035.4+/-116.9 nkat x L(-1), were similar to those of control group. At the same time, TBE and MTT analysis indicated that there was no significant cell viability difference between electroporation groups and control group.
This single-pulse electroporation procedure performed at low voltage (220-400 V) but with high capacitance (950 microF) is one of the optimal choices to introduce foreign genes into primary rat hepatocyte.
研究电穿孔对原代大鼠肝细胞的影响，并优化将外源基因导入原代肝细胞的电穿孔条件。
采用低电压（220 - 400 V）但高电容（500 - 950微法）的单脉冲程序。根据电穿孔条件将肝细胞分为4组：I组，220 V和500微法；II组，220 V和950微法；III组，400 V和950微法，以及IV组。对照组为新鲜分离的肝细胞，并在与电穿孔组相同的条件下直接培养。通过台盼蓝排斥法（TBE）和MTT分析检测电穿孔对原代大鼠肝细胞的影响。此外，通过生化测定法测量培养肝细胞上清液中的白蛋白（Alb）、丙氨酸转氨酶（ALT）和乳酸脱氢酶（LDH）。
孵育后第1天至第15天，各电穿孔组的原代大鼠肝细胞外观正常，与对照组相同。TBE染色显示，电穿孔组和对照组均有轻微的肝细胞损伤且存活率高。培养3、7、11和15天时，肝细胞活力分别约为92.6±2.5%、89.5±3.3%、82.0±3.5%和74.3±1.2%。MTT分析表明，各电穿孔组肝细胞活力之间无显著差异，且与对照组相似。电穿孔后第36小时，对照组上清液中的Alb、ALT和LDH分别为5.3±0.1 g·L⁻¹、183.7±8.4 nkat·L⁻¹和896.8±58.5 nkat·L⁻¹；II组分别为5.7±0.1 g·L⁻¹、215.4±16.7 nkat·L⁻¹和1063.8±51.8 nkat·L⁻¹；III组分别为5.8±0.2 g·L⁻¹、217.1±8.4 nkat·L⁻¹和1063.8±10.0 nkat·L⁻¹。统计学分析，II组和III组的蛋白水平显著高于对照组（P<0.05），而I组的蛋白产生量、Alb、ALT和LDH分别为5.3±0.2 g·L⁻¹、205.4±3.3 nkat·L⁻¹和1035.4±116.9 nkat·L⁻¹，与对照组相似。同时，TBE和MTT分析表明，电穿孔组和对照组之间的细胞活力无显著差异。
这种采用低电压（220 - 400 V）但高电容（950微法）的单脉冲电穿孔程序是将外源基因导入原代大鼠肝细胞的最佳选择之一。
