Published online Oct 15, 2002. doi: 10.3748/wjg.v8.i5.893
Revised: May 10, 2002
Accepted: May 15, 2002
Published online: October 15, 2002
AIM: To investigate the effects of electroporation on primary rat hepatocyte and to optimize the electroporation conditions introducing foreign genes into primary hepatocytes.
METHODS: A single-pulse procedure was performed at low voltage (220-400 V) but with high capacitance (500-950 μF). Hepatocytes were divided into 4 groups according to the electroporation conditions: group I, 220 V and 500 μF; group II, 220 V and 950 μF; group III, 400 V and 950 μF, 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.
RESULTS: 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 d, hepatocyte viability was approximatly 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·L-1, 183.7 ± 8.4 nkat·L-1 and 896.8 ± 58.5 nkat·L-1; those of group II were 5.7 ± 0.1 g·L-1, 215.4 ± 16.7 nkat·L-1 and 1063.8 ± 51.8 nkat·L-1; and those of group III were 5.8 ± 0.2 g·L-1, 217.1 ± 8.4 nkat·L-1 and 1063.8 ± 10.0 nkat·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·L-1, 205.4 ± 3.3 nkat·L-1 and 1035.4 ± 116.9 nkat·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.
CONCLUSION: This single-pulse electroporation procedure performed at low voltage (220-400 V) but with high capacitance (950 μF) is one of the optimal choices to introduce foreign genes into primary rat hepatocyte.