摘要
AIM: To investigate the effects of electroporation on primaryrat hepatocyte and to optimize the electroporation conditionsintroducing foreign genes into primary hepatocytes.METHODS: A single-pulse procedure was performed at Iowvoltage (220-400 V) but with high capacitance (500-950 μF).Hepatocytes were divided into 4 groups according to theelectroporation conditions: group Ⅰ, 220 V and 500 μF;group Ⅱ, 220 Vand 950 μF; group Ⅲ, 400 V and 950 μF,and group Ⅳ.The control group was freshly isolatedhepatocytes and directly cultured under the same conditionsas those of electroporation groups. The effects ofelectroporation on primary rat hepatocytes were detectedby trypan blue exclusion (TBE) and MTT analysis. Besides,albumin (AIb), alanine transaminase (ALT) and lactatedehydrogenase (LDH) in the supernatants of culturedhepatocytes were measured by biochemical assay.RESULTS: Between day 1 and day 15 after incubation,primary rat hepatocytes of each electroporation groupappeared normal, being the same with those of controlgroup. TBE staining showed that slight hepatocyte damageand high survival rate were found in the electroporationgroups and the control group. Cultured for 3, 7, 11 and 15days, 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 hepatocyteshad no significant difference between each electroporationgroup, and those were similar to that of control group. Atthe 36th hour after electroporation, AIb, ALT and LDH in thesupernatants 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 Ⅱwere 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 5.80.2 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 significantlyhigher than those of control group (P＜0.05), whereas theprotein production of group Ⅰ, AIb, 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 significantcell viability difference between electroporation groups andcontrol group.CONCLUSION: This single-pulse electroporation procedureperformed at Iow voltage (220-400 V) but with highcapacitance (950 μF) is one of the optimal choices tointroduce foreign genes into primary rat hepatocyte.
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 days,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,AIb,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 Ⅲwere significantly higher than those of control group(P<0.05),whereas the protein production of group Ⅰ,AIb,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.
基金
the National Natural Science Foundation of China,No.39670340
