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目的采用监测荧光报告基因表达的策略,探索应用电穿孔法进行稳定转染的适合悬浮淋巴细胞的电场强度。基因转染技术广泛应用于基因表达调控,基因功能,信号转导和药物筛选研究,已经成为当前生命科学的基本操作之一。外源性DNA导入哺乳动物或人细胞有两种类型:瞬时转染和稳定转染。天生趋于悬浮的细胞非常难转染,多种方法都效率低下。而电穿孔法理论上可用于各种细胞,尤其对于悬浮细胞十分有效。本文就悬浮生长的T淋巴细胞的电穿孔法转染条件之一(电场强度)进行优化探讨。方法用荧光报告基因质粒转入急性白血病T淋巴细胞系,根据报告荧光活性单位值和蛋白浓度判断不同电场强度(250,270,290V/cm)对悬浮细胞的最佳电穿孔转染条件。结果随电压升高,原始荧光报告基因表达加强,270和290V无明显差异,存活细胞数量依次降低。相对荧光单位,表明相同细胞数量存活时,转染效率按照250,270,290V顺序依次升高。中等强度的270V电压,既保证了足够多的转染体存活,又可得到满意的高表达基因荧光信号。结论根据荧光蛋白表达和蛋白吸光度判断,950μF电容和0.4cm的放电杯中转染淋巴细胞,电场强度270 V/cm的效率最佳。
OBJECTIVE: To detect the electric field strength of suitable suspension cells for stable transfection by electroporation using the strategy of monitoring the expression of fluorescent reporter gene. Gene transfection technology is widely used in gene expression regulation, gene function, signal transduction and drug screening research, which has become one of the basic operations of current life sciences. There are two types of exogenous DNA introduced into mammalian or human cells: transient transfection and stable transfection. Cells that are inherently lean in suspension are very difficult to transfect and many are inefficient. The electroporation method can theoretically be used for various cells, especially for suspension cells. In this paper, one of the electroporation transfection conditions (electric field intensity) of T-lymphocytes growing in suspension was optimized. Methods Fluorescent reporter plasmids were transfected into T lymphocytes of acute leukemia and the optimal electroporation conditions for suspension cells were determined according to reported fluorescence activity units and protein concentrations. Results With the increase of voltage, the expression of the original fluorescent reporter gene was strengthened. There was no significant difference between 270 and 290V and the number of viable cells decreased. Relative fluorescence units, indicating that the same cell number survival, transfection efficiency in accordance with the order of 250,270,290 V increased. The medium voltage of 270V not only ensures enough transfectants to survive, but also achieves satisfactory fluorescent signals of highly expressed genes. Conclusion According to the expression of fluorescent protein and protein, the optimal electric field intensity of 270 V / cm was obtained when the cells were transfected with 950μF capacitor and 0.4cm discharge cup.