鼠粒细胞-巨噬细胞集落刺激因子基因转染小鼠黑色素瘤B16F1细胞

Cationic Lipid Mediated the Transaction of mGM-CSF Gene into Murine Melanoma B16F1 Cell Line

目的 :探讨基因转移载体脂质体介导转染的鼠粒细胞 巨噬细胞集落刺激因子 (mGM CSF)基因在B16F1细胞的表达动态及表达产物的生物活性。方法 :脂质体和质粒DNA复合后 ,转染 10 6B16F1细胞 ,连续 5d换细胞培养液 ,并检测其中的mGM CSF含量。采用 [3 HTdR]渗入法 ,测定DA1G细胞和转染后第一天收获的以 1:10对倍稀释到 1:6 40的上清液及 [3 HTdR]混合培养后 ,渗入到DA1G细胞的 [3 HTdR]量 (cpm) ,以分析表达产物的生物活性。结果 :转染B16F1细胞的mGM CSF基因得到有效表达。但随着时间的推移 ,表达由第 1天的 135ng/ml下降到第 5天的 42 .2ng/ml。渗入到DA1G细胞的 [3 HTdR]量 (cpm)随上清液的稀释而逐步下降 ,由 1:10稀释度的9371下降到 1:6 40时的 32 5 1。和DMEM混合培养的DA1G细胞的 [3 HTdR]渗入量 (cpm)仅为 472。结论 :脂质体作为基因转移载体可有效地介导mGM CSF基因转染B16F1细胞 。

Objective:To examine the expression kinetics and bioactivity of expressed product of mGMCSF gene transfected into B16F1 cells with cationic lipid as a vector in order to explore the potential application of this gene transfer approach.Methods:The lipid-DNA complex was added to B16F1 cells of 10 6.The culture medium of cells was replaced each day for 5 days and mGMCSF expression level was assayed.The expressed mGMCSF was bioassayed by calculating incorporation rate(cpm) of into DA1G cells when DA1G cells were cultivated with diluted culture medium of mGMCSF gene transfected B16F1 cells and . Results: The mGMCSF was efficiently expressed in B16F1 cells and the expression level decreased from 135 ng/ml on day 1 to 42.2 ng/ml on day 5.The incorporation rate(cpm) of into DA1G cells decreased from 9371 at dilution of 1:10 to 3251 at dilution of 1:640,suggesting the expressed mGMCSF had bioactivity. Conclusion:Cationic lipid could efficiently mediate the transfection of mGMCSF gene into B16F1 cells and the transfected mGMCSF gene is expressed with bioactivity.This suggested that the approach of using cationic lipid as gene transfer vector should have potentially applicable promise.