摘要
目的优选出pMaxGFP导入人肝癌HepG2细胞中磷酸钙转染法的应用条件。方法取对数生长期的人肝癌HepG2细胞,采用磷酸钙转染法将pMaxGFP导入HepG2细胞,转染前1 h换不同血清浓度的新鲜培养液培养HepG2细胞,血清浓度分别为无血清、1%、2%、5%、10%、15%。转染48 h后以倒置荧光显微镜观察绿色荧光蛋白表达,评估转染效率,同时以原位台盼蓝染色检测转染后的细胞存活率,优选出最适血清浓度。在最适血清浓度条件下,磷酸钙转染法将pMaxGFP转染贴壁状态和悬浮状态的HepG2细胞,共分为A、B、C、D、E 5组,分别代表转染贴壁状态、悬浮状态3×104细胞/孔、4×104细胞/孔、5×104细胞/孔、6×104细胞/孔。转染48 h后在荧光倒置显微镜下观察绿色荧光蛋白表达,评估转染效率,同时以原位台盼蓝染色检测转染后的细胞存活率,优选出最适的细胞状态和细胞密度。结果 48 h后,无血清组HepG2细胞大量死亡,而其他组有较少部分细胞死亡。1%、2%、5%、10%、15%血清浓度的转染效率分别为30.92%±1.29%、30.10%±1.05%、21.27%±0.63%、19.10%±0.51%、10.44%±1.42%,血清浓度为1%、2%时的转染效率明显高于其他血清浓度,P<0.05;1%、2%、5%、10%、15%血清浓度的细胞存活率分别为76.01%±0.94%、89.68%±0.68%、92.38%±1.02%、93.36%±1.35%、93.49%±0.91%,血清浓度为2%时的细胞存活率明显高于1%血清浓度。在转染培养基血清浓度为2%条件下,A、B、C、D、E组转染效率分别为30.74%±0.50%、6.38%±0.28%、31.41%±0.42%、30.94%±0.47%、1.41%±0.27%,A、C、D组转染效率明显高于B、E组,P﹤0.05;A、B、C、D、E组细胞存活率分别为91.76%±0.99%、88.40%±1.29%、88.38%±1.08%、87.48%±0.78%、64.45%±0.55%,A、B、C、D组细胞存活率明显高于E组。结论优选出pMaxGFP导入人肝癌HepG2细胞中的磷酸钙转染法应用条件为转染培养基中血清浓度2%、悬浮状态下细胞密度为4×104细胞/孔或5×104细胞/孔。
Objective To optimize the application conditions of calcium phosphate transfection method when pMaxG-FP is delivered to human hepatoma HepG2 cells.Methods pMaxGFP was delivered to human hepatoma HepG2 cells of logarithmic growth phase by calcium phosphate transfection method.1 h before transfection, different serum concentrations of fresh medium which were null, 1%, 2%, 5%, 10%and 15%were changed to every culture wells.After 48 h, invert-ed fluorescence microscope was used to observe the expression of GFP to evaluate the transfection efficiency, while in situ try pan blue staining to detect cell viability.Thus, the optimal serum concentration was found.Then, under the condition of optimal serum concentration, the following experiment was divided into A, B, C, D, E five groups, namely adherent state, suspended 3 ＆#215;104 cells /well, 4 ＆#215;104 cells /well, 5 ＆#215;104 cells /well, 6 ＆#215;104 cells /well.After 48 h, inverted fluorescence microscope was used to observe the expression of GFP to evaluate the transfection efficiency, while in situ try pan blue staining to detect cell viability.Then the best state of cell and its density were optimized in the same way.Results＆amp;nbsp;After 48 h, there were a great number of dead cells in the serum-free group, while a little in the other group.In the 1%,2%, 5%, 10%, 15% serum concentration group, transfection efficiency was 30.92%±1.29%, 30.10%±1.05%, 21.27%±0.63%, 19.10%±0.51%, 10.44%±1.42%, respectively.Transfection efficiency was significantly higher than the other groups when the serum concentration was 1%and 2%, P〈0.05;in the 1%, 2%, 5%, 10%, 15%serum concentration group, cell viability was 76.01%±0.94%, 89.68%±0.68%, 92.38%±1.02%, 93.36%±1.35%, 93.49%±0.91%, respectively.Cell viability in the 1%serum concentration group was significantly higher than the 2%. Under the condition of 2%serum concentration, transfection efficiency of A, B, C, D, E group was 30.74%±0.50%, 6.38%±0.28%, 31.41%±0.42%, 30.94%±0.47%, 1.41%±0.27%, respectively.A, C and D groups were sig-nificantly higher than B and E groups, P〈0.05;cell viability of A, B, C, D, E group was 91.76% ±0.99%, 88.40%±1.29%, 88.38% ±1.08%, 87.48%±0.78%, 64.45% ±0.55%, respectively.A, B, C and D groups were sig-nificantly higher than E group.Conclusion The application conditions of calcium phosphate transfection method when pMaxGFP is delivered to human hepatoma HepG2 cells are 2%serum concentration and 4 ＆#215;104 cells/well or 5 ＆#215;104 cells/well when suspending transfection.
出处
《山东医药》
CAS
2014年第35期23-26,共4页
Shandong Medical Journal
基金
湖南省科技厅课题资助项目(98SSY1003)
关键词
磷酸钙转染法
细胞转染
细胞存活率
细胞状态
calcium phosphate transfection method
cell transfection
cell viability
the state of cell
