RNA干扰对HepG2细胞AFP基因表达的影响

发布时间：2019-02-13 03:20

【摘要】：目的:研究 RNA 干扰(RNAi)技术对 HepG2 细胞 AFP 基因表达的影响,以及抑制 AFP 表达后对 HepG2 细胞增殖及凋亡作用的影响。方法:第一部分 RNA 干扰(RNA interference RNAi)抑制 HepG2 细胞 AFP 基因表达方法的建立。 1.目标基因序列的选择及质粒载体构建,经Blast 软件查对及序列同源性分析,选择两条目标基因序列——AFP1 与AFP2,构建质粒载体 pGenesil-1-AFP1(含绿色荧光蛋白编码序列)及pGenesil-2-AFP2。2.用阳离子脂质体 META/质粒载体 pGenesil-1-AFP1 不同剂量及浓度配比转染 HepG2 细胞,荧光显微镜观察转染效率,并检测细胞培养上清液 AFP 的量的变化,筛选出最佳阳离子脂质体 META/质粒载体pGenesil-1-AFP1 配比。第二部分 RNA 干扰技术对 HepG2 细胞 AFP 基因表达的影响。1. 转染试剂 METAFECTENE/质粒载体混合液配制及转染细胞。2.采用微粒子化学发光免疫分析法检测转染后 24,48,72 小时培养上清液中AFP的含量。3. 采用间接免疫细胞化学技术观察处理组和对照组细胞内AFP基因表达情况,了解在细胞水平 pGenesil-AFP-siRNA 对 AFP 表达的抑制作用。第三部分 siRNA 抑制 AFP 表达对 HepG2 细胞增殖及凋亡的影响。分别应用 MTT 比色法,DNA Fragmentation 及检测细胞内 cAMP 水平来观察。结果:1. 筛选出最佳的阳离子脂质体META/质粒载体pGenesil-1-AFP1转染复合物的配比为 8μl:2.5μg。 2. 转染 pGenesil-1-siAFP1 与pGenesil-2-siAFP2 的实验组与对照组比较,AFP 含量下降明显(P0.01);pGenesil-1-siAFP1 实验组与 pGenesil-2-siAFP2 实验组比较,AFP 含量在各时段均显著下降。 3. pGenesil-1-siAFP1 的实验组干扰后 DNAFragmentation 观察无细胞凋亡,但是 MTT 比色法发现 siRNA-AFP1 的实验组对细胞增殖有明显的抑制作用,对细胞增殖抑制率最高达 51.35%。。而且实验组细胞内的 cAMP 浓度(15.64μmol/L)明显低于各对照组细胞内的cAMP 浓度(P0.01)。结论: RNA 干扰技术能有效地抑制 HepG2 细胞 AFP 基因表达,这种方法不仅对体外研究 AFP 基因功能有一定意义,而且为进一步研究应用 RNA 干扰技术抗肿瘤治疗提供了依据。
[Abstract]:Aim: to study the effect of RNA interference (RNAi) on the expression of AFP gene in HepG2 cells and the effect of AFP inhibition on the proliferation and apoptosis of HepG2 cells. Methods: the first part of RNA interference (RNA interference RNAi) inhibition of HepG2 cells AFP gene expression method. 1. Selection of target gene sequence and construction of plasmid vector. Two target gene sequences, AFP1 and AFP2, were selected by Blast software and sequence homology analysis. Construction of plasmid Vector pGenesil-1-AFP1 (containing Green fluorescent protein coding sequence) and pGenesil-2-AFP2.2. HepG2 cells were transfected with cationic liposome META/ plasmid pGenesil-1-AFP1 in different dosages and concentrations. The transfection efficiency was observed by fluorescence microscope, and the changes of the amount of AFP in the supernatant of cell culture were detected. The optimal ratio of cationic liposome META/ vector pGenesil-1-AFP1 was selected. The second part is the effect of RNA interference on the expression of AFP gene in HepG2 cells. 1. Transfection reagent METAFECTENE/ plasmid vector mixture prepared and transfected cells. 2. Microparticle chemiluminescence immunoassay was used to detect the content of AFP in the supernatant of culture supernatant for 72 hours after transfection. Indirect immunocytochemical technique was used to observe the expression of AFP gene in the cells of the treatment group and the control group, and to understand the inhibitory effect of pGenesil-AFP-siRNA on the expression of AFP at the cellular level. The third part is the effect of siRNA on the proliferation and apoptosis of HepG2 cells by inhibiting the expression of AFP. MTT colorimetric method was used to detect, DNA Fragmentation and the intracellular cAMP level was measured. Results: 1. The optimal ratio of cationic liposome META/ plasmid vector pGenesil-1-AFP1 transfection complex was 8 渭 l: 2.5 渭 g. 2. Compared with the control group, the AFP content of the experimental group transfected with pGenesil-1-siAFP1 and pGenesil-2-siAFP2 decreased significantly (P0.01). The content of AFP in pGenesil-1-siAFP1 group was significantly lower than that in pGenesil-2-siAFP2 group. 3. No apoptosis was observed by DNAFragmentation in the experimental group of pGenesil-1-siAFP1, but MTT colorimetric assay showed that the experimental group of siRNA-AFP1 had obvious inhibitory effect on cell proliferation, and the highest inhibition rate was 51.35%. The intracellular cAMP concentration (15.64 渭 mol/L) in the experimental group was significantly lower than that in the control group (P0.01). Conclusion: RNA interference technique can effectively inhibit the expression of AFP gene in HepG2 cells. This method not only has a certain significance for studying the function of AFP gene in vitro, but also provides a basis for further study on the application of RNA interference technique in antitumor therapy.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R363

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