pEGFP-C1-P53质粒的构建及其与纳米羟基磷灰石复合物转染鼻咽癌细胞的实验研究

发布时间：2018-04-27 23:01

本文选题：羟基磷灰石 + P53　；参考：《中南大学》2010年硕士论文

【摘要】： 目的：探讨经聚乙烯亚胺修饰的纳米羟基磷灰石-P53复合物转染鼻咽癌细胞(HNE2系)的效果。方法：(1)构制pEGFP-C1-P53质粒；(2)用pEGFP-C1-P53质粒和经聚乙烯亚胺修饰的纳米羟基磷灰石(简称：PEI-nHA)混合制成羟基磷灰石-P53复合物,用于鼻咽癌HNE2系细胞的转染；然后检测转染后的鼻咽癌HNE2细胞中P53转染效率。(3) RT-PCR和Western-blot实验检测纳米羟基磷灰石和脂质体2000介导P53基因在基因和蛋白质水平的表达。结果：(1)在转染实验中转染后的空白组(单纯质粒和HNE2细胞混合)在荧光显微镜下检测未见荧光,脂质体2000组在荧光显微镜下检测可见40%左右细胞内具有绿色荧光,纳米载体组分成62.5ug/ml、125ug/ml、250ug/ml、500ug/ml四种浓度转染,在荧光显微镜下检测经四种不同浓度纳米材料介导转染HNE2细胞后,62.5ug/ml浓度的PEI-nHA转染效率约为4%,125 ug/ml浓度的PEI-nHA转染效率约为6%,250ug/ml浓度的PEI-nHA转染效率约为5%,500 ug/ml浓度的PEI-nHA转染效率约为1%。(2) RT-PCR和Western-blot实验经脂质体2000、纳米羟基磷灰石介导转染进鼻咽癌HNE2细胞均可检测到P53在基因和蛋白水平的表达,经脂质体2000介导转染的HNE2细胞的P53基因和蛋白表达水平较纳米羟基磷灰石载体介导的明显增强。结论：(1)经聚乙烯亚胺修饰的纳米羟基磷灰石介导转染鼻咽癌HNE2细胞的转染率较脂质体2000偏低,有待进一步研究。
[Abstract]:Aim: to investigate the effect of poly (imine) modified nano-hydroxyapatite-p53 complex transfected into nasopharyngeal carcinoma cell line (HNE2). Methods the pEGFP-C1-P53 plasmids were constructed by 1) the pEGFP-C1-P53 plasmids were mixed with polyethylene imide modified nano-hydroxyapatite to form hydroxyapatite p53 complexes, which were used for transfection of nasopharyngeal carcinoma (NPC) HNE2 cells. Then the transfection efficiency of p53 in nasopharyngeal carcinoma (HNE2) cells was detected by RT-PCR and Western-blot. The expression of p53 gene mediated by nano-hydroxyapatite and liposome 2000 was detected at the gene and protein levels. Results in the transfection experiment, no fluorescence was detected in the blank group (simple plasmids and HNE2 cells mixed), while in the liposome 2000 group, about 40% of the cells showed green fluorescence under the fluorescence microscope. The nanocarrier group was transfected into four concentrations of 62.5ug / ml 125ugrml 250ugpml / ml 500ugrml, After transfection of HNE2 cells with four different concentrations of nanomaterials, the transfection efficiency of PEI-nHA was about 42.5 渭 g / ml, and the transfection efficiency of PEI-nHA was about 62.5 ug/ml / ml. The transfection efficiency of PEI-nHA at 250ug-1 / ml concentration was about 5500 ug/ml / ml PEI-nHA transfection efficiency. The efficiency of RT-PCR and Western-blot experiments was about 1: 1. RT-PCR and Western-blot were transfected into HNE2 cells of nasopharyngeal carcinoma by liposome 2000. the expression of p53 at gene and protein level could be detected by nano-hydroxyapatite transfection into nasopharyngeal carcinoma HNE2 cells. The expression level of p53 gene and protein in HNE2 cells transfected with liposome 2000 was significantly higher than that mediated by nano-hydroxyapatite vector. Conclusion the transfection efficiency of HNE2 cells mediated by polyethylene imine modified nano-hydroxyapatite is lower than that of liposome 2000, which needs further study.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.63

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