纳米羟基磷灰石介导p53基因抗肿瘤的研究

发布时间：2018-04-22 20:24

本文选题：纳米羟基磷灰石 + 癌症基因治疗　；参考：《浙江理工大学》2012年硕士论文

【摘要】：羟基磷灰石是一种由钙离子、磷酸根离子、羟基组成的磷酸钙复合物，分子式为Ca_(10)(OH)_2(PO_4)_6，它是构成天然骨、牙的主要成分。羟基磷灰石具有良好的生物活性和生物相容性。其中，纳米尺度的羟基磷灰石对肿瘤细胞的生长有遏制作用，而对正常细胞基本无毒副作用。纳米粒子通过细胞吞噬作用进入细胞；同时，羟基磷灰石颗粒在癌细胞偏酸性环境中发生降解释放出携带的目的基因。因此，纳米羟基磷灰石可以作为一种新型的无机非病毒癌症基因治疗载体。本研究中，首次将纳米羟基磷灰石颗粒用作介导p53基因转染癌细胞的基因治疗载体。首先试验设计、合成了三种不同粒径的纳米HAp颗粒（L-HAp，M-HAp，S-HAp），平均粒径分别为3.8μm，141nm，39nm。该三种颗粒均为球型，且表面电位相同。其次，将反转录得到的p53基因构建到真核表达载体pEGFP-C1（表达绿色荧光蛋白）上，构成重组质粒pEGFP-C1-p53（表达GFP-P53融合蛋白）。然后，将上述两者在一定条件下温浴、复合形成HAp-pEGFP-C1-p53复合物。最后，，分别体外转染人癌细胞A549，Huh7，HeLa和人正常细胞QSG-7701，Beas-2B，从细胞生物学水平评价纳米HAp颗粒作为癌症基因治疗载体的安全性和有效性。本试验中合成的三种不同粒径的纳米HAp颗粒均能介导p53基因转染癌细胞，表达相应的蛋白。流式细胞仪检测结果显示纳米HAp颗粒可以转染5%-10%的癌细胞；而转染正常细胞的效率约为0.5%，即几乎不能转染正常细胞或转染正常细胞后几乎不能表达GFP-P53融合蛋白；其中M-HAp的转染效率略高于L-HAp和S-HAp。HAp-pEGFP-C1-p53复合物转染癌细胞后可以诱导癌细胞凋亡，对癌细胞的杀伤效果达到80%以上。MTT实验和结晶紫染色结果显示，纳米HAp颗粒对正常细胞没有明显的细胞毒性，具有较好的生物相容性。综上结果，本研究为纳米HAp颗粒作为安全有效的无机非病毒基因治疗载体系统和进一步完善基因载体设计提供了理论依据。
[Abstract]:Hydroxyapatite is a calcium phosphate compound consisting of calcium ions, phosphate ions and hydroxyl groups. The molecular formula is Ca_ (10) (OH) _2 (PO_4) _6. It is the main component of the natural bone and teeth. Hydroxyapatite has a good bioactivity and biocompatibility. There is no toxic side effect on normal cells. Nanoparticles enter cells through cell phagocytosis; at the same time, hydroxyapatite particles degrade in the acidic environment of cancer cells to release the target genes. Therefore, nano hydroxyapatite can be used as a new type of inorganic non virus cancer gene therapy carrier.
In this study, nano hydroxyapatite particles were used for the first time as a gene therapy carrier to mediate p53 gene transfection to cancer cells. First, three different particle sizes of nano HAp particles (L-HAp, M-HAp, S-HAp) were synthesized. The average particle size was 3.8 m, 141nm, 39nm., the three particles were all spherical and the surface potential was the same. Second, reverse transcription. The obtained p53 gene was constructed on the eukaryotic expression vector, pEGFP-C1 (expressing green fluorescent protein), and formed a recombinant plasmid pEGFP-C1-p53 (expression of GFP-P53 fusion protein). Then, the above two were bathing under certain conditions to form a HAp-pEGFP-C1-p53 complex. Finally, the transfection of human cancer cells A549, Huh7, HeLa and human normal cells QSG- in vitro, respectively. 7701, Beas-2B, evaluate the safety and efficacy of HAp nanoparticles as a cancer gene therapy vector from the cellular biological level.
In this experiment, three different particle sizes of nano HAp particles can mediate p53 gene transfection to cancer cells and express corresponding proteins. Flow cytometry results show that nano HAp particles can transfect 5%-10% cancer cells, and the efficiency of transfection of normal cells is about 0.5%, that is, it is almost impossible to transfect normal cells or transfect normal cells. The GFP-P53 fusion protein could not be expressed, in which the transfection efficiency of M-HAp was slightly higher than that of L-HAp and S-HAp.HAp-pEGFP-C1-p53 complex transfected to cancer cells. The killing effect on cancer cells was more than 80%.MTT experiments and crystal violet staining results showed that nano HAp particles had no obvious cytotoxicity to normal cells. It has good biocompatibility. In summary, this study provides a theoretical basis for the nano HAp particles as a safe and effective non viral gene therapy carrier system and to further improve the design of gene carrier.

【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

【共引文献】