壳聚糖修饰的羟基磷灰石纳米粒载体介导BMP-2转染成骨细胞及对其增殖、分化的影响
发布时间：2018-11-08 20:17
【摘要】：目的：通过用壳聚糖（chitosan,CS）修饰的羟基磷灰石(hydroxyapatite, HA)纳米粒载体携带骨形态发生蛋白2（Bone morphogenetic protein2,BMP2）基因转染成骨细胞,研究CS-HA对成骨细胞的毒性反应、转染后成骨细胞内BMP-2基因表达情况及对细胞增殖与分化的影响，为基因修饰的成骨细胞的临床研究奠定基础。 方法：复苏成骨细胞株并进行传代培养；用壳聚糖对羟基磷灰石纳米粒进行表面修饰；采用MTT法检测HA-CS纳米粒载体对成骨细胞的毒性反应；用HA-CS纳米粒载体转染技术,将BMP-2目的基因导入成骨细胞,评估转染效率并采用RT-PCR法鉴定BMP-2的表达;同时检测细胞增殖情况及碱性磷酸酶（alkaline phoaphatase，ALP）活性。 结果：MTT结果提示HA-CS纳米粒混悬液浓度在500μg/ml以下对成骨细胞生长的影响与对照组比较差异无统计学意义；其转染效率虽不及同等条件下的脂质体但优于没经修饰的HA; RT-PCR结果显示转染后同对照组及空质粒组比较成骨细胞内BMP-2稳定表达并表达增强;同时也促进了成骨细胞的增殖及增加了ALP的活性。 结论：HA-CS纳米粒在一定浓度下对成骨细胞无毒性作用,作为输送载体可以安全地将目的基因BMP-2导入成骨细胞并稳定表达，，同时促进了成骨细胞的增殖与分化。
[Abstract]:Aim: to study the toxic effect of chitosan,CS on osteoblasts by transfection of hydroxyapatite (hydroxyapatite, HA) nanoparticles carrying bone morphogenetic protein-2 (Bone morphogenetic protein2,BMP2 gene into osteoblasts. The expression of BMP-2 gene in osteoblasts after transfection and its effect on cell proliferation and differentiation lay a foundation for the clinical study of genetically modified osteoblasts. Methods: resuscitation osteoblasts were subcultured, hydroxyapatite nanoparticles were modified with chitosan, and the toxicity of HA-CS nanoparticles to osteoblasts was detected by MTT method. The BMP-2 target gene was transfected into osteoblasts by HA-CS nanoparticles transfection technique. The transfection efficiency was evaluated and the expression of BMP-2 was identified by RT-PCR method. The proliferation and alkaline phosphatase (alkaline phoaphatase,ALP) activity were also detected. Results: the results of MTT showed that there was no significant difference in the effect of the concentration of HA-CS nanoparticles suspension below 500 渭 g/ml on the growth of osteoblasts compared with the control group. The transfection efficiency was lower than that of liposomes under the same conditions, but better than that of unmodified HA; RT-PCR. The results showed that the stable expression and expression of BMP-2 in osteoblasts after transfection were higher than those in control group and empty plasmid group. It also promoted the proliferation of osteoblasts and increased the activity of ALP. Conclusion: HA-CS nanoparticles have no toxic effect on osteoblasts at a certain concentration. As a carrier, the target gene BMP-2 can be safely transferred into osteoblasts and expressed stably, and the proliferation and differentiation of osteoblasts can be promoted at the same time.
【学位授予单位】：佳木斯大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08
[Abstract]:Aim: to study the toxic effect of chitosan,CS on osteoblasts by transfection of hydroxyapatite (hydroxyapatite, HA) nanoparticles carrying bone morphogenetic protein-2 (Bone morphogenetic protein2,BMP2 gene into osteoblasts. The expression of BMP-2 gene in osteoblasts after transfection and its effect on cell proliferation and differentiation lay a foundation for the clinical study of genetically modified osteoblasts. Methods: resuscitation osteoblasts were subcultured, hydroxyapatite nanoparticles were modified with chitosan, and the toxicity of HA-CS nanoparticles to osteoblasts was detected by MTT method. The BMP-2 target gene was transfected into osteoblasts by HA-CS nanoparticles transfection technique. The transfection efficiency was evaluated and the expression of BMP-2 was identified by RT-PCR method. The proliferation and alkaline phosphatase (alkaline phoaphatase,ALP) activity were also detected. Results: the results of MTT showed that there was no significant difference in the effect of the concentration of HA-CS nanoparticles suspension below 500 渭 g/ml on the growth of osteoblasts compared with the control group. The transfection efficiency was lower than that of liposomes under the same conditions, but better than that of unmodified HA; RT-PCR. The results showed that the stable expression and expression of BMP-2 in osteoblasts after transfection were higher than those in control group and empty plasmid group. It also promoted the proliferation of osteoblasts and increased the activity of ALP. Conclusion: HA-CS nanoparticles have no toxic effect on osteoblasts at a certain concentration. As a carrier, the target gene BMP-2 can be safely transferred into osteoblasts and expressed stably, and the proliferation and differentiation of osteoblasts can be promoted at the same time.
【学位授予单位】：佳木斯大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08
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