超顺磁氧化铁纳米粒介导bFGF转染猫角膜内皮细胞

发布时间：2018-01-18 13:13

本文关键词：超顺磁氧化铁纳米粒介导bFGF转染猫角膜内皮细胞　出处：《苏州大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的用经过多聚赖氨酸修饰的超顺磁氧化铁纳米粒子作为基因载体,将携带碱性成纤维生长因子的绿色荧光蛋白质粒pEGFP-C1-bFGF转染至体外培养的猫角膜内皮细胞,并初步研究其转染效果,探讨超顺磁氧化铁纳米粒子作为一种新型基因载体进行体外角膜内皮细胞基因转染的可行性。本实验分两部分: 第一部分:猫角膜内皮细胞培养及鉴定。第二部分:超顺磁氧化铁纳米粒介导pEGFP-C1-bFGF基因转染猫角膜内皮细胞及其表达的观察。方法第一部分:无菌条件下取猫龄小于3个月的猫眼球,沿角膜缘剪开取角膜片,按揭取后弹力层培养法进行角膜内皮细胞的原代及传代培养。倒置显微镜下观察不同阶段培养的细胞的形态,用台盼蓝染色计数各代活细胞的百分数。并用NSE抗体对传代培养的细胞进行鉴定,观察传代细胞是否保持有角膜内皮细胞的特性。第二部分:用多聚赖氨酸为修饰物,使多聚赖氨酸通过静电作用结合于超顺磁氧化铁纳米粒子表面,获得多聚赖氨酸-超顺磁氧化铁纳米粒子复合物(SPIONs—PLL)。借助含有报告基因的真核表达载体pEGFP-C1,用SPIONs—PLL作为基因载体,将bFGF基因转染至体外培养的猫角膜内皮细胞。在倒置荧光显微镜下观察转染效率,并继续观察bFGF基因表达情况。结果第一部分:体外成功培养原代猫角膜内皮细胞,并传到第4代。其中原代与1代细胞生长良好,细胞呈铺路石样向周边铺展扩增,大部细胞形态呈规则多边形、生长状态好,活细胞的百分数分别为99.8%,99.1%。而从2代开始的角膜内皮细胞贴壁和增殖能力开始减弱,传至3代细胞形态变大不规则、胞质内空泡渐增多,细胞数量渐少,活细胞的百分数分别为98.2%,94.3%,91.3%。传代培养的猫角膜内皮细胞用NSE鉴定,胞质内出现抗体NSE的阳性表达。第二部分:获得密度均匀的SPIONs—PLL悬浮溶液。由于SPIONs—PLL在中性溶液中带正电,颗粒彼此间存在静电斥力,可以抵消部分重力作用,所以SPIONs—PLL在久置后仍处于悬浮状态。并且成功将pEGFP-C1-bFGF基因转染入猫角膜内皮细胞,48 h后荧光显微镜下可见特异性的绿色荧光,未转染的细胞未见绿色荧光,转染细胞与未转染细胞均生长良好。经western blot检测,证明转染携带有bFGF基因质粒的细胞中bFGF基因表达显著增高。结论 1、体外利用揭取后弹力层法能够成功培养出角膜内皮细胞并传代,随着细胞的传代细胞增殖能力减弱,继续传代能力差。传代细胞经NSE鉴定呈阳性表达,证明本实验所培养的角膜内皮细胞为神经嵴来源的细胞。 2、经多聚赖氨酸修饰的超顺磁氧化铁纳米粒子可作为基因载体将pEGFP-C1-bFGF基因转染入猫角膜内皮细胞并得到表达,转染的细胞生长良好。且超顺磁氧化铁纳米粒子所具有的超顺磁性可达到在外加磁场条件下提高转染效率的效果。说明超顺磁氧化铁纳米粒子可作为一种新型基因载体在基因工程细胞转染方面具有较大的可行性。
[Abstract]:objective
After using poly-L-lysine modified superparamagnetic iron oxide nanoparticles as gene carrier, will carry the basic culture of fibroblast growth factor GFP plasmid pEGFP-C1-bFGF was transfected into in vitro cat corneal endothelial cells, and to study the effect of transfection, superparamagnetic iron oxide nanoparticles as a novel gene vector for gene in vitro cornea the feasibility of transfection of endothelial cells.
The experiment is divided into two parts:
Part 1: culture and identification of corneal endothelial cells in cat's cornea.
The second part: Ultraparamagnetic iron oxide nanoparticles mediated pEGFP-C1-bFGF gene transfection in cat corneal endothelial cells and their expression.
Method
The first part: sterilely cat age less than 3 months of the cat eye, along the limbus cornea cut to take the mortgage, Descemet culture method of corneal endothelial cells and cultured in different stages. Observe the morphology of cultured cells under the inverted microscope, the percentage of each generation counting with trypan. Cell blue staining. And NSE antibody on cultured cells were identified and observed whether cell maintain characteristics of corneal endothelial cells.
The second part: the use of poly lysine as modifier, the poly lysine by electrostatic interaction combined with superparamagnetic iron oxide nanoparticles, obtain polylysine superparamagnetic iron oxide nanoparticle composites (SPIONs - PLL). The eukaryotic expression vector pEGFP-C1 containing reporter gene, SPIONs PLL as gene vector, bFGF gene was transfected into in vitro cultured cat corneal endothelial cells. The transfection efficiency was observed under the inverted fluorescence microscope, and to observe the expression of bFGF gene.
Result
The first part: the primary cultured cat corneal endothelial cells were cultured successfully, and spread to the fourth generation. One original and 1 generation cells grew well, cells showed cobblestone amplification to the surrounding spreading of the cells showed irregular polygons, growth state, percentage of living cells was 99.8% 99.1%., and from the beginning of the 2 generation the corneal endothelial cell adherence and proliferation ability began to weaken, spread to the 3 generation cells became larger and irregular, cytoplasmic vacuoles gradually increased, the number of cells gradually less, percentage of living cells were 98.2%, 94.3%, 91.3%. were cultured cat corneal endothelial cells were identified by NSE, the positive expression of NSE cell antibody in the matter.
The second part: SPIONs PLL suspended solution density uniform. Because the SPIONs - PLL in neutral solution are positively charged, electrostatic repulsion between particles, can be partially offset the effect of gravity, so SPIONs PLL in long after the suspension is still in the state. And the success of the pEGFP-C1-bFGF gene was transfected into cat corneal endothelial cells, 48 h under the fluorescence microscope after visible specific green fluorescence, the green fluorescence of untransfected cells were transfected cells and untransfected cells were growing well. By Western blot assay, the expression of bFGF that transfected with bFGF gene plasmid genes was significantly increased.
conclusion
1, in vitro by stripping Descemet method successfully cultured corneal endothelial cells proliferation and passage, with the reduced ability of cell to cell, passage ability. Cells were identified by NSE positive expression, prove the cultured corneal endothelial cells from neural crest cells.
2, the poly lysine modified superparamagnetic iron oxide nanoparticles can be used as a gene vector pEGFP-C1-bFGF gene transfection in cat corneal endothelial cells and expression of transfected cells grew well. And super paramagnetic iron oxide superparamagnetic nanoparticles can improve transfection efficiency in the presence of an external magnetic field effect. Superparamagnetic iron oxide nanoparticles can be used as a new gene carrier has great feasibility in gene engineering cell transfection.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R77

【相似文献】