丝素蛋白作为促血管生长基因载体的研究
发布时间：2018-07-20 16:46
【摘要】：血管的再生是组织工程及原位组织再生领域面临的关键问题。利用转基因技术，将促进血管再生的生长因子基因导入目的细胞并使其表达，可有效促进血管生成。VEGF165和Ang-1是两种最重要的促血管再生生长因子。VEGF作用于血管形成的早期，促进原始血管网的形成，Ang-1则作用于其后的血管改建、塑形，促进形成成熟的且具有空间结构的血管网。联合使用VEGF165和Ang-1双基因转染细胞，则既有利于相互协调共同促进血管的形成，又可使新生血管获得持久稳定的结构。为了将目的基因导入靶细胞，需要依靠基因传递载体。分子量为25kDa的支链PEI是迄今最成功的非病毒高分子基因载体，其表面带有高密度的正电荷，转染效率较高，但细胞毒性较大。丝素蛋白是一种具有良好细胞相容性和可降解性的蛋白质，丝素分子带负电，不能直接包装DNA，但是能够屏蔽PEI多余的正电荷，减小细胞毒性。同时柞蚕丝素蛋白内存在细胞特异性粘附序列RGD，可以与血管内皮细胞、成纤维细胞等细胞表面的受体发生特异性相互作用。因此，用丝素和PEI共同包装质粒DNA，作为基因传递载体，可以通过细胞表面受体介导的特异性相互作用及其内吞作用，代替PEI和细胞的非特异性静电相互作用，以降低细胞毒性，提高转染效率。 本文以丝素和PEI共同作为VEGF165和Ang-1双基因共表达质粒的传递载体，研究与PEI单独作为传递载体相比，载体的形态、结构、理化性质的变化，及其对细胞毒性、转染效率的影响，并对其影响机制进行初步探讨。 首先，通过静电吸附方法制备PEI/DNA复合物， DNA的含量为2μg/ml。用琼脂糖凝胶电泳确定能完全包被质粒时，PEI与DNA的N/P比大于3。MTT法证明，细胞培养液中PEI含量在1-5μg/ml时，细胞存活率都在90%以上，细胞毒性较低。PEI含量为3μg/ml时（其中，PEI/DNA复合物的含量为5μg/ml，DNA的含量为2μg/ml），荧光强度最强，转染效率最大，且细胞毒性较低。 其次，分别用柞蚕丝素（Antheraea pernyi silk fibroin,ASF）和家蚕丝素(Bombyxmori silk fibroin, BSF)包被PEI/DNA，得到ASF/PEI/DNA和BSF/PEI/DNA复合物。用X-射线能谱分析了复合物表面元素的含量变化，证明蚕丝丝素可以结合到PEI/DNA表面。原子力显微镜（AFM）和激光粒度仪(DLS)测试结果显示ASF和BSF都可以包被PEI/DNA形成纳米级颗粒，PEI/DNA的平均粒径为274.3±3.4nm，ASF/PEI/DNA的平均粒径是337.6±7.4nm，SF/PEI/DNA复合物的粒径与PEI/DNA相比有所增大。ASF/PEI/DNA和BSF/PEI/DNA的表面电位与PEI/DNA相比有明显降低。SF/PEI/DNA三元复合物能抵抗核酸酶的降解。 最后，分别用ASF/PEI/DNA和BSF/PEI/DNA转染L929细胞，激光共聚焦显微镜（LSCM）观察转染情况，流式细胞仪检测绿色荧光蛋白（GFP）阳性细胞比率。结果表明，ASF/PEI/DNA复合物的转染效率比PEI/DNA复合物的高。转染CHL细胞后，出现了相似的结果。MTT法测试结果显示ASF/PEI/DNA组的细胞存活率大于PEI/DNA组的细胞存活率。用ASF与PEI共包被DNA后，一方面能够通过受体介导的内吞作用，增强复合物对细胞的靶向性；另一方面能降低复合物的细胞毒性，使细胞生长状况和功能良好，有利于转录和翻译，从而提高VEGF-Ang-1双基因共表达质粒的基因转染效率。细胞经转染携带双基因片段的质粒后，VEGF的分泌比同种条件下的携带单基因的质粒组分泌的VEGF多。同一时间点，ASF/PEI/DNA组细胞比其它组细胞分泌的VEGF多。证明用ASF与PEI共同包被DNA，不仅能够提高转染效率，而且转染后的VEGF基因能够正常表达、分泌VEGF。 本文首次用柞蚕丝素蛋白和PEI共同作为VEGF165和Ang-1双基因共表达传递载体转染成纤维细胞，研究表明，与单纯使用PEI相比，，能有效提高转染效率并降低细胞毒性，为组织工程及原位组织再生领域的血管再生提供了一种新的基因传递载体。
[Abstract]:The regeneration of blood vessels is the key problem in tissue engineering and in situ tissue regeneration. Using transgenic technology, the growth factor genes that promote vascular regeneration are introduced into the target cells and expressed, which can effectively promote the angiogenesis of.VEGF165 and Ang-1, the two most important vascular regeneration growth factor.VEGF in angiogenesis. In the early stage, the formation of the original vascular network was promoted, and Ang-1 acted on the subsequent remodeling, shaping, and promoting the formation of a mature and spatially structured vascular network. The combined use of VEGF165 and Ang-1 double genes to transfect cells would be beneficial to the coordination and common promotion of vascular formation, and to make new blood vessels lasting and stable structure. The target gene is introduced into the target cell, and it needs to rely on the gene transfer carrier. The branched chain PEI with molecular weight of 25kDa is the most successful non viral high molecular gene carrier. Its surface has high density positive charge, high transfection efficiency and large cytotoxicity. Silk fibroin is a kind of protein with good cytocompatibility and degradability. The fibroin molecule is negative and can not directly package DNA, but it can shield the excess positive charge of PEI and reduce the cytotoxicity. At the same time, tussah silk fibroin protein is stored in the cell specific adhesion sequence RGD, which can interact with the receptors on the surface of vascular endothelial cells, fibroblasts and other cells. Therefore, the silk fibroin and PEI are used together to package the substance. The granular DNA, as a gene delivery carrier, can replace the non specific electrostatic interaction of PEI and cells through specific interaction and endocytosis mediated by cell surface receptors, in order to reduce cytotoxicity and improve transfection efficiency.
In this paper, we used silk fibroin and PEI as the carrier of VEGF165 and Ang-1 double gene co expression plasmid, and studied the changes in the morphology, structure and physicochemical properties of the carrier as the carrier of PEI, and the effect on the cytotoxicity and transfection efficiency of the vector, and the mechanism of its influence was discussed.
First, the PEI/DNA complex was prepared by electrostatic adsorption. When the content of DNA was 2 u g/ml., the N/P ratio of PEI and DNA was more than 3.MTT method. The survival rate of the cell culture solution was more than 90% when the PEI content was 1-5 u g/ml, and the cytotoxicity of.PEI was 3 mu g/ml (among them, PEI/) The content of DNA complex is 5 g/ml, and the content of DNA is 2 g/ml. The fluorescence intensity is the strongest, and the transfection efficiency is the largest and the cytotoxicity is low.
Secondly, Antheraea pernyi silk fibroin (ASF) and family silk fibroin (Bombyxmori silk fibroin, BSF) were wrapped by PEI/DNA, and ASF/PEI/DNA and BSF/PEI/DNA complexes were obtained. The results of laser particle size analyzer (DLS) test show that both ASF and BSF can be wrapped by PEI/DNA to form nanoscale particles. The average particle size of PEI/DNA is 274.3 + 3.4nm, the average particle size of ASF/PEI/DNA is 337.6 + 7.4nm. The particle size of SF/PEI/DNA complex is larger than PEI/DNA, and the surface potential of BSF is significantly lower than that of the BSF. .SF/PEI/DNA three complex can resist the degradation of nuclease.
Finally, transfection of L929 cells with ASF/PEI/DNA and BSF/PEI/DNA and laser confocal microscopy (LSCM) were used to observe the transfection situation. The ratio of green fluorescent protein (GFP) positive cells was detected by flow cytometry. The results showed that the transfection efficiency of ASF/PEI/DNA complex was higher than that of PEI/DNA complex. After transfection of CHL cells, a similar result was found in.MTT method. The results showed that the cell survival rate of the ASF/PEI/DNA group was greater than that in the PEI/DNA group. After the co package of DNA with ASF and PEI, the cell viability was enhanced by the receptor mediated endocytosis, and the cytotoxicity of the complex was reduced, the cell growth and function were good, and it was beneficial to the transcription and development. In order to improve the gene transfection efficiency of the VEGF-Ang-1 double gene co expression plasmid, the VEGF secreted more than VEGF carrying the single gene plasmid group under the same condition after transfection of the plasmid carrying the double gene fragment. At the same time, the ASF/PEI/DNA group cells secreted more VEGF than the other groups. It was proved that ASF and PEI were used together. Coated DNA can not only improve the transfection efficiency, but also transfect the VEGF gene to express normally and secrete VEGF..
In this paper, the first use of tussah silk fibroin protein and PEI as a VEGF165 and Ang-1 double gene co expression transmission vector transfection into fibroblasts. The study shows that compared with the simple use of PEI, it can effectively improve the transfection efficiency and reduce the cytotoxicity, which provides a new gene delivery load for tissue engineering and in situ tissue regeneration. Body.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：TS141;R318.0
本文编号：2134136
[Abstract]:The regeneration of blood vessels is the key problem in tissue engineering and in situ tissue regeneration. Using transgenic technology, the growth factor genes that promote vascular regeneration are introduced into the target cells and expressed, which can effectively promote the angiogenesis of.VEGF165 and Ang-1, the two most important vascular regeneration growth factor.VEGF in angiogenesis. In the early stage, the formation of the original vascular network was promoted, and Ang-1 acted on the subsequent remodeling, shaping, and promoting the formation of a mature and spatially structured vascular network. The combined use of VEGF165 and Ang-1 double genes to transfect cells would be beneficial to the coordination and common promotion of vascular formation, and to make new blood vessels lasting and stable structure. The target gene is introduced into the target cell, and it needs to rely on the gene transfer carrier. The branched chain PEI with molecular weight of 25kDa is the most successful non viral high molecular gene carrier. Its surface has high density positive charge, high transfection efficiency and large cytotoxicity. Silk fibroin is a kind of protein with good cytocompatibility and degradability. The fibroin molecule is negative and can not directly package DNA, but it can shield the excess positive charge of PEI and reduce the cytotoxicity. At the same time, tussah silk fibroin protein is stored in the cell specific adhesion sequence RGD, which can interact with the receptors on the surface of vascular endothelial cells, fibroblasts and other cells. Therefore, the silk fibroin and PEI are used together to package the substance. The granular DNA, as a gene delivery carrier, can replace the non specific electrostatic interaction of PEI and cells through specific interaction and endocytosis mediated by cell surface receptors, in order to reduce cytotoxicity and improve transfection efficiency.
In this paper, we used silk fibroin and PEI as the carrier of VEGF165 and Ang-1 double gene co expression plasmid, and studied the changes in the morphology, structure and physicochemical properties of the carrier as the carrier of PEI, and the effect on the cytotoxicity and transfection efficiency of the vector, and the mechanism of its influence was discussed.
First, the PEI/DNA complex was prepared by electrostatic adsorption. When the content of DNA was 2 u g/ml., the N/P ratio of PEI and DNA was more than 3.MTT method. The survival rate of the cell culture solution was more than 90% when the PEI content was 1-5 u g/ml, and the cytotoxicity of.PEI was 3 mu g/ml (among them, PEI/) The content of DNA complex is 5 g/ml, and the content of DNA is 2 g/ml. The fluorescence intensity is the strongest, and the transfection efficiency is the largest and the cytotoxicity is low.
Secondly, Antheraea pernyi silk fibroin (ASF) and family silk fibroin (Bombyxmori silk fibroin, BSF) were wrapped by PEI/DNA, and ASF/PEI/DNA and BSF/PEI/DNA complexes were obtained. The results of laser particle size analyzer (DLS) test show that both ASF and BSF can be wrapped by PEI/DNA to form nanoscale particles. The average particle size of PEI/DNA is 274.3 + 3.4nm, the average particle size of ASF/PEI/DNA is 337.6 + 7.4nm. The particle size of SF/PEI/DNA complex is larger than PEI/DNA, and the surface potential of BSF is significantly lower than that of the BSF. .SF/PEI/DNA three complex can resist the degradation of nuclease.
Finally, transfection of L929 cells with ASF/PEI/DNA and BSF/PEI/DNA and laser confocal microscopy (LSCM) were used to observe the transfection situation. The ratio of green fluorescent protein (GFP) positive cells was detected by flow cytometry. The results showed that the transfection efficiency of ASF/PEI/DNA complex was higher than that of PEI/DNA complex. After transfection of CHL cells, a similar result was found in.MTT method. The results showed that the cell survival rate of the ASF/PEI/DNA group was greater than that in the PEI/DNA group. After the co package of DNA with ASF and PEI, the cell viability was enhanced by the receptor mediated endocytosis, and the cytotoxicity of the complex was reduced, the cell growth and function were good, and it was beneficial to the transcription and development. In order to improve the gene transfection efficiency of the VEGF-Ang-1 double gene co expression plasmid, the VEGF secreted more than VEGF carrying the single gene plasmid group under the same condition after transfection of the plasmid carrying the double gene fragment. At the same time, the ASF/PEI/DNA group cells secreted more VEGF than the other groups. It was proved that ASF and PEI were used together. Coated DNA can not only improve the transfection efficiency, but also transfect the VEGF gene to express normally and secrete VEGF..
In this paper, the first use of tussah silk fibroin protein and PEI as a VEGF165 and Ang-1 double gene co expression transmission vector transfection into fibroblasts. The study shows that compared with the simple use of PEI, it can effectively improve the transfection efficiency and reduce the cytotoxicity, which provides a new gene delivery load for tissue engineering and in situ tissue regeneration. Body.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：TS141;R318.0
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