丝素蛋白作为促血管生长基因载体的研究
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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。
Revascularization is the key challenge in tissue engineering and in situ tissueregeneration. Transgenic technology can import angiogenesis growth factor genes intotarget cells to express certain growth factor protein and contribute to angiogenesis.Vascular endothelial growth factor (VEGF165) and angiopoietin-1(Ang-1) have beenidentified as two key angiogenesis growth factors. VEGF promotes the formation ofprimitive vascular network in the early formation of blood vessels; Ang-1plays a role inthe vascular reconstruction and shaping, as well as promotes the formation of a matureand spatial structure of vascular network. Combination of VEGF165and Ang-1can notonly enhance angiogenesis but also obtain stable blood vessels. Gene delivery vector isneeded for importing gene into target cells. The branched polyethylenimine (PEI,25kDa)has been the most successful non-viral gene vector. Due to its high positive charge, PEIhas high transfection efficiency, but it has high cytotoxicity. Silk fibroin (SF) is a naturalprotein and presents excellent cell biocompatibility and biodegradability. As an anionicpolymer, SF can not combine with DNA directly. It can bind to the cationic PEI/DNAcomplex electrostatically to modify the complex surface and lower cytotoxicity. At thesame time, ASF has tripeptide sequence RGD which is known to exhibit specificinteractions with cells. It can interact specifically with receptors of vascular endothelialcells and fibroblast to replace the non-specific electrostatic interaction between PEI andcells.
This paper develops SF and PEI as VEGF165and Ang-1gene delivery vector. Theshape, structure, physical characteristics, cytotoxicity and transfection efficiency wasstudied compared with PEI/DNA. Its mechanism was also discussed.
First, PEI/DNA complex was prepared (DNA2μg/ml). When N/P is above3, PEIcan entrap DNA completely. MTT assay demonstrate that survival rate of cells is morethan90%when PEI is1-5μg/ml. When PEI is3μg/ml (DNA2μg/ml), GFP-positivepercentage is highest. Here PEI/DNA is5μg/ml (3μg PEI和2μg DNA), which canboth obtain highest transfection efficiency and lowest cell cytotoxicity.
ASF and BSF were used to encapsulate PEI/DNA to obtain ASF/PEI/DNA andBSF/PEI/DNA complexes. EDS can test the wt%of different elements on the surfaceof complexes and demonstrated ASF can bind to the cationic PEI/DNA complex. Theresults of AFM and DLS indicated ASF and BSF can both entrap PEI/DNA. PEI/DNAshowed274.3±3.4nm particle size and ASF/PEI/DNA showed337.6±7.4nm particlesize. The addition of silk fibroin to the PEI/DNA complex decreased its ξ-potentials.ASF/PEI/DNA complex can avoid hydrolysis.
L929cells were added ASF/PEI/DNA and BSF/PEI/DNA, transfection wasobserved by LSCM and GFP-positive percentage was detected in the flow cytometer.Results showed that transfection efficiency of ASF/PEI/DNA was higher than that ofPEI/DNA. The results of CHL cells were the same.1d and2d after transfection,GFP-positive percentage of ASF/PEI/DNA (60:3:2) was higher than PEI/DNA. Theresults shows ASF and PEI can raise transfection efficiency of VEGF-Ang-1DNA.Cell cytotoxicity of ASF/PEI/DNA, PEI/DNA and BSF/PEI/DNA was examined.Survival rate of cells with ASF/PEI/DNA was higher than that of PEI/DNA. Theaddition of VEGF165and Ang-1genes raised VEGF more greatly than the addition ofVEGF165. ASF/PEI/DNA group showed the most VEGF than any other group. Thisindicates ASF and PEI can not only raise transfection efficiency, but also can makeVEGF gene express and secrete VEGF.
This paper uses Antheraea pernyi silk fibroin and PEI for the first time to serve asdelivery vector for VEGF165and Ang-1genes. The results showed improvedtransfection efficiency and low cytotoxicity, providing a new non-viral gene vector fortissue engineering and in situ tissue regeneration.
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。
Revascularization is the key challenge in tissue engineering and in situ tissueregeneration. Transgenic technology can import angiogenesis growth factor genes intotarget cells to express certain growth factor protein and contribute to angiogenesis.Vascular endothelial growth factor (VEGF165) and angiopoietin-1(Ang-1) have beenidentified as two key angiogenesis growth factors. VEGF promotes the formation ofprimitive vascular network in the early formation of blood vessels; Ang-1plays a role inthe vascular reconstruction and shaping, as well as promotes the formation of a matureand spatial structure of vascular network. Combination of VEGF165and Ang-1can notonly enhance angiogenesis but also obtain stable blood vessels. Gene delivery vector isneeded for importing gene into target cells. The branched polyethylenimine (PEI,25kDa)has been the most successful non-viral gene vector. Due to its high positive charge, PEIhas high transfection efficiency, but it has high cytotoxicity. Silk fibroin (SF) is a naturalprotein and presents excellent cell biocompatibility and biodegradability. As an anionicpolymer, SF can not combine with DNA directly. It can bind to the cationic PEI/DNAcomplex electrostatically to modify the complex surface and lower cytotoxicity. At thesame time, ASF has tripeptide sequence RGD which is known to exhibit specificinteractions with cells. It can interact specifically with receptors of vascular endothelialcells and fibroblast to replace the non-specific electrostatic interaction between PEI andcells.
This paper develops SF and PEI as VEGF165and Ang-1gene delivery vector. Theshape, structure, physical characteristics, cytotoxicity and transfection efficiency wasstudied compared with PEI/DNA. Its mechanism was also discussed.
First, PEI/DNA complex was prepared (DNA2μg/ml). When N/P is above3, PEIcan entrap DNA completely. MTT assay demonstrate that survival rate of cells is morethan90%when PEI is1-5μg/ml. When PEI is3μg/ml (DNA2μg/ml), GFP-positivepercentage is highest. Here PEI/DNA is5μg/ml (3μg PEI和2μg DNA), which canboth obtain highest transfection efficiency and lowest cell cytotoxicity.
ASF and BSF were used to encapsulate PEI/DNA to obtain ASF/PEI/DNA andBSF/PEI/DNA complexes. EDS can test the wt%of different elements on the surfaceof complexes and demonstrated ASF can bind to the cationic PEI/DNA complex. Theresults of AFM and DLS indicated ASF and BSF can both entrap PEI/DNA. PEI/DNAshowed274.3±3.4nm particle size and ASF/PEI/DNA showed337.6±7.4nm particlesize. The addition of silk fibroin to the PEI/DNA complex decreased its ξ-potentials.ASF/PEI/DNA complex can avoid hydrolysis.
L929cells were added ASF/PEI/DNA and BSF/PEI/DNA, transfection wasobserved by LSCM and GFP-positive percentage was detected in the flow cytometer.Results showed that transfection efficiency of ASF/PEI/DNA was higher than that ofPEI/DNA. The results of CHL cells were the same.1d and2d after transfection,GFP-positive percentage of ASF/PEI/DNA (60:3:2) was higher than PEI/DNA. Theresults shows ASF and PEI can raise transfection efficiency of VEGF-Ang-1DNA.Cell cytotoxicity of ASF/PEI/DNA, PEI/DNA and BSF/PEI/DNA was examined.Survival rate of cells with ASF/PEI/DNA was higher than that of PEI/DNA. Theaddition of VEGF165and Ang-1genes raised VEGF more greatly than the addition ofVEGF165. ASF/PEI/DNA group showed the most VEGF than any other group. Thisindicates ASF and PEI can not only raise transfection efficiency, but also can makeVEGF gene express and secrete VEGF.
This paper uses Antheraea pernyi silk fibroin and PEI for the first time to serve asdelivery vector for VEGF165and Ang-1genes. The results showed improvedtransfection efficiency and low cytotoxicity, providing a new non-viral gene vector fortissue engineering and in situ tissue regeneration.
引文
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