絲素蛋白作為促血管生長基因載體的研究
發(fā)布時(shí)間：2018-07-20 16:46
【摘要】：血管的再生是組織工程及原位組織再生領(lǐng)域面臨的關(guān)鍵問題。利用轉(zhuǎn)基因技術(shù)，將促進(jìn)血管再生的生長因子基因?qū)肽康募?xì)胞并使其表達(dá)，可有效促進(jìn)血管生成。VEGF165和Ang-1是兩種最重要的促血管再生生長因子。VEGF作用于血管形成的早期，促進(jìn)原始血管網(wǎng)的形成，Ang-1則作用于其后的血管改建、塑形，促進(jìn)形成成熟的且具有空間結(jié)構(gòu)的血管網(wǎng)。聯(lián)合使用VEGF165和Ang-1雙基因轉(zhuǎn)染細(xì)胞，則既有利于相互協(xié)調(diào)共同促進(jìn)血管的形成，又可使新生血管獲得持久穩(wěn)定的結(jié)構(gòu)。為了將目的基因?qū)氚屑?xì)胞，需要依靠基因傳遞載體。分子量為25kDa的支鏈PEI是迄今最成功的非病毒高分子基因載體，其表面帶有高密度的正電荷，轉(zhuǎn)染效率較高，但細(xì)胞毒性較大。絲素蛋白是一種具有良好細(xì)胞相容性和可降解性的蛋白質(zhì)，絲素分子帶負(fù)電，不能直接包裝DNA，但是能夠屏蔽PEI多余的正電荷，減小細(xì)胞毒性。同時(shí)柞蠶絲素蛋白內(nèi)存在細(xì)胞特異性粘附序列RGD，可以與血管內(nèi)皮細(xì)胞、成纖維細(xì)胞等細(xì)胞表面的受體發(fā)生特異性相互作用。因此，用絲素和PEI共同包裝質(zhì)粒DNA，作為基因傳遞載體，可以通過細(xì)胞表面受體介導(dǎo)的特異性相互作用及其內(nèi)吞作用，代替PEI和細(xì)胞的非特異性靜電相互作用，以降低細(xì)胞毒性，提高轉(zhuǎn)染效率。 本文以絲素和PEI共同作為VEGF165和Ang-1雙基因共表達(dá)質(zhì)粒的傳遞載體，研究與PEI單獨(dú)作為傳遞載體相比，載體的形態(tài)、結(jié)構(gòu)、理化性質(zhì)的變化，及其對細(xì)胞毒性、轉(zhuǎn)染效率的影響，并對其影響機(jī)制進(jìn)行初步探討。 首先，通過靜電吸附方法制備PEI/DNA復(fù)合物， DNA的含量為2μg/ml。用瓊脂糖凝膠電泳確定能完全包被質(zhì)粒時(shí)，PEI與DNA的N/P比大于3。MTT法證明，細(xì)胞培養(yǎng)液中PEI含量在1-5μg/ml時(shí)，細(xì)胞存活率都在90%以上，細(xì)胞毒性較低。PEI含量為3μg/ml時(shí)（其中，PEI/DNA復(fù)合物的含量為5μg/ml，DNA的含量為2μg/ml），熒光強(qiáng)度最強(qiáng)，轉(zhuǎn)染效率最大，且細(xì)胞毒性較低。 其次，分別用柞蠶絲素（Antheraea pernyi silk fibroin,ASF）和家蠶絲素(Bombyxmori silk fibroin, BSF)包被PEI/DNA，得到ASF/PEI/DNA和BSF/PEI/DNA復(fù)合物。用X-射線能譜分析了復(fù)合物表面元素的含量變化，證明蠶絲絲素可以結(jié)合到PEI/DNA表面。原子力顯微鏡（AFM）和激光粒度儀(DLS)測試結(jié)果顯示ASF和BSF都可以包被PEI/DNA形成納米級顆粒，PEI/DNA的平均粒徑為274.3±3.4nm，ASF/PEI/DNA的平均粒徑是337.6±7.4nm，SF/PEI/DNA復(fù)合物的粒徑與PEI/DNA相比有所增大。ASF/PEI/DNA和BSF/PEI/DNA的表面電位與PEI/DNA相比有明顯降低。SF/PEI/DNA三元復(fù)合物能抵抗核酸酶的降解。 最后，分別用ASF/PEI/DNA和BSF/PEI/DNA轉(zhuǎn)染L929細(xì)胞，激光共聚焦顯微鏡（LSCM）觀察轉(zhuǎn)染情況，流式細(xì)胞儀檢測綠色熒光蛋白（GFP）陽性細(xì)胞比率。結(jié)果表明，ASF/PEI/DNA復(fù)合物的轉(zhuǎn)染效率比PEI/DNA復(fù)合物的高。轉(zhuǎn)染CHL細(xì)胞后，出現(xiàn)了相似的結(jié)果。MTT法測試結(jié)果顯示ASF/PEI/DNA組的細(xì)胞存活率大于PEI/DNA組的細(xì)胞存活率。用ASF與PEI共包被DNA后，一方面能夠通過受體介導(dǎo)的內(nèi)吞作用，增強(qiáng)復(fù)合物對細(xì)胞的靶向性；另一方面能降低復(fù)合物的細(xì)胞毒性，使細(xì)胞生長狀況和功能良好，有利于轉(zhuǎn)錄和翻譯，從而提高VEGF-Ang-1雙基因共表達(dá)質(zhì)粒的基因轉(zhuǎn)染效率。細(xì)胞經(jīng)轉(zhuǎn)染攜帶雙基因片段的質(zhì)粒后，VEGF的分泌比同種條件下的攜帶單基因的質(zhì)粒組分泌的VEGF多。同一時(shí)間點(diǎn)，ASF/PEI/DNA組細(xì)胞比其它組細(xì)胞分泌的VEGF多。證明用ASF與PEI共同包被DNA，不僅能夠提高轉(zhuǎn)染效率，而且轉(zhuǎn)染后的VEGF基因能夠正常表達(dá)、分泌VEGF。 本文首次用柞蠶絲素蛋白和PEI共同作為VEGF165和Ang-1雙基因共表達(dá)傳遞載體轉(zhuǎn)染成纖維細(xì)胞，研究表明，與單純使用PEI相比，，能有效提高轉(zhuǎn)染效率并降低細(xì)胞毒性，為組織工程及原位組織再生領(lǐng)域的血管再生提供了一種新的基因傳遞載體。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：TS141;R318.0
本文編號：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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：TS141;R318.0
【參考文獻(xiàn)】
相關(guān)期刊論文 前5條
1 黨時(shí)鵬;王如興;張學(xué)光;張燕;王明元;郭冬萍;李肖蓉;;聚乙烯亞胺修飾的白蛋白微泡轉(zhuǎn)染CHO細(xì)胞實(shí)驗(yàn)研究[J];南京醫(yī)科大學(xué)學(xué)報(bào)(自然科學(xué)版);2010年05期
2 張璇;潘仕榮;馮敏;李子俊;張未;羅昕;;PEG-PEI共聚物介導(dǎo)VEGF165基因轉(zhuǎn)染及對內(nèi)皮細(xì)胞生長的影響[J];生物化學(xué)與生物物理進(jìn)展;2007年10期
3 馮敏,李蓓;聚乙烯亞胺-聚甲基丙烯酸甲酯陽離子納米粒介導(dǎo)基因轉(zhuǎn)移的研究[J];藥學(xué)學(xué)報(bào);2005年10期
4 傅曉源;張浩偉;謝灝;丁誠;彭鍇;張陽德;;聚乙烯亞胺載基因納米顆粒的制備及其相關(guān)特性的研究[J];中國現(xiàn)代醫(yī)學(xué)雜志;2008年24期
5 馮敏;王玉潔;彭輝;;聚乙烯亞胺-乳酸羥基乙酸共聚物陽離子納米粒介導(dǎo)宮頸癌細(xì)胞基因轉(zhuǎn)染效率的研究[J];中國藥學(xué)雜志;2007年08期
相關(guān)博士學(xué)位論文 前3條
1 李達(dá);基于聚乙烯亞胺為骨架的非病毒轉(zhuǎn)基因載體的研究[D];浙江大學(xué);2007年
2 周現(xiàn)鋒;基于超分子組裝構(gòu)建基因疫苗的非病毒載體[D];吉林大學(xué);2007年
3 孫西洋;新型納米復(fù)合載體的構(gòu)建及其介導(dǎo)NK4基因在治療乳腺癌中的應(yīng)用[D];中國醫(yī)科大學(xué);2010年
相關(guān)碩士學(xué)位論文 前2條
1 石海飛;血管生成素促進(jìn)組織工程真皮支架血管化的研究[D];浙江大學(xué);2005年
2 劉鐵連;Ang-1或/和VEGF165轉(zhuǎn)基因細(xì)胞修飾的再生絲素膜誘導(dǎo)血管形成效應(yīng)及分子機(jī)制研究[D];蘇州大學(xué);2009年
本文編號：2134136
本文鏈接：http://sikaile.net/yixuelunwen/swyx/2134136.html
最近更新
教材專著
