装载促血管再生基因的丝素支架构建及其对真皮再生的影响
发布时间：2018-10-22 11:59
【摘要】：促进微血管再生,加快血管化速度,对于提高皮肤真皮再生支架的存活率至关重要。本文将含有核定位短肽的鱼精蛋白(PS)偶联到富含精氨酸-甘氨酸-天门冬氨酸(RGD)三肽序列和组氨酸(His)的柞蚕丝素蛋白(ASF)上,构建同时具有细胞靶向性、内涵体逃逸及细胞核定位多功能的可生物降解新型阳离子化丝素(AP)。通过(AP+PEI)共包被和AP/ASF/PEI层层包被VEGF及Ang-1双基因共表达质粒将质粒p DNA负载到丝素支架上,得到装载促血管再生基因的丝素支架。利用(AP+PEI)共包被和AP/ASF/PEI层层包被基因载体传递促血管再生的VEGF165-Ang-1双基因共表达质粒原位转染细胞,促进被转染细胞持续、局部地分泌VEGF、Ang-1等血管再生所需的生长因子,加速支架的血管化速度,提高真皮再生支架的成活率。首先,在EDC介导下,使柞蚕丝素蛋白的羧基与鱼精蛋白的氨基发生偶联反应生成酰胺键,建立对柞蚕丝素进行阳离子化改性的技术。测试显示,当柞蚕丝素与鱼精蛋白质量比为100/10时,柞蚕丝素的Zeta电位从-5.2 m V上升到+15.5 mv,等电点p I从4.2变为8.7。当AP/p DNA质量比大于4/2时能将p DNA完全包裹,且复合物表面带正电荷。(AP+PEI)共包被法和AP/ASF/PEI层层包被法均能高效得包裹p DNA,并保护p DNA免受核酸酶的降解。(AP+PEI)/p DNA复合物表面Zeta电位为+20~+30 m V,平均直径分布为200~400 nm。AP/ASF/PEI/p DNA复合物表面Zeta电位分为+15~+25m V,平均直径分布为400~600 nm。(AP+PEI)共包被法和AP/ASF/PEI层层包被法均能介导质粒p DNA成功转染EA.hy926细胞,并表现出较高的转染效率和较低的细胞毒性。AP/ASF/PEI层层包被法的转染效率略高于(AP+PEI)共包被法。采用冷冻干燥法将(AP+PEI)/p DNA((30+10)/2)和AP/ASF/PEI/p DNA(45/45/10/2)复合物装载到丝素支架中,制备基因活性丝素支架。复合物在支架内分散良好,细胞在该支架内部正常粘附和生长。(AP+PEI)/p DNA((30+10)/2)和AP/ASF/PEI/p DNA(45/45/10/2)复合物能成功将VEGF165-Ang-1双基因共表达质粒导入目的细胞,导入的基因能正常地表达并分泌VEGF,且随时间的延长VEGF分泌量显著增多。该基因活性丝素支架体外能转染鸡胚尿囊膜上的细胞,并刺激其生成丰富的血管,且血管形态呈多分支的弥漫放射状,血管数目及大小都明显高于未装载基因活性物质的丝素多孔材料组。进一步,将该丝素基因活性支架植入SD大鼠背部全层皮肤缺损创面,体内实验结果显示该支架炎症反应轻微,能良好地与周围组织融合。周围组织及真皮修复细胞迁入材料内部,支架可引导新生组织的长入和毛细血管形成,并促进表皮成活。随着修复时间的延长,支架内新生毛细血管和胶原纤维的生成量明显比单纯丝素多孔支架内多。在创面修复早期,该基因活性支架内VEGF、Ang-1、CD31、α-SMA、PDGF、b FGF和v WF因子的阳性表达率都比未装载基因的丝素多孔支架高,进一步说明装载(AP+PEI)/p DNA((30+10)/2)和AP/ASF/PEI/p DNA(45/45/10/2)基因活性物质的丝素支架,能够原位转染周围细胞并表达和分泌目的基因对应的VEGF和Ang-1因子,为组织修复细胞的迁移、粘附、生长及功能发挥创造良好的微环境,进而促进血管再生,进而促进真皮组织的修复重建。
[Abstract]:It is essential to promote microvascular regeneration, accelerate blood vessel velocity, and to improve survival rate of dermal tissue regeneration stent. In this paper, protamine (PS) containing the short peptide of nuclear localization is coupled to a silk fibroin (ASF) rich in arginine-glycine-aspartic acid (MBP) tripeptide sequence and histidine (His), which is constructed with cell targeting property at the same time. A novel biodegradable cationic silk fibroin (AP) for the escape and nuclear localization of the connotation body. The plasmid p-DNA was loaded onto the silk fibroin scaffold by co-coating of (AP + PEI) and AP/ ASF/ PEI layer-coated VEGF and Ang-1 double gene to obtain silk fibroin stent loaded with angiogenic genes. By using (AP + PEI) co-coating and AP/ ASF/ PEI layer-coated gene vector to transfer the VEGF165-Ang-1 double gene co-expressing plasmid to transfect cells in-situ to promote the sustained and local secretion of VEGF, Ang-1 and other vascular regeneration required growth factors, accelerating the blood vessel speed of the stent, and the survival rate of the dermis regeneration bracket is improved. Firstly, in EDC-mediated reaction, the amino acid of silk fibroin and the amino group of protamine were coupled with amino group of protamine, and then the technology of cationic modification of silk fibroin was established. When the mass ratio of silk fibroin to protamine was 100/ 10, the Zeta potential of silk fibroin increased from -5. 2m V to + 1.5 m, and the isoelectric point p I changed from 4. 2 to 8. 7. When the mass ratio of AP/ p DNA is greater than 4/ 2, the p DNA can be completely wrapped, and the surface of the complex is positively charged. (AP + PEI) co-coating method and AP/ ASF/ PEI layer-by-layer coating method can efficiently package p-DNA and protect p-DNA from the degradation of nucleases. (AP + PEI)/ p DNA complex surface Zeta potential is + 20 ~ + 30m V, the mean diameter distribution is 200 ~ 400 nm. The surface Zeta potential of AP/ ASF/ PEI/ p DNA complex is divided into + 15 ~ + 25m V, and the mean diameter distribution is 400 ~ 600nm. (AP + PEI) co-coating method and AP/ ASF/ PEI layer coating method can mediate plasmid p-DNA transfection into EA. hy926 cells, and show higher transfection efficiency and lower cytotoxicity. The transfection efficiency of AP/ ASF/ PEI layer coating was slightly higher than that of AP + PEI. A gene active silk fibroin scaffold was prepared by loading (AP + PEI)/ p DNA (30 + 10)/ 2) and AP/ ASF/ PEI/ p DNA (45/ 45/ 10/ 2) complex into silk fibroin scaffold by lyophilization. the composite is well dispersed within the stent and the cells are normally adhered and grown within the stent. (AP + PEI)/ p DNA (30 + 10)/ 2) and AP/ ASF/ PEI/ p DNA (45/ 45/ 10/ 2) can successfully introduce the VEGF165-Ang-1 double gene co-expression plasmid into the target cell, and the introduced gene can express and secrete VEGF normally, and the increase of VEGF secretion over time is significantly increased. The gene active silk fibroin scaffold can be used for in vitro transfection of the cells on the chick embryo chorioallantoic membrane and stimulate the cells to generate abundant blood vessels, and the vascular morphology is in a multi-branch diffuse radial manner, and the number and the size of the blood vessels are significantly higher than that of the silk fibroin porous material group which does not have the active substances of the gene. Furthermore, the silk fibroin gene active scaffold was implanted into the skin defect wound on the back of SD rats. The surrounding tissues and the dermis repair cells migrate into the interior of the material, the scaffold can guide the long entry of the new tissue and the formation of capillaries, and promote the survival of the epidermis. With the prolongation of repair time, the production of new capillaries and collagen fibers in scaffold was significantly higher than that in porous scaffold of pure silk fibroin. In the early stage of wound repair, the positive expression rate of VEGF, Ang-1, CD31, p38-SMA, PDGF, b FGF and v WF in the gene active scaffold was higher than that of the unloaded silk fibroin porous scaffold, and further explained the silk fibroin scaffold of the active substances of the loading (AP + PEI)/ p DNA (30 + 10)/ 2) and AP/ ASF/ PEI/ p DNA (45/ 45/ 10/ 2). VEGF and Ang-1 factor corresponding to the target gene can be transfected into the surrounding cells in situ, and the migration, adhesion, growth and function of the tissue repair cells can be used to create a good microenvironment, thereby promoting blood vessel regeneration and further promoting the restoration and reconstruction of the dermal tissue.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318.08
本文编号：2287096
[Abstract]:It is essential to promote microvascular regeneration, accelerate blood vessel velocity, and to improve survival rate of dermal tissue regeneration stent. In this paper, protamine (PS) containing the short peptide of nuclear localization is coupled to a silk fibroin (ASF) rich in arginine-glycine-aspartic acid (MBP) tripeptide sequence and histidine (His), which is constructed with cell targeting property at the same time. A novel biodegradable cationic silk fibroin (AP) for the escape and nuclear localization of the connotation body. The plasmid p-DNA was loaded onto the silk fibroin scaffold by co-coating of (AP + PEI) and AP/ ASF/ PEI layer-coated VEGF and Ang-1 double gene to obtain silk fibroin stent loaded with angiogenic genes. By using (AP + PEI) co-coating and AP/ ASF/ PEI layer-coated gene vector to transfer the VEGF165-Ang-1 double gene co-expressing plasmid to transfect cells in-situ to promote the sustained and local secretion of VEGF, Ang-1 and other vascular regeneration required growth factors, accelerating the blood vessel speed of the stent, and the survival rate of the dermis regeneration bracket is improved. Firstly, in EDC-mediated reaction, the amino acid of silk fibroin and the amino group of protamine were coupled with amino group of protamine, and then the technology of cationic modification of silk fibroin was established. When the mass ratio of silk fibroin to protamine was 100/ 10, the Zeta potential of silk fibroin increased from -5. 2m V to + 1.5 m, and the isoelectric point p I changed from 4. 2 to 8. 7. When the mass ratio of AP/ p DNA is greater than 4/ 2, the p DNA can be completely wrapped, and the surface of the complex is positively charged. (AP + PEI) co-coating method and AP/ ASF/ PEI layer-by-layer coating method can efficiently package p-DNA and protect p-DNA from the degradation of nucleases. (AP + PEI)/ p DNA complex surface Zeta potential is + 20 ~ + 30m V, the mean diameter distribution is 200 ~ 400 nm. The surface Zeta potential of AP/ ASF/ PEI/ p DNA complex is divided into + 15 ~ + 25m V, and the mean diameter distribution is 400 ~ 600nm. (AP + PEI) co-coating method and AP/ ASF/ PEI layer coating method can mediate plasmid p-DNA transfection into EA. hy926 cells, and show higher transfection efficiency and lower cytotoxicity. The transfection efficiency of AP/ ASF/ PEI layer coating was slightly higher than that of AP + PEI. A gene active silk fibroin scaffold was prepared by loading (AP + PEI)/ p DNA (30 + 10)/ 2) and AP/ ASF/ PEI/ p DNA (45/ 45/ 10/ 2) complex into silk fibroin scaffold by lyophilization. the composite is well dispersed within the stent and the cells are normally adhered and grown within the stent. (AP + PEI)/ p DNA (30 + 10)/ 2) and AP/ ASF/ PEI/ p DNA (45/ 45/ 10/ 2) can successfully introduce the VEGF165-Ang-1 double gene co-expression plasmid into the target cell, and the introduced gene can express and secrete VEGF normally, and the increase of VEGF secretion over time is significantly increased. The gene active silk fibroin scaffold can be used for in vitro transfection of the cells on the chick embryo chorioallantoic membrane and stimulate the cells to generate abundant blood vessels, and the vascular morphology is in a multi-branch diffuse radial manner, and the number and the size of the blood vessels are significantly higher than that of the silk fibroin porous material group which does not have the active substances of the gene. Furthermore, the silk fibroin gene active scaffold was implanted into the skin defect wound on the back of SD rats. The surrounding tissues and the dermis repair cells migrate into the interior of the material, the scaffold can guide the long entry of the new tissue and the formation of capillaries, and promote the survival of the epidermis. With the prolongation of repair time, the production of new capillaries and collagen fibers in scaffold was significantly higher than that in porous scaffold of pure silk fibroin. In the early stage of wound repair, the positive expression rate of VEGF, Ang-1, CD31, p38-SMA, PDGF, b FGF and v WF in the gene active scaffold was higher than that of the unloaded silk fibroin porous scaffold, and further explained the silk fibroin scaffold of the active substances of the loading (AP + PEI)/ p DNA (30 + 10)/ 2) and AP/ ASF/ PEI/ p DNA (45/ 45/ 10/ 2). VEGF and Ang-1 factor corresponding to the target gene can be transfected into the surrounding cells in situ, and the migration, adhesion, growth and function of the tissue repair cells can be used to create a good microenvironment, thereby promoting blood vessel regeneration and further promoting the restoration and reconstruction of the dermal tissue.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318.08
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