裝載促血管再生基因的絲素支架構(gòu)建及其對真皮再生的影響
發(fā)布時(shí)間：2018-10-22 11:59
【摘要】：促進(jìn)微血管再生,加快血管化速度,對于提高皮膚真皮再生支架的存活率至關(guān)重要。本文將含有核定位短肽的魚精蛋白(PS)偶聯(lián)到富含精氨酸-甘氨酸-天門冬氨酸(RGD)三肽序列和組氨酸(His)的柞蠶絲素蛋白(ASF)上,構(gòu)建同時(shí)具有細(xì)胞靶向性、內(nèi)涵體逃逸及細(xì)胞核定位多功能的可生物降解新型陽離子化絲素(AP)。通過(AP+PEI)共包被和AP/ASF/PEI層層包被VEGF及Ang-1雙基因共表達(dá)質(zhì)粒將質(zhì)粒p DNA負(fù)載到絲素支架上,得到裝載促血管再生基因的絲素支架。利用(AP+PEI)共包被和AP/ASF/PEI層層包被基因載體傳遞促血管再生的VEGF165-Ang-1雙基因共表達(dá)質(zhì)粒原位轉(zhuǎn)染細(xì)胞,促進(jìn)被轉(zhuǎn)染細(xì)胞持續(xù)、局部地分泌VEGF、Ang-1等血管再生所需的生長因子,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介導(dǎo)下,使柞蠶絲素蛋白的羧基與魚精蛋白的氨基發(fā)生偶聯(lián)反應(yīng)生成酰胺鍵,建立對柞蠶絲素進(jìn)行陽離子化改性的技術(shù)。測試顯示,當(dāng)柞蠶絲素與魚精蛋白質(zhì)量比為100/10時(shí),柞蠶絲素的Zeta電位從-5.2 m V上升到+15.5 mv,等電點(diǎn)p I從4.2變?yōu)?.7。當(dāng)AP/p DNA質(zhì)量比大于4/2時(shí)能將p DNA完全包裹,且復(fù)合物表面帶正電荷。(AP+PEI)共包被法和AP/ASF/PEI層層包被法均能高效得包裹p DNA,并保護(hù)p DNA免受核酸酶的降解。(AP+PEI)/p DNA復(fù)合物表面Zeta電位為+20~+30 m V,平均直徑分布為200~400 nm。AP/ASF/PEI/p DNA復(fù)合物表面Zeta電位分為+15~+25m V,平均直徑分布為400~600 nm。(AP+PEI)共包被法和AP/ASF/PEI層層包被法均能介導(dǎo)質(zhì)粒p DNA成功轉(zhuǎn)染EA.hy926細(xì)胞,并表現(xiàn)出較高的轉(zhuǎn)染效率和較低的細(xì)胞毒性。AP/ASF/PEI層層包被法的轉(zhuǎn)染效率略高于(AP+PEI)共包被法。采用冷凍干燥法將(AP+PEI)/p DNA((30+10)/2)和AP/ASF/PEI/p DNA(45/45/10/2)復(fù)合物裝載到絲素支架中,制備基因活性絲素支架。復(fù)合物在支架內(nèi)分散良好,細(xì)胞在該支架內(nèi)部正常粘附和生長。(AP+PEI)/p DNA((30+10)/2)和AP/ASF/PEI/p DNA(45/45/10/2)復(fù)合物能成功將VEGF165-Ang-1雙基因共表達(dá)質(zhì)粒導(dǎo)入目的細(xì)胞,導(dǎo)入的基因能正常地表達(dá)并分泌VEGF,且隨時(shí)間的延長VEGF分泌量顯著增多。該基因活性絲素支架體外能轉(zhuǎn)染雞胚尿囊膜上的細(xì)胞,并刺激其生成豐富的血管,且血管形態(tài)呈多分支的彌漫放射狀,血管數(shù)目及大小都明顯高于未裝載基因活性物質(zhì)的絲素多孔材料組。進(jìn)一步,將該絲素基因活性支架植入SD大鼠背部全層皮膚缺損創(chuàng)面,體內(nèi)實(shí)驗(yàn)結(jié)果顯示該支架炎癥反應(yīng)輕微,能良好地與周圍組織融合。周圍組織及真皮修復(fù)細(xì)胞遷入材料內(nèi)部,支架可引導(dǎo)新生組織的長入和毛細(xì)血管形成,并促進(jìn)表皮成活。隨著修復(fù)時(shí)間的延長,支架內(nèi)新生毛細(xì)血管和膠原纖維的生成量明顯比單純絲素多孔支架內(nèi)多。在創(chuàng)面修復(fù)早期,該基因活性支架內(nèi)VEGF、Ang-1、CD31、α-SMA、PDGF、b FGF和v WF因子的陽性表達(dá)率都比未裝載基因的絲素多孔支架高,進(jìn)一步說明裝載(AP+PEI)/p DNA((30+10)/2)和AP/ASF/PEI/p DNA(45/45/10/2)基因活性物質(zhì)的絲素支架,能夠原位轉(zhuǎn)染周圍細(xì)胞并表達(dá)和分泌目的基因?qū)?yīng)的VEGF和Ang-1因子,為組織修復(fù)細(xì)胞的遷移、粘附、生長及功能發(fā)揮創(chuàng)造良好的微環(huán)境,進(jìn)而促進(jìn)血管再生,進(jìn)而促進(jìn)真皮組織的修復(fù)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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R318.08
本文編號：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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R318.08
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