發(fā)布時(shí)間：2018-06-06 17:49

  本文選題：基質(zhì)細(xì)胞衍生因子-1α + 聚電解質(zhì)多層膜　； 參考：《上海交通大學(xué)》2014年博士論文

【摘要】：第一部分大鼠脫細(xì)胞主動(dòng)脈帶瓣管道的構(gòu)建目的:瓣膜替換手術(shù)是目前對(duì)于心臟瓣膜疾病最為有效的治療手段。但臨床上所使用的人工瓣膜在生物相容性或耐久性方面存在著一定的缺陷。而組織工程瓣膜(TEHV)具有自我修復(fù)、重構(gòu)以及在體內(nèi)生長(zhǎng)的優(yōu)點(diǎn),有可能成為傳統(tǒng)人工瓣膜的替代者。本研究主要比較了不同脫細(xì)胞方法對(duì)于大鼠主動(dòng)脈帶瓣管道的脫細(xì)胞效果以及保護(hù)細(xì)胞外基質(zhì)的能力,以尋求更好的辦法來(lái)制備組織工程瓣膜支架。材料和方法:無(wú)菌獲取大鼠主動(dòng)脈帶瓣管道,隨機(jī)分為四組,根據(jù)脫細(xì)胞方案不同分為0.25%Triton-X100+0.25%脫氧膽酸鈉(SD)組(TS組)、1%十二烷基磺酸鈉組(SDS組)、0.5%Triton-X100+0.5%SD+0.5%SDS(STS組),未脫細(xì)胞的帶瓣管道作為對(duì)照組。處理完成后,通過(guò)組織學(xué)HE染色評(píng)估脫細(xì)胞效果以及纖維結(jié)構(gòu)的保存情況,采用DNA含量測(cè)定定量分析核酸殘留情況,并確定最佳的脫細(xì)胞方案,最后通過(guò)掃描電鏡評(píng)估進(jìn)一步明確脫細(xì)胞效果以及纖維支架排列情況。結(jié)果:TS組以及SDS組帶瓣管道細(xì)胞成分去除不完全,纖維結(jié)構(gòu)破壞明顯,而STS組帶瓣管道脫細(xì)胞完全,纖維結(jié)構(gòu)得到很好地保留。結(jié)論:聯(lián)合應(yīng)用Triton-X100、SD以及SDS制備的大鼠脫細(xì)胞主動(dòng)脈帶瓣管道細(xì)胞成分去除完全,纖維結(jié)構(gòu)完整,為后續(xù)研究提供了良好的瓣膜支架材料。第二部分PEM修飾大鼠脫細(xì)胞主動(dòng)脈帶瓣管道及其體外相容性研究目的:脫細(xì)胞心臟瓣膜支架是構(gòu)建組織工程人工瓣膜的支架材料之一。然而脫細(xì)胞心臟瓣膜支架殘留的致凝性、免疫原性以及體內(nèi)重構(gòu)的緩慢導(dǎo)致其出現(xiàn)了早期衰敗。為了克服這些缺陷以及促進(jìn)再細(xì)胞化,構(gòu)建具有良好生物相容性以及使用壽命的組織工程瓣膜,我們采用肝素-SDF-1α聚電解質(zhì)多層膜(PEM)涂層大鼠脫細(xì)胞主動(dòng)脈帶瓣管道,并且體外檢測(cè)其血小板和細(xì)胞相容性。材料和方法:肝素以及SDF-1α交替dip-coating在供體的U型主動(dòng)脈帶瓣管道表面形成PEM。通過(guò)免疫熒光檢測(cè)明確肝素-SDF-1αPEM在帶瓣管道表面的包被。通過(guò)血小板粘附實(shí)驗(yàn)以及LDH測(cè)定評(píng)估其抗血小板功能。分離、培養(yǎng)大鼠骨髓間充質(zhì)干細(xì)胞(BMSCs),評(píng)估表面修飾對(duì)BMSCs粘附、增殖以及遷移能力的影響。結(jié)果:免疫熒光檢測(cè)顯示PEM包被在大鼠脫細(xì)胞帶瓣管道表面。體外研究表明肝素-SDF-1αPEM顯著降低了血小板在帶瓣管道上的粘附從而改善了管道的血小板相容性,同時(shí)BMSCs在帶管道上的粘附、增殖以及遷移能力也得到了顯著增強(qiáng)。結(jié)論:肝素-SDF-1αPEM能夠涂層在大鼠脫細(xì)胞帶瓣管道表面,經(jīng)過(guò)修飾的帶瓣管道表現(xiàn)出較好的血液相容性,同時(shí)BMSCs在管道上的粘附、增殖以及遷移能力也得到了明顯改善。第三部分PEM修飾大鼠脫細(xì)胞主動(dòng)脈帶瓣管道體內(nèi)再細(xì)胞化研究目的:我們之前的研究已經(jīng)證實(shí)了肝素-SDF-1αPEM修飾的大鼠脫細(xì)胞主動(dòng)脈帶瓣管道的生物相容性,而肝素-SDF-1αPEM修飾的脫細(xì)胞帶瓣管道的再細(xì)胞化特性尚不明確。在本部分研究中,我們將修飾后的脫細(xì)胞帶瓣管道移植到大鼠的腎下腹主動(dòng)脈,評(píng)價(jià)其體內(nèi)的功能以及再細(xì)胞化情況。材料和方法:體內(nèi)研究中,我們將包被以及未包被肝素-SDF-1αPEM的脫細(xì)胞帶瓣管移植到大鼠的腎下腹主動(dòng)脈。12只大鼠被分為兩組,PEM-DVC組(n=6):采用肝素-SDF-1αPEM修飾的脫細(xì)胞帶瓣管道進(jìn)行移植;Un DVC組(n=6):采用未包被的脫細(xì)胞帶瓣管道進(jìn)行移植。為了評(píng)估管道移植后的功能,我們?cè)谝浦残g(shù)后第1天以及4周時(shí)進(jìn)行超聲檢測(cè),同時(shí)在術(shù)后2周以及4周時(shí)進(jìn)行Micro-CT檢測(cè)。在帶瓣管道移植后4周時(shí)將移植的帶瓣管道取出,通過(guò)組織學(xué)以及免疫組織化學(xué)檢測(cè)明確纖維結(jié)構(gòu)的保存以及再細(xì)胞化情況。結(jié)果:不同時(shí)間點(diǎn)的超聲以及Micro-CT檢測(cè)結(jié)果顯示PEM-DVC組以及Un DVC組帶瓣管道移植后均保持通暢,無(wú)明顯血栓、狹窄以及動(dòng)脈瘤形成。移植4周后帶瓣管道組織學(xué)檢測(cè)顯示兩組帶瓣管道膠原以及彈力纖維結(jié)構(gòu)均完整。但是,熒光免疫組化結(jié)果表明PEM-DVC在體內(nèi)能夠更好的自體內(nèi)皮化并能夠促進(jìn)內(nèi)皮祖細(xì)胞在管道的募集。結(jié)論:肝素-SDF-1αPEM修飾脫細(xì)胞帶瓣管道能夠促進(jìn)管道內(nèi)膜層的再內(nèi)皮化,使得管道擁有理想的血液相容性以及生理功能,為組織工程瓣膜的構(gòu)建提供新的理論基礎(chǔ)。
[Abstract]:The construction of the first part of the rat aorta with the aortic valve: valve replacement is the most effective treatment for heart valve disease. However, there are some defects in the biocompatibility or durability of the prosthetic valve used clinically, and the tissue Cheng Banmo (TEHV) is self repairing, reconstructing, and reconstructing as well. The advantages of growth in the body may be a substitute for the traditional artificial valve. This study mainly compares the decellular removal efficiency of the aortic valve conduit and the ability to protect the extracellular matrix in rat aorta, in order to find a better way to prepare the tissue engineering valve scaffold. Materials and methods: aseptic acquisition of rat main. The arterial valve tube was divided into four groups randomly. According to the different cell schemes, they were divided into 0.25%Triton-X100+0.25% deoxycholic acid sodium (SD) group (group TS), 1% sodium alkyl sulfonate group (group SDS), 0.5%Triton-X100+0.5%SD+0.5%SDS (group STS), and no acellular valve tube as the control group. After the treatment was completed, the cell removal efficiency was evaluated by histological HE staining. The preservation of fruit and fiber structure, quantitative analysis of nucleic acid residue by DNA content, and the best method of cell removal. Finally, the effect of cell removal and the arrangement of fibrous scaffold were further clarified by scanning electron microscope. Results: the removal of cell components in group TS and SDS group was incomplete and the fibrous structure was destroyed. It was found that the STS group was completely deformed and the fiber structure was well preserved. Conclusion: the combined use of Triton-X100, SD and SDS for the removal of the cell components of the rat aorta with the aortic valve was complete and the fiber structure was complete, which provided a good membrane scaffold material for the follow-up study. Second parts of the PEM modified rat were the main decellular host. Objective to study the compatibility of arterial valved conduit and in vitro Objective: the acellular heart valve scaffold is one of the scaffolding materials for the construction of tissue engineered prosthetic valves. However, the early decay of the decellular cardiac valve scaffold remains caused by the deactivation, immunogenicity, and the slow reconstruction of the body. Cytochemistry, construction of tissue engineering valve with good biocompatibility and life expectancy, we used heparin -SDF-1 alpha polyelectrolyte multilayer film (PEM) to coat the rat aortic valve ducts, and in vitro test its platelet and cell compatibility. Materials and methods: hepatin and SDF-1 alpha alternating dip-coating in the donor's U type initiative PEM. was detected by immunofluorescence to determine the inclusion of heparin -SDF-1 alpha PEM on the surface of the petal tube by immunofluorescence. The platelet adhesion test and LDH assay were used to evaluate the antiplatelet function. The rat bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured to evaluate the effect of surface repair on the adhesion, proliferation and migration of BMSCs. Results: the immunofluorescence test showed that the PEM packet was on the surface of the rat decellular valve tube. In vitro study showed that heparin -SDF-1 alpha PEM significantly reduced the adhesion of platelets on the valve tube and improved the platelets compatibility of the pipes. Meanwhile, the adhesion, proliferation and migration ability of BMSCs in the pipeline were also significantly enhanced. The heparin -SDF-1 alpha PEM can be coated on the surface of the rat decellularized petal tube, and the modified band tube shows good blood compatibility. At the same time, the adhesion, proliferation and migration of BMSCs on the pipe have also been obviously improved. Third the aim of the re cell study of the main artery petal conduit of the PEM modified rats: Our previous study has demonstrated the biocompatibility of the heparin -SDF-1 alpha PEM modified rat aortic valved conduit, while the re cell characteristics of the heparin -SDF-1 alpha PEM modified decellular valve tube are not clear. In this study, we transplanted the modified deacellular ducts to the lower abdominal initiative of rats. Pulse, evaluation of the function and re cell situation in the body. Materials and methods: in the study in vivo, we were divided into two groups,.12 rats,.12 rats were divided into two groups, group PEM-DVC (n=6): heparin -SDF-1 alpha PEM modified acellular conduit. Un DVC group (n=6): transplantation of uncovered acellular conduit. In order to evaluate the function of the tube after transplantation, we performed ultrasound examination at first and 4 weeks after the transplantation, and performed the Micro-CT test at 2 and 4 weeks after the operation. The graft conduit was removed at 4 weeks after the valve transplantation. The results of ultrasound and Micro-CT detection at different time points showed that the PEM-DVC group and the Un DVC group were all smooth after the tube transplantation, and there was no obvious thrombus, stenosis and the formation of aneurysm. 4 weeks after transplantation, the histological examination of the petal pipe showed that two The results showed that PEM-DVC could improve endothelialization in the body and promote the recruitment of endothelial progenitor cells in the pipeline. Conclusion: heparin -SDF-1 alpha PEM modification of decellular ducts can promote the re endothelialization of the tube intima and make the pipeline own The ideal blood compatibility and physiological function provide a new theoretical basis for tissue engineering valve construction.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R654.2;R318.11

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 史嘉瑋;董念國(guó);孫宗全;;Immobilization of Decellularized Valve Scaffolds with Arg-Gly-Asp-containing Peptide to Promote Myofibroblast Adhesion[J];Journal of Huazhong University of Science and Technology(Medical Sciences);2009年04期

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本文編號(hào)：1987591