本文選題：心血管植入生物材料 + 抗凝血　； 參考：《西南交通大學》2012年博士論文

【摘要】：心血管植入材料面臨的最主要的問題是凝血和血栓發(fā)生,因此提高這類生物材料的生物相容性是非常重要和有意義的。目前,通過物理或者化學的方法在材料表面固定抗凝和促內(nèi)皮細胞粘附的生物分子已經(jīng)被證實能夠較好的抑制血栓發(fā)生和促進內(nèi)皮化,但是多數(shù)研究都集中在改善血液相容性或者加速內(nèi)皮化的某一方面,還很少有同時兼顧抗凝和促內(nèi)皮化表面構(gòu)建的研究,而且基本未見有同時兼顧多種生物學功能表面構(gòu)建的報道。 本文選擇具有較好生物相容性的Ti作為改性基礎(chǔ),主要采用兩種不同的生物化修飾方法在其表面同時固定具有抗凝和促內(nèi)皮化的生物分子：肝素和纖連蛋白。首先研究了Ti表面肝素和纖連蛋白的層層自組裝(LBL),并對自組裝膜的生物相容性進行了評價；其次,在此基礎(chǔ)上,發(fā)展出了單分子自組裝肝素和纖連蛋白的方法,借助APTE硅烷化單層以及靜電吸引力的作用,在Ti表面構(gòu)建肝素/纖連蛋白(Hep/Fn)復合生物膜,并對實驗條件進行優(yōu)化。研究了在不同pH (pH7, pH4)以及在有無EDC催化下的Hep/Fn復合生物膜的穩(wěn)定性和生物功能性,包括血液相容性、內(nèi)皮細胞相容性、炎癥相容性以及抗平滑肌細胞增生性能。最后對所構(gòu)建的Hep/Fn復合生物膜的生物學功能的機理進行了探討。綜合采用石英晶體微天平(QCM-D)、水接觸角分析、傅立葉變換紅外光譜(FTIR)、酶聯(lián)免疫吸附實驗(ELISA)、免疫熒光染色分析、掃描電子顯微鏡(SEM)、原子力顯微鏡(AFM)、X射線光電子能譜(XPS)等方法對Hep/Fn復合膜的制備過程、生物學活性、成分和性質(zhì)分別進行了定性和定量表征；通過血小板粘附和激活實驗、凝血時間(APTT、PT)實驗、纖維蛋白原吸附和變性檢測、內(nèi)皮細胞培養(yǎng)實驗、巨噬細胞和細胞因子檢測實驗以及平滑肌細胞培養(yǎng)實驗,全面評價了所構(gòu)建的復合膜的生物相容性。并通過體外PBS浸泡法以及流動腔實驗對制備的復合膜的穩(wěn)定性以及血小板粘附和內(nèi)皮細胞附著力進行了初步評價；最后綜合借助酶聯(lián)免疫吸附法、電化學法和等溫滴定量熱的方法對相關(guān)機理問題進行了初步研究。全文主要結(jié)果如下： 1.肝素和纖連蛋白可以LBL方式組裝在Ti表面且具有一定的穩(wěn)定性和較好的血液相容性。但是Hep/Fn自組裝膜的內(nèi)皮細胞相容性略差,可能是因為多層膜中肝素的釋放所致。LBL構(gòu)建的多層膜中生物分子釋放的控制對于其生物學功能的實現(xiàn)是重要的決定條件。 2.獲得了血液相容性和內(nèi)皮細胞相容性均較好的pH4體系下構(gòu)建的Hep/Fn復合表面。定量及定性表征顯示,盡管肝素和纖連蛋白在不同構(gòu)建體系(pH7,pH4,EDC/NHS)下樣品表面的量無顯著差異,但是pH4下肝素結(jié)合ATIII的能力最好,纖連蛋白細胞結(jié)合位點(RGD)的暴露最多,因此復合物中的生物分子均保持了較好的生物活性,這也是導致后續(xù)生物學實驗結(jié)果差異的主要原因之一。 3.在pH4條件下形成的Hep/Fn復合膜具有較好的穩(wěn)定性和生物活性。流動狀態(tài)的下證實此復合生物膜具有較好的血液相容性,且表面粘附的內(nèi)皮細胞具有較高的穩(wěn)定性,這為將來的體內(nèi)應用提供了重要的參考依據(jù)。 4.培養(yǎng)內(nèi)皮細胞和巨噬細胞后的Hep/Fn修飾的樣品上的細胞因子表達(TNF-a, MCP-1和IL-1B)要比純Ti上面的低,共固定Hep/Fn復合物可以減少巨噬細胞的粘附量,從而可以明顯的提高材料的抗炎癥性能；而且,Hep/Fn復合物可以有效的抑制平滑肌細胞的遷移和增殖,從而抑制植入材料上的內(nèi)膜增生,但是卻不會影響內(nèi)皮細胞的粘附和生長。研究發(fā)現(xiàn),Hep/Fn復合物促進內(nèi)皮化和抑制平滑肌細胞增生的機制不同,其機制跟肝素結(jié)合到纖連蛋白上導致纖連蛋白構(gòu)象變化有關(guān)。 5.綜合利用酶聯(lián)免疫法、電化學和等溫滴定量熱法研究了不同pH體系下構(gòu)建的Hep/Fn表面的肝素和纖連蛋白的生物學活性,并對兩種分子的相互作用進行了分析,對樣品表面吸附血漿蛋白行為進行了綜合分析和比較,同時也研究了肝素和纖連蛋白在溶液中的相互作用過程。獲得了對前期不同pH條件下Hep/Fn復合物不同生物學行為較好的解釋,對于深入了解不同pH條件下的Hep/Fn表面的不同生物學行為具有十分重要的參考意義和價值。
[Abstract]:The most important problems faced by cardiovascular implant materials are coagulation and thrombosis, so improving the biocompatibility of such biomaterials is very important and meaningful. At present, biomaterials with physical or chemical methods to immobilization anticoagulant and promote endothelial cell adhesion on the surface of the material have been proved to be able to inhibit the blood. Thrombus occurs and promotes endothelialization, but most studies focus on some aspects of improving blood compatibility or accelerating endothelialization. There are few studies that have both anticoagulant and endothelialization surface construction, and there have been no reports of various biological functional surface construction at the same time.
In this paper, we chose Ti with good biocompatibility as a modification basis, mainly using two different biological modification methods to immobilization of anticoagulant and endothelialized biomolecules on the surface of them: heparin and fibronectin. First, the layer self assembly (LBL) of heparin and fibronectin on the surface of Ti was studied, and the biology of self assembled monolayers was studied. The compatibility was evaluated. Secondly, a single molecule self-assembled heparin and fibronectin were developed on this basis. With the help of APTE silane monolayer and electrostatic attraction, heparin / fibronectin (Hep/Fn) composite biofilm was constructed on the Ti surface, and the experimental conditions were optimized. The study on different pH (pH7, pH4) and at the same time was carried out. The stability and bioactivity of the Hep/Fn composite biofilms with or without EDC catalysis, including blood compatibility, endothelial cell compatibility, inflammatory compatibility and anti smooth muscle cell proliferation. Finally, the mechanism of the biological function of the constructed Hep/Fn composite biofilm was discussed. Quartz crystal microbalance (QCM-D), water was used. Contact angle analysis, Fu Liye transform infrared spectroscopy (FTIR), enzyme linked immunosorbent assay (ELISA), immunofluorescence staining, scanning electron microscopy (SEM), atomic force microscopy (AFM) and X ray photoelectron spectroscopy (XPS) were used to characterize and quantitatively characterize the biological activity, composition and properties of the Hep/Fn composite membranes, respectively. Through platelet adhesion and activation experiments, coagulation time (APTT, PT) experiments, fibrinogen adsorption and denaturation detection, endothelial cell culture experiment, macrophage and cytokine detection experiments, and smooth muscle cell culture experiments, the biocompatibility of the composite membrane was evaluated comprehensively. In addition, the PBS immersion method and the flow chamber were used in vitro. The stability of the composite membranes and the platelet adhesion and adhesion of endothelial cells were preliminarily evaluated by the experiment. Finally, the related mechanism problems were preliminarily studied with the help of enzyme linked immunosorbent assay, electrochemical method and isothermal titration calorimetry. The main results are as follows:
1. heparin and fibronectin can be assembled on the surface of Ti with LBL and have a certain stability and good blood compatibility. But the compatibility of the endothelial cells of the Hep/Fn self assembled membrane is slightly poor. It may be because the release of biomolecules in the multilayer membrane constructed by the release of heparin in the multilayer membrane is the result of the realization of its biological function. It is an important determinant.
2. the Hep/Fn composite surface constructed under the pH4 system with good blood compatibility and endothelial cell compatibility was obtained. Quantitative and qualitative characterization showed that although heparin and fibronectin had no significant difference on the surface of the samples under different construction systems (pH7, pH4, EDC/NHS), the ability of heparin to bind ATIII under pH4 was the best, and fibronectin cell junction was the best. The RGD is exposed most, so the biomolecules in the complex maintain good biological activity, which is one of the main reasons for the differences in the results of subsequent biological experiments.
3. the Hep/Fn composite membrane formed under the condition of pH4 has good stability and biological activity. The flow state confirms that the composite biofilm has good blood compatibility and the surface adhered endothelial cells have high stability. This provides an important reference for future application in vivo.
4. the expression of cytokine expression (TNF-a, MCP-1 and IL-1B) on Hep/Fn modified samples after cultured endothelial cells and macrophages is lower than that on pure Ti. Co immobilization of Hep/Fn complex can reduce the adhesion of macrophages, which can obviously improve the anti-inflammatory properties of the material; moreover, the Hep/Fn complex can effectively inhibit the smooth muscle of the smooth muscle. The migration and proliferation of cells inhibit intimal hyperplasia on the implanted material, but it does not affect the adhesion and growth of endothelial cells. It is found that the mechanism of the Hep/Fn complex promotes endothelialization and inhibits the proliferation of smooth muscle cells. The mechanism is related to the conformation of fibronectin to the fibronectin, which is combined with heparin.
5. the biological activity of heparin and fibronectin on the surface of Hep/Fn under different pH systems was studied by enzyme linked immunosorbent assay, electrochemistry and isothermal titration calorimetry, and the interaction between the two molecules was analyzed. The adsorption of plasma protein on the surface of the sample was analyzed and compared, and the heparin and the heparin were also studied. The interaction process of fibronectin in the solution has obtained a good explanation for different biological behavior of Hep/Fn complexes under different pH conditions. It is of great reference significance and value for understanding the different biological behavior of Hep/Fn surface under different pH conditions.
【學位授予單位】：西南交通大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R318.08

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