發(fā)布時(shí)間：2018-08-17 14:51

【摘要】：家蠶絲素蛋白具有優(yōu)良的生物相容性，其用作組織工程支架等生物醫(yī)用材料前景廣闊。但未經(jīng)交聯(lián)的絲素材料易溶于水，難以實(shí)際應(yīng)用。尋求一種使絲素蛋白材料的生物相容性和物理性能都較好，且可調(diào)控其生物降解速率的新的交聯(lián)手段，是絲蛋白生物材料領(lǐng)域的迫切需求。酪氨酸酶是一種含銅的金屬酶，，廣泛分布于微生物、動(dòng)植物及人體中，與生物體合成色素直接相關(guān)。本文用酪氨酸酶催化絲素大分子間的交聯(lián)反應(yīng)，制備再生絲素蛋白材料，對(duì)交聯(lián)絲素材料的結(jié)構(gòu)、物理性能以及體外酶降解行為進(jìn)行系統(tǒng)性的研究，并以絲素膜為載體進(jìn)行L929細(xì)胞體外培養(yǎng)，研究交聯(lián)絲素膜的細(xì)胞相容性。 首先，用流延法制備酪氨酸酶催化交聯(lián)的絲素膜。熱水溶失率的測(cè)試結(jié)果表明交聯(lián)后絲素膜的耐水性得到顯著提高，膜的交聯(lián)度隨著酪氨酸酶添加量的增加而有所增大。交聯(lián)膜的紅外吸收光譜顯示，在酪氨酸酶的催化作用下，絲素蛋白的酪氨酸殘基氧化為鄰苯醌，并與自由氨基發(fā)生分子間和分子內(nèi)的交聯(lián)。氨基酸分析結(jié)果表明，經(jīng)交聯(lián)后膜內(nèi)酪氨酸和賴氨酸含量減少，進(jìn)一步說明酪氨酸酶能夠催化絲素蛋白中的酪氨酸和賴氨酸發(fā)生相互作用從而有效地交聯(lián)絲素蛋白。X-射線衍射結(jié)果表明交聯(lián)絲素膜仍以無(wú)定形結(jié)構(gòu)為主，與未經(jīng)交聯(lián)的絲素膜相比，凝聚態(tài)無(wú)顯著變化。絲素膜經(jīng)酪氨酸酶催化交聯(lián)后，斷裂強(qiáng)度有所提高，斷裂伸長(zhǎng)率變化不明顯。 其次，用冷凍干燥法制備酪氨酸酶催化交聯(lián)的多孔材料。研究表明，經(jīng)酪氨酸酶催化交聯(lián)后，絲素多孔材料的熱水溶失率明顯降低。紅外吸收光譜、X-射線衍射和熱分析結(jié)果表明，交聯(lián)后的多孔材料以無(wú)定形結(jié)構(gòu)為主。交聯(lián)多孔材料的孔徑、孔隙率隨著絲素溶液濃度的增大和冷凍溫度的降低而減小。 再次，選擇膠原酶IA作為體外催化絲素蛋白材料水解的模型酶，研究不同交聯(lián)度的絲素膜的體外生物降解行為。結(jié)果表明，隨著絲素膜交聯(lián)度的增大，其降解速率顯著減緩，降解產(chǎn)物中的游離氨基酸含量減少。通過調(diào)節(jié)交聯(lián)度，可以調(diào)控絲素膜的生物降解速率。 最后，通過體外細(xì)胞培養(yǎng)研究了L929細(xì)胞在酪氨酸酶催化交聯(lián)絲素膜上的粘附、生長(zhǎng)和增殖狀態(tài)。用熒光倒置顯微鏡觀察了細(xì)胞生長(zhǎng)狀態(tài)，測(cè)定細(xì)胞黏附率，使用MTT比色法和考馬斯亮藍(lán)染色法測(cè)定細(xì)胞活力。結(jié)果表明交聯(lián)絲素膜能夠支持細(xì)胞的黏附、生長(zhǎng)和增殖，酪氨酸酶催化交聯(lián)膜與乙醇處理的純絲素膜上的細(xì)胞活力無(wú)顯著性差異。 通過本文的研究，為制備物理性能和生物相容性較好，且可調(diào)控其生物降解速率的絲素蛋白材料，提供了一種新的交聯(lián)手段。
[Abstract]:Silk fibroin protein has good biocompatibility and is widely used as biomedical materials such as tissue engineering scaffold. But the uncrosslinked silk fibroin material is soluble in water and difficult to be applied in practice. It is an urgent need in the field of silk protein biomaterials to seek a new cross-linking method which can make silk fibroin materials have good biocompatibility and physical properties and can regulate their biodegradation rate. Tyrosinase is a copper-containing metalloenzyme widely distributed in microorganisms animals and plants and human body and directly related to biosynthetic pigments. In this paper, regenerated fibroin materials were prepared by using tyrosinase to catalyze cross-linking reaction between fibroin macromolecules. The structure, physical properties and enzymatic degradation behavior of cross-linked fibroin materials in vitro were systematically studied. L929 cells were cultured with silk fibroin membrane in vitro to study the cytocompatibility of cross-linked fibroin membrane. Firstly, the fibroin membrane catalyzed by tyrosinase was prepared by casting method. The results of hot water solubilization test showed that the water resistance of silk fibroin membrane increased significantly after crosslinking, and the crosslinking degree of silk fibroin membrane increased with the increase of tyrosinase content. The IR absorption spectra of the cross-linked membrane showed that the tyrosine residue of silk fibroin was oxidized to o-benzoquinone under the catalysis of tyrosinase and cross-linked with free amino groups both intramolecular and intramolecular. The results of amino acid analysis showed that the contents of tyrosine and lysine decreased after crosslinking. It is further demonstrated that tyrosinase can catalyze the interaction between tyrosine and lysine in fibroin, thus effectively crosslinking fibroin. X- ray diffraction results indicate that the cross-linked fibroin membrane is still amorphous. Compared with the uncrosslinked fibroin membrane, there was no significant change in the condensed state. When the fibroin membrane was crosslinked with tyrosinase, the breaking strength increased and the elongation at break did not change obviously. Secondly, the porous materials catalyzed by tyrosinase were prepared by freeze-drying method. The results showed that the hot water solution loss rate of fibroin porous materials decreased obviously after the crosslinking catalyzed by tyrosinase. The results of X-ray diffraction and thermal analysis show that the cross-linked porous materials are mainly amorphous. The pore size and porosity of crosslinked porous materials decrease with the increase of fibroin concentration and the decrease of freezing temperature. Thirdly, collagenase IA was selected as the model enzyme to catalyze the hydrolysis of silk fibroin in vitro, and the biodegradation behavior of silk fibroin membrane with different crosslinking degree was studied in vitro. The results showed that with the increase of the crosslinking degree of silk fibroin film, the degradation rate of silk fibroin film decreased significantly, and the content of free amino acid in the degradation product decreased. The biodegradation rate of silk fibroin membrane can be regulated by adjusting the crosslinking degree. Finally, the adhesion, growth and proliferation of L929 cells on the membrane catalyzed by tyrosinase were studied by cell culture in vitro. Cell growth was observed by fluorescence inverted microscope and cell adhesion was measured. Cell viability was determined by MTT colorimetry and Coomassie brilliant blue staining. The results showed that the cross-linked fibroin membrane could support cell adhesion, growth and proliferation. There was no significant difference in cell viability between the cross-linked membrane catalyzed by tyrosinase and the pure fibroin membrane treated with ethanol. This study provides a new cross-linking method for the preparation of silk fibroin material with good physical properties and biocompatibility and which can regulate its biodegradation rate.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R318.08

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本文編號(hào)：2187992