重组纤维连接蛋白羧基端细胞结合域增强骨髓基质干细胞黏附力的实验研究
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摘要
组织工程心脏瓣膜的体外构建首先要将种子细胞成功地种植到瓣膜支架上,细胞与材料的相互作用是组织工程研究的主要领域之一,细胞必须与材料黏附,才能进行迁移、分化和增殖。目前所使用的生物材料有人工合成材料和天然材料,大多属于生物“隋性”材料,不能为种子细胞的附着和生长提供良好的生物界面。为了使种子细胞快速、牢固地黏附到支架上,需对材料进行表面改性。常用的方法有表面修饰法、化学改性法、等离子体法等。支架表面固定蛋白质进行表面修饰是目前最为常用的方法,其中物理吸附是最简单、易行的方法。
细胞外基质(extra cellular matrix,ECM)是由多种大分子成分形成的具有一定组织性的网络结构,主要包括胶原、蛋白多糖及非胶原糖蛋白。纤维连接蛋白(fibronectin,Fn)是ECM中重要的糖蛋白,它具有同时与ECM中多种成分相结合的特点,并促进ECM其他成分的沉积,而且Fn分子中的某些结构域能识别细胞表面的整合素受体,并与之结合,所以Fn是细胞黏附的桥梁和纽带,在细胞的黏附过程中起着至关重要的作用。但天然Fn存在来源不足、有传染性隐患、可引起免疫反应等缺点,另外Fn是大分子糖蛋白,由2335个氨基酸组成,采用基因转染、蛋白表达的方法技术难度巨大,获取几乎是不可能的,从而限制了其在组织工程研究中的广泛应用。重组纤维连接蛋白羧基端细胞结合域(FnCTD_(64))由607个氨基酸构成,是Fn分子中靠近羧基端的细胞结合位点,Fn有四个细胞结合位点,FnCTD_(64)中就含有三个,与Fn相比,它具有来源充足、无传染性、不引起免疫反应等特点。用FnCTD_(64)预涂心瓣膜支架是否也能增强种子细胞的黏附力,目前国内外尚未见报道。为此,我们以96孔板和去细胞猪主动脉瓣叶作为载体,预涂Fn、BSA及本实验室表达、纯化的重组蛋白FnCTD_(64),然后种植BMSCs,观察预涂蛋白对细胞黏附的影响,以寻找增强细胞黏附力的有效方法。
主要方法:
1.重组纤维连接蛋白羧基端细胞结合域(FnCTD64)真核表达:抽提人胎脑总RNA,使用RT-PCR技术扩增获得FnCTD64基因序列,构建表达FnCTD64蛋白的重组腺病毒Ad.FnCTD64。重组腺病毒h转染293细胞后,通过离子交换和分子筛纯化细胞
The cells are seeded on the scaffold is the first step of tissue engineering heart valves(TEHVs) constructed in vitro. The cells must adhesion on the scaffold firstly and then can migrate, differentiate and proliferate. Currently there are two kinds of biomaterial: artificial synthesized material and natural material. They can not provide appropriate interface to the cells to attach and grow. In order to promote cells adhesion, biomaterial surface need to be decorated, there are many techniques to do this, for example, modified surface method, chemical transforming method, plasma method et el. It is the most common method that the proteins are fixed onto the scaffold surface, physical attaching means is the simplest and easiest way.Extra cellular matrix is composed of many great molecules, included: collagens, proteoglycans, glycoproteins. Finbronectin is important glycoprotein, it can combine with many components in the ECM and boost other components to deposit, moreover some structure domains in Fn can recognize integrin receptors and combine with it. So Fn is like a bridge of cell adhesion and it is very important, carboxy-terminal cell-binding domain of fibronectin is made up of 607 amino acids and is the biding cell sites near the carboxy terminal. There are four biding cell sites in the Fn molecular, three of them is in the FnCTD_64, the function of the sites is to promote cells attaching and migrating. Objective: To study the methods promoting the adhesion of bone marrow stromal cells(BMSCs). The study try to prepare to construct tissue engineering heart valves in vitro. Methods:1. Expression and purification of recombinant carboxy-terminal cell-bindingdomain of fibronectin with eukaryotic gene expression system: The mRNA was isolated from human fetal brain using standard procedures. Then, FnCTD64 DNA was synthesized and amplified by RT-PCR. Thereafter, FnCTD64 DNA was implanted into cosmid vector pAxcAwt and constructed the recombinant adenovirus coding for FnCTD64 gene (Ad.FnCTD64). finally, recombinant FnCTD64 protein was purificated using
细胞外基质(extra cellular matrix,ECM)是由多种大分子成分形成的具有一定组织性的网络结构,主要包括胶原、蛋白多糖及非胶原糖蛋白。纤维连接蛋白(fibronectin,Fn)是ECM中重要的糖蛋白,它具有同时与ECM中多种成分相结合的特点,并促进ECM其他成分的沉积,而且Fn分子中的某些结构域能识别细胞表面的整合素受体,并与之结合,所以Fn是细胞黏附的桥梁和纽带,在细胞的黏附过程中起着至关重要的作用。但天然Fn存在来源不足、有传染性隐患、可引起免疫反应等缺点,另外Fn是大分子糖蛋白,由2335个氨基酸组成,采用基因转染、蛋白表达的方法技术难度巨大,获取几乎是不可能的,从而限制了其在组织工程研究中的广泛应用。重组纤维连接蛋白羧基端细胞结合域(FnCTD_(64))由607个氨基酸构成,是Fn分子中靠近羧基端的细胞结合位点,Fn有四个细胞结合位点,FnCTD_(64)中就含有三个,与Fn相比,它具有来源充足、无传染性、不引起免疫反应等特点。用FnCTD_(64)预涂心瓣膜支架是否也能增强种子细胞的黏附力,目前国内外尚未见报道。为此,我们以96孔板和去细胞猪主动脉瓣叶作为载体,预涂Fn、BSA及本实验室表达、纯化的重组蛋白FnCTD_(64),然后种植BMSCs,观察预涂蛋白对细胞黏附的影响,以寻找增强细胞黏附力的有效方法。
主要方法:
1.重组纤维连接蛋白羧基端细胞结合域(FnCTD64)真核表达:抽提人胎脑总RNA,使用RT-PCR技术扩增获得FnCTD64基因序列,构建表达FnCTD64蛋白的重组腺病毒Ad.FnCTD64。重组腺病毒h转染293细胞后,通过离子交换和分子筛纯化细胞
The cells are seeded on the scaffold is the first step of tissue engineering heart valves(TEHVs) constructed in vitro. The cells must adhesion on the scaffold firstly and then can migrate, differentiate and proliferate. Currently there are two kinds of biomaterial: artificial synthesized material and natural material. They can not provide appropriate interface to the cells to attach and grow. In order to promote cells adhesion, biomaterial surface need to be decorated, there are many techniques to do this, for example, modified surface method, chemical transforming method, plasma method et el. It is the most common method that the proteins are fixed onto the scaffold surface, physical attaching means is the simplest and easiest way.Extra cellular matrix is composed of many great molecules, included: collagens, proteoglycans, glycoproteins. Finbronectin is important glycoprotein, it can combine with many components in the ECM and boost other components to deposit, moreover some structure domains in Fn can recognize integrin receptors and combine with it. So Fn is like a bridge of cell adhesion and it is very important, carboxy-terminal cell-binding domain of fibronectin is made up of 607 amino acids and is the biding cell sites near the carboxy terminal. There are four biding cell sites in the Fn molecular, three of them is in the FnCTD_64, the function of the sites is to promote cells attaching and migrating. Objective: To study the methods promoting the adhesion of bone marrow stromal cells(BMSCs). The study try to prepare to construct tissue engineering heart valves in vitro. Methods:1. Expression and purification of recombinant carboxy-terminal cell-bindingdomain of fibronectin with eukaryotic gene expression system: The mRNA was isolated from human fetal brain using standard procedures. Then, FnCTD64 DNA was synthesized and amplified by RT-PCR. Thereafter, FnCTD64 DNA was implanted into cosmid vector pAxcAwt and constructed the recombinant adenovirus coding for FnCTD64 gene (Ad.FnCTD64). finally, recombinant FnCTD64 protein was purificated using
引文
1. Chirgwin, JM. Przybala AE, Mac Donald RJ, et al. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry, 1979, 18: 5294-5498.
2. Piotr C, Nicoletta S. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenoi-chloroform extraction. Biochem, 1987, 162: 156-159.
3. Miyake S, Makimuru M, Kanegae Y, et al. Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome. Proc Natl Acad Sci USA, 1996, 93: 1320-4.
4.吴乃虎 编著 基因工程原理上册.第二版.北京:科学技术出版社,1998:322.
5.F.奥斯伯 等著,颜子颖 等译.精编分子生物学实验指南.北京:科学技术出版社,1998:276.
6. Saito I, Oya Y, Yamamoto K, et al. Construction of nondefective adenovirus type 5 bearing a 2.8 kilobase hepatitis B virus DNA near the right end of its genome. J Virol, 1985, 54: 711-719.
7. Chinnadurai G, Chinnadurai S, Brusca J. Physical mapping of a large-plaque mutation of adenovirus type 2. J Virol, 1979, 32: 623-628.
8. Sharp PA, Moore C, Haverty JL. The infectivity of adenovirus 5 DNA-protein complex. Virology, 1976, 54: 442-456.
9. Saito I, Oya Y, Yamamoto K, et al. Construction of nondefective adenovirus type 5 bearing a 2.8 kilobase hepatitis B virus DNA near the right end of its genome. J Virol, 1985, 54: 711-719.
10. Lochmuller H, Jani A, Huard J, et al. Emergence of early region 1 conding replication-competent adenovirus in stocks of replicationg-defective adenovirus recombinants (delta E1+delta E3) during multiple passages in 293 cells. Hum Gene Ther. 1994, 5(12): 1485-1491.
11.师治贤,王俊德编著.《生物大分子的液相色谱分离和制备》(第二版),pp131—134。北京:科学出版社,1996。
2. Piotr C, Nicoletta S. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenoi-chloroform extraction. Biochem, 1987, 162: 156-159.
3. Miyake S, Makimuru M, Kanegae Y, et al. Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome. Proc Natl Acad Sci USA, 1996, 93: 1320-4.
4.吴乃虎 编著 基因工程原理上册.第二版.北京:科学技术出版社,1998:322.
5.F.奥斯伯 等著,颜子颖 等译.精编分子生物学实验指南.北京:科学技术出版社,1998:276.
6. Saito I, Oya Y, Yamamoto K, et al. Construction of nondefective adenovirus type 5 bearing a 2.8 kilobase hepatitis B virus DNA near the right end of its genome. J Virol, 1985, 54: 711-719.
7. Chinnadurai G, Chinnadurai S, Brusca J. Physical mapping of a large-plaque mutation of adenovirus type 2. J Virol, 1979, 32: 623-628.
8. Sharp PA, Moore C, Haverty JL. The infectivity of adenovirus 5 DNA-protein complex. Virology, 1976, 54: 442-456.
9. Saito I, Oya Y, Yamamoto K, et al. Construction of nondefective adenovirus type 5 bearing a 2.8 kilobase hepatitis B virus DNA near the right end of its genome. J Virol, 1985, 54: 711-719.
10. Lochmuller H, Jani A, Huard J, et al. Emergence of early region 1 conding replication-competent adenovirus in stocks of replicationg-defective adenovirus recombinants (delta E1+delta E3) during multiple passages in 293 cells. Hum Gene Ther. 1994, 5(12): 1485-1491.
11.师治贤,王俊德编著.《生物大分子的液相色谱分离和制备》(第二版),pp131—134。北京:科学出版社,1996。