Study On Effect Of Carboxy-terminal Cell-binding Domain Of Fibronectin On Adhesion Of Bone Marrow Stromal Cells

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 FnCTD₆4, 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...