Method for cell adhesion and wound healing
a cell adhesion and wound healing technology, applied in the field of cell adhesion and wound healing, can solve the problems of unclear cell biological and molecular biological mechanisms of wound healing, and the expression of ig-h3 in normal dermal tissues and dermal wounds has not yet been firmly established, and achieve the effect of blocking cell adhesion
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example 1
Identification of Cell Adhesion Activity of RGD-Independent βig-h3 Proteins
[0062]1-1: Production of Recombinant βig-h3 Protein
[0063]In order to find the domains of βig-h3 which have, in practice, cell adhesion and spreading activity, the C-terminal terminal sequence Arg-Gly-Asp (RGD), known as a ligand recognition sequence for several integrins, was examined for effect on the cell adhesion property of βig-h3. In this regard, an RGD-deleted recombinant βig-h3 protein (βigh3-ΔRGD) and a wild-type recombinant βig-h3 protein (βigh3-WT) were prepared.
[0064]First, the full-length human βig-h3 cDNA cloned in pBluescript (pBsβig-h3) was digested with NdeI and BglII. The DNA fragment was subcloned into the EcoRV-EcoRI site of pET-29b(+) (Novagen Inc.). βigh3-WT was prepared by introducing a 1351 bp NcoI fragment excised from βig-h3 cDNA into the NcoI site of this clone. βigh3-ΔRGD was derived from βigh3-WT by cutting out a 3′-fragment of the βigh3-WT plasmid with AoCI and NotI followed by bl...
experimental example 1
Identification of Cell Surface Receptor of βig-h3 Involved in Cell Adhesion Activity of βig-h3
1-1: Identification of Cell Adhesion Activity Using Matrix Peptide and Reagent
[0071]In order to identify cell surface receptors involved in the cell adhesion activity of βig-h3 protein, an inhibition assay was performed using various reagents.
[0072]Initially, plastic culture dishes were coated with 10 μg / ml fibronectin, βigh3-WT or βigh3-ΔRGD. HCE cells were preincubated for 30 min in media containing 5 mM EDTA, 100 μg / ml βigh3-WT, 100 μg / ml igh3-ΔRGD, 1 mM RGD, 1 mM RGE or 100 μg / ml fibronectin, or none of them, and then assayed for cell adhesion as in Example 1.
[0073]Cell adhesion to βig-h3 was significantly inhibited by βig-h3 itself, RGD peptide and EDTA, and partially inhibited by fibronectin and EGTA, while being not inhibited by RGE peptide. Cell adhesion to fibronectin was also significantly inhibited by fibronectin itself, RGD peptide and EDTA, and partially inhibited by βig-h3 and...
example 2
Identification of Domains Essential to Cell Adhesion Activity of βig-h3
[0081]In an attempt to identify essential amino acids conferring cell adhesion activity of βig-h3, an examination was made to determine whether each repeat domain is capable of mediating cell adhesion.
[0082]Four recombinant proteins corresponding respectively to four repeat domain were prepared: four βig-h3 cDNA fragments encoding amino acids 129-241, 237-377, 368-506, and 498-637, respectively, were amplified by PCR and cloned into the EcoRV-XhoI site of pET-29b(+) and the resulting four expression vectors, named pβig-h3 D-I, pβig-h3 D-II, pβig-h3 D-III, and pβig-h3 D-IV, were used to prepare the recombinant proteins, as shown in FIG. 7. E. coli transformants with the expression vectors pβig-h3 D-II and pβig-h3 D-IV were designated E. coli BL21 / Hisβ-g and E. coli BL21 / Hisβ-e and deposited in the Korean Collection for Type Culture of Korea Research Institute of Bioscience and Biotechnology (KRIBB) with accession ...
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