Preparation Of A Fusion Protein Aimed Collagen Under Endothelium And Its Effect On EPCs Homing Injured Vessel

BackgroundThe repair of injured vessel intima mainly relies on the local endothelium at the margin of the injured position and the endothelial progenitor cell in the circulation. The endothelial proliferation in close vicinity to the injured position usually promote its changes of phenotype and (or) dysfunction. But EPCs can become endothelium in the micro-environment in vivo and play its roles normally. So implantation of EPCs is one of the most perspective method in the area of treatment of injured vessel intima. But because there is lack of specific guided signal, the low gathering rate in the injured area of EPCs leads low efficacy of cell implantation. If some kind of molecular can guide EPCs to the injured vessel specifically, the efficacy of cell implantation can be boosted. It is known that platelet can cohere to the injured vessel specifically. If EPCs are indeed with this kind of special coherence ability through some kind of technology, we can solve the problem.The most important molecular inducing the coherence of platelets to sub-endothelial matrix is von Willebrand factor (vWF).And the key area of vWF relevant to the coherence is A3 domain (vWF A3).When the intima was damaged, A3 combined with the exposure collagen under the endothelium immediately, and then platelet was cohered by another domain of vWF, which is called A1. It is possible to EPCs possessing the coherence ability such as platelets if it has a A3 domain in the surface of cells.Expression of vWF A3 can be completed by gene transfection. The challenge is how to control the protein expressing in the surface of the cells, but not in the cells. The newly Imerging technology called protein tranfection provides strategy to solve this problem. The interest protein is expressed coalescing with lycosyl-phosphatidylinositol (GPI) by gene engineering technology, so it can be anchored at the surface of the cell membrane and do not cross over the double phospholipids Decker of membrane.It can be anticipated to gain EPCs with vWF A3 in the surface by this technology and find a good method to promote the orient moving of EPCs to the injured intima.ObjectiveThe purpose of this study was to gain EPCs with vWF A3 in the surface, which can cohere with the collagen under the injured endothelium specifically and to search if it has any efficacy on orient moving and recovery potency.Method1. Plasmid vetor of vWF A3 fusion protein which contained GPI structure expressing in eukaryotic cells was constructed.The A3 domain of von Willebrand factor and exon of GPI signal peptide were gained respectively by RT-PCR. And then FLAG tag was added into the upstream of GPI code by PCR. After cut and ligated by enzyme, vWF A3 code and GPI code were inserted into the plasmid vector pcDNA3.1(-) simultaneously.2. Recombination protein rGPI- vWF A3 was expressed and purified.The plasmid pcDNA3.1(-)-vWF A3-GPI was transfected into CHO cells with aid of lipotransfection method. After G418 screening, positive clones were chosen by FCM. Immunofluorescence laser confocal microscopy was use to observed these cells after crawled on the slide. PI-PLC was used for treating these cells, and the changes of the fusion protein on the surface of the cells were detected after treatment. Protein was purified by immunoaffinity chromatography, and assessed by SDS-PAGE and Western blot.3.Bioactivity of the recombination protein vWF A3-GPI was analyzed.Binding activity of recombination protein vWF A3-GPI conjunction with collagen was analyzed with ELISA. Anchoring effect of vWF A3-GPI to EPC membrane was also analyzed by FCM. After modified by vWF A3-GPI, the ability of proliferation and conjunction of EPC was analyzed with method of CCK8.4.The distribution of implanting EPCs/vWF A3 was quantitative analyzed in the animal model of injury of carotid artery.The distribution of implanting EPCs/vWF A3 was quantitative analyzed in the animal model of injury of carotid artery by both 3H-TdR marker and GFP marker. Thus, we could detect that if EPC modified by vWF A3 could promote recruiting to the area of injured arterial.5.Promoting effect on vascular repair was detected . The reendothelialization of injured arterial was observed by staining of Evan Blue. The thickness and area of neointima, the area of primitive lumina, the area of narrow lumina were observed by staining of HE.Result1.There were two florescence traps of total RNA product from human endothelial cells and human MNCs, which contained 670bp and 150bp respectively.2.The recombinant plasmid pcDNA3.1(-)-vWF A3-GPI was analyses by enzyme cutting which confirmed that the length of inserted fragment was 80bp. Compared with the sequencing by Blast, the gene sequence was correct and the tag of FLAG was added, which could be used for the next experiment of expression and purification.3.The recombined plasmid was transfected to CHO cells and a high expression clone was obtained by G418. Its positive rate was dramatically decline from 99.95% to 5.7%; There was a 60KD fusion protein in Western blot analysis. It was mainly expressed on cell surface observed by immunofluorescence laser confocal microscopy. The reslut of SDS-PAGE showed that its molecular mass was 60KD and had a FLAG marker.4.The best incubation time and concentration was 2h and 400ng/ml decided by the ELISA test. After 12h, the incorporation rate of fusion protein was still above 89%. After anchored, EPCs could bind collagen and digested PI-PLC.5.The proliferation ability of EPCs was not affected by anchoring. After injected into test animals, EPCs could enhance reendothelialization and the endothelialization rate was elevated from 20.3±2.1% to 85.7±5.1. The percentage ratio of cell contribution (injured vessel/normal vessel) was elected from 4.0 to 27.4. Green cells could be observed by fluorescence in the injured site after transplantation. The extent of hyperplasia degraded from 75±19.3% to 21±3.6%.Conclusion1.GPI-signal gene sequence was successfully cloned from the DNA obtained from human MNCs and vWF A3 gene was cloned from RNA obtained from human endothelial cell, the plasmid vector pcDNA3.1(-)-vWF A3-GPI was also constructed successfully.2.Fusion protein vWF A3-GPI was expressed by transfected CHO cells and purified immunoaffinity chromatography. It's molecular was 60KD. The fusion protein was mostly expressed on cell surface and was digested by PI-PLC from cell membrane. When expressing fusion protein, CHO can bind collagen.3.With the protein transfer technique, vWF A3-GPI fusion protein was stably anchored on the surface of EPCs, and the surface protein showed its biological activities of collagen conjunction.4. EPCs could orient to injured sites, that were helpful to promote endothelium recovery of injured carotid artery. Anchored by vWF A3-GPI, the ratio of EPCs homing to injured vascular could elevate and these EPCs could promote endothelium recovery and inhibit hyperplasia of intima in mice.5.The study found a new approach to propare a'homing molecule-anchoring'EPCs with protein transfer technique. The results provided experimental evidence to support the feasibility and effectiveness of this novel approach.