| Cardiovascular disease（CVDs）is the leading cause of death in humans.For coronary artery bypass surgery in these patients,small-diameter tissue-engineered blood vessels（TEBVs）,especially those less than 6mm in diameter,are in great clinical demand.Due to the lack of functional endothelium,small-caliber TEBVs are prone to thrombosis,chronic inflammation and endometrial hyperplasia after transplantation,leading to failure of transplantation.Endothelium,as the most important component of vascular function,plays an important role in maintaining long-term vascular patency.Therefore,rapid endothelialization of TEBVs is an important strategy to inhibit post-transplant thrombosis,chronic inflammation and endometrial hyperplasia.After TEBV implantation,monocytes were recruited to the graft site and transformed into macrophages,which are involved in angiogenesis.Using the physiological functions of macrophages,a cellular bioreactor was constructed in TEBV to transform macrophages through the delivery and transfection of microRNA（miR）mediated by graphene quantum dots（GQD）.Rapid endothelialization of TEBVs can be achieved by the continuous secretion of vascular endothelial growth factor（VEGF）and the promotion of endothelial cell migration（ECs）by the modified macrophages in the vascular transplantation area.The main research contents and conclusions of this paper are as follows:（1）Biocompatibility test of GQDs.The morphology of GQDs was detected by transmission electron microscopy（TEM）,and the results showed that the diameter of GQDs was about 50-100 nm.The results of infrared spectrum showed the existence of amino functional groups in GQDs-NH2.CCK-8 cell activity assay and flow apoptosis assay were used to test the toxicity of GQDs on macrophages and endothelial cells.The optimum concentration of GQDs was found.（2）The delivery,transfection and protection of miR-150 by GQDs.The uptake of GQDs by endothelial cells,smooth muscle cells and macrophages was detected and compared by transmission electron microscopy（TEM）in vitro,which confirmed the phagocytosis of GQDs by macrophages.Endothelial cells and smooth muscle cells were not found.After the cross linking of miR150 with GQDs through sulfo-LC-SPDP,macrophages were transfected in vitro with fluorescently labeled GQDs-miR150-cy3.Fluorescence confocal assay showed that the transfection rate of GQDs on macrophages was about 70%.In addition,miR are easily degraded during delivery in vivo.GQDs-miR150-cy3 was cross-linked to collagen slides,and RNase containing PBS was used to simulate blood flow scour in parallel plate flow cavity experiment,so as to detect the anti-blood flow scour effect of GQDs-miR150 system.Experimental results showed that GQDs could protect miR-resistant blood flow erosion.The results of qPCR tested about co-incubation of RNase and GQDs-miR150-cy3 indicated that GQDs had a protective effect on the loaded miRs.After delivery,miR150 exerts biological regulation on macrophages.The results of WB、Elisa and Transwell analysis showed that miR150 could promote the secretion of VEGF and promote the migration of endothelial cells by increasing the expression of HIF-1α.（3）Construction of GQDs-miR150-modified TEBVs with bioreactors.Acellular matrix vascular scaffolds were prepared from the carotid arteries of normal rats,and GQDs-miR150-cy3 was modified to the inner and outer surfaces of the scaffolds.The results of HE staining and masson staining showed that there were no cells in the acellular vascular stent,and the main component was collagen.Fluorescence imaging results showed that GQDs-miR150-cy3 was uniformly modified on both the inner and outer surfaces of blood vessels.After carotid artery transplantation in rats,the transplanted blood vessels were removed 3 days,7 days,14 days and 30 days after transplantation,respectively,and were observed by scanning electron microscopy and stained with HEstaining and CD31immunofluorescence staining in frozen sections.The results of HEstaining and CD31immunofluorescence staining showed that GQDs-miR150-modified TEBVs had endothelial growth on the 7 day after transplantation,and a relatively complete endothelium was formed 30 days after transplantation.At 90 days after transplantation,CT results of small animals showed that GQDs-miR150-modified TEBVs showed a higher patency rate,and HE staining and immunofluorescence staining showed peripheral vascular trophoblastic vessels and nerve growth. |
