The Study On The Transplantation Mechanism Of The Scaffold From Heterogeneous Small Vessel And The CD34～+ Cell Biological Specific Property

The study on the transplantation mechanism of thescaffold from heterogenous small vessel andthe CD34~+ cell biological specific propertyPrefaceVascular transplantation plays an important role in the domain of organtransplantation surgery and becomes an effective method in the therapy of vascu-lar disease. Many vascular disease patients have gained new life by autologousor homologous vascular transplantation. However, two factors are seriously im-peding the progress of the clinical application and extension of vascular trans-plantation:①The sum of autologous or homologous donor vessels is limited, e-ven influencing the blood supply of corresponding tissue and organ after somelarge donor vessels were cut out.②The toxicant and adverse effects of immuno-suppressive agent. For solving the shortage of donor vessels, people begin tostudy of heterogeneous donor. But after heterogeneous transplantation, acute im-munorejection is intense. How to reduce the immunogenicity of heterogeneousvessels to minimal level and lengthen the lifespan of the transplant in host body,has become a key deciding if the heterogeneous transplantation could success. Itis urgent for us to research and develop the ideal tissue-engineering blood ves-sel, which has extensive resource, with unobstructed, antithrombotic, histocom-patible, vigor, durable, operable and economical characteristics. There are twonecessary conditions in the reestablishment of engineering vessels:①vascularscaffold materials;②vascular seed ceils. Vascular scaffold materials can beclassified as natural scaffold and man-made polymer generally. The former areoften obtained directly from tissue and organ of mammals, and after specialtreatment, it can act as a scaffold for implanting seed cells to reestablishment ofengineering vessels. Receptor endothelial cells are often used as seed cells. But the culture of endothelial cells needs a long time and cultured endothelial cellsbecome aging quickly after subculture. The CD34~+ cells in bone marrow can dif-ferentiate into endothelial Cells. If implanting the CD34~+ cells into the heteroge-neous blood vessel scaffold, and making cell proliferation in the scaffold suc-cessfully, it would become possible to reestablishment of novel tissue-engineer-ing vessels. This research is mainly to discuss the feasibility of heterogeneoussmall blood vessel scaffold for experimental transplant application and the biolog-ical characteristics of the CD34~+ seed ceils, and to provide the experimental ba-sis for reestablishing tissue-engineering small blood vessels, as well as to de-velop a new way for future clinical blood vessel transplantation.Materials and Methods1. The select of experimental animal: We use ninety five Wistar rats, fe-male and male is not limited ,weighting 318.50g-588.81g, and sixty sevenJapanese white rabbit, female and male no limited, weighting 1.75kg-3.50kg.The experimental animals were divided into four groups:①normal controlgroup: 5 Wistar rats and 7 Japanese white rabbit②xenogenic scaffold from va-sotransplantation group: 45 Wistar rats③xenogenic small vessels transplanta-tion group: 45 Wistar rats④xenogenic host group: 60 Japanese white rabbit,each side of the ear is one case,total 120 cases.2. On the base of using routine tissue staining technique, TritonX-100shed cell intravascular stent method, Romeis elasticity fibers staining method,Mallory-Heidenhain collagen fibers staining method, De Rio-Horetga reticu-lar fiber staining method and electron microscopy were used to observe the endo-thelial cells and smooth muscle cells of correlate vessel wall and immunohisto-chemistry technique was utilized to detect antigenicity. After the treatment oftaking off ceils by tissue engineering technique, a natural scaffold from vesselwill be established.3. We use microsurgery vascular anastomosis technique, xenogenic scaffoldfrom vessel and xenogenic small vessels were anastomosed with parasitifer arteryby invaginate at proximal, end while the end to end anastomosis were perfomred in the distal end.4. We use affinity histochemistry method, to detect the expression of angio-cellular respond gene and the expression of CD34~+ cell and CD34 antigen. Thenusing CD34 antibody as a media, adhereing the CD34~+ cell in bone marrow andnatural scaffold from vessel, lastly a CD34~+ type natural scaffold from vessel isestablished.5. We apply cell culture technique by adding endothelial cell inductor tocultivate bone marrow cell. We can use the vessel to perform different speciesvascular transplantation.Result1. The tunica intima and tunica media of scaffold from vessel are spongi-ness, collagen fibers, elastic fiber and reticulate fiber are ranked like stereoreticulodromous. Collagen fibers are the essential component.2. After the transplantation of natural scaffold from vessel for 24 hours,blood vessel pulsation of Japanese white rabbit can be touched, without bloodoutleakage,α—galaclosyl residues can be detected on membranes of endotheli-al cell in central artery of Japanese white rabbit and in Wistar rat caudal artery.3. After the transplantation storey between donator blood vessel and acceptorblood vessel, immediately patency rate is 100％. Through 24 hours continuousobservation on postoperation blood current, the unobstructed time of xenogenicscaffold from vessel is 46 hours and 47 minutes, the unobstructed time of xeno-genic small blood vessel is 14 hours. 100 days after transplantation, xenogenicscaffold form vessel still keep its shape and structure.4. 10 min after the donate scaffold from vessel were transplanted to the re-ceptor blood vessel, the receptor blood vessel already has the expression of Egr-1, then goes to steady. The xenogenic small blood vessel still has the expres-sion of Egr-130 days after transplantation.5. The maximum diameter of endothelial cell is 8.32 1.04 m, The maxi-mum width is 5.79 0.68 m, The ratio of diameter and width is 1.45 0.22. Thedistance between two neighboring cells is 6.42 2.45 m, they are connected like a string-of-beads by line structure. The natural scaffold from vessel wall, isthin, velvet, white, lumina tumbling.6. The endomembrane line up much fiber, tunica media is mainly collagenfibers. CD34 can be detected in the marrow cell of Japanese white rabbit. Thereare silk like and cobweb shape structure in the nucleated cell of peripheral bloodof Japanese white rabbit.Conclusions1. Acellular tissue matrix of caudal artery of Wistar rats is used as a smallblood vessel scaffold in this experiment. The scaffold is mainly constructed bycollagen fiber, with elastic fiber and reticular fiber arranged in three-dimen-sional network.2. The expression ofα-Gal of endothelial ceils and smooth muscle cells ispositive in the central artery of Japanese white rabbit, as well as in the caudalartery of Wistar rat. The expression ofα-Gal is not only located in the cellmembrane, but also in the cell nuclei. In the caudal artery of Wistar rat, theexpression ofα-Gal is higher than that in the central artery of Japanese whiterabbit. Most importantly, the expression ofα-Gal can not be detected in the a-cellular tissue matrix.3. 100 days after transplantation, xenogenic scaffold form vessel were stillkeeping its shape and structure.4. The transplantation stomy between donator blood vessel and acceptorblood vessel, immediately patent rate is 100％. Through 24 hours continuousobservation on postoperation blood current, the unobstructed time of xenogenicscaffold from vessel is 46 hours and 47 minutes; the unobstructed time of xeno-genic small blood vessel is 14 hours. Xenogenic vessel scaffold transplantation isbetter than xenogenic small blood vessel transplantation.5. The thrombus of xenogenic scaffold from vessel and xenogenic smallblood vessel after transplantation is mainly mixed type thrombus. The recanaliza-tion would occur after operation, and gradually organize, granulation tissue e-merge. 6. Telescoping anastomosis method is better than conventional end-to-end anastomosis method for heterogeneous small vessel scaffold transplantation.7. After xenogenic small vessel scaffold transplantation or xenogenic smallblood vessel transplantation, the expression of Egr-1 soon exist in the recipientdistal-side artery as well as the xenogenic small blood vessel graft. But withoutexpression of Egr-1 can be found in xenogenic small vessel scaffold during 24hours of transplantation.8. After xenogenic small vessel scaffold transplantation, the expression ofEgr-1 changed from strong to weak in endothelial cell and contractile fiber cellsof the recipient distal-side artery of xenogenic scaffold. On the contrary, theexpression of Egr-1 increased gradually after small blood vessel transplanta-tion.9. There are much CD34~+ cells in the bone marrow of Japanese white rab-bit. According to the cell size, CD34~+ cell can be divided into large and smallgroups.10. In the marginal ear vein of Japanese white rabbit, some endothelial likecells are found. These cells are connected each other by filament structure. Thefilament structure might be a type of cell junction.11. After culture of nucleated cells isolated from peripheral blood of Japa-nese white rabbit, many cell clones formed. Between these clones, there aremany filaments with network arrangement. These structures might be relevant totransfer of information between cell clones.