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Anti-CD34 Antibody Surface Modification on Decellularized and Photooxidated Bovine Jugular Vein Matrix and It’s Reendothelialization

Author: ZhaoXiangDong
Tutor: HuTieHui
School: Central South University
Course: Surgery
Keywords: Acellular photo-oxidation Bovine jugular vein (BJV) Mouse anti-human CD34 antibody FITC-labeled goat anti- mouse IgG SANPAH The cattle jugular vein ( BJVC ) CD34 antibody Human umbilical vein endothelial cell line ( of HUVEC , CRL- 2480 ) Endothelialization In vitro In vivo
CLC: R392
Type: PhD thesis
Year: 2008
Downloads: 62
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Abstract


ChapterⅠFabrication and Preliminary Assessment of Mouse Anti-human CD34 Grafting onto Decellularized and Photooxidated Bovine Jugular Vein Matrix IntimaObjective:To investigate the suitable reactive concentration and ratio of photocrosslinking mouse anti-human CD34 onto the decellularized and photooxidated bovine jugular vein(BJV)matrix intima.Methods:BJV were decellularized and photooxidated,then divided into 16 groups randomly.The different concentration solutions of mouse anti-human CD34 and photochemical crosslinker SANPAH were prepared.The reaction between them were performed with 4 different mole ratios in 2 hours away from light.100μl reacted solution of them were dropped onto the prepared BJVC,then irradiated under 365nm ultraviolet ray for 5 minutes.The BJV were rinsed in a shaking machine for 6 hours.The fast frozen sections of the specimens were observed under the fluorescent microscope,after having been dropping goat anti-mouse IgG FITC-labeled.The brigtness of samples were measured and compared between two groups by using Image Pro Plus software.The grafting effect was viewed by different fluorescent brightness,so the suitable reactive concentration and ratio were educed.Results:The brighter mean and integrated fluorescence optical density on the side of endangium was viewed on the SANPAH photocrosslinking group.The higher the concentrations of mouse anti-human CD34 and SANPAH were,the brighter the mean and integrated fluorescence optical density was.But when the concentration of them was higher than 0.665×10-2mM,the difference of fluorescent brightness was not so clear.In the same concentration,the fluorescence was brightest if the reactive mole ratio between them was 1:20.Conclusion:mouse anti-human CD34 could be covalently coated onto the BJVC endangium by photochemical cross-linker SANPAH.The optimal concentration of mouse anti-human CD34 and SANPAH and reactive mole ratio between them were 0.665×10-2mM and 1:20 respectively. ChapterⅡReendothelialization on bovine jugular vein matrix modified with Mouse Anti-human CD34 in vitroObjective:To investigate the reendothelialization on bovine jugular vein matrix surface modified with mouse anti-human CD34 in vitroMethods:Decellularized and photooxidated bovine jugular vein was cut into 24 pieces of diameter 1.6cm,which were as large as the well in the 24-well culture plate.Mouse anti-human CD34 was photocrosslinked (mouse anti-human CD34 group)onto the surface of 12 BJVC pieces with the same procedure as the first chapter mentioned,12 rest untreated pieces as control.Human umbilical vein endothelial cells(HUVEC, CRL-2480)were implanted on these pieces with 5×105/ml density and cultured for 7 days.The cells were departed with enzyme from the surface of these pieces at 1st,3th,5th,7th,their numbers were counted.The slices of BJVC implanted with cells were HE stained and pictured.The difference between two groups was compared to verify whether BJVC modified with mouse anti-human CD34 could promote the cell adhesion and proliferation.Results:(1)Cells counting:The cells number in mouse anti-human CD34 group was more than control group at 1st,3rd,5th.7thday(p<0.05). (2)At 1stday,the cells arrayed intensively and not spreaded completely in each group’s surface.At 3rdday,the cells on BJVC surface decreased apparently and disrupt among themselves in control group,while the mouse anti-human CD34 group gained a cell-layer well.At 5thand 7thday, this phenomenon was more obvious.But this phenomenon was no different between 5thand 7thday respectively.Conclusions:mouse anti-human CD34 surface modification could promote the cell adhesion and prolification on BJVC in vitro. ChapterⅢReendothelialization on bovine jugular vein matrix modified with Mouse Anti-human CD34 in vivoObjective:To investigate the reendothelialization on bovine jugular vein matrix surface modified with mouse anti-human CD34 in vivoMethods:1.24 dogs were divided into mouse anti-human CD34 modified group and control group equally.BJVCs were implanted between their right ventricle and pulmonary artery.Running suture technique was performed.The dogs were fed with Warfarin of 1.25mg/d respectively.The conducts were harvested at 10thday,20thday,1st,2nd month postoperatively.Gross view was achieved.The samples were HE stained and scanned under scanning electron microscope and tested ofⅧfactor dyeing with immunohistochemistry method.The cell numbers of endangium and the thickness of neointima were measured and compared between two groups by using Image Pro Plus software.Results:(1)Gross view:The conducts were harvested from IVC of the rats.Adherence with surrounding tissue was slight.The color of methylthioninium chloride disappeared and the patches turned white and were still soft.The inner side of all conducts was smooth.No thrombus was found in both two groups.The suture line was seen clearly.A thickening lamella was viewed on all patches.(2)HE(hematoxylin-eosin)stain:BJVC treated with photooxidation could inhibit cell infiltration.The infiltration occurred firstly in anastomotic area and was not through the whole midpiece wall after 2 months in both groups except in a very small area.Inside the neointima there were more cells in mouse anti-human CD34 group than control group.As soon as the neointima was shucked,cells were seen on BJVC surface in mouse anti-human CD34 group while few cells in control group at 10thday postoperatively by HE stain.Endothelioid cells were viewed in mouse anti-human CD34 group at 10thday and it completely covered neointima at 20thdays While this phenomenon occurred at 2nd month in control group.(3)Ⅷfactor staining:Positive staining was viewed at 10thday in mouse anti-human CD34 group,stronger positive at 20thday postoperatively,and there was neocapillary in neointima at 10thday postoperatively.There were fewer cells on the neointimal surface at 3rd month in control group.Negative staining still existed at 2ndmonth in control group postoperatively,though lots of cells were seen on the neointimal surface at this time.(4)Scanning electron microscope:Lots of endothelioid cells along with the direction of blood flow were observed but there was still distance between the cells in mouse anti-human CD34 group at 10thday.A layer of endothelial cells were compacted and well-arrayed,the cell junction was tight at 20thday in mouse anti-human CD34 group.There was only cellulose and macromolecule substance on the neointima in control group at these times.Though it was seems that there were cells under the surface but the endothelial cell covering was not observed.(5)Thickness of neointima:The neointima was thicker in mouse anti-human CD34 group than in control group at 10thday postoperatively,but it was not significant(P>0.05).It was thinner in mouse anti-human CD34 group than in control at 20thday,1st,2ndmonth postoperatively(P<0.01).In general,the neointima turned thicker when time went on,which got to the peak at 1stmonth and then went down in both group.The thickness of neointima at 20thday decreased to the level of that at 10thday postoperatively in mouse anti-human CD34 group, while the thickness of neointima was still large at 2ndmonth in control group.Conclusions:mouse anti-human CD34 surface modification could promote the cell adhesion and prolification on BJVC in vivo.Fast reendothelialization was achieved on the BJVC patches modified with mouse anti-human CD34 in vivo.Even still accompanying with intima hyperplasia,but the thickness of neointima was slighter than that untreated with mouse anti-human CD34.

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