CD34抗体表面修饰去细胞光氧化牛颈静脉再内皮化的研究
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摘要
第一章牛颈静脉基质材料的制备及表面CD34抗体修饰的研究
目的:通过按不同摩尔比,用光化学交联剂SANPAH将不同浓度鼠抗人CD34抗体接枝于经过去细胞和光氧化后的牛颈静脉(BJV)表面的初步研究,明确接枝鼠抗人CD34抗体的最佳浓度和与SANPAH的最佳反应摩尔比,探讨CD34抗体光化学偶联法修饰去细胞光氧化牛颈静脉基质的方法。
方法:按4个不同摩尔比,SANPAH和4个不同浓度鼠抗人CD34抗体进行反应后,经紫外线照射光化学接枝于经过去细胞和光氧化后的BJV上,各组进行快速冰冻切片,滴加FITC标记的羊抗鼠IgG,然后在荧光显微镜下照相,用Image-pro plus 5.0对图像进行分析,比较不同摩尔比和浓度反应、结合的荧光效果,从而初步推断出最佳反应和结合的摩尔比和浓度。
结果:随着鼠抗人CD34抗体浓度的升高,荧光累积光密度、平均光密度,整体上是越来越强,但是当浓度高于0.665×10~(-2)mM时,荧光差别不是很明显;当鼠抗人CD34抗体和SANPAH的反应摩尔比为1:20时,荧光最强。
结论:最佳的鼠抗人CD34抗体和SANPAH反映接枝的浓度是0.665×10~(-2)mM,最佳的摩尔比是1:20。
第二章CD34抗体表面修饰去细胞光氧化牛颈静脉体外再内皮化研究
目的:探讨鼠抗人CD34抗体表面修饰后的去细胞光氧化牛颈静脉体外促进再内皮化的情况。
方法:牛颈静脉经过去细胞和光氧化交联后,裁成与24孔培养板孔径相同大小的、直径1.6cm的血管片,用光化学偶联剂SANPAH采用第一章的方法将CD34抗体接枝到牛颈静脉血管片上,以未接枝CD34抗体的作为空白对照。在其上面种植人脐静脉内皮细胞株(HUVEC,CRL-2480)5×10~5/ml,静态培养,取第1,3,5,7日标本,一半血管片表面细胞消化并记数,另一半标本切片HE染色,对比CD34抗体接枝后和空白对照组血管片表面细胞粘附数目和HE的染色结果,以探讨接枝CD34抗体体外能否促进细胞粘附和生长。
结果:(1)细胞计数:细胞培养第1、3、5、7日,CD34抗体组细胞数目均多于对照组(p＜0.05),(2)标本石蜡包埋后切片HE染色结果:第1天各组血管片表面细胞排列密集,紊乱,未完全铺开;第3天,对照组血管表面细胞数量减少明显,有断裂出现;CD34抗体组细胞连接成片,呈单层排布;第5,7日这种现象更明显,第7日两组细胞数与本组第5日无明显差异。
结论:CD34抗体表面修饰的牛颈静脉,体外能促进内皮细胞的粘附与生长。
第三章CD34抗体表面修饰去细胞光氧化牛颈静脉体内再内皮化研究
目的:探讨鼠抗人CD34抗体表面修饰后的去细胞光氧化牛颈静脉体内促进再内皮化的情况。
方法:24只犬随机分入处理组和对照组,将CD34抗体表面修饰和未修饰的血管植入犬右室流出道与肺动脉主干之间,采用连续缝合的方式进行旁路重建,术后两组均给予阿托伐他汀10mg/kg╱天和1.25mg/天剂量的华法林喂养,分别于术后10天、20天、1月、2月取出血管,标本进行石蜡包埋HE染色、Ⅷ因子免疫组织化学、透射电镜和扫描电镜检测,取普通显微镜40倍下的HE染色的血管片标本照相,用Image-pro plus 5.0对图像进行测量内膜厚度,计算平均值,两组间进行比较;取普通显微镜40倍Ⅷ因子免疫组织化学血管标本照相,Image-pro plus 5.0对图像进行细胞计数,评价内皮化程度。
结果:(1)组织大体观结果:间植血管质软,周围粘连较轻,颜色变白,亚甲兰的蓝色已完全消失。剖开见血管表面光滑,瓣膜组织菲薄,开闭好,无血栓,其上附着一薄层增厚组织,薄而透明,为新生内膜组织。
(2)HE染色结果:经过去细胞光氧化交联的BJVC植入体内,细胞浸润受到抑制,浸润全层首先在血管吻合区域发生,术后10天两组中间区域均未能浸润全层,只是在小部分个别区域全层BJVC有细胞相连;CD34抗体组在新生内膜中有更多的细胞,而对照组则主要还是纤维素等不定型成分。新生内膜在吻合区域薄,中间较厚,剔除新生内膜后的HE切片显示:术后10天,CD34抗体组BJVC表面有细胞粘附,对照组几乎没有细胞;CD34抗体组术后20天即能在内膜面看到内皮样细胞覆盖,术后1个月完整覆盖补片,对照组则需要术后2个月才能看到内膜面较多的细胞覆盖,Image-pro plus 5.0细胞计数抗体组较对照组多,内皮化程度高,统计学有显著差异。
(3)Ⅷ因子染色结果:CD34抗体表面修饰后的血管片植入大鼠体内10天后Ⅷ因子染色即呈阳性,而且新生内膜中已经形成新生微血管,20天后阳性更强;对照组20天后内膜面只有很少的细胞,Ⅷ因子染色是阴性,2个月后虽然内膜面虽有较多的细胞,但是Ⅷ因子染色仍是阴性。
(4)扫描电镜结果:CD34抗体组术后10天内膜面可见较多的上皮样细胞顺血流方向排列,但是细胞之间有间距,术后20天内膜面附着一层排列致密、整齐的内皮细胞,细胞间可见形成的牢固连接;对照组术后20天内膜面仍是增厚的纤维素样物质,下面似有细胞突起,可能为平滑肌或成纤维细胞,表面无内皮样细胞覆盖,只是粘附一些大分子物质和散在的一些小细胞。
(5)内膜厚度的比较:术后第10天,CD34抗体组新生内膜较对照组厚,但是没有统计学意义(P＞0.05),随后的20天、1个月、2个月CD34抗体组新生内膜均较对照组薄,而且有统计学意义(P＜0.01)。两组新生内膜总体上说随着时间的延长,厚度越来越大,术后20天时达到高峰,1个月即开始下降,CD34抗体组术后20天新生内膜厚度有波折,其在2个月时下降到术后10天的水平,而对照组厚度值仍然较大。
结论:CD34抗体表面修饰的牛颈静脉,体内能快速达到内皮化的效果,虽然仍然伴有内膜的增生,但是新生内膜的厚度显著薄于未经CD34抗体修饰的牛颈静脉。
ChapterⅠFabrication and Preliminary Assessment of Mouse Anti-human CD34 Grafting onto Decellularized and Photooxidated Bovine Jugular Vein Matrix Intima
Objective: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~(-2)mM,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~(-2)mM and 1:20 respectively.
ChapterⅡReendothelialization on bovine jugular vein matrix modified with Mouse Anti-human CD34 in vitro
Objective:To investigate the reendothelialization on bovine jugular vein matrix surface modified with mouse anti-human CD34 in vitro
Methods: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 5×10~5/ml density and cultured for 7 days.The cells were departed with enzyme from the surface of these pieces at 1~(st),3~(th),5~(th),7~(th),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 1~(st),3~(rd),5~(th).7~(th)day(p<0.05). (2)At 1~(st)day,the cells arrayed intensively and not spreaded completely in each group's surface.At 3~(rd)day,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 5~(th)and 7~(th)day, this phenomenon was more obvious.But this phenomenon was no different between 5~(th)and 7~(th)day 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 vivo
Objective:To investigate the reendothelialization on bovine jugular vein matrix surface modified with mouse anti-human CD34 in vivo
Methods: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 10~(th)day,20~(th)day,1~(st),2~(nd) 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 10~(th)day postoperatively by HE stain.Endothelioid cells were viewed in mouse anti-human CD34 group at 10~(th)day and it completely covered neointima at 20~(th)days While this phenomenon occurred at 2~(nd) month in control group.
(3)Ⅷfactor staining:Positive staining was viewed at 10~(th)day in mouse anti-human CD34 group,stronger positive at 20~(th)day postoperatively,and there was neocapillary in neointima at 10~(th)day postoperatively.There were fewer cells on the neointimal surface at 3~(rd) month in control group.Negative staining still existed at 2~(nd)month 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 10~(th)day.A layer of endothelial cells were compacted and well-arrayed,the cell junction was tight at 20~(th)day 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 10~(th)day postoperatively,but it was not significant(P>0.05).It was thinner in mouse anti-human CD34 group than in control at 20~(th)day,1~(st),2~(nd)month postoperatively(P<0.01).In general,the neointima turned thicker when time went on,which got to the peak at 1~(st)month and then went down in both group.The thickness of neointima at 20~(th)day decreased to the level of that at 10~(th)day postoperatively in mouse anti-human CD34 group, while the thickness of neointima was still large at 2~(nd)month 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.
目的:通过按不同摩尔比,用光化学交联剂SANPAH将不同浓度鼠抗人CD34抗体接枝于经过去细胞和光氧化后的牛颈静脉(BJV)表面的初步研究,明确接枝鼠抗人CD34抗体的最佳浓度和与SANPAH的最佳反应摩尔比,探讨CD34抗体光化学偶联法修饰去细胞光氧化牛颈静脉基质的方法。
方法:按4个不同摩尔比,SANPAH和4个不同浓度鼠抗人CD34抗体进行反应后,经紫外线照射光化学接枝于经过去细胞和光氧化后的BJV上,各组进行快速冰冻切片,滴加FITC标记的羊抗鼠IgG,然后在荧光显微镜下照相,用Image-pro plus 5.0对图像进行分析,比较不同摩尔比和浓度反应、结合的荧光效果,从而初步推断出最佳反应和结合的摩尔比和浓度。
结果:随着鼠抗人CD34抗体浓度的升高,荧光累积光密度、平均光密度,整体上是越来越强,但是当浓度高于0.665×10~(-2)mM时,荧光差别不是很明显;当鼠抗人CD34抗体和SANPAH的反应摩尔比为1:20时,荧光最强。
结论:最佳的鼠抗人CD34抗体和SANPAH反映接枝的浓度是0.665×10~(-2)mM,最佳的摩尔比是1:20。
第二章CD34抗体表面修饰去细胞光氧化牛颈静脉体外再内皮化研究
目的:探讨鼠抗人CD34抗体表面修饰后的去细胞光氧化牛颈静脉体外促进再内皮化的情况。
方法:牛颈静脉经过去细胞和光氧化交联后,裁成与24孔培养板孔径相同大小的、直径1.6cm的血管片,用光化学偶联剂SANPAH采用第一章的方法将CD34抗体接枝到牛颈静脉血管片上,以未接枝CD34抗体的作为空白对照。在其上面种植人脐静脉内皮细胞株(HUVEC,CRL-2480)5×10~5/ml,静态培养,取第1,3,5,7日标本,一半血管片表面细胞消化并记数,另一半标本切片HE染色,对比CD34抗体接枝后和空白对照组血管片表面细胞粘附数目和HE的染色结果,以探讨接枝CD34抗体体外能否促进细胞粘附和生长。
结果:(1)细胞计数:细胞培养第1、3、5、7日,CD34抗体组细胞数目均多于对照组(p＜0.05),(2)标本石蜡包埋后切片HE染色结果:第1天各组血管片表面细胞排列密集,紊乱,未完全铺开;第3天,对照组血管表面细胞数量减少明显,有断裂出现;CD34抗体组细胞连接成片,呈单层排布;第5,7日这种现象更明显,第7日两组细胞数与本组第5日无明显差异。
结论:CD34抗体表面修饰的牛颈静脉,体外能促进内皮细胞的粘附与生长。
第三章CD34抗体表面修饰去细胞光氧化牛颈静脉体内再内皮化研究
目的:探讨鼠抗人CD34抗体表面修饰后的去细胞光氧化牛颈静脉体内促进再内皮化的情况。
方法:24只犬随机分入处理组和对照组,将CD34抗体表面修饰和未修饰的血管植入犬右室流出道与肺动脉主干之间,采用连续缝合的方式进行旁路重建,术后两组均给予阿托伐他汀10mg/kg╱天和1.25mg/天剂量的华法林喂养,分别于术后10天、20天、1月、2月取出血管,标本进行石蜡包埋HE染色、Ⅷ因子免疫组织化学、透射电镜和扫描电镜检测,取普通显微镜40倍下的HE染色的血管片标本照相,用Image-pro plus 5.0对图像进行测量内膜厚度,计算平均值,两组间进行比较;取普通显微镜40倍Ⅷ因子免疫组织化学血管标本照相,Image-pro plus 5.0对图像进行细胞计数,评价内皮化程度。
结果:(1)组织大体观结果:间植血管质软,周围粘连较轻,颜色变白,亚甲兰的蓝色已完全消失。剖开见血管表面光滑,瓣膜组织菲薄,开闭好,无血栓,其上附着一薄层增厚组织,薄而透明,为新生内膜组织。
(2)HE染色结果:经过去细胞光氧化交联的BJVC植入体内,细胞浸润受到抑制,浸润全层首先在血管吻合区域发生,术后10天两组中间区域均未能浸润全层,只是在小部分个别区域全层BJVC有细胞相连;CD34抗体组在新生内膜中有更多的细胞,而对照组则主要还是纤维素等不定型成分。新生内膜在吻合区域薄,中间较厚,剔除新生内膜后的HE切片显示:术后10天,CD34抗体组BJVC表面有细胞粘附,对照组几乎没有细胞;CD34抗体组术后20天即能在内膜面看到内皮样细胞覆盖,术后1个月完整覆盖补片,对照组则需要术后2个月才能看到内膜面较多的细胞覆盖,Image-pro plus 5.0细胞计数抗体组较对照组多,内皮化程度高,统计学有显著差异。
(3)Ⅷ因子染色结果:CD34抗体表面修饰后的血管片植入大鼠体内10天后Ⅷ因子染色即呈阳性,而且新生内膜中已经形成新生微血管,20天后阳性更强;对照组20天后内膜面只有很少的细胞,Ⅷ因子染色是阴性,2个月后虽然内膜面虽有较多的细胞,但是Ⅷ因子染色仍是阴性。
(4)扫描电镜结果:CD34抗体组术后10天内膜面可见较多的上皮样细胞顺血流方向排列,但是细胞之间有间距,术后20天内膜面附着一层排列致密、整齐的内皮细胞,细胞间可见形成的牢固连接;对照组术后20天内膜面仍是增厚的纤维素样物质,下面似有细胞突起,可能为平滑肌或成纤维细胞,表面无内皮样细胞覆盖,只是粘附一些大分子物质和散在的一些小细胞。
(5)内膜厚度的比较:术后第10天,CD34抗体组新生内膜较对照组厚,但是没有统计学意义(P＞0.05),随后的20天、1个月、2个月CD34抗体组新生内膜均较对照组薄,而且有统计学意义(P＜0.01)。两组新生内膜总体上说随着时间的延长,厚度越来越大,术后20天时达到高峰,1个月即开始下降,CD34抗体组术后20天新生内膜厚度有波折,其在2个月时下降到术后10天的水平,而对照组厚度值仍然较大。
结论:CD34抗体表面修饰的牛颈静脉,体内能快速达到内皮化的效果,虽然仍然伴有内膜的增生,但是新生内膜的厚度显著薄于未经CD34抗体修饰的牛颈静脉。
ChapterⅠFabrication and Preliminary Assessment of Mouse Anti-human CD34 Grafting onto Decellularized and Photooxidated Bovine Jugular Vein Matrix Intima
Objective: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~(-2)mM,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~(-2)mM and 1:20 respectively.
ChapterⅡReendothelialization on bovine jugular vein matrix modified with Mouse Anti-human CD34 in vitro
Objective:To investigate the reendothelialization on bovine jugular vein matrix surface modified with mouse anti-human CD34 in vitro
Methods: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 5×10~5/ml density and cultured for 7 days.The cells were departed with enzyme from the surface of these pieces at 1~(st),3~(th),5~(th),7~(th),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 1~(st),3~(rd),5~(th).7~(th)day(p<0.05). (2)At 1~(st)day,the cells arrayed intensively and not spreaded completely in each group's surface.At 3~(rd)day,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 5~(th)and 7~(th)day, this phenomenon was more obvious.But this phenomenon was no different between 5~(th)and 7~(th)day 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 vivo
Objective:To investigate the reendothelialization on bovine jugular vein matrix surface modified with mouse anti-human CD34 in vivo
Methods: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 10~(th)day,20~(th)day,1~(st),2~(nd) 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 10~(th)day postoperatively by HE stain.Endothelioid cells were viewed in mouse anti-human CD34 group at 10~(th)day and it completely covered neointima at 20~(th)days While this phenomenon occurred at 2~(nd) month in control group.
(3)Ⅷfactor staining:Positive staining was viewed at 10~(th)day in mouse anti-human CD34 group,stronger positive at 20~(th)day postoperatively,and there was neocapillary in neointima at 10~(th)day postoperatively.There were fewer cells on the neointimal surface at 3~(rd) month in control group.Negative staining still existed at 2~(nd)month 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 10~(th)day.A layer of endothelial cells were compacted and well-arrayed,the cell junction was tight at 20~(th)day 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 10~(th)day postoperatively,but it was not significant(P>0.05).It was thinner in mouse anti-human CD34 group than in control at 20~(th)day,1~(st),2~(nd)month postoperatively(P<0.01).In general,the neointima turned thicker when time went on,which got to the peak at 1~(st)month and then went down in both group.The thickness of neointima at 20~(th)day decreased to the level of that at 10~(th)day postoperatively in mouse anti-human CD34 group, while the thickness of neointima was still large at 2~(nd)month 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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