【作者】 乔刚；

【导师】 胡建国；

【作者基本信息】 中南大学 ， 外科学， 2007， 博士

【摘要】 第一章基质材料的制备及表面RGD修饰的研究目的：探讨RGD光化学偶联法修饰去细胞光氧化牛颈静脉基质的方法，以及修饰的RGD肽在体内存留情况。方法：1．制备去细胞、光氧化交联的牛颈静脉基质材料，裁减成1×1cm~2大小的48个血管片，随机分入12个组中；将RGD-FITC和光偶合剂SANPAH分别配制成0.12mM、0.6mM、1.2mM和6mM四种浓度的溶液，按照1：0、1：1和1：10的三个反应摩尔比在避光下反应2小时，制成12组反应液。每组100μl反应液滴加在血管片上，365nm的紫外线照射5分钟，引发光化学偶联，将RGD共价结合到牛颈静脉血管片上，震荡漂洗6小时，样品制成快速冰冻切片，荧光显微镜下观察。通过观察基质材料表面不同浓度、不同摩尔比反应接枝后的荧光差别，从而初步推断出合适的反应和结合浓度。2．以浓度为0.6mM、反应摩尔比为1：1的FITC-RGD和SANPAH，同法制备表面修饰后的牛颈静脉血管片(实验组)，物理吸附RGD的血管片作为对照组，8只SD大鼠随机平均分入上述两组中，将血管片分别植入两组大鼠下腔静脉，于术后5天、10天、15天、20天取出标本，进行快速冰冻切片，观察并照相留底，以了解荧光标记的RGD是否能经受体内血流的冲击和细胞的吞噬而仍然存在。结果：应用光偶联剂后，血管内膜面有一层较强的荧光，随着RGD和SANPAH的浓度的升高，荧光整体上是越来越强，但是二者的浓度高于0.6mM时，荧光差别不明显；相同浓度下当二者的反应摩尔比为1：1时，荧光最强。经过光化学偶联后的FITC-RGD肽在体内20天仍然存在，只是荧光略有衰减，单纯涂覆的RGD组绿色荧光5天即完全消失。结论：光偶联剂SANPAH能够实现RGD与去细胞光氧化牛颈静脉的化学偶联，RGD和SANPAH较为合适的接枝反应浓度是0.6mM，反应摩尔比是1：1，光化学交联方法接枝的RGD在体内保留时间至少超过20天，体内存留效果良好。第二章RGD表面修饰BJV后体外、体内的再内皮化研究目的：探讨RGD表面修饰后的牛颈静脉体内和体外促进再内皮化的情况。方法：1．体外细胞黏附生长：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，用光偶联剂SANPAH采用第一章的方法将RGD接枝到牛颈静脉血管片上，以未接枝RGD的作为空白对照。在其上面种植人脐静脉内皮细胞株细胞(CRL-2480)5×10~5／ml，静态培养5天，取1，3，5天标本消化血管片表面细胞并记数，标本切片HE染色，对比RGD接枝后和空白对照组血管片表面细胞黏附数目和HE的染色结果，以探讨接枝RGD体外能否促进细胞黏附和生长。2．再内皮化的体内实验：8只SD大鼠平均分入处理组和对照组，将RGD表面修饰和未修饰的血管片植入下腔静脉，采用连续缝合的方式进行补片，术后两组均给予10mg／kg／天剂量的阿托伐他汀喂养，分别于术后5天、10天、15天、20天取出血管片，标本进行石蜡包埋、HE染色、扫描电镜和Ⅷ因子免疫组织化学检测，取普通显微镜40倍下的HE染色的血管片留底照片，在Image Pro Plus图像测量软件上测量内膜厚度，计算平均值，两组间进行比较。结果：1．体外培养：(1)细胞记数：细胞培养第1、3、5天，RGD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；RGD组细胞连接成片，呈单层排布；第5天这种现象更明显。2．体内实验：(1)组织大体观结果：血管片隆起于下腔静脉表面，质软，周围粘连较轻，颜色变白，亚甲兰的蓝色已完全消失。剖开下腔静脉后见补片表面光滑，无血栓，缝线清晰可见，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：经过光氧化交联的BJV植入体内，细胞浸润受到抑制，浸润全层首先在血管片吻合区域发生，20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BJV有细胞相连；RGD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5天，RGD组BJV表面有细胞黏附，对照组几乎没有细胞；RGD组5天即能在内膜面看到内皮样细胞覆盖，10天完整覆盖补片，对照组则需要20天才能看到内膜面较多的细胞覆盖。(3)Ⅷ因子染色结果：RGD表面修饰后的血管片植入大鼠体内5天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，10天阳性更强；对照组5～15天内膜面只有很少的细胞，Ⅷ因子染色是阴性，20天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：RGD组术后5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10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10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，只是黏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5天，RGD组新生内膜较对照组厚，但是没有统计学意义(P＞0.5)，随后的10天、15天、20天RGD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15天时达到高峰，20天即开始下降，RGD组术后10天新生内膜厚度有波折，其在20天时下降到术后5天的水平，而对照组厚度值仍然较大。结论：RGD表面修饰的牛颈静脉，体外能促进内皮细胞的黏附与生长，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RGD修饰的牛颈静脉。更多还原

【Abstract】 PartⅠFabrication of decellularized and photooxidated bovine jugular vein matrix & RGD surface modificationObjective:To investigate the mode of photocrosslinking RGD peptide onto the decellularized and photooxidated bovine jugular vein matrix surface and the persistence of RGD in vivo.Methods:1. Bovine jugular vein was decellularized and photooxidated, then cut into 48 pieces of 1×1cm~2 and divided into 12 groups randomly. The solutions of RGD covalent labelled with FITC and photochemical crosslinker SANPAH were prepared, their concentrations were 0.12mM, 0.6mM, 1.2mM and 6mM. The reaction between them were performed with 3 different mole ratios in 2 hours away from light, which were 1∶0, 1∶1 and 1∶10. 100μl reacted solution of them were dropped onto the prepared BJV pieces, then irradiated under 365nm ultraviolet ray for 5 minutes. The pieces were rinsed in a shaking machine for 6 hours. The fast frozen sections of the specimens were observed under the fluorescent microscope. The grafting effect was viewed by different fluorescent brightness, so the suitable reactive concentration and ratio were educed. This reactive concentration and ratio were selected for further experiment in vivo as below.2. Four RGD modified BJV patches were prepared which were treated with RGD-FITC and SANPAH of 0.6mM and 1∶1 mole ratio by the same method as above mentioned(SANPAH group). Another four patches were only coated with RGD-FITC directly(control group). Eight Sprague-Dawley rats (SD rats) were divided into this two groups equally. All the patches were transplanted into murine inferior vena cava wall and harvested at the 5th, 10th, 15th, 20th day postoperatively. Their fast frozen sections were observed and pictured under fluorescent microscope as above procedure.Results: The brighter fluorescence on the side of endangium was viewed on the SANPAH photocrosslinking group. The higher the concentrations of RGD and SANPAH were, the brighter the fluorescence was. But when the concentration of them was higher than 0.6mM, 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∶1. In vivo fluorescence still existed after 20 days postoperatively in SANPAH group even though attenuating slightly. At 5th day postoperatively, the fluorescence disapeared in RGD group. Conclusion: RGD peptide could be covalent grained onto the BJVC surface by photochemical cross-linker SANPAH. The optimal concentration of RGD and SANPAH and reactive mole ratio between them were 0.6mM and 1∶1. The persistence time of RGD treated with photochemical cross-linker was over 20 days at least in vivo. 10~5/ml density and cultured for 5 days. The cells were departed with enzyme from the surface of these pieces at 1st, 3th, 5th, their numbers were counted. The slices of BJV implanted with cells were HE stained and pictured. The difference between two groups was compared to verify whether BJV modified with RGD could promote the cell adhesion and proliferation.2. In vivo: Eight SD rats were divided into RGD modified group and control group equally. BJV patches were implanted in their IVC wall. Continuous suture technique was performed. The rats were fed with atorvastatin of 10mg/kg/d. The patches were harvested at 5th, 10th, 15th, 20th day postoperatively. Gross view was achieved. The samples were HE stained and scanned under scanning electron microscope and tested ofⅧfactor dyeing with immunohistochemistry method. The thickness of neointima were measured and compared between two groups by using Image Pro Plus software.Results:1. In vitro: (1) Cells counting: The cells number in RGD group was more than control group at 1st, 3rd, 5th day (p＜0.05). (2) At 1st day, the cells arrayed intensively and not spreaded completely in each group’s surface. At 3rd day, the cells on BJV surface decreased apparently and disrupt among themselves in control group, while the RGD group gained a cell-layer well. At 5th day, this phenomenon was more obvious. 2.In vivo: (1) Gross view: The patches 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 patches 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: BJV treated with photooxidation could inhibit cell infiltration. The infiltration occurred firstly in anastomotic area and was not through the whole midpiece wall after 20 days in both two groups except in a very small area. Inside the neointima there were more cells in RGD group than control group. As soon as the neointima was shucked, cells were seen on BJV surface in RGD group while few cells in control group at 5th day postoperatively by HE stain. Endothelioid cells were viewed in RGD group at 5th day and it completely covered neointima at 10th day. While this phenomenon occurred at 20th day in control group.(3)Ⅷfactor staining: Positive staining was viewed at 5th day in RGD group, stronger positive at 10th day postoperatively, and there was neocapillary in neointima at 5th day postoperatively. There were fewer cells on the neointimal surface at 15th in control group. Negative staining still existed at 20th 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 RGD group at 5th day. A layer of endothelial cells were compacted and well-arrayed, the cell junction was tight at 10th day in RGD 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 RGD group than in control group at 5th day postoperatively, but it was not significant(P＞0.5). It was thinner in RGD group than in control at 10th, 15th, 20th day postoperatively(P≤0.01). In general, the neointima turned thicker when time went on, which got to the peak at 15th day and then went down in both group. The thickness of neointima at 20th day decreased to the level of that at 5th day postoperatively in RGD group, while the thickness of neointima was still large at 20th day in control group.Conclusions:RGD surface modification could promote the cell adhesion and prolification on BJV in vivo. Fast reendothelialization was achieved on the BJV patches modified with RGD in vivo. Even still accompanying with intima hyperplasia, but the thickness of neointima was slighter than that untreated with RGD.更多还原