CBD-bFGF结合胶原支架材料促进犬膀胱重建修复的实验研究

发布时间：2018-05-03 14:14

本文选题：组织工程 + 碱性成纤维细胞生长因子　；参考：《第三军医大学》2014年硕士论文

【摘要】：先天性疾病、炎症、感染、肿瘤以及创伤等可导致膀胱结构和功能的缺失与损害，而传统的肠道代膀胱会引起多种并发症，如酸碱代谢紊乱、感染、结石甚至恶变等。组织工程技术的发展为膀胱重建带来了全新的方法。目前，构建的膀胱组织植入体内后的快速血管化是组织工程膀胱面临的主要问题，利用在支架材料上复合生长因子为组织工程膀胱体内充分快速血管化提供了一种选择。碱性成纤维细胞生长因子（basic fibroblast growth factor，bFGF）对创伤愈合、组织修复、血管形成起着重要促进作用，但将bFGF简单吸附到材料上再植入体内，体液的浸润和流动造成因子扩散，导致修复效果有限。因此，我们在bFGF的N端偶联胶原结合区（Collagen binding domain，CBD），使其具有结合胶原能力，在体内可以定点、持续释放再生信号分子，同时外源的胶原支架材料也为细胞增殖和组织再生提供了三维空间。本课题以大动物比格犬进行膀胱重建实验，，研究内容包括以下两个部分：第一部分：比格犬膀胱损伤模型制作及组织工程膀胱的重建。将由中科院遗传与发育研究所提供的CBD-bFGF和胶原支架材料，制备成CBD-bFGF/Collagen材料及PBS/Collagen材料。13只雄性成年比格犬分为假手术组（Sham operated，n=3）、单纯胶原组（PBS/Collagen, n=5）和CBD-bFGF复合胶原材料组（CBD-bFGF/Collagen, n=5），取下腹部旁正中切口暴露膀胱，切除50%～60%膀胱，分别用CBD-bFGF/Collagen材料及PBS/Collagen材料进行膀胱重建。术后1月内单纯胶原组及CBD-bFGF复合胶原材料组各有一条比格犬死亡，尸检后提示原因为膀胱严重感染导致膀胱破裂，进一步引起腹腔感染而死亡。各组剩下比格犬全部存活，无尿漏、感染发生。第二部分：两种修复材料对比格犬膀胱重建修复作用评价。术后6月，我们对所有比格犬的膀胱进行了形态学、组织学及功能学检测，评价两种修复材料对比格犬膀胱重建修复作用。尿动力学分析结果：膀胱最大容量和顺应性在PBS/Collagen组、CBD-bFGF/Collagen组、Sham operated组依次增加，3组间比较差异有统计学意义（P 0.05），而CBD-bFGF/Collagen组和Sham operated组逼尿肌最大压力比较差异无统计学意义（P0.05）。静脉肾盂造影及逆行膀胱造影结果：肾脏、输尿管正常显影，膀胱轮廓清晰，形态正常。大体标本及组织学观察结果：PBS/Collagen组膀胱有憩室及小梁形成，上皮层正常，黏膜下层组织增生，肌层再生不良；而CBD-bFGF/Collagen组仅有少量小梁形成，显示了与对照组相似的正常3层组织结构。综上所述，在比格犬膀胱重建修复实验中，CBD-bFGF/Collagen修复材料能够更好的促进膀胱组织再生和功能恢复，采用CBD-bFGF结合胶原支架材料构建的组织工程膀胱是理想的膀胱替代修复材料。
[Abstract]:Congenital diseases, inflammation, infection, tumor and trauma can lead to the loss and damage of bladder structure and function. Traditional intestinal replacement of bladder can cause many complications, such as acid-base metabolic disorder, infection, stone and even malignant change. The development of tissue engineering technology has brought a new method for bladder reconstruction. At present, the rapid vascularization of the constructed bladder tissue after implantation is the main problem facing the tissue engineering bladder. Using the composite growth factor on the scaffold material provides a choice for the full rapid vascularization in the tissue engineering bladder. Basic fibroblast growth factor (bFGF) plays an important role in wound healing, tissue repair and angiogenesis. However, bFGF is simply adsorbed on the material and implanted into the body. The infiltration and flow of body fluids lead to factor diffusion. Resulting in limited repair effect. Therefore, we have the ability of binding collagen in the N-terminally coupled collagen binding region of bFGF, Collagen binding domain, so that we can continuously release regenerative signaling molecules at specific sites in the body. At the same time, exogenous collagen scaffolds also provide three-dimensional space for cell proliferation and tissue regeneration. In this study, the bladder reconstruction experiment was carried out with a large animal Beagle dog. The research includes the following two parts: The first part: the model of bladder injury in Beagle dog and the reconstruction of tissue engineering bladder. CBD-bFGF and collagen scaffolds to be provided by the Institute of Genetics and Development of the Chinese Academy of Sciences, A total of 13 male adult Beagle dogs were prepared into CBD-bFGF/Collagen and PBS/Collagen materials. They were divided into sham operated group (sham operated), collagen group (CBD-bFGF/Collagen / Collagen5) and CBD-bFGF composite glue raw material group (CBD-bFGF- / Collagen, nnbFGF / Collagen). The central incision of lower abdomen was taken to expose bladder, and 50 60% of bladder was removed. CBD-bFGF/Collagen and PBS/Collagen materials were used for bladder reconstruction. One beagle died in the collagen group and one in the CBD-bFGF raw material group within one month after the operation. The postmortem revealed that severe infection of the bladder resulted in rupture of the bladder and further death in the abdominal cavity. All the remaining Beagle dogs in each group survived, had no urine leakage and infection. Part two: evaluation of the effect of two kinds of restoration materials on bladder reconstruction in Beagle dog. Six months after operation, we examined the bladder morphology, histology and function of all Beagle dogs, and evaluated the effects of two kinds of restoration materials on bladder reconstruction in Beagle dogs. The results of urodynamic analysis showed that the maximal bladder volume and compliance were significantly increased in PBS/Collagen group (CBD-bFGF / Collagen group), but there was no significant difference in maximal detrusor pressure between CBD-bFGF/Collagen group and Sham operated group (P 0.05). The results of intravenous pyelography and retrograde cystography: normal development of kidney and ureter, clear outline of bladder and normal shape. The results of gross specimen and histological observation showed that the bladder had diverticulum and trabecular formation in the group of w: PBS / Collagen, normal epithelium, hyperplasia of submucous tissue and poor regeneration of myometrium, while in CBD-bFGF/Collagen group, only a few trabeculae were formed. A normal 3-layer structure similar to that of the control group was shown. In conclusion, CBD-bFGFr / Collagen can promote bladder tissue regeneration and function recovery better in the bladder reconstruction experiment of Beagle dog. The tissue engineering bladder constructed with CBD-bFGF combined with collagen scaffold is an ideal bladder replacement material.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R699.5

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