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Experimental studies on development of the artificial esophagus utilizing cell-seeding method and EGF

细胞接种法和EGF研制人工食管的实验研究

基本信息

批准号：
62570616
负责人：
ANDO Nobutoshi
金额：
$ 1.28万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1989
项目状态：
已结题

项目摘要

To prevent anastomotic leakages and stricture formations of the artificial esophagus,the inner surface of the prosthesis should be epithelized immediately after implantation. A silicone-coated stainless steal mesh tube was used as the esophageal prothesis,and was implanted under the skin of canine back,wrapping with a prepared latissmus dorsi muscle flap. Two weeks later,the inner surface of the prosthesis was covered with good granulations. As the first trial of epithelizing the inner surface of the prothesis,the skin flap was induced to the inside of the prothesis,and EGF was injected into the prothesis. However thr prothesis suffered intedion frequently and the epithelization was not satisfactory one. Therefore,cell-seeding method was applied. At first,epidermal cells derived enzymatically from the skin were seeded into the lumen of the prosthesis. But epidermal cells were not able to be implanted. The failure of cell-seeding was considered to be attributable to a lack of seeded cells,so cell culture of epidermal cells harvested enzymatically from skin was tried in order to increase cell counts. The method of cell is same as under: 1.Skin was excised. 2.Incubated in DMEM with disease at 37ﾟC for 3 hrs. 3. Epidermis was separated from dermis. 4.Epidermal cells were separated after trypsinzation. 5.Epidermal cells were cultured in KGM. 10 to 14 days later,when epidermal cells were cultured to confluency in flask,trypsinization was done to obtain epidermal cells suspension. Then cultured epidermal cells suspension were seeded into inner surface of the prosthesis. Some of the cultured epidermal cells were able to be implanted on the granulations of the prosthesis. Microscopically,layers of stratified squamous epithelium were seen on the granulations. Based on these results, the epithelization on the inner-surface of the prosthesis is considered to be possible by seeding technique of cultured epidermal cells.

为了防止人工食管吻合口瘘和狭窄的形成，假体的内表面应在植入后立即上皮化。采用硅胶涂层不锈钢网状管作为食管假体，植入犬背部皮下，用制备好的背阔肌肌瓣包裹。两周后，假体的内表面被良好的颗粒覆盖。作为修复体内表面上皮化的第一次尝试，将皮瓣引至修复体内侧，并将EGF注入修复体内。但修复体易发生内分泌，上皮化效果不理想。因此，采用细胞接种方法。首先，将酶促来源于皮肤的表皮细胞接种到假体的内腔中。但是表皮细胞不能被植入。细胞接种的失败被认为是由于缺乏接种的细胞，因此尝试从皮肤酶促收获的表皮细胞的细胞培养以增加细胞计数。细胞培养方法同下：1.切取皮肤。2.在37 ° C下在具有疾病的DMEM中孵育3小时。3.将表皮与真皮分离。4.胰蛋白酶消化后分离表皮细胞。5.表皮细胞在KGM中培养。培养10 ~ 14 d后，待表皮细胞在培养瓶中汇合后，用胰蛋白酶消化，获得表皮细胞悬液。然后将培养的表皮细胞悬液接种于假体内表面。一些培养的表皮细胞能够植入假体的颗粒上。显微镜下，在颗粒上观察到复层鳞状上皮层。基于这些结果，认为通过培养的表皮细胞的接种技术在假体的内表面上上皮化是可能的。