细菌纤维素作为角膜组织工程支架的研究

发布时间：2018-01-30 21:12

本文关键词： 组织工程角膜细菌纤维素角膜基质细胞　出处：《吉林大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的：以细菌纤维素为支架构建组织工程角膜基质,研究其在体内外的生物相容性和降解性。方法：将大耳白兔角膜基质细胞进行体外分离、培养和扩增后,种植到BC膜上继续培养,细胞爬满后移植到同种同体基质囊袋中。术后定期应用裂隙灯、前节OCT和Cs-4检查移植后兔眼的组织形态学改变和复合膜的降解情况,同时观察兔眼有无炎症反应和新生血管的发生。分别于术后2、5、7个月,用角膜剪无菌摘除右眼角膜,置于4%戊二醛固定,进行HE染色、免疫组化和扫描电镜观察。结果：兔角膜基质细胞在BC上生长良好。术后1周兔眼无刺激症状,植片处无新生血管。术后1个月可见较深长的新生血管贯穿植片,但角膜未水肿,植片周围的角膜组织透明性有所好转。2个月时,角膜呈半透明,复合膜片大小有所缩减,新生血管较1个月时好转。7个月时角膜新生血管变少变细,其透明性好转。前节OCT和共焦显微镜检查：移植术后1周角膜基质细胞-BC复合膜密度明显高于正常角膜组织,术后5月时复合膜边界模糊,密度接近正常角膜组织,说明复合膜发生降解。术后2月共焦显微镜显示植片周围的内皮细胞数量和形态大致正常。术后7月基质细胞密度下降,偶见神经纤维。病理检查：术后5月时,角膜上皮细胞增生,排列紊乱,基质细胞—BC复合膜的纤维结构与正常角膜基质有区别,两者粘连不紧密,有炎细胞浸润,角膜组织无坏死和溶解。术后7月时,角膜上皮细胞仍增生,但排列规整,复合膜内未见空泡,有细胞结构,说明BC没有发生剧烈降解,BC两侧和内部可见与角膜基质细胞相同的细胞结构,周围有少量炎症细胞浸润,角膜组织未见溶解坏死。免疫组化检查：移植术后2、5、7月,三只大耳白兔的复合膜均呈现α-SMA染色阳性,说明在体内基质细胞被活化为肌成纤维细胞,但随移植时间的推移,基质细胞的活化数量逐渐减少。扫描电镜显示复合膜与正常角膜组织结构界限清晰,术后7个月时仍能见到复合膜。结论： BC的细胞相容性良好,构建角膜基质细胞-BC复合膜成功。BC具有可降解性,但BC膜在体内降解速度较慢。角膜新生血管、肌成纤维细胞的增殖与角膜透明性相关。
[Abstract]:Objective: Tissue engineered corneal stroma was constructed with bacterial cellulose as scaffold to study its biocompatibility and biodegradability in vitro and in vivo. Methods: Rabbit corneal stromal cells were isolated in vitro, cultured and expanded, then cultured on BC membrane, then transplanted into homologous matrix bag after seeding. Slit lamp was used regularly after operation. Anterior segment OCT and Cs-4 were used to examine the histomorphologic changes and the degradation of composite membrane in rabbit eyes after transplantation, and to observe whether there were inflammatory reaction and angiogenesis in rabbit eyes at 2 and 7 months postoperatively. The cornea of right eye was removed with corneal scissors and fixed with 4% glutaraldehyde for HE staining, immunohistochemistry and scanning electron microscopy. Results: Rabbit corneal stromal cells grew well on BC. There were no irritating symptoms in rabbit eyes and no neovascularization in the grafts 1 week after operation. A longer neovascularization was observed 1 month after operation, but corneal edema was not observed. The transparency of corneal tissue around the grafts was improved. At 2 months, the cornea was translucent, the size of composite membrane was reduced, the neovascularization was better than that at 1 month, and the corneal neovascularization became smaller and thinner at 7 months. Its transparency has improved. Anterior segment OCT and confocal microscopy: the density of corneal stromal cell-BC composite membrane was significantly higher than that of normal corneal tissue at 1 week after transplantation. On May, the boundary of composite membrane was blurred and the density was close to normal corneal tissue. The results showed that the number and morphology of endothelial cells around the grafts were normal on February, and the density of stromal cells decreased in July, and the nerve fibers were occasionally seen. Pathological examination: on May, corneal epithelial cells proliferated and arranged in disorder. The fibrous structure of stromal cell-BC composite membrane was different from that of normal corneal stroma, the adhesion between them was not close and inflammatory cells infiltrated. On July, corneal epithelial cells were still proliferated, but arranged regularly. There were no vacuoles and cellular structure in the composite membrane, indicating that BC did not degrade violently. The same cellular structure was observed in both sides and inside of BC with a small amount of inflammatory cells infiltrating around it and no necrosis in corneal tissue. Immunohistochemical examination: on July, the composite membrane of three rabbits showed 伪 -SMA staining, indicating that stromal cells were activated into myofibroblasts in vivo. However, the activation of stromal cells decreased gradually with the development of transplantation time, and the boundary between the composite membrane and the normal corneal tissue structure was clear by scanning electron microscope, and the composite membrane could still be seen 7 months after transplantation. Conclusion: The cytocompatibility of BC was good. The successfully constructed corneal stromal cell-BC composite membrane was biodegradable, but the degradation rate of BC membrane in vivo was slow and corneal neovascularization. Proliferation of myofibroblasts is related to corneal transparency.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R779.6

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