胶原基含糖半互穿网络聚合角膜替代物体内外生物相容性研究

发布时间：2018-06-17 10:55

本文选题：胶原基含糖聚合半互穿网络角膜替代物 + 生物相容性　；参考：《天津医科大学》2012年硕士论文

【摘要】：研究目的评价胶原基含糖半互穿网络角膜替代物在体外以及兔角膜组织中的生物相容性,以探讨其作为角膜支架材料的可行性。方法第一部分：体外生物相容性研究,选择中国大耳白兔1只,雌雄不限,2.0-2.5kg,由天津医科大学基础实验动物中心提供,排除角膜疾患。胶原基含糖半互穿网络角膜替代物材料由天津大学材料科学与技术学院提供。材料性能：直径12mm,厚度300μm,白光透过率90%以上,平衡含水量90%以上,抗张强度0.6±0.1mpa,体外胶原酶(12微克／毫升)降解试验,30小时内剩余质量在40%以上。取材：速眠新(静松灵、乙二胺四乙酸(EDTA)、盐酸二氢埃托啡和氟哌啶醇的复方制剂)和氯胺酮进行肌肉注射麻醉,兔眼周围予以清洁消毒铺巾,开睑器开眼,抗生素盐水冲洗结膜囊,在显微镜下用隧道刀对角膜缘上皮进行剖切,剪下角膜缘上皮组织,再剪成1mm×1mm大小的组织块均匀铺于胶原基含糖半互穿网络聚合角膜替代材料及半透膜上。倒置显微镜对体外构建的角膜上皮细胞生长速度,形态等进行观察,两周后将培养细胞行苏木精一伊红(HE)染色。第二部分：体内生物相容性研究,实验组48眼,单眼前板层植入胶原基含糖半互穿网络角膜替代物材料,对照组48眼行同种异体角膜前板层移植,其余24只白兔为板层角膜供体。角膜移植术后3天、7天、15天、1个月、3个月、6个月进行肉眼及裂隙灯观察,观察内容包括：角膜透明度及新生血管情况,术后3天、7天、15天、1个月、3个月、6个月分别处死每组动物中的8只,术眼固定于10%甲醛溶液中,行HE染色在光镜下进行兔角膜组织病理学观察及上皮细胞标志蛋白K3的免疫组化检测。评分结果采用Wileoxon符号秩和检验判断实验组和对照组在角膜透明度及新生血管情况方面有无统计学差异。结果第一部分 1.倒置显微镜观察,5天左右组织块细胞向周围融合成单层,细胞呈膜状生长,生长界限明显,成沙滩样外观,细胞间相互紧密连接,接近组织块的区域可呈复层生长,持续培养2周,细胞呈复层生长。气液界面培养可以形成连续的上皮细胞层,细胞和载体之间连接较牢固。 2.HE染色显示体外构建的角膜上皮具有复层扁平上皮的结构。第二部分 1.术后实验组及对照组角膜透明度逐渐升高,术后3天、7天、15天两组透明度比较差异有统计学意义(P0.05),术后1个月、3个月及6个月与对照组比较,差异无统计学意义(P0.05)。 2.实验组角膜新生血管评分在术后3天,7天、15天、1个月随时间延长逐渐升高,至术后1个月达最高,后逐渐降低。对照组角膜新生血管评分在术后3天、7天、15天逐渐升高,术后15天达到最高,后逐渐减退。统计分析显示,两组术后各时间点角膜新生血管评分比较,术后3天及术后1个月、3个月、6个月差异无统计学意义(P0.05),其余时间点差异均有统计学意义(P0.05)。 3.术后3天,实验组角膜中央上皮层大部分缺损,周边上皮正在向中央生长,均匀红染的胶原物质覆盖表面,基质层水肿,炎性细胞在植片与切口交界处明显。术后7天,角膜表面单层上皮细胞局部覆盖,基质层内可见逐渐降解的胶原物质,浅层基质胶原纤维排列紊乱,并且大量成纤维细胞,散在少量淋巴细胞。术后15天,浅层基质仍有较多的成纤维细胞,沉积的胶原纤维排列仍不规则。术后1个月,角膜上皮覆盖完全,浅基质层胶原纤维排列趋于规则,但仍较正常基质紊乱。术后3个月,上皮修复良好,浅基质层胶原纤维走行与下方胶原纤维排列趋于一致。至术后6个月,角膜上皮修复,胶原纤维排列规则,部分区域存在小的胶原紊乱区。对照组术后3天,角膜上皮局部缺损,浅基质水肿明显,成纤维细胞略有增加。术后7天角膜上皮修复良好,基质水肿减轻,成纤维细胞数量增加较明显。术后15天,上皮完整,基质层胶原纤维排列基本规则,成纤维细胞数量有所减少。术后1个月,胶原纤维排列趋于一致,局部欠规则。术后3个月及6个月,细胞层次清楚,基质层胶原排列规则。 4.免疫组化显示术后6个月角膜上皮细胞特异性标志蛋白K3检测为阳性表达。结论胶原基含糖半互穿网络角膜替代物在体外支持角膜上皮生长,细胞间连接紧密,形成连续的细胞上皮层。该材料在兔角膜组织内能支持角膜上皮的生长,促进活性角膜基质的改建,下一步我们将继续提高角膜材料体内移植的稳定性,加强其机械强度,降低材料的免疫性。胶原基含糖半互穿网络角膜替代物具有较好的体内外生物相容性,进一步改进后有望成为一种新型的角膜移植支架材料。
[Abstract]:Purpose of study

To evaluate the biocompatibility of corneal substitutes of collagen - based semi - interpenetrating network in vitro and in rabbit corneal tissue to investigate the feasibility of using corneal substitutes as corneal scaffold materials .

method

The first part : In vitro biocompatibility study , 1 male white rabbit was selected , the male and female were not limited , 2.0 - 2.5kg were provided by Tianjin Medical University Foundation Experimental Animal Center . The material properties were as follows : 12 mm in diameter , 300 渭m in thickness , 90 % of white light transmittance , 90 % of balance water content , 0.6 卤 0.1mpa in vitro collagenase and 12 ug / ml in vitro .

In the second part : In vivo biocompatibility study , 48 eyes of the experimental group were implanted with collagen - based semi - interpenetrating network corneal substitutes . The other 24 white rabbits were observed with naked eyes and slit lights at 3 days , 7 days , 15 days , 1 month , 3 months and 6 months .

Results

the first portion

1 . After 5 days , the cells were fused into a single layer , the cells were in the form of film - like growth , the growth boundary was obvious , the appearance of the beach - like appearance was observed , the cells were closely connected with each other , the cells close to the tissue mass could grow in a complex layer , and the cells were grown for 2 weeks .

2.HE staining showed that the corneal epithelium constructed in vitro had the structure of stratified squamous epithelium .

the second part

1 . The corneal transparency of the experimental group and the control group increased gradually , and the difference of the two groups was statistically significant ( P0.05 ) at 3 days , 7 days and 15 days after the operation , and the difference was not statistically significant ( P0.05 ) .

2 . The score of corneal neovascularization in experimental group increased gradually after 3 days , 7 days , 15 days and 1 month after operation . The score of corneal neovascularization in the control group increased gradually after 3 days , 7 days and 15 days after operation .

3 days after operation , the central epithelial layer of the cornea of the experimental group was mostly defect , the peripheral epithelium was growing in the center , the stroma layer was edema , the inflammatory cells were more obvious at the junction of the graft and the incision . After the operation , the corneal epithelium was repaired well , and the number of fibroblasts in the superficial stroma was decreased . After the operation , the corneal epithelium was repaired well , and the number of fibroblasts in the superficial stroma was decreased .

4 . The expression of specific marker protein K3 in corneal epithelial cells was detected by immunohistochemistry in 6 months after operation .

Conclusion

in ord to promote that growth of corneal epithelium and to promote the alteration of active corneal stroma , we will continue to improve the stability of corneal epithelium transplantation , strengthen its mechanical strength and reduce the immunity of the material .
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R779.6

【参考文献】