纤维蛋白凝胶用于兔角膜缘上皮细胞移植培养的研究

发布时间：2018-05-22 09:43

本文选题：角膜缘干细胞缺乏 + 动物模型　；参考：《华中科技大学》2012年硕士论文

【摘要】：【目的】研究成功构建兔全角膜缘干细胞缺乏模型的方法和时间，观察造模后各时间点模型眼角膜的病理结构。【方法】用手术刀360°切除角膜缘内1mm外2mm厚约100~150μm的上皮组织和剩余的全部中央角膜上皮组织及其浅层基质，，以角膜混浊、角膜新生血管和角膜上皮荧光素钠染色为观察指标，分别统计术后第2~5周时的模型得分，并将各时期成功模型眼标本进行HE染色、PAS染色和间接免疫荧光染色。【结果】术后第2~5周时的模型成功率分别为12.5%、62.5%、81.3%和87.5%，卡方检验结果示术后第3周造模成功率明显高于第2周，且差异有统计学意义（Χ2=8.533，P=0.003），而第3周与第4周相比、第4周与第5周相比、第3周与第5周相比差异均无统计学意义。造模成功的平均时间是3.21±0.80周。成功模型眼的典型外观可见角膜明显混浊、角膜新生血管和荧光素钠染色阳性。病理切片HE染色示角膜基质水肿、较多炎症细胞浸润和新生血管。PAS染色仅在第5周时可见杯状细胞。造模成功的模型眼角膜在各时间点CK3染色均为阴性。【结论】手术切除角膜缘及全部中央角膜上皮和浅层基质可以成功构建全角膜缘干细胞缺乏的动物模型，模型成功的平均时间为3.2周。【目的】研究兔角膜缘干细胞在纤维蛋白凝胶上的最佳接种方式及其生长情况。【方法】取角膜缘上皮组织块进行细胞培养，MTT法测定细胞相对活性，并用针对抗角蛋白3（CK3）的单克隆抗体AE5和抗增殖性细胞核抗原单克隆抗体（PCNA）进行间接免疫荧光法来鉴定角膜缘干细胞。将传第二代的细胞分A、B两组分别接种到纤维蛋白凝胶表面和凝胶体内进行培养，在接种后第5天、10天和15天时将凝胶溶解离心后计数细胞数目，比较两种接种方式下细胞的增殖力。【结果】兔角膜缘干细胞传第一代时在接种后第三天细胞增殖活性最强，间接免疫荧光染色：CK3染色胞浆阳性，PCNA染色胞核阳性。以凝胶为载体培养干细胞时，第5d时A组细胞数低于B组，第10d和第15d时A组细胞数均高于B组。【结论】以纤维蛋白凝胶为载体培养角膜缘干细胞时将细胞接种在凝胶表面比包埋在凝胶体内更有利于细胞增殖，适合用于角膜缘干细胞移植培养的研究。
[Abstract]:[objective] to study the method and time of establishing rabbit limbal stem cell deficiency model successfully, and to observe the pathological structure of corneal model at different time points. [methods] the corneal opacity, corneal neovascularization and fluorescein natrium staining of corneal neovascularization and corneal epithelium were observed with the surgical scalpel 360 掳excision of 100 ~ 150 渭 m 2mm outside the limbus of cornea and all remaining central corneal epithelium and its superficial stroma. The model scores at the 2nd week after operation were counted and the successful eye specimens were stained with HE staining pas and indirect immunofluorescence staining. [results] the success rates of the models at the second week after operation were 81.3% and 87.5%, respectively. The results of chi-square test showed that the success rate of modeling at the third week after operation was significantly higher than that at the second week, and the difference was statistically significant (X ~ (2) 8.53 ~ (3) P ~ (0. 003), but the third week compared with the fourth week. There was no significant difference between week 4 and week 5, week 3 and week 5. The average time of success was 3.21 卤0.80 weeks. The typical appearance of successful model eyes showed corneal opacity, corneal neovascularization and positive staining of fluorescein sodium. He staining showed corneal stroma edema, more inflammatory cell infiltration and neovascularization. Pas staining showed goblet cells only at the 5th week. The CK3 staining of the model cornea was negative at all time points. [conclusion] the animal model of limbal stem cell deficiency can be successfully constructed by excision of limbus cornea and all central corneal epithelium and superficial stroma. The average successful time of the model is 3.2 weeks. Objective: to study the optimal inoculation and growth of rabbit limbal stem cells on fibrin gel. [methods] the relative activity of corneal limbal epithelium was measured by MTT assay. The corneal limbal stem cells were identified by indirect immunofluorescence method using monoclonal antibody AE5 and anti-proliferating cell nuclear antigen monoclonal antibody PCNA. The second passage of cells were divided into two groups: group A and B were inoculated into fibrin gel and cultured in vivo. The cells were dissolved and centrifuged on the 5th and 15th days after inoculation, and the number of cells was counted. The proliferative power of cells was compared between the two inoculation methods. [results] the proliferative activity of rabbit corneal limbal stem cells was the highest on the third day after inoculation, and the cytoplasm was positive with cytoplasm and PCNA with indirect immunofluorescence staining. The number of stem cells in group A was lower than that in group B on day 5, and the number of cells in group A was higher than that in group B on day 10 and day 15. [conclusion] when cultured limbal stem cells with fibrin gel as carrier, seeding the cells on the gel surface is more beneficial to cell proliferation than embedding in the gel, so it is suitable for the study of corneal limbal stem cell transplantation culture.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R779.65

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