本文選題：角膜緣干細胞缺乏 + 動物模型　； 參考：《華中科技大學》2012年碩士論文

【摘要】：【目的】研究成功構(gòu)建兔全角膜緣干細胞缺乏模型的方法和時間，觀察造模后各時間點模型眼角膜的病理結(jié)構(gòu)。 【方法】用手術(shù)刀360°切除角膜緣內(nèi)1mm外2mm厚約100~150μm的上皮組織和剩余的全部中央角膜上皮組織及其淺層基質(zhì)，，以角膜混濁、角膜新生血管和角膜上皮熒光素鈉染色為觀察指標，分別統(tǒng)計術(shù)后第2~5周時的模型得分，并將各時期成功模型眼標本進行HE染色、PAS染色和間接免疫熒光染色。 【結(jié)果】術(shù)后第2~5周時的模型成功率分別為12.5%、62.5%、81.3%和87.5%，卡方檢驗結(jié)果示術(shù)后第3周造模成功率明顯高于第2周，且差異有統(tǒng)計學意義（Χ2=8.533，P=0.003），而第3周與第4周相比、第4周與第5周相比、第3周與第5周相比差異均無統(tǒng)計學意義。造模成功的平均時間是3.21±0.80周。成功模型眼的典型外觀可見角膜明顯混濁、角膜新生血管和熒光素鈉染色陽性。病理切片HE染色示角膜基質(zhì)水腫、較多炎癥細胞浸潤和新生血管。PAS染色僅在第5周時可見杯狀細胞。造模成功的模型眼角膜在各時間點CK3染色均為陰性。 【結(jié)論】手術(shù)切除角膜緣及全部中央角膜上皮和淺層基質(zhì)可以成功構(gòu)建全角膜緣干細胞缺乏的動物模型，模型成功的平均時間為3.2周。 【目的】研究兔角膜緣干細胞在纖維蛋白凝膠上的最佳接種方式及其生長情況。 【方法】取角膜緣上皮組織塊進行細胞培養(yǎng)，MTT法測定細胞相對活性，并用針對抗角蛋白3（CK3）的單克隆抗體AE5和抗增殖性細胞核抗原單克隆抗體（PCNA）進行間接免疫熒光法來鑒定角膜緣干細胞。將傳第二代的細胞分A、B兩組分別接種到纖維蛋白凝膠表面和凝膠體內(nèi)進行培養(yǎng)，在接種后第5天、10天和15天時將凝膠溶解離心后計數(shù)細胞數(shù)目，比較兩種接種方式下細胞的增殖力。 【結(jié)果】兔角膜緣干細胞傳第一代時在接種后第三天細胞增殖活性最強，間接免疫熒光染色：CK3染色胞漿陽性，PCNA染色胞核陽性。以凝膠為載體培養(yǎng)干細胞時，第5d時A組細胞數(shù)低于B組，第10d和第15d時A組細胞數(shù)均高于B組。 【結(jié)論】以纖維蛋白凝膠為載體培養(yǎng)角膜緣干細胞時將細胞接種在凝膠表面比包埋在凝膠體內(nèi)更有利于細胞增殖，適合用于角膜緣干細胞移植培養(yǎng)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 擄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 鹵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.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R779.65

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