本文選題：膠原基含糖聚合半互穿網(wǎng)絡角膜替代物 + 生物相容性�。� 參考：《天津醫(yī)科大學》2012年碩士論文

【摘要】：研究目的 評價膠原基含糖半互穿網(wǎng)絡角膜替代物在體外以及兔角膜組織中的生物相容性,以探討其作為角膜支架材料的可行性。 方法 第一部分：體外生物相容性研究,選擇中國大耳白兔1只,雌雄不限,2.0-2.5kg,由天津醫(yī)科大學基礎實驗動物中心提供,排除角膜疾患。膠原基含糖半互穿網(wǎng)絡角膜替代物材料由天津大學材料科學與技術學院提供。材料性能：直徑12mm,厚度300μm,白光透過率90%以上,平衡含水量90%以上,抗張強度0.6±0.1mpa,體外膠原酶(12微克／毫升)降解試驗,30小時內(nèi)剩余質(zhì)量在40%以上。取材：速眠新(靜松靈、乙二胺四乙酸(EDTA)、鹽酸二氫埃托啡和氟哌啶醇的復方制劑)和氯胺酮進行肌肉注射麻醉,兔眼周圍予以清潔消毒鋪巾,開瞼器開眼,抗生素鹽水沖洗結膜囊,在顯微鏡下用隧道刀對角膜緣上皮進行剖切,剪下角膜緣上皮組織,再剪成1mm×1mm大小的組織塊均勻鋪于膠原基含糖半互穿網(wǎng)絡聚合角膜替代材料及半透膜上。倒置顯微鏡對體外構建的角膜上皮細胞生長速度,形態(tài)等進行觀察,兩周后將培養(yǎng)細胞行蘇木精一伊紅(HE)染色。 第二部分：體內(nèi)生物相容性研究,實驗組48眼,單眼前板層植入膠原基含糖半互穿網(wǎng)絡角膜替代物材料,對照組48眼行同種異體角膜前板層移植,其余24只白兔為板層角膜供體。角膜移植術后3天、7天、15天、1個月、3個月、6個月進行肉眼及裂隙燈觀察,觀察內(nèi)容包括：角膜透明度及新生血管情況,術后3天、7天、15天、1個月、3個月、6個月分別處死每組動物中的8只,術眼固定于10%甲醛溶液中,行HE染色在光鏡下進行兔角膜組織病理學觀察及上皮細胞標志蛋白K3的免疫組化檢測。評分結果采用Wileoxon符號秩和檢驗判斷實驗組和對照組在角膜透明度及新生血管情況方面有無統(tǒng)計學差異。 結果 第一部分 1.倒置顯微鏡觀察,5天左右組織塊細胞向周圍融合成單層,細胞呈膜狀生長,生長界限明顯,成沙灘樣外觀,細胞間相互緊密連接,接近組織塊的區(qū)域可呈復層生長,持續(xù)培養(yǎng)2周,細胞呈復層生長。氣液界面培養(yǎng)可以形成連續(xù)的上皮細胞層,細胞和載體之間連接較牢固。 2.HE染色顯示體外構建的角膜上皮具有復層扁平上皮的結構。 第二部分 1.術后實驗組及對照組角膜透明度逐漸升高,術后3天、7天、15天兩組透明度比較差異有統(tǒng)計學意義(P0.05),術后1個月、3個月及6個月與對照組比較,差異無統(tǒng)計學意義(P0.05)。 2.實驗組角膜新生血管評分在術后3天,7天、15天、1個月隨時間延長逐漸升高,至術后1個月達最高,后逐漸降低。對照組角膜新生血管評分在術后3天、7天、15天逐漸升高,術后15天達到最高,后逐漸減退。統(tǒng)計分析顯示,兩組術后各時間點角膜新生血管評分比較,術后3天及術后1個月、3個月、6個月差異無統(tǒng)計學意義(P0.05),其余時間點差異均有統(tǒng)計學意義(P0.05)。 3.術后3天,實驗組角膜中央上皮層大部分缺損,周邊上皮正在向中央生長,均勻紅染的膠原物質(zhì)覆蓋表面,基質(zhì)層水腫,炎性細胞在植片與切口交界處明顯。術后7天,角膜表面單層上皮細胞局部覆蓋,基質(zhì)層內(nèi)可見逐漸降解的膠原物質(zhì),淺層基質(zhì)膠原纖維排列紊亂,并且大量成纖維細胞,散在少量淋巴細胞。術后15天,淺層基質(zhì)仍有較多的成纖維細胞,沉積的膠原纖維排列仍不規(guī)則。術后1個月,角膜上皮覆蓋完全,淺基質(zhì)層膠原纖維排列趨于規(guī)則,但仍較正常基質(zhì)紊亂。術后3個月,上皮修復良好,淺基質(zhì)層膠原纖維走行與下方膠原纖維排列趨于一致。至術后6個月,角膜上皮修復,膠原纖維排列規(guī)則,部分區(qū)域存在小的膠原紊亂區(qū)。對照組術后3天,角膜上皮局部缺損,淺基質(zhì)水腫明顯,成纖維細胞略有增加。術后7天角膜上皮修復良好,基質(zhì)水腫減輕,成纖維細胞數(shù)量增加較明顯。術后15天,上皮完整,基質(zhì)層膠原纖維排列基本規(guī)則,成纖維細胞數(shù)量有所減少。術后1個月,膠原纖維排列趨于一致,局部欠規(guī)則。術后3個月及6個月,細胞層次清楚,基質(zhì)層膠原排列規(guī)則。 4.免疫組化顯示術后6個月角膜上皮細胞特異性標志蛋白K3檢測為陽性表達。 結論 膠原基含糖半互穿網(wǎng)絡角膜替代物在體外支持角膜上皮生長,細胞間連接緊密,形成連續(xù)的細胞上皮層。該材料在兔角膜組織內(nèi)能支持角膜上皮的生長,促進活性角膜基質(zhì)的改建,下一步我們將繼續(xù)提高角膜材料體內(nèi)移植的穩(wěn)定性,加強其機械強度,降低材料的免疫性。膠原基含糖半互穿網(wǎng)絡角膜替代物具有較好的體內(nèi)外生物相容性,進一步改進后有望成為一種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 鹵 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 .
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R779.6

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