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

【摘要】：研究目的 評(píng)價(jià)膠原基含糖半互穿網(wǎng)絡(luò)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(xué)基礎(chǔ)實(shí)驗(yàn)動(dòng)物中心提供,排除角膜疾患。膠原基含糖半互穿網(wǎng)絡(luò)角膜替代物材料由天津大學(xué)材料科學(xué)與技術(shù)學(xué)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(qiáng)度0.6±0.1mpa,體外膠原酶(12微克／毫升)降解試驗(yàn),30小時(shí)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(fù)方制劑)和氯胺酮進(jìn)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(jié)膜囊,在顯微鏡下用隧道刀對角膜緣上皮進(jìn)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(luò)聚合角膜替代材料及半透膜上。倒置顯微鏡對體外構(gòu)建的角膜上皮細(xì)胞生長速度,形態(tài)等進(jìn)行觀察,兩周后將培養(yǎng)細(xì)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(shí)驗(yàn)組48眼,單眼前板層植入膠原基含糖半互穿網(wǎng)絡(luò)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(shù)后3天、7天、15天、1個(gè)月、3個(gè)月、6個(gè)月進(jìn)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(shù)后3天、7天、15天、1個(gè)月、3個(gè)月、6個(gè)月分別處死每組動(dòng)物中的8只,術(shù)眼固定于10%甲醛溶液中,行HE染色在光鏡下進(jìn)行兔角膜組織病理學(xué)觀察及上皮細(xì)胞標(biāo)志蛋白K3的免疫組化檢測。評(píng)分結(jié)果采用Wileoxon符號(hào)秩和檢驗(yàn)判斷實(shí)驗(yàn)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(jì)學(xué)差異。 結(jié)果 第一部分 1.倒置顯微鏡觀察,5天左右組織塊細(xì)胞向周圍融合成單層,細(xì)胞呈膜狀生長,生長界限明顯,成沙灘樣外觀,細(xì)胞間相互緊密連接,接近組織塊的區(qū)域可呈復(fù)層生長,持續(xù)培養(yǎng)2周,細(xì)胞呈復(fù)層生長。氣液界面培養(yǎng)可以形成連續(xù)的上皮細(xì)胞層,細(xì)胞和載體之間連接較牢固。 2.HE染色顯示體外構(gòu)建的角膜上皮具有復(fù)層扁平上皮的結(jié)構(gòu)。 第二部分 1.術(shù)后實(shí)驗(yàn)組及對照組角膜透明度逐漸升高,術(shù)后3天、7天、15天兩組透明度比較差異有統(tǒng)計(jì)學(xué)意義(P0.05),術(shù)后1個(gè)月、3個(gè)月及6個(gè)月與對照組比較,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 2.實(shí)驗(yàn)組角膜新生血管評(píng)分在術(shù)后3天,7天、15天、1個(gè)月隨時(shí)間延長逐漸升高,至術(shù)后1個(gè)月達(dá)最高,后逐漸降低。對照組角膜新生血管評(píng)分在術(shù)后3天、7天、15天逐漸升高,術(shù)后15天達(dá)到最高,后逐漸減退。統(tǒng)計(jì)分析顯示,兩組術(shù)后各時(shí)間點(diǎn)角膜新生血管評(píng)分比較,術(shù)后3天及術(shù)后1個(gè)月、3個(gè)月、6個(gè)月差異無統(tǒng)計(jì)學(xué)意義(P0.05),其余時(shí)間點(diǎn)差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。 3.術(shù)后3天,實(shí)驗(yàn)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(xì)胞在植片與切口交界處明顯。術(shù)后7天,角膜表面單層上皮細(xì)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(xì)胞,散在少量淋巴細(xì)胞。術(shù)后15天,淺層基質(zhì)仍有較多的成纖維細(xì)胞,沉積的膠原纖維排列仍不規(guī)則。術(shù)后1個(gè)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(shù)后3個(gè)月,上皮修復(fù)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(shù)后6個(gè)月,角膜上皮修復(fù),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(shù)后3天,角膜上皮局部缺損,淺基質(zhì)水腫明顯,成纖維細(xì)胞略有增加。術(shù)后7天角膜上皮修復(fù)良好,基質(zhì)水腫減輕,成纖維細(xì)胞數(shù)量增加較明顯。術(shù)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(xì)胞數(shù)量有所減少。術(shù)后1個(gè)月,膠原纖維排列趨于一致,局部欠規(guī)則。術(shù)后3個(gè)月及6個(gè)月,細(xì)胞層次清楚,基質(zhì)層膠原排列規(guī)則。 4.免疫組化顯示術(shù)后6個(gè)月角膜上皮細(xì)胞特異性標(biāo)志蛋白K3檢測為陽性表達(dá)。 結(jié)論 膠原基含糖半互穿網(wǎng)絡(luò)角膜替代物在體外支持角膜上皮生長,細(xì)胞間連接緊密,形成連續(xù)的細(xì)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(jìn)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(qiáng)其機(jī)械強(qiáng)度,降低材料的免疫性。膠原基含糖半互穿網(wǎng)絡(luò)角膜替代物具有較好的體內(nèi)外生物相容性,進(jìn)一步改進(jìn)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(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R779.6

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