本文選題：家兔　切入點(diǎn)：角膜內(nèi)皮細(xì)胞　出處：《中國(guó)海洋大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

【摘要】：角膜內(nèi)皮盲是一種常見(jiàn)的角膜病,盡管可以通過(guò)角膜移植來(lái)治愈,但由于角膜的捐獻(xiàn)數(shù)量極其有限和患者的排斥反應(yīng)而使許多患者無(wú)法重見(jiàn)光明。人工角膜組織工程技術(shù)是當(dāng)今治療角膜內(nèi)皮盲等的研究熱點(diǎn),其亟需解決的關(guān)鍵問(wèn)題之一是如何獲得足量的角膜內(nèi)皮細(xì)胞。而角膜內(nèi)皮細(xì)胞的規(guī)�；w外培養(yǎng)是獲得大量角膜內(nèi)皮細(xì)胞的理想途徑,也是人工角膜的重要研究?jī)?nèi)容之一,對(duì)于角膜病理生理學(xué)、藥理毒理學(xué)、免疫學(xué)、分子生物學(xué)研究均具有重要意義。但到目前為止,仍沒(méi)見(jiàn)到有關(guān)家兔角膜內(nèi)皮細(xì)胞未經(jīng)轉(zhuǎn)染而成功建系的報(bào)道。為了探索哺乳動(dòng)物角膜內(nèi)皮細(xì)胞的建系技術(shù),以便為組織工程化人工角膜提供足量的細(xì)胞材料,本文以家兔角膜內(nèi)皮為材料,對(duì)角膜內(nèi)皮細(xì)胞的體外培養(yǎng)、細(xì)胞系的建立及其鑒定展開(kāi)了研究。 為了成功啟動(dòng)家兔角膜內(nèi)皮細(xì)胞的體外培養(yǎng),本文取活家兔的角膜,用氯化汞溶液和慶大霉素依次進(jìn)行消毒,經(jīng)0.25%胰蛋白酶液部分處理角膜內(nèi)皮層后,將角膜內(nèi)皮面朝下貼于24孔培養(yǎng)板中,補(bǔ)加含有硫酸軟骨素、硫酸軟骨素氧化降解物、羧甲基殼多糖、N-乙酰葡萄糖鹽酸鹽、氨基葡萄糖鹽酸鹽、家兔角膜基質(zhì)細(xì)胞培養(yǎng)液、牛眼生素、EGF和bFGF的DMEM-F12培養(yǎng)液(20%胎牛血清),置入37℃、5%CO_2培養(yǎng)箱中培養(yǎng)。為了獲得純凈的角膜內(nèi)皮細(xì)胞,每隔12～48小時(shí)進(jìn)行揭膜和轉(zhuǎn)孔再培養(yǎng),進(jìn)而成功啟動(dòng)了家兔角膜內(nèi)皮細(xì)胞的原代培養(yǎng)。原代培養(yǎng)中的角膜內(nèi)皮細(xì)胞多為四角形或多邊形,一周后逐漸伸展成為多角形的內(nèi)皮樣,三周后細(xì)胞長(zhǎng)成單層,此時(shí)細(xì)胞飽滿、透光性好。待角膜內(nèi)皮細(xì)胞長(zhǎng)成單層后,便采用胰蛋白酶消化法進(jìn)行傳代培養(yǎng)。傳代培養(yǎng)中的細(xì)胞,開(kāi)始時(shí)絕大多數(shù)為多角形內(nèi)皮樣形態(tài),但隨著傳代次數(shù)增加,逐漸轉(zhuǎn)變?yōu)閮?nèi)皮樣與成纖維樣兩種細(xì)胞形態(tài)共存的生長(zhǎng)狀態(tài)。傳至第49代的角膜內(nèi)皮細(xì)胞,主要呈現(xiàn)為成纖維樣細(xì)胞形態(tài),其生長(zhǎng)與分裂狀況良好。此時(shí),細(xì)胞群體的倍增時(shí)間為2.24天,
[Abstract]:Corneal endothelium blindness is a common corneal disease, although it can be cured by corneal transplantation. However, because of the extremely limited amount of corneal donation and the rejection of patients, many patients can not see the light again. Tissue engineering technology of artificial cornea is a hot research topic in the treatment of corneal endothelium blindness. One of the key problems that need to be solved is how to obtain enough corneal endothelial cells, and the in vitro culture of corneal endothelial cells is an ideal way to obtain a large number of corneal endothelial cells, which is also one of the important research contents of corneal prosthesis. For corneal pathophysiology, pharmacology, toxicology, immunology, molecular biology, but so far, In order to explore the technique of establishing corneal endothelial cells in mammals in order to provide enough cellular materials for tissue engineered cornea, there is still no report about the successful establishment of rabbit corneal endothelial cells without transfection. In this paper, rabbit corneal endothelium was used as the material to study the culture, establishment and identification of corneal endothelial cells in vitro. In order to successfully initiate the rabbit corneal endothelial cell culture in vitro, the rabbit cornea was harvested and disinfected with mercuric chloride solution and gentamicin in turn. The corneal endothelium was partially treated with 0.25% trypsin solution. The corneal endothelium was pasted face down to the 24-well culture plate, supplemented with chondroitin sulfate, chondroitin sulfate oxidative degradation, carboxymethyl chitosan N-acetyl glucose hydrochloride, glucosamine hydrochloride, rabbit corneal stromal cell culture medium. In order to obtain pure corneal endothelial cells, 20% fetal bovine serum was cultured in 37 鈩，

本文編號(hào)：1649359