【摘要】：目的：體外分離、培養(yǎng)人脂肪間充質(zhì)干細胞與兔角膜內(nèi)皮細胞，了解這兩種細胞體外增值生長特性，并通過共培養(yǎng)誘導(dǎo)人脂肪間充質(zhì)干細胞向角膜內(nèi)皮樣細胞分化，檢測其功能性蛋白質(zhì)-水通道蛋白1（Aquaporin1，AQP1)的表達，以探討誘導(dǎo)后的人脂肪間充質(zhì)干細胞作為替代角膜內(nèi)皮細胞進行移植手術(shù)的種子細胞的可行性。 方法： 1、從醫(yī)院手術(shù)室獲取人體腹部脂肪，通過膠原酶消化、離心等方法分離提取人脂肪間充質(zhì)干細胞進行培養(yǎng)，觀察及描繪細胞生長狀態(tài)。取P3細胞，用流式細胞儀做表面抗原鑒定。 2、揭膜法獲取新西蘭大白兔眼后彈力層及內(nèi)皮細胞層，置于細胞培養(yǎng)皿中進行培養(yǎng)，觀察及描繪角膜內(nèi)皮細胞生長狀態(tài)。取P1細胞，免疫組織化學(xué)鑒定角膜內(nèi)皮細胞的相對特異性蛋白質(zhì)-神經(jīng)特異性烯醇化酶(NSE)。 3、將兔角膜內(nèi)皮細胞和人脂肪間充質(zhì)干細胞分別接種于Transwell上、下兩室，共培養(yǎng)10d后，觀察誘導(dǎo)后人脂肪間充質(zhì)干細胞的形態(tài)，免疫組織化學(xué)鑒定AQP1的表達。 結(jié)果： 1、體外培養(yǎng)的原代人脂肪間充質(zhì)干細胞7-9d后呈典型的成纖維細胞樣，12-14d后鋪滿瓶底約80%左右，傳代細胞增值明顯快于原代細胞；細胞生長曲線呈“S”形，3-5d為對數(shù)生長期；流式細胞儀結(jié)果顯示細胞表面抗原CD44陽性率為93.7%，CD34陽性率為1.6%。 2、揭膜法培養(yǎng)的原代兔角膜內(nèi)皮細胞，5d后并形成良好的單層，呈“鋪路石子”狀，10d后呈漩渦狀排列，中央細胞呈三角形及短梭形，傳代細胞形態(tài)多為六邊形或多邊形；細胞生長曲線基本呈“S”形，2-5d為對數(shù)生長期；免疫組織化學(xué)檢測細胞NSE表達，胞漿見棕黃色粗大顆粒，染色陽性。 3、人脂肪間充質(zhì)干細胞與兔角膜內(nèi)皮細胞于Transwell共培養(yǎng)系統(tǒng)中培養(yǎng)10d后，細胞形態(tài)無明顯改變，免疫組織化學(xué)檢測誘導(dǎo)后的人脂肪間充質(zhì)干細胞AQP1表達，胞膜染色呈陽性。 結(jié)論：通過上述方法可以獲得活性較好且純度較高的人脂肪間充質(zhì)干細胞和兔角膜內(nèi)皮細胞；利用Transwell共培養(yǎng)系統(tǒng)可以成功誘導(dǎo)人脂肪間充質(zhì)干細胞表達角膜內(nèi)皮細胞功能性蛋白質(zhì)AQP1；誘導(dǎo)后的人脂肪間充質(zhì)干細胞將來很有可能作為移植治療角膜內(nèi)皮病變和損傷的種子細胞。
[Abstract]:Objective: to isolate and culture human adipose mesenchymal stem cells and rabbit corneal endothelial cells in vitro, to understand the value-added growth characteristics of these two kinds of cells in vitro, and to induce human adipose mesenchymal stem cells to differentiate into corneal endothelial cells by co-culture. The expression of aquaporin 1 (Aquaporin1,AQP1), a functional protein, was detected to explore the feasibility of inducing human adipose mesenchymal stem cells as seed cells to replace corneal endothelial cells. Methods: 1. Human abdominal fat was obtained from hospital operating room. Human adipose mesenchymal stem cells were isolated and extracted by collagenase digestion and centrifugation, and the cell growth status was observed and described. 2. P3 cells were identified by flow cytometry. 2. The posterior elastic layer and endothelial cell layer of New Zealand white rabbits were obtained by debunking method and cultured in cell culture dish to observe and depict the growth state of corneal endothelial cells. Nerve specific enolase (NSE)., a relatively specific protein in corneal endothelial cells, was identified by immunohistochemistry in P1 cells. 3. Rabbit corneal endothelial cells and human adipose mesenchymal stem cells were inoculated on Transwell and co-cultured for 10 days. The morphology of induced human adipose mesenchymal stem cells was observed and the expression of AQP1 was identified by immunohistochemistry. Results: 1. The primary human adipose mesenchymal stem cells cultured in vitro showed typical fibroblasts after 7 and 9 days, and about 80% of them were covered at the bottom of the bottle after 12 to 14 days. The increment of the passage cells was significantly faster than that of the primary cells. The cell growth curve was "S" and the logarithmic growth period was 3 to 5 days, and the positive rate of cell surface antigen CD44 was 93.7% and 1.6%, respectively. 2. The primary rabbit corneal endothelial cells cultured by debunking method formed a good monolayer after 5 days, in the shape of "paving stone", 10 days later, the central cells were triangular and short fusiform, and most of the passage cells were hexagonal or polygons. The cell growth curve was basically "S" shape, and the logarithmic growth period was 2 鈮，

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