人脂肪间充质干细胞与兔角膜内皮细胞的体外分离培养及共培养研究
发布时间：2019-05-20 18:26
【摘要】：目的：体外分离、培养人脂肪间充质干细胞与兔角膜内皮细胞，了解这两种细胞体外增值生长特性，并通过共培养诱导人脂肪间充质干细胞向角膜内皮样细胞分化，检测其功能性蛋白质-水通道蛋白1（Aquaporin1，AQP1)的表达，以探讨诱导后的人脂肪间充质干细胞作为替代角膜内皮细胞进行移植手术的种子细胞的可行性。 方法： 1、从医院手术室获取人体腹部脂肪，通过胶原酶消化、离心等方法分离提取人脂肪间充质干细胞进行培养，观察及描绘细胞生长状态。取P3细胞，用流式细胞仪做表面抗原鉴定。 2、揭膜法获取新西兰大白兔眼后弹力层及内皮细胞层，置于细胞培养皿中进行培养，观察及描绘角膜内皮细胞生长状态。取P1细胞，免疫组织化学鉴定角膜内皮细胞的相对特异性蛋白质-神经特异性烯醇化酶(NSE)。 3、将兔角膜内皮细胞和人脂肪间充质干细胞分别接种于Transwell上、下两室，共培养10d后，观察诱导后人脂肪间充质干细胞的形态，免疫组织化学鉴定AQP1的表达。 结果： 1、体外培养的原代人脂肪间充质干细胞7-9d后呈典型的成纤维细胞样，12-14d后铺满瓶底约80%左右，传代细胞增值明显快于原代细胞；细胞生长曲线呈“S”形，3-5d为对数生长期；流式细胞仪结果显示细胞表面抗原CD44阳性率为93.7%，CD34阳性率为1.6%。 2、揭膜法培养的原代兔角膜内皮细胞，5d后并形成良好的单层，呈“铺路石子”状，10d后呈漩涡状排列，中央细胞呈三角形及短梭形，传代细胞形态多为六边形或多边形；细胞生长曲线基本呈“S”形，2-5d为对数生长期；免疫组织化学检测细胞NSE表达，胞浆见棕黄色粗大颗粒，染色阳性。 3、人脂肪间充质干细胞与兔角膜内皮细胞于Transwell共培养系统中培养10d后，细胞形态无明显改变，免疫组织化学检测诱导后的人脂肪间充质干细胞AQP1表达，胞膜染色呈阳性。 结论：通过上述方法可以获得活性较好且纯度较高的人脂肪间充质干细胞和兔角膜内皮细胞；利用Transwell共培养系统可以成功诱导人脂肪间充质干细胞表达角膜内皮细胞功能性蛋白质AQP1；诱导后的人脂肪间充质干细胞将来很有可能作为移植治疗角膜内皮病变和损伤的种子细胞。
[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，
本文编号：2481861
[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，
本文编号：2481861
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