| 3D共培养模型对体外培养兔角膜缘干细胞的影响 |
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| 引用本文： | 周瑛,孙秋萍,蔡岩,高晓唯.3D共培养模型对体外培养兔角膜缘干细胞的影响[J].眼科新进展,2019,0(4):331-335. |
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| 作者姓名： | 周瑛 孙秋萍 蔡岩 高晓唯 |
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| 作者单位： | 830000 新疆维吾尔自治区乌鲁木齐市，新疆医科大学（周瑛，高晓唯）；830000 新疆维吾尔自治区乌鲁木齐市，解放军第四七四医院眼科（周瑛，孙秋萍，蔡岩，高晓唯） |
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| 基金项目： | 新疆维吾尔自治区科技支疆项目(编号:201491171);解放军第四七四医院重点扶持科研项目(编号:2017474006;2018474008)~~ |
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| 摘 要： | 目的探讨三维(three-dimension,3D)共培养模型对体外扩增兔角膜缘干细胞(limbal stem cells,LSCs)的作用。方法体外培养兔LSCs,以牙周膜干细胞(human periodontal stem cell,HPDLSCs)作为饲细胞建立共培养模型,根据载体不同分为3组,纤维蛋白胶组:将兔LSCs、HPDLSCs、纤维蛋白胶3D共培养;羊膜组:将兔LSCs、HPDLSCs、羊膜3D共培养;对照组:将兔LSCs、HPDLSCs共培养。共培养5 d后,用倒置显微镜、HE染色、扫描电镜观察兔LSCs的细胞形态,采用免疫荧光染色检测各组兔LSCs阳性标志物p63的表达,并比较各组p63的阳性率。结果共培养5 d后,倒置显微镜、HE染色观察细胞形态,羊膜组、纤维蛋白胶组细胞排列紧密,呈大小不等的团块状集落,细胞大小基本一致;对照组细胞排列相对稀疏;扫描电镜下观察见纤维蛋白胶组和羊膜组细胞与载体贴附良好。免疫荧光染色显示纤维蛋白胶组、羊膜组和对照组LSCs的p63阳性率分别为(69.93±8.76)%、(78.36±8.56)%和(58.59±8.31)%,三组间总体比较差异有统计学意义(F=9.43,P=0.000)。两两比较中纤维蛋白胶组、羊膜组LSCs的p63阳性率均大于对照组,差异均有统计学意义(均为P<0.05)。纤维蛋白胶组、羊膜组的p63阳性率比较,差异无统计学意义(P>0.05)。结论以纤维蛋白胶、羊膜作为载体,HPDLSCs作为饲细胞的3D共培养模型有助于体外扩增兔LSCs。
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| 关 键 词： | 角膜缘干细胞 纤维蛋白胶 羊膜 3D共培养 |
Effect of three-dimensional co-culture model on rabbit corneal limbal stem cells culture in vitro |
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| ZHOU Ying,SUN Qiu-Ping,CAI Yan,GAO Xiao-Wei.Effect of three-dimensional co-culture model on rabbit corneal limbal stem cells culture in vitro[J].Recent Advances in Ophthalmology,2019,0(4):331-335. |
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| Authors: | ZHOU Ying SUN Qiu-Ping CAI Yan GAO Xiao-Wei |
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| Affiliation: | Xinjiang Medical University（ZHOU Ying，GAO Xiao-Wei），Urumqi 830000，Xinjiang Uygur Autonomous Region，China；Ophthalmic Center，No.474 Hospital of Chinese PLA（ZHOU Ying，SUN Qiu-Ping，CAI Yan，GAO Xiao-Wei），Urumqi 830000，Xinjiang Uygur Autonomous Region，China |
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| Abstract: | Objective To investigate the effect of three-dimensional（3D） co-culture model on the expansion of rabbit limbal stem cells in vitro.Methods The rabbit limbal stem cells （LSCs） were cultured in vitro，and the human periodontal stem cells （HPDLSCs） were served as the feeder cells to establish of co-culture model.According to different carriers，the cells were divided into three groups:the fibrin glue group，in which LSCs were co-cultured with fibrin glue and HPDLSCs；the amniotic membrane group，in which LSCs were co-cultured with amniotic membrane and HPDLSCs；and the control group，in which LSCs were co-cultured only with the HPDLSCs.After co-culture for 5 days，we used inverted microscope，hematoxylin and eosin staining and scanning electron microscopy to observe the cells morphology，and then used the immunofluorescence staining to detect the expression of the LSCs positive marker p63，followed by the comparison of the positive rate of p63 in each group.Results After co-cultured for five days，the results from inverted microscope and hematoxylin and eosin staining showed that cells in the fibrin glue group and the amniotic membrane group were uniform and serried arrangement，and presented cluster-like colonies with varying sizes，and cell sizes were uniformly，whereas cells in the control group were relatively sparse.Scanning electron microscopy showed that the cells were well adhered to the carrier in the fibrin glue group and the amniotic membrane group.Immunofluorescence staining showed that the positive rates of p63 in the fibrin glue group，the amniotic membrane group，the control group were （69.93±8.76）％，（78.36±8.56）％，（58.59±8.31）％，respectively，and there was significant difference among the three groups （F=9.43，P=0.000）.In pairwise comparison，the positive rates of p63 in the fibrin glue group and amniotic membrane group were both higher than those of the control group，and there was significant differences （both P<0.05），but there was no significant difference in the positive rate of p63 between the fibrin glue group and amniotic membrane group （P>"0.05）.Conclusion The co-culture 3D model is helpful for in vivo expansion of rabbit LSCs. |
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| Keywords: | limbal stem cells fibin glue amniotic membrane 3D co-culture |
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