摘要
目的:建立一种模拟糖尿病视网膜病变(DR)血管内皮细胞与神经细胞相互作用的二维体外模型。方法:将大鼠视网膜微血管内皮细胞(RMECs)与神经节细胞(RGCs)通过0.4 μm聚碳酯膜微孔小室进行共培养。按照培养液葡萄糖浓度为分为高糖组和对照组。高糖组和对照组培养液的葡萄糖浓度分别为50.0 mM和5.5 mM。采用荧光倒置显微镜成像观察细胞形态；应用细胞增殖实验检测细胞的增殖情况；采用划痕实验和迁移实验检测细胞迁移的能力；采用管腔形成实验检测管腔的形成能力；使用碘化丙啶－烟酸己可碱实验观察细胞凋亡的情况。细胞迁移数目、凋亡率、管腔形成数量及交叉点数均符合正态分布，以(±s)表示，两组间比较采用独立样本t检验；细胞增殖力的光密度(OD)值和细胞迁移率为偏态分布，以中位数和四分位数描述，组间比较采用秩和检验。结果:共培养模型细胞培养48 h后，共培养模型上室RGCs呈单层椭圆样贴壁生长，下室RMECs较不规则，均呈单层铺路石样生长，细胞间呈紧密连接状态。高糖组和对照组RMECs的OD值分别为1.163(1.142，1.188)和0.812(0.805，0.817)；两组RGCs的OD值分别为0.723(0.709，0.759)和1.934(1.803，2.030)，组间比较的差异均有统计学意义(Z＝－6.069，－5.940；P<0.05)。高糖组和对照组RMECs的细胞迁移率分别为[53.64(37.69，65.88)%]和[32.25(22.34，42.85)%]，前者迁移能力强于后者，组间比较的差异有统计学意义(Z＝－2.841，P<0.05)。高糖组和对照组RMECs的迁移数分别为(164±31)个和(113±16)个；细胞凋亡率分别为(14.30±4.29)%和(9.65±3.43)%；管腔形成数分别为(23±5)个和(19±4)个；管腔交叉点数分别为(11±3)个和(13±2)个。两组RMECs迁移数、管腔形成数及凋亡率比较的差异均有统计学意义(t＝－4.653，－2.137，－2.929；P<0.05)；管腔交叉点数比较的差异无统计学意义(t＝1.424，P>0.05)。高糖组和对照组RGCs细胞凋亡率分别为(6.91±1.15)%和(4.87±1.70)%。两组细胞凋亡率比较的差异有统计学意义(t＝－2.446，P<0.05)。结论:本研究建立的RMECs与RGCs藕联二维体外共培养模型具有与单细胞培养相同的表观，在高糖环境下，RMECs与RGCs的活性、凋亡、RMECs的迁移能力及管腔形成能力均表现为同步的病理生理改变，为DR的进一步机制研究奠定了模型基础。
Abstract
Objective:To establish an 2D in vitro simulations, which model the interactions between retinal vascular endothelial cells (RMECs) and ganglion cells (RGC) under simulated diabetic retinopathy (DR) condition.Methods:The rat RMECs and RGC were co-cultured in the Transwell (0.4 μm), cells were divided into the control group (5.5 mM ) and high glucose group (50.0 mM) according to the concentration of glucose in the culture medium. Cell morphology was observed by fluorescence inverted microscope imaging; cell proliferation assay, scratch and transwell migration assay as well as the lumen formation assay used to detect the capacity of the proliferation, migration, and lumen formation of RMECs and RGC. The apoptosis of cells was observed by PI/Hoechest experiment. The number of the cell migration, formed lumens and lumen intersections as well as the apoptosis rate in the two groups conformed to a normal distribution, were described as ±s, and compared by t testing. The optical density (OD) value of cell proliferation and cell migration rate in the two groups conformed to a non-normal distribution, were described using median and interquartile range, and comared by the Mann-Whitney U test.Results:In the co-culture model, the RGCs in the upper chamber showed adherent growth as a oval-like monolayer, while the RMECs in the lower chamber were more irregular, as a single layer of paving stones, and tightly connected between cells. The OD value of cell proliferation in RMECs of the high glucose group and control group were 1.163 (1.142, 1.188) and 0.812 (0.805, 0.817); the OD value of RGCs in two groups were 0.723 (0.709, 0.759) and 1.934 (1.803, 2.030). There were both statistically significant differences in the OD value of cell proliferation of two cells between two groups (Z=-6.069, -5.940; P<0.05). The migration ability of RMECs in the high glucose group and control group were [53.64 (37.69, 65.88)]% and [32.25(22.34, 42.85)%], which the former group better than latter group and there was a significant difference between two groups (Z=-2.841, P<0.05). The migration score of RMECs in two groups were (164±31) and (113±16); the apoptosis rate were (14.30±4.29)% and (9.65±3.43)%; the number of lumen formation were (23±5) and (19±4); lumen intersections were (11±3) and (13±2), respectively. There were all statistically significant differences in the migration score, the number of lumen formation, and the apoptosis rate between two groups (t=-4.653, -2.137, -2.929; P<0.05); non-significant difference in the lumen intersections between groups (t=1.424, P>0.05). The apoptosis rate of RGCs in two groups were (6.91±1.15) % and (4.87±1.70)% with statistically significant differences between two groups (t=-2.446, P<0.05).Conclusions:The 2D co-culture model of RMECs and RGCs has shown that there were the same performance as that of single cell culture in vitro. Under the high glucose environment, the cell activity, apoptosis, migration ability, and lumen formation ability of RMECs and the cell activity and apoptosis of RGCs with synchronous physiological and pathological changes, which laying a model foundation for further mechanism research of DR.
基金项目
国家自然科学基金面上项目(81170859)
国家自然科学基金面上项目(82070988)
国家自然科学基金国际合作重点项目(81561128015)
国家科技部国家重点研发计划项目(2016YFC1305604)
万方数据