有序排列微模板培养角膜基质细胞及其细胞生物学变化

Keratocyte alignment culture on microgrooved patterns and cellular biology

目的探讨CO2激光打标机处理的有序排列微模板培养兔角膜基质细胞的生长特征及其细胞生物学变化。方法用CO2激光打标机刻制的聚苯乙烯有序排列微模板培养兔角膜基质细胞,模板沟槽划线间隔距离0.25mm者为窄间隔组,间隔距离1mm者为宽间隔组,平板组作为对照组。将角膜基质细胞悬液以1×105/mL细胞密度接种于培养板中,倒置显微镜下观察细胞生长情况,苏木精-伊红染色观察细胞排列的形态学差异,免疫荧光法检测培养板上细胞的波形蛋白,实时监测显微镜下观察24h窄间隔组细胞的接触指引特性及细胞的动态生长过程。结果培养第1天倒置显微镜下见3组培养细胞贴壁生长状态无明显区别,窄间隔组和宽间隔组细胞逐渐围绕沟槽接触指引生长,表现为有序排列,且窄间隔组较宽间隔组明显。苏木精-伊红染色和免疫荧光染色显示培养的细胞在窄间隔组沟槽的接触指引下呈有序排列,呈现10余层细胞排列的平行板层结构。3组细胞波形蛋白免疫荧光染色均呈阳性反应。实时监测显微镜下可见窄间隔组细胞体部首先贴壁,在接触指引下生长,而细胞在干燥环境下的凋亡始于细胞伪足突起处。结论利用CO2激光打标机制备的有序排列微模板可获得有序排列的角膜基质细胞,有利于在接近生理状态条件下角膜基质层的构建。

Background The regular lamellar array of cornea stroma is the basis of maintaining the normal physiological function of cornea.To culture the keratocytes with parallel arrangement is key for tissue engineer cornea technique.The known method to culture keratocytes is complex.Objective The present study aimed to investigate the property of rabbit corneal keratocytes cultured in vitro under the physiological status,alignment of keratocyte cultured in microgrooved patterns of polystyrene plates.Methods Micropatterned polystyrene cell culture plates were prepared by CO2 laser marking machines.The alignment patterns were designed as alternating grooves and ridges,including 0.25 mm lineation space （narrow groove group） and 1 mm lineation space（wide groove group）,and no alignment pattern worked as control group.The cultured plates were sterilized in epoxy ethane.Corneal keratocytes were isolated from New Zealand white rabbit corneas using collagenase type digestion method and cultured in DMEM containing 10% fetal bovine serum.The suspension of the second passage of keratocytes was inoculated on the culture plates at a density of 1×10^5 cells/mL.Morphology of cultured cells was observed under the phase contrast microscope daily.The cellular growth and contact guidance property were investigated with HE staining and immunofluorescence staining of Vimentin.Cells in narrow groove group was incubated at 37 ℃ in a closed dry 5% CO2 environment and the processes of cellular adherence,contact guidance proliferation and apoptosis of 24 hour time-lapse were observed under the real-time microscope.Results Keratocytes in groove groups grew along the grooves,but cells in control group showed no alignment pattern.Histological evaluation of HE staining and immunofluorescence staining showed that keratocytes in groove groups had contact guidance feature and elongated on the surface with microgrooved patterns and aligned along that patterns.The cells of three groups presented the positive response for Vimentin.Compared to the wide group,the cells in narrow group showed more obvious growing characteristics of alignment.The images of cells in narrow group under the real-time microscope confirmed that cells proliferated along the arranged grooves to abide by the contact guidance.The cellular adherence was firstly observed in cell body,but its apoptosis occurred firstly in cell foot processes.Conclusion Keratocytes cultured on microgrooved surfaces have alignment pattern,especially in the pattern of 0.25 mm lineation space.Keratocytes show contact guidance feature in alignment growth and close to the physiological status,which is suitable to the three-dimensional corneal stroma reconstruction in vitro.