原子力显微镜观察角膜内皮细胞诱导后诱导多能干细胞的形态学变化

摘要

Background Induced pluripotent stem cells (iPSCs)can differentiate into various types of somatic cells without causing ethical controversy and immune rejection in clinical activity,which is similar to differentiation ability of embryonic stem cells.So,iPSCs may be used as seed cells for tissue engineering corneal endothelial reconstruction.Objective The present study was to survey the morphologic change of iPSCs after coculture with corneal endothelium cells(CECs) under the atomic force microscopy(AFM).Methods Rabbit CECs and human MMC-iPSCs were isolated and cultured respectively.The iPSCs were identified with the marker by immunochemistry.iPSCs passaged for 7 days were then cultured with 60％ confluent CECs to establish the co-culture model.The surface morphology and cellular membrane ultrastructure of differentiated iPSCs after induced by CECs were examined by AFM combination with inverted microscope,and compared with CECs and undifferentiated iPSCs.Results Thelengthand width were(66.93±10.48)μm and (44.85 ± 8.14) μm in CECs,(12.51±1.40)μm and (10.93 ±1.69) μm in uninduced iPSCs,and(36.12±10.29) μm and(31.53±9.65)μm in CECs-induced iPSCs.Both the length and width values of CECs-induced iPSCs were statistically bigger than those uninduced iPSCs,with significant differences between them (P＜0.05),but no significant difference was seen in the width valne of CECs-induced iPSCs in comparison with CECs(P＞0.05).The convex structure of CECs cytomembrane surface showed the digitation in shape with the size and height(2.11 ± 1.03) μm and (115.68±92.08) nm respectively,and the concave structure of cytomembrane surface of CECs was fenestrae-like depression and the size was (1.49 ± 0.65) μm.The numerical valuc of mean square root roughness (Rq)and average roughness (Ra)of cytomembrane surface of CECs were(39.20±7.82)nm and (30.37±5.32)nm respectively.The convex surface of cytomembrane of iPSCs was granular-like in shape with size and height(0.39±0.22)μm and(13.11±9.18)nm respectively.The concave surface of cytomembrane of iPSCs was worm-eaten-like concave with the size(0.34±0.18)μm.The numerical value of Rq and Ra of geometrical parameters of cytomembrane surface of iPSCs were (26.60 ± 4.93)nm and (9.97 ± 3.78) nm respectively.The convex surface of cytomembrane of induced iPSCs was digital-like in shape with the size and height (1.91±0.76) μm and(106.55±77.27) nm respectively.The concave surface of cytomembrane of induced iPSCs was fenestrae-like depression and the size of concave was(1.6l±1.25) μm.The numerical value of Rq and Ra on surface of cytomembrane of induced iPSCs was (57.33± 12.80) nm and (43.63± 11.17) nm respectively.The numerical values of the size and height of convex,the size of concave,Rq and Ra on surface of cytomembrane in induced iPSCs were statistically bigger than in iPSCs(P＜0.05)and were not significant differences in comparison with CECs (P＞0.05).Conclusions Morphology of iPSCs translate toward the CECs after induce for 7 days under the AFM.This outcome lays the foundation for further study on iPSCs.%背景 诱导多能干细胞(iPSCs)具有类似胚胎干细胞的分化能力,可以分化为全身各种类型的体细胞,同时不会引起伦理学问题和免疫排斥反应,因此iPSCs有可能作为组织工程角膜内皮重建的种子细胞. 目的 利用原子力显微镜(AFM)观察人iPSCs与兔角膜内皮细胞(CECs)混合共培养后分化iPSCs的形态学变化,并找出其细胞膜超微结构变化的规律.方法 分别培养兔CECs和人MMC-iPSCs细胞系,用免疫组织化学法测定iPSCs细胞的标志物以鉴定iPSCs细胞.将传代后7d的iPSCs与60％融合的CECs在内皮细胞培养基中共培养建立混合共培养模型,利用AFM结合倒置显微镜观察CECs及共培养前后iPSCs的形貌和超微结构变化.结果 CECs的长度和宽度分别为(66.93±10.48) μm和(44.85±8.14) μm,共培养前未分化的iPSCs的长度和宽度分别为(12.51±1.40)μm和(10.93±1.69) μm,共培养后分化的iPSCs长度和宽度分别为(36.12±10.29) μm和(31.53±9.65) μm.分化的iPSCs长度和宽度均大于共培养前的iPSCs,差异均有统计学意义(P＜0.05).分化的iPSCs宽度与CECs比较差异无统计学意义(P＞0.05).CECs细胞膜表面突起的最大宽度和高度分别为(2.11±1.03)μm和(115.68±92.08) nm,膜表面凹陷为窗孔样凹陷,最大宽度为(1.49±0.65) μm,膜表面结构几何参数均方根粗糙度(Rq)为(39.20±7.82) nm,平均粗糙度(Ra)为(30.37±5.32)nm;未分化的iPSCs细胞膜表面突起为颗粒状,突起的最大宽度和高度分别为(0.39±0.22) μm和(13.11±9.18)nm,膜表面凹陷为虫蚀样,凹陷的最大宽度为(0.34±0.18) μn,Rq和Ra分别为(26.60±4.93)nm和(9.97±3.78) nm;分化的iPSCs细胞膜表面突起为指状,突起的最大宽度和高度分别为(1.91±0.76) μm和(106.55±77.27)nm,膜表面凹陷为窗孔样,凹陷的最大宽度为(1.61± 1.25) μm,Rq为(57.33±12.80) nm,Ra为(43.63±11.17)nm.分化的iPSCs细胞膜表面突起最大宽度和高度、凹陷最大宽度、Rq、Ra均大于共培养前未分化的iPSCs,差异均有统计学意义(P＜0.05),与CECs比较差异均无统计学意义(P＞0.05). 结论 AFM能够敏感地观察到iPSCs与CECs接触共培养7d后iPSCs向CECs方向转化的形态学变化,为进一步研究iPSCs向CECs的分化提供了研究基础.