中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2012年
11期
976-981
,共6页
招志毅%陈建苏%钟敬祥%谭美华%李善义%戴应
招誌毅%陳建囌%鐘敬祥%譚美華%李善義%戴應
초지의%진건소%종경상%담미화%리선의%대응
诱导多能干细胞%角膜内皮细胞%混合共培养%分化%原子力显微镜
誘導多能榦細胞%角膜內皮細胞%混閤共培養%分化%原子力顯微鏡
유도다능간세포%각막내피세포%혼합공배양%분화%원자력현미경
Induced pluripotent stem cell%Corneal endothelium cell%Mix co-culture%Differentiation%Atomic force microscopy
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.
揹景 誘導多能榦細胞(iPSCs)具有類似胚胎榦細胞的分化能力,可以分化為全身各種類型的體細胞,同時不會引起倫理學問題和免疫排斥反應,因此iPSCs有可能作為組織工程角膜內皮重建的種子細胞. 目的 利用原子力顯微鏡(AFM)觀察人iPSCs與兔角膜內皮細胞(CECs)混閤共培養後分化iPSCs的形態學變化,併找齣其細胞膜超微結構變化的規律.方法 分彆培養兔CECs和人MMC-iPSCs細胞繫,用免疫組織化學法測定iPSCs細胞的標誌物以鑒定iPSCs細胞.將傳代後7d的iPSCs與60％融閤的CECs在內皮細胞培養基中共培養建立混閤共培養模型,利用AFM結閤倒置顯微鏡觀察CECs及共培養前後iPSCs的形貌和超微結構變化.結果 CECs的長度和寬度分彆為(66.93±10.48) μm和(44.85±8.14) μm,共培養前未分化的iPSCs的長度和寬度分彆為(12.51±1.40)μm和(10.93±1.69) μm,共培養後分化的iPSCs長度和寬度分彆為(36.12±10.29) μm和(31.53±9.65) μm.分化的iPSCs長度和寬度均大于共培養前的iPSCs,差異均有統計學意義(P＜0.05).分化的iPSCs寬度與CECs比較差異無統計學意義(P＞0.05).CECs細胞膜錶麵突起的最大寬度和高度分彆為(2.11±1.03)μm和(115.68±92.08) nm,膜錶麵凹陷為窗孔樣凹陷,最大寬度為(1.49±0.65) μm,膜錶麵結構幾何參數均方根粗糙度(Rq)為(39.20±7.82) nm,平均粗糙度(Ra)為(30.37±5.32)nm;未分化的iPSCs細胞膜錶麵突起為顆粒狀,突起的最大寬度和高度分彆為(0.39±0.22) μm和(13.11±9.18)nm,膜錶麵凹陷為蟲蝕樣,凹陷的最大寬度為(0.34±0.18) μn,Rq和Ra分彆為(26.60±4.93)nm和(9.97±3.78) nm;分化的iPSCs細胞膜錶麵突起為指狀,突起的最大寬度和高度分彆為(1.91±0.76) μm和(106.55±77.27)nm,膜錶麵凹陷為窗孔樣,凹陷的最大寬度為(1.61± 1.25) μm,Rq為(57.33±12.80) nm,Ra為(43.63±11.17)nm.分化的iPSCs細胞膜錶麵突起最大寬度和高度、凹陷最大寬度、Rq、Ra均大于共培養前未分化的iPSCs,差異均有統計學意義(P＜0.05),與CECs比較差異均無統計學意義(P＞0.05). 結論 AFM能夠敏感地觀察到iPSCs與CECs接觸共培養7d後iPSCs嚮CECs方嚮轉化的形態學變化,為進一步研究iPSCs嚮CECs的分化提供瞭研究基礎.
배경 유도다능간세포(iPSCs)구유유사배태간세포적분화능력,가이분화위전신각충류형적체세포,동시불회인기윤리학문제화면역배척반응,인차iPSCs유가능작위조직공정각막내피중건적충자세포. 목적 이용원자력현미경(AFM)관찰인iPSCs여토각막내피세포(CECs)혼합공배양후분화iPSCs적형태학변화,병조출기세포막초미결구변화적규률.방법 분별배양토CECs화인MMC-iPSCs세포계,용면역조직화학법측정iPSCs세포적표지물이감정iPSCs세포.장전대후7d적iPSCs여60％융합적CECs재내피세포배양기중공배양건립혼합공배양모형,이용AFM결합도치현미경관찰CECs급공배양전후iPSCs적형모화초미결구변화.결과 CECs적장도화관도분별위(66.93±10.48) μm화(44.85±8.14) μm,공배양전미분화적iPSCs적장도화관도분별위(12.51±1.40)μm화(10.93±1.69) μm,공배양후분화적iPSCs장도화관도분별위(36.12±10.29) μm화(31.53±9.65) μm.분화적iPSCs장도화관도균대우공배양전적iPSCs,차이균유통계학의의(P＜0.05).분화적iPSCs관도여CECs비교차이무통계학의의(P＞0.05).CECs세포막표면돌기적최대관도화고도분별위(2.11±1.03)μm화(115.68±92.08) nm,막표면요함위창공양요함,최대관도위(1.49±0.65) μm,막표면결구궤하삼수균방근조조도(Rq)위(39.20±7.82) nm,평균조조도(Ra)위(30.37±5.32)nm;미분화적iPSCs세포막표면돌기위과립상,돌기적최대관도화고도분별위(0.39±0.22) μm화(13.11±9.18)nm,막표면요함위충식양,요함적최대관도위(0.34±0.18) μn,Rq화Ra분별위(26.60±4.93)nm화(9.97±3.78) nm;분화적iPSCs세포막표면돌기위지상,돌기적최대관도화고도분별위(1.91±0.76) μm화(106.55±77.27)nm,막표면요함위창공양,요함적최대관도위(1.61± 1.25) μm,Rq위(57.33±12.80) nm,Ra위(43.63±11.17)nm.분화적iPSCs세포막표면돌기최대관도화고도、요함최대관도、Rq、Ra균대우공배양전미분화적iPSCs,차이균유통계학의의(P＜0.05),여CECs비교차이균무통계학의의(P＞0.05). 결론 AFM능구민감지관찰도iPSCs여CECs접촉공배양7d후iPSCs향CECs방향전화적형태학변화,위진일보연구iPSCs향CECs적분화제공료연구기출.
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.
