人羊膜上皮细胞构建组织工程角膜上皮的实验研究
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摘要
目的①建立体外培养及标记人羊膜上皮细胞(HAECs)的方法；②探讨人羊膜上皮细胞作为“种子细胞”向角膜上皮细胞转分化的可能性；③应用原子力显微镜(AFM)从细胞形貌变化上探讨人羊膜上皮细胞向角膜上皮细胞转分化的可能性；④通过动物活体移植研究构建人羊膜上皮细胞-兔角膜基质组织工程角膜进行眼表重建的可行性。
方法①取足月产人羊膜,采用酶消化法获得HAECs进行原代和传代培养,对培养的细胞进行形态学观察和鉴定,并观察荧光染料DAPI标记HAECs的效果；②HAECs与兔角膜基质细胞共培养2w,采用CK3+12免疫细胞荧光法鉴定诱导后的细胞；HAECs分别接种在兔和人表层角膜基质片上采用气液界面法培养2w,CK3+12免疫组织化学染色和免疫组织荧光染色鉴定诱导后的HAECs；③应用AFM分别对共培养前后的人羊膜上皮细胞的形态结构和表面超微结构进行观察,并与人角膜上皮细胞(HCECs)对比,分析细胞形貌的变化；④构建的HAECs-兔角膜基质组织工程角膜移植治疗干细胞缺乏的动物模型,从眼表观察、组织病理学及细胞生物学等多方面观察其疗效。
结果①HAECs体外培养呈铺路石样外观,核／浆比率小,连接成片。细胞形态为多角形,角蛋白keritin阳性表达,CK3+12阴性表达；DAPI荧光标记率100%,2w后标记率无改变,荧光强度减弱；②共培养2w的HAECs免疫荧光阳性表达CK3+12；基质片上HAECs形成4-5层细胞复层结构,与正常角膜上皮结构相似,免疫荧光和免疫组化均阳性表达CK3+12；③AFM观察,共培养2w后HAECs细胞核中央由山谷样外观转变成类似HCECs的山峰样外观；④组织工程HAECs-兔角膜基质角膜缘移植术后4w、组织工程HAECs-兔角膜基质中央角膜移植术后4w和组织工程HAECs-兔角膜基质全板层角膜移植术后2w,角膜植片保持透明,HE染色见角膜复层上皮,与正常角膜上皮结构相似,免疫荧光和免疫组化均阳性表达CK3+12。
结论①体外可成功进行HAECs原代和传代培养,DAPI可作为有效的HAECs体外标记物；②HAECs在兔角膜基质细胞或兔／人角膜基质片诱导培养条件下可以向角膜上皮细胞转分化；③ATM从形态学方面客观上进一步证实与兔角膜基质细胞共培养可以成功诱导HAECs向CECs转分化；④构建的HAECs-兔角膜基质组织工程角膜移植可以重建角膜上皮层。
Objective①To establish a method of culturing and labelling human amniotic epithelial cells (HAECs) in vitro.②To explore the feasibility of HAECs differentiated into corneal epithelium cells as "seed cells".③To investigate the transdifferentiation of HAECs into corneal epithelium cells using atomic force microcopy (AFM) on morphology.④To evaluate the effect of the HAECs-rabbit corneal stroma tissue engineering cornea on ocular reconstruction in animal models.
Methods①HAECs were isolated from serologically screened donor human placenta using enzyme digestion and cultivated in culture medium for primary culture and passage. The cultured HAECs were observed on morphology, identified by immunocytochemical methods and investigated the effect of DAPI fluorescence labelling for 2 weeks.②HAECs were co-cultured with rabbit corneal stroma cells for 2 weeks, identified by immunofluorescence staining for CK3+12. HAECs were seeds onto rabbit or human corneal stroma, and incubated in culture medium for 2 weeks using air-liquid interface culture methods, identified by immunohistostaining for CK3+12.③The characteristic of morphology and ultra-microstucture of HAECs before and after co-culture were investigated using AFM, compared to human corneal epithelial cells (HCECs).④HAECs-rabbit corneal stroma tissue engineering cornea transplantation were operated for ocular surface restoration in different animal models with limbal stem cell deficiency. The effect was assesses by ocular surface observation, histopathology and cell biology.
Results①HAECs immediately harvested from the fresh placenta could be cultured in culture media. HAECs became a cobble-stone-like shape with a low N/C ratio and formed a sheet with obviously discernible intercellular junctions. The cultured polygon HAECs displayed positive immunoreactivity to keratin and negative to CK3+12. DAPI fluorescence labelling ratio of the cultured cell was 100%, the ratio had no change but fluorescence intensity decreased after 2 weeks.②The co-cultured HAECs displayed positive immunoreactivity to CK3+12. HAECs on the corneal stroma have 4-5 layers, same as normal corneal epithelium, displayed positive immunoreactivity to CK3+12.③The ATM images revealed that the nucleus center shape of the co-cultured HAEC changed from valley appearance to HCEC-like mountain peak appearance.④HAECs-rabbit corneal stroma tissue engineering cornea grafts were transparency during observation period, with corneal epithelium-like stratified epithelium and CK3+12 positive expression.
Conclusions①HAECs can be successfully primary cultured and passaged in vitro. DAPI is an effective fluorescence marker for HAECs.②HAECs could be transformed into HCECs induced by rabbit corneal stroma cells or rabbit/human corneal stroma.③The visualized data obtained from AFM for HAECs can prove further the co-cultured HAECs with corneal stroma cells transformed into HCECs successfully on morphology.④HAECs-rabbit corneal stroma tissue engineering cornea grafts can reconstruct corneal epithelium.
方法①取足月产人羊膜,采用酶消化法获得HAECs进行原代和传代培养,对培养的细胞进行形态学观察和鉴定,并观察荧光染料DAPI标记HAECs的效果；②HAECs与兔角膜基质细胞共培养2w,采用CK3+12免疫细胞荧光法鉴定诱导后的细胞；HAECs分别接种在兔和人表层角膜基质片上采用气液界面法培养2w,CK3+12免疫组织化学染色和免疫组织荧光染色鉴定诱导后的HAECs；③应用AFM分别对共培养前后的人羊膜上皮细胞的形态结构和表面超微结构进行观察,并与人角膜上皮细胞(HCECs)对比,分析细胞形貌的变化；④构建的HAECs-兔角膜基质组织工程角膜移植治疗干细胞缺乏的动物模型,从眼表观察、组织病理学及细胞生物学等多方面观察其疗效。
结果①HAECs体外培养呈铺路石样外观,核／浆比率小,连接成片。细胞形态为多角形,角蛋白keritin阳性表达,CK3+12阴性表达；DAPI荧光标记率100%,2w后标记率无改变,荧光强度减弱；②共培养2w的HAECs免疫荧光阳性表达CK3+12；基质片上HAECs形成4-5层细胞复层结构,与正常角膜上皮结构相似,免疫荧光和免疫组化均阳性表达CK3+12；③AFM观察,共培养2w后HAECs细胞核中央由山谷样外观转变成类似HCECs的山峰样外观；④组织工程HAECs-兔角膜基质角膜缘移植术后4w、组织工程HAECs-兔角膜基质中央角膜移植术后4w和组织工程HAECs-兔角膜基质全板层角膜移植术后2w,角膜植片保持透明,HE染色见角膜复层上皮,与正常角膜上皮结构相似,免疫荧光和免疫组化均阳性表达CK3+12。
结论①体外可成功进行HAECs原代和传代培养,DAPI可作为有效的HAECs体外标记物；②HAECs在兔角膜基质细胞或兔／人角膜基质片诱导培养条件下可以向角膜上皮细胞转分化；③ATM从形态学方面客观上进一步证实与兔角膜基质细胞共培养可以成功诱导HAECs向CECs转分化；④构建的HAECs-兔角膜基质组织工程角膜移植可以重建角膜上皮层。
Objective①To establish a method of culturing and labelling human amniotic epithelial cells (HAECs) in vitro.②To explore the feasibility of HAECs differentiated into corneal epithelium cells as "seed cells".③To investigate the transdifferentiation of HAECs into corneal epithelium cells using atomic force microcopy (AFM) on morphology.④To evaluate the effect of the HAECs-rabbit corneal stroma tissue engineering cornea on ocular reconstruction in animal models.
Methods①HAECs were isolated from serologically screened donor human placenta using enzyme digestion and cultivated in culture medium for primary culture and passage. The cultured HAECs were observed on morphology, identified by immunocytochemical methods and investigated the effect of DAPI fluorescence labelling for 2 weeks.②HAECs were co-cultured with rabbit corneal stroma cells for 2 weeks, identified by immunofluorescence staining for CK3+12. HAECs were seeds onto rabbit or human corneal stroma, and incubated in culture medium for 2 weeks using air-liquid interface culture methods, identified by immunohistostaining for CK3+12.③The characteristic of morphology and ultra-microstucture of HAECs before and after co-culture were investigated using AFM, compared to human corneal epithelial cells (HCECs).④HAECs-rabbit corneal stroma tissue engineering cornea transplantation were operated for ocular surface restoration in different animal models with limbal stem cell deficiency. The effect was assesses by ocular surface observation, histopathology and cell biology.
Results①HAECs immediately harvested from the fresh placenta could be cultured in culture media. HAECs became a cobble-stone-like shape with a low N/C ratio and formed a sheet with obviously discernible intercellular junctions. The cultured polygon HAECs displayed positive immunoreactivity to keratin and negative to CK3+12. DAPI fluorescence labelling ratio of the cultured cell was 100%, the ratio had no change but fluorescence intensity decreased after 2 weeks.②The co-cultured HAECs displayed positive immunoreactivity to CK3+12. HAECs on the corneal stroma have 4-5 layers, same as normal corneal epithelium, displayed positive immunoreactivity to CK3+12.③The ATM images revealed that the nucleus center shape of the co-cultured HAEC changed from valley appearance to HCEC-like mountain peak appearance.④HAECs-rabbit corneal stroma tissue engineering cornea grafts were transparency during observation period, with corneal epithelium-like stratified epithelium and CK3+12 positive expression.
Conclusions①HAECs can be successfully primary cultured and passaged in vitro. DAPI is an effective fluorescence marker for HAECs.②HAECs could be transformed into HCECs induced by rabbit corneal stroma cells or rabbit/human corneal stroma.③The visualized data obtained from AFM for HAECs can prove further the co-cultured HAECs with corneal stroma cells transformed into HCECs successfully on morphology.④HAECs-rabbit corneal stroma tissue engineering cornea grafts can reconstruct corneal epithelium.
引文
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