聚己内酯/明胶纳米纤维膜在组织工程化视网膜色素上皮构建中的应用

Appilcation of polycaprolactone/collagen nanofibrous membranes in constituting tissue engineering retinal pigment epithelium

目的探索聚己内酯/明胶纳米纤维复合膜在构建组织工程化视网膜色素上皮(RPE)中的应用前景。方法采用自发分化法将人胚胎干细胞(ESC)诱导为视网膜色素上皮(ESC-RPE)细胞,并通过流式细胞术检测ESC-RPE细胞特定细胞标志物的表达情况;采用静电纺丝法制备聚己内酯(PCL)纳米纤维膜和PCL/明胶纳米纤维复合膜,扫描电子显微镜下观察2种纳米纤维膜的表面形貌,并测定2种支架的接触角;利用细胞计数试剂盒-8(CCK-8)法检测2种纳米纤维膜浸提液对ESC-RPE细胞活力的影响;将ESC-RPE细胞培养在PCL/明胶纳米纤维复合膜上,通过免疫荧光法检测组织工程化RPE特异性标志物的表达。结果ESC-RPE细胞不表达多能性标志物OCT4,阳性表达RPE特异性标志物小眼畸形相关转录因子(MITF)、RPE65和Bestrophin。PCL纳米纤维膜和PCL/明胶纳米纤维复合膜均呈纤维多孔状,2种支架的接触角分别为(97.2±3.1)°和(13.6±2.4)°,2种支架的浸提液对ESC-RPE细胞的生长均无显著影响。培养在PCL/明胶纳米纤维复合膜上的ESC-RPE细胞阳性表达特异性标志物MITF和ZO-1。结论PCL/明胶纳米纤维复合膜具有良好的亲水性和生物相容性,并且可支持ESC-RPE细胞的生长,有望应用于组织工程化RPE的构建。

Objective To explore the potential of polycaprolactone(PCL)/gelatin nanofibrous composite membrane in constituting tissue engineering retinal pigment epithelium(RPE).Methods The embryonic stem cells(ESCs)were differentiated into RPE(ESC-RPE)cells by spontaneous differentiation methods.Flow cytometry was used to detect the expression of specific RPE markers in ESC-RPE cells.The nanofiber membrane of PCL and PCL/gelatin nanofibrous composite membrane were prepared by electrospinning.The topography and contact angle of the two membranes were detected.The viability of ESC-RPE cells treated by the extracts of the two kinds of membranes was detected by cell counting kit-8(CCK-8)method.After the ESC-RPE cells were cultured on the PCL/gelatin nanofibrous composite membrane,immunostaining was used to detect the expression of specific RPE markers of tissue engineering RPE.Results ESC-RPE cells were negative for pluripotent marker OCT4,but positive for microphthalmia-associated transcription factor(MITF),RPE65 and Bestrophin.Both the nanofiber membrane of PCL and PCL/gelatin nanofibrous composite membrane were porous and the contact angle were(97.2±3.1)°and(13.6±2.4)°,respectively.The extracts of the two kinds of membranes showed no significant effect on the growth of ESC-RPE cells.The tissue engineering RPE based on PCL/gelatin nanofibrous composite membrane positively expressed MITF and ZO-1.Conclusions PCL/gelatin nanofibrous composite membrane shows good hydrophilicity and biocompatibility.Besides,the membrane is favor for the growth of ESC-RPE cells and holds the potential to constitute tissue engineering RPE.