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自发和诱导分化产生的人胚胎干细胞源性视网膜色素上皮补体H因子的表达

Expression of Complement Factor H in Human Embryonic Stem Cell-derived Retinal Pigment Epithelium

  • 摘要:
      目的  研究人胚胎干细胞(hESC)分别经自发分化和诱导分化产生的视网膜色素上皮细胞(RPE)表达及分泌补体替代通路调控蛋白补体H因子(CFH)的情况。
      方法  将hESC分别按照临床试验中所采用的自发分化法和利用生长因子与小分子药物诱导分化的方法获得hESC-RPE,在第3代hESC-RPE培养至第4~5周后,采用实时荧光定量PCR和免疫荧光进行鉴定,酶联免疫吸附(ELISA)法检测CFH的分泌水平。对照组为ARPE-19细胞系。
      结果  hESC通过自发分化和诱导分化均可获得在细胞形态及特异性基因表达方面与人原代RPE高度相似的细胞。自发分化产生的视网膜色素上皮细胞(sdRPE)和诱导分化产生的视网膜色素上皮细胞(iRPE)的CFH mRNA相对表达量均高于对照组ARPE-19细胞系(P<0.05)。在24 h、48 h和72 h的条件培养基中,sdRPE组的CFH分泌量高于iRPE组(P=0.000 2),且sdRPE和iRPE均高于ARPE-19细胞系(P<0.000 1)。
      结论  两种分化方式来源的hESC-RPE均可表达并分泌CFH,提示hESC-RPE可能具有一定的调控补体替代通路的能力。

     

    Abstract:
      Objective  To study the expression and secretion of alternative complement pathway regulator complement factor H (CFH) in spontaneously produced or induced human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE).
      Methods  RPE cells were acquired by spontaneous differentiation from hESC (sdRPE), a source of hESC-RPE, according to the method used in clinical trials. RPE cells were also acquired under the induction of growth factors and small molecules for 14 d (iRPE). Acquired cells were kept culturing for 3 month for maturation. All differentiated cells(P3)were cultivated for 4-5 weeks prior to characterization with qRT-PCR and immunofluorescence. Secretion levels of CFH were investigated by ELISA. ARPE-19 cell line was served as control.
      Results  Both sdRPE and iRPE showed high similarity in cell morphology and the pattern of specific gene expression with human RPE. The relative CFH mRNA expression levels of both sdRPE and iRPE were significantly higher than that of ARPE-19 (P<0.05). The CFH secretion levels of sdRPE in the 24 h-, 48 h- and 72 h-culture medium were higher than those of iRPE (P=0.000 2); and this CFH secretion levels of both sdRPE and iRPE were higher than that of the ARPE-19 cell line (P<0.000 1).
      Conclusion  Both sdRPE and iRPE derived by different differentiation methods expressed and secreted CFH, suggesting that hESC-RPE may have certain ability to regulate the alternative complement pathway.

     

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