人畸胎瘤细胞株PA-1胚胎干细胞生物学特性的鉴定

Identification of embryonic stem cells's biological characteristics of human teratocarcinoma cell line PA-1

目的鉴定人畸胎瘤细胞株PA-1的胚胎干细胞生物学特性。方法用碱性磷酸酶染色法鉴定体外培养PA-1细胞的分化水平,用逆转录聚合酶链式反应(RT-PCR)法检测PA-1细胞多能性基因Nanog、Oct4和Sox2的表达情况。通过悬滴培养法使PA-1细胞形成拟胚体,用RT-PCR法检测拟胚体分化的三胚层特异性基因的表达。通过SCID小鼠体内成瘤实验,用HE染色和免疫组化鉴定PA-1细胞体内接种后三胚层组织的分化。结果体外贴壁培养PA-1细胞碱性磷酸酶染色呈深紫色,PA-1细胞能表达多能性基因Nanog,Oct4和Sox2。悬滴培养PA-1细胞可形成拟胚体(EB),且能够表达三胚层特异性基因(内胚层:Sox17,AFP;中胚层:C-actin,Brachyury;外胚层:Sox1,Pax6)。PA-1细胞接种至SCID小鼠皮下可以形成畸胎瘤,HE染色可见神经外胚层组织,免疫组化可见三胚层细胞表面标志抗原:GFAP(外胚层)、Vimentin(中胚层)及Desmin(内胚层)均呈阳性表达。结论 PA-1细胞在贴壁培养过程中能够保持未分化/低分化状态,并表达多能性基因; PA-1细胞在体内及体外分化实验中均能分化出三胚层的细胞或组织;证明PA-1细胞具有一定的胚胎干细胞生物学特性。

Objective To identify the biological characteristics of embryonic stem cells of human teratocarcinoma cell line PA-1. Methods The differentiation of PA-1 cells in vitro was identified by alkaline phosphatase staining. The expression of pluripotency genes Nanog,Oct4 and Sox2 in PA-1 cells was detected by reverse transcription-polymerase chain reactionRT-PCR) . Cells were used as the embryoid body and the expression of the three germ-layer-specific genes in embryoid-derived embryos was detected by RT-PCR . The tumorigenicity of SCID mice was evaluated by HE staining and immunohistochemistry for identification of the tissue differentiation. Results PA-1 cells were stained dark purple with alkaline phosphatase in vitro. PA-1 cells expressed pluripotency genes Nanog,Oct4 and Sox2 ) and the three germ layer specific genes endoderm: Sox17,AFP; mesoderm: C-actin,Brachyury; ectoderm: Sox1,Pax6) ; PA-1 cells can form teratoma by inoculating into SCID mice subcutaneously ; neuroectodermal tissues can be found by HE staining and the surface markers such as GFAP ectoderm) ,Vimentin mesoderm) and Desmin endoderm) were all expressed in immunohistochemistry. Conclusion PA-1 cells can maintain undifferentiated /poorly differentiated state and express pluripotency genes during adherent culture. PA-1 cells can differentiate into three germ layer cells or tissues in vivo and in vitro differentiation experiments. The result illustrated that PA-1 cells have some biological characteristics of embryonic stem cell.