目的 利用携带腺苷三磷酸结合盒转运子A3(ABCA3)复合杂合突变的新生儿呼吸窘迫综合征(NRDS)患儿的外周血单核细胞(PBMCs)，建立符合研究需求的诱导多能干细胞(iPSCs)细胞系。 方法 细胞实验研究。采集患儿的外周静脉血并分离出PBMCs，体外培养，用携带重编程因子的非整合性仙台病毒载体转染PBMCs，对所产生的iPSCs细胞系进行染色体核型分析。采用免疫荧光技术、流式细胞术检测干细胞多能性标志物、验证其分化潜能，采用Sanger测序对基因突变进行分析，另外还行短串联重复序列(STR)位点分析，聚合酶链式反应(PCR)和琼脂糖凝胶电泳法检测病毒残留。 结果 对建立的iPSCs细胞系行核型分析显示为正常的二倍体46，XY核型，免疫荧光技术显示干细胞多能性标志物OCT4、SSEA4、Nanog及Sox2染色呈阳性。流式细胞术检测干细胞多能性标志物，显示TRA-1-60、SSEA-4和OCT4的表达。诱导向三胚层分化后用免疫荧光技术对外胚层(PAX-6)、中胚层(Brachyury)和内胚层(甲胎蛋白)标志物进行免疫荧光染色，结果显示均为阳性。Sanger测序显示c。3997_3998del及c。3137C>T复合杂合突变。STR位点分析结果显示其来源于患儿PBMCs，PCR和琼脂糖凝胶电泳法未检测到仙台病毒残留。 结论 本研究用ABCA3复合杂合突变患儿的PBMCs建立了iPSCs细胞系，为ABCA3基因突变所致的NRDS发病机制的研究、治疗药物筛选及细胞治疗奠定了基础。 Objective Induced pluripotent stem cells (iPSCs) cell lines were established using peripheral blood mononuclear cells (PBMCs) from a patient suffering from neonatal respiratory distress syndrome (NRDS) who carried Adenosine triphosphate-binding cassette transporter A3 (ABCA3) compound heterozygous mutations。 Methods Cell experimental research。Peripheral venous blood was collected and PBMCs were isolated and cultured in vitro。 PBMCs were transfected with non-integrated Sendai vector carrying reprogramming factors。The chromosome karyotypes of the established iPSCs were analyzed。Immunofluorescence and flow cytometry were used to detect pluripotency markers of stem cells and verify their differentiation potential。Sanger sequencing was performed to analyze gene mutations。In addition， short tandem repeat (STR) analysis was performed， polymerase chain reaction(PCR) and agarose gel electrophoresis were used to detect virus residual。 Results Karyotype analysis of established iPSCs cell lines showed normal diploid 46， XY karyotype。Immunofluorescence showed positive staining of stem cell pluripotency markers OCT4， SSEA4， Nanog and Sox2。Flow cytometry was used to detected stem cell pluripotency markers and showed expression of TRA-1-60， SSEA-4 and OCT4。After differentiation into all three germ layers， immunofluorescence was performed to detect ectoderm (Pax-6)， mesoderm (Brachyury) and endoderm alpha-fetoprotein markers， and the results showed positive staining， which confirmed that the iPSCs had the potential to differentiate。Sanger sequencing showed c。 3997_3998del and c。 3137C>T compound heterozygous mutations。STR analysis showed they originate from PBMCs， and no Sendai virus residual was detected by PCR and agarose gel electrophoresis。 Conclusions In this study， PBMCs from patient carrying ABCA3 compound heterozygous mutations was used to establish iPSCs cell lines。The research lays a foundation for the study of pathogenesis， therapeutic drug screening and cell therapy of NRDS caused by ABCA3 gene mutations。
Generation of an induced pluripotent stem cell line from a patient with surfactant metabolism dysfunction carryingABCA3 mutations
Objective Induced pluripotent stem cells (iPSCs) cell lines were established using peripheral blood mononuclear cells (PBMCs) from a patient suffering from neonatal respiratory distress syndrome (NRDS) who carried Adenosine triphosphate-binding cassette transporter A3 (ABCA3) compound heterozygous mutations. Methods Cell experimental research.Peripheral venous blood was collected and PBMCs were isolated and cultured in vitro. PBMCs were transfected with non-integrated Sendai vector carrying reprogramming factors.The chromosome karyotypes of the established iPSCs were analyzed.Immunofluorescence and flow cytometry were used to detect pluripotency markers of stem cells and verify their differentiation potential.Sanger sequencing was performed to analyze gene mutations.In addition, short tandem repeat (STR) analysis was performed, polymerase chain reaction(PCR) and agarose gel electrophoresis were used to detect virus residual. Results Karyotype analysis of established iPSCs cell lines showed normal diploid 46, XY karyotype.Immunofluorescence showed positive staining of stem cell pluripotency markers OCT4, SSEA4, Nanog and Sox2.Flow cytometry was used to detected stem cell pluripotency markers and showed expression of TRA-1-60, SSEA-4 and OCT4.After differentiation into all three germ layers, immunofluorescence was performed to detect ectoderm (Pax-6), mesoderm (Brachyury) and endoderm alpha-fetoprotein markers, and the results showed positive staining, which confirmed that the iPSCs had the potential to differentiate.Sanger sequencing showed c. 3997_3998del and c. 3137C>T compound heterozygous mutations.STR analysis showed they originate from PBMCs, and no Sendai virus residual was detected by PCR and agarose gel electrophoresis. Conclusions In this study, PBMCs from patient carrying ABCA3 compound heterozygous mutations was used to establish iPSCs cell lines.The research lays a foundation for the study of pathogenesis, therapeutic drug screening and cell therapy of NRDS caused by ABCA3 gene mutations.
万方数据
