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目的建立唐氏综合征患者永生细胞系,为唐氏综合征的基因诊断提供标准品。方法采集唐氏综合征患者外周血,采用 EB 病毒转化外周血 B 淋巴细胞和加环孢霉素 A 抑制 T 淋巴细胞的技术,建立永生淋巴母细胞系。用细胞遗传学方法、实时荧光定量 PCR 及短串联重复序列(STR)多态性分析技术检测其建系前后的遗传特性及稳定性。结果建系成功,经过传代、冻存和复苏,建系后第10代染色体核型和 DNA 标志未发生改变。建系前后染色体核型均为47,XX,+21。多重荧光相对定量 PCR 鉴定,唐氏综合征患者外周血原代细胞和转化后第10代细胞株的△Ct 值均在-2.55±3s 范围内,与正常对照相比△Ct 值范围无重叠。21号染色体上的 STR 基因座 D21S11多态性分析表明,唐氏综合征患者外周血原代细胞和转化后第10代细胞株均有同样的3种基因型。结论染色体核型和 DNA 分析表明,唐氏综合征患者永生细胞系建立前后遗传特点一致,且稳定,可用作基因诊断试剂盒产品质量检测、质量控制的标准品。
Objective To establish an immortal cell line in patients with Down’s syndrome and provide a standard for the genetic diagnosis of Down’s syndrome. Methods Peripheral blood samples from patients with Down’s syndrome were collected. EBV-transformed peripheral B lymphocytes and cyclosporin A-suppressed T lymphocytes were used to establish immortal lymphoblastoid cell lines. The genetic characteristics and stability before and after its establishment were detected by cytogenetics, real-time fluorescence quantitative PCR and short tandem repeat (STR) polymorphism analysis. Results The establishment of a successful line, after passage, cryopreservation and recovery, after the establishment of the tenth generation of chromosome karyotype and DNA markers did not change. Chromosome karyotypes were 47, XX, +21 before and after the establishment of the department. Multiplex fluorescence quantitative PCR showed that the △ Ct values of peripheral blood primary cells and the transformed cells of the 10 generation in Down Syndrome were all within -2.55 ± 3s, there was no overlap of △ Ct values with the normal controls. The STR loci D21S11 polymorphism analysis on chromosome 21 showed that there were 3 genotypes in the peripheral blood primary cells and the 10th generation transformed cells in Down’s syndrome. Conclusion Chromosome karyotypes and DNA analysis show that the immortalized cell lines of Down Syndrome patients have consistent and stable genetic characteristics before and after establishment and can be used as a standard for product quality testing and quality control of gene diagnostic kits.