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目的建立家族性甲状腺乳头状癌(FPTC)永生化细胞系,为研究家族性非髓样甲状腺癌(FNMTC)提供新的途径。方法选取FPTC患者的肿瘤标本,采用消化法分离原代细胞。使用改良的DMEM/F12培养基,添加促甲状腺激素(TSH)、三碘甲状腺原氨酸(T3)、表皮细胞生长因子(EGF)及氢化可的松等进行培养。在原代细胞早期分2种方式导入外源基因SV40T/TERT用于诱导永生化。利用RT-PCR检测甲状腺过氧化物酶(TPO)、甲状腺球蛋白(TG)、促甲状腺激素受体(TSHR)、钠/碘共同转运体(NIS)等基因的表达,同时利用免疫荧光技术检测TPO及磷脂酰肌醇蛋白聚糖-3(GPC3)蛋白的表达。提取该患者外周血DNA,进行突变基因的检测。提取细胞基因组PCR扩增后测序,进行突变基因的检测。结果分离的FPTC原代细胞呈梭形或不规则多角形贴壁生长;细胞生长6个月,转染SV40T组(标记为FPTC-S)传代至p26,FPTC细胞传代至p23,转染SV40T+h TERT组(标记为FPTC-ST)传代至p19,细胞增殖能力较好;FPTC-S与FPTC-ST细胞均能够在基因水平稳定表达TPO、TG与TSHR;患者外周血中存在MLH1 R217C胚系突变;原代细胞存在BRAF V600E突变;原代细胞及永生化的细胞系中均存在MLH1 R217基因突变。结论本研究初步建立了FPTC永生化细胞系,且该细胞系中存在MLH1 R217C及BRAF V600E突变,该细胞系将为研究以上突变及其相互作用关系提供研究模型。
Objective To establish an immortalized human thyroid papillary carcinoma cell line (FPTC) and provide a novel approach for the study of familial non-myeloid thyroid carcinoma (FNMTC). Methods Tumor specimens from patients with FPTC were selected and primary cells were isolated by digestion. The cultured DMEM / F12 medium was added with TSH, T3, EGF and hydrocortisone. Introduction of exogenous gene SV40T / TERT in 2 ways early in primary cells was used to induce immortalization. The expressions of thyroid peroxidase (TPO), thyroglobulin (TG), thyrotropin receptor (TSHR) and sodium / iodine co-transporter (NIS) were detected by RT-PCR and detected by immunofluorescence TPO and Glypican-3 (GPC3) protein expression. The patient’s peripheral blood DNA was extracted for the detection of the mutant gene. The cell genome was extracted and sequenced after PCR amplification to detect the mutant gene. Results The isolated FPTC primary cells grew into spindle or irregular polygons. The cells were grown for 6 months and then transfected into SV40T group (labeled as FPTC-S). The FPTC cells were passaged to p23 and transfected into SV40T + hTERT group (labeled as FPTC-ST), the cell proliferation ability was better; both FPTC-S and FPTC-ST cells could stably express TPO, TG and TSHR at the gene level; there were MLH1 R217C germline Mutation; BRAF V600E mutation exists in primary cells; MLH1 R217 gene mutation exists in primary cells and immortalized cell lines. Conclusion In this study, an immortalized FPTC cell line was initially established and MLH1 R217C and BRAF V600E mutations were present in this cell line. This cell line will provide a research model for studying the above mutations and their interactions.