MLH1 R217C/BRAF V600E 突变型家族性甲状腺乳头状癌永生化细胞系的建立及鉴定

摘要： 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+hTERT 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。

Establishing and identification of an immortal cell line of familial papillary thyroid carcinoma with MLH1 R217C/BRAF V600E mutations

Abstract: Objective To establishing an immortal cell line of familial papillary thyroid carcinoma (FPTC), and explore a new approach for studying familial non-medullary thyroid carcinoma (FNMTC). Methods The specimen from a patient with FPTC was selected, separated, and the primary cells were cultured using DMEM/F12 medium (with TSH, T3,EGF and hydrocortisone). To inducing cell immortalization, the exogenous genes SV40T/TERT were transfected into cells by two ways. RT-PCR was used to detect the expressions of thyroid peroxidase (TPO), thyroid globulin (TG), thyroid stimulating hormone receptor (TSHR) and sodium/iodide co- transporter (NIS). Immunofluorescence method was used to detect the expressions of TPO and GPC3. In order to detect the genomic mutations, the peripheral blood DNA of the patient wasextracted. The cell genome was detected. Results The FPTC cells adhered to the plate and showed an irregular polygon shape. The cells can stably grow for six months, FPTC-S (with SV40T transfected) passaged to p26, FPTC cells passaged to p23 and FPTC-ST (with SV40T/TERT transfected) passaged to p19. Both FPTC-S and FPTC-ST can stably express TPO,TG and TSHR in mRNA level. MLH1 R217C mutation existed in the peripheral blood of the patient, and BRAF V600E mutation existed in the primary cultured cells. Either the primary or the immortal cells showed MLH1 R217C mutation.Conclusion This study preliminarily established an immortal cell line of familial papillary thyroid carcinoma with MLH1 R217C and BRAF V600E mutations. This cell line provides a research model for studying these mutations in FPTC.