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目的探索人甲状腺乳头状癌原代细胞的体外长期培养方法及其监测鉴定手段。方法常规分离甲状腺乳头状癌细胞,并用含10%胎牛血清、L-谷氨酰胺和20 ng/ml人重组表皮生长因子(EGF)的高糖型DMEM培养液培养,分别在不同时间观察甲状腺乳头状癌细胞生长情况,测定培养液中的甲状腺球蛋白(Tg)和甲状腺过氧化物酶(TPO)水平,用免疫细胞化学方法检测Tg的表达。结果培养约8 d甲状腺乳头状癌细胞长满约80%的培养瓶底,符合传代标准;在培养45 d内生长良好,经5~6传代后细胞逐渐老化。细胞培养液中Tg含量随培养时间延长呈抛物线形变化,培养约14 d Tg达到峰值(985.2μg/L);TPO在细胞培养液中始终未检测到。免疫荧光染色结果显示,体外培养甲状腺乳头状癌原代细胞的Tg表达阳性。结论应用本研究建立的培养条件可以使人甲状腺乳头状癌原代细胞传5~6代,细胞存活约45 d。建立了简便、系统的监测及鉴定甲状腺乳头状癌细胞生物活性手段,提示在14 d左右该细胞用于基因研究较为合适,为研究甲状腺乳头状癌发生、发展机理及其特征提供了一种合适替代工具。
Objective To explore the long-term culture method of human thyroid papillary carcinoma primary cells in vitro and its monitoring and identifying methods. Methods Thyroid papillary carcinoma cells were isolated routinely and cultured in high glucose DMEM containing 10% fetal bovine serum, L-glutamine and 20 ng / ml recombinant human epidermal growth factor (EGF). The thyroid The growth of papillary carcinoma cells was measured. The levels of thyroglobulin (Tg) and thyroid peroxidase (TPO) in the culture medium were measured. The expression of Tg was detected by immunocytochemistry. Results About 8 days, papillary thyroid carcinoma cells grew up to about 80%, which met the passage criteria. The cells grew well within 45 days of culture. The cells gradually aged after 5 ~ 6 passages. The content of Tg in the cell culture fluid changed parabolically with the prolongation of culture time, reaching the peak value of about 14 d after the culture (985.2 μg / L). TPO was not detected in the cell culture solution. Immunofluorescence staining showed that in vitro cultured primary papillary thyroid carcinoma cells showed positive Tg expression. Conclusion The culture conditions established in this study can make human primary papillary thyroid carcinoma cell passage 5 to 6 generations, cell survival of about 45 d. The establishment of a simple and systematic monitoring and identification of papillary thyroid cancer cell biological activity means that the cells at about 14 d for gene research is more appropriate for the study of thyroid papillary carcinoma, mechanism of development and its characteristics provide a suitable Alternative tools.