目的 探讨海藻-甘草含药血清对人甲状腺正常细胞Nthy-ori-3-1增殖的作用，为进一步研究海藻与甘草配伍抗甲状腺肿大的作用机制奠定基础。方法 体外培养Nthy-ori-3-1细胞，使用不同浓度的促甲状腺激素受体人单克隆抗体M22作用于Nthy-ori-3-1细胞不同时间，观察M22作用的最佳浓度及时间。制备海藻-甘草含药血清，M22刺激细胞24 h后，分别给予5%、10%、15%、20%海藻-甘草含药血清干预24 h，采用CCK-8法检测各组细胞增殖情况。流式细胞术检测细胞周期分布和凋亡情况；透射电镜观察细胞形态；免疫荧光检测微管相关蛋白轻链3（microtubule-associated protein light chain3，LC3）表达情况；Western blotting检测Beclin1蛋白表达。结果 M22明显刺激Nthy-ori-3-1细胞增殖，0.125 μg/mL的M22作用24 h时促增殖作用最佳（P<0.01）。经海藻-甘草含药血清干预后，细胞增殖明显抑制（P<0.05、0.01），细胞呈现明显的凋亡形态学变化，G₀/G₁期细胞比例明显升高（P<0.01），G₂/M期细胞比例无显著性差异，细胞凋亡率明显上升（P<0.01），LC3平均荧光强度增加，Beclin1蛋白表达水平明显升高（P<0.01）。结论 M22可刺激Nthy-ori-3-1细胞增殖，海藻-甘草含药血清抑制M22诱导的Nthy-ori-3-1细胞增殖，促进其凋亡，可能通过调节自噬来发挥抑制Nthy-ori-3-1细胞增殖的作用，以达到抗甲状腺肿大的疗效。

Objective To investigate the effect of Haizao (Sargassum)-Gancao (Glycyrrhizae Radix et Rhizoma) containing serum on proliferation of human thyroid cells Nthy-ori-3-1, and lay a foundation for further research on the anti-goiter mechanism of Sargassum-Glycyrrhizae Radix et Rhizoma combination. Methods Nthy-ori-3-1 cells were cultured in vitro, different concentrations of thyroid stimulating hormone receptor human monoclonal antibody M22 were used to act on Nthy-ori-3-1 cells at different times, and the optimal concentration and time of M22 were observed. Sargassum-Glycyrrhizae Radix et Rhizoma containing serum were prepared, after cells were stimulated by M22 for 24 h, 5%, 10%, 15% and 20% Sargassum-Glycyrrhizae Radix et Rhizoma containing serum were given for intervention for 24 h. CCK-8 method was used to detect cell proliferation in each group. The cell cycle distribution and apoptosis of each group were detected by flow cytometry; Cells morphology was observed by transmission electron microscopy; The expression of microtubule-associated protein light chain 3 (LC3) was detect by immunofluorescence; The expression of Beclin1 protein was detected by Western blotting. Results M22 significantly stimulated the proliferation of Nthy-ori-3-1 cells, and the proliferation was best at 0.125 μg/mL concentration of M22 for 24 h (P < 0.01). After intervention with Sargassum-Glycyrrhizae Radix et Rhizoma containing serum, cell proliferation was significantly inhibited (P < 0.05, 0.01), cells showed obvious morphological changes of apoptosis, and the proportion of G₀/G₁ phase cells was significantly increased (P < 0.01), there was no significant difference in the proportion of G₂/M phase cells, and the apoptosis rate was significantly increased (P < 0.01), the mean fluorescence intensity of LC3 was increased, Beclin1 protein expression level was significantly increased (P < 0.01). Conclusion M22 can stimulate the proliferation of Nthy-ori-3-1 cells, Sargassum-Glycyrrhizae Radix et Rhizoma containing serum can inhibit the proliferation of M22-induced Nthy-ori-3-1 cells and promote apoptosis, which may play a role on inhibiting the proliferation of Nthy-ori-3-1 cells by regulating autophagy, so as to achieve the effect of anti-goiter.

R285.5

国家自然科学基金资助项目（82104411）