甲亢宁胶囊含药血清对M22刺激FRTL-5细胞增殖的影响及自噬在其中的作用  

Effect of Jiakangning Capsule Containing Serum on M22-induced Proliferation of FRTL-5 Cells and the Role of Autophagy in it

作　　者：魏军平[1] 郑慧娟[1] 李敏[1] 王秋虹[1] 魏璠[1] 柏力萄 陈舒雅 WEI Jun-ping;ZHENG Hui-juan;LI Min;WANG Qiu-hong;WEI Fan;BAI Li-tao;CHEN Shu-ya(Department of Endocrinology,Guang＇anmen Hospital,China Academy of Chinese Medical Sciences,Beijing 100053)

机构地区：[1]中国中医科学院广安门医院内分泌科,北京100053

出　　处：《中国中西医结合杂志》2018年第10期1222-1228,共7页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：国家自然科学基金面上项目(No.81573961)

摘　　要：目的探讨甲亢宁胶囊含药血清对甲状腺刺激性抗体(TSAb)单克隆抗体M22刺激Fisher大鼠甲状腺细胞系(FRTL-5)增殖作用和自噬的影响。方法体外培养FRTL-5细胞,采用不同浓度及时间观察M22刺激细胞增殖的量效关系,并确定作用最佳浓度及时间并制备甲亢宁胶囊含药血清。将FRTL-5细胞分为模型组(M22刺激24 h+15%空白大鼠血清),中药低、中、高剂量组(M22刺激24 h后5%、10%、15%甲亢宁胶囊含药血清干预24 h),正常组(15%空白大鼠血清)。采用CCK8法、酶联免疫竞争法检测各组细胞增殖情况及上清液中c AMP水平。确定高剂量(15%)甲亢宁胶囊含药血清抑制细胞增殖作用最强后再将FRTL-5细胞分为正常组(15%空白大鼠血清)、模型组(M22刺激24 h+15%空白大鼠血清)和中药组(M22刺激24 h后15%甲亢宁胶囊含药血清干预24 h),透射电镜下观察各组细胞内自噬体的变化,Western blot检测各组细胞自噬相关LC3及Beclin1蛋白水平。结果 M22对FRTL-5细胞的增殖影响呈现明显量效关系,确定1μg/m L M22于24 h增殖作用最佳。与正常组比较,模型组FRTL-5细胞增殖活性明显增强(P <0. 01),细胞上清液中c AMP释放量增加(P <0. 01),自噬相关蛋白LC3Ⅱ/Ⅰ和Beclin1表达减少(P<0. 01);与模型组比较,中药中剂量组和高剂量组FRTL-5细胞增殖被抑制(P <0. 01),上清液中c AMP释放量减少(P <0. 05,P <0. 01),中药低剂量组作用差异无统计学意义(P>0. 05),中药组自噬关键蛋白LC3Ⅱ/Ⅰ、Beclin1表达均增高(P <0. 01)。透射电镜提示,模型组较正常组自噬小体的数量减少,而中药组较模型组增多。结论 M22可促进FRTL-5细胞增殖且导致细胞自噬异常,而中药甲亢宁胶囊可通过改善其自噬异常,进而发挥抑制FRTL-5细胞增殖的作用。Objective To investigate the effect of a serum containing Jiakangning（ JKN） capsule on the proliferation and autophagy of Fisher rat thyroid（ FRTL-5） cell line stimulated by monoclonal thyroid-stimulating antibody（ TSAb） M22. Methods FRTL-5 cells were cultured in vitro. The dose-effect relationship of M22 on stimulation of cell proliferation was observed and recorded for different concentrations and time periods. The FRTL-5 cells were divided into model group（ M22 stimulation for 24 h ＋ 15% serum of healthy rats）,low,medium and high JKN groups（ M22 stimulation for 24 h ＋ administration of 5%,10%,and 15% JKN for 24 h,respectively） and normal group（ 15% serum of healthy rats）. The CCK8 and enzyme-linked immunosorbent assays were used to detect cell proliferation and c AMP level in the supernatants,respectively. It was determined that the highest dose（ 15%） of JKN had the strongest inhibitory effect on cell proliferation; therefore,FRTL-5 cells in this group were further divided intonormal group（ 15% serum of healthy rats）,model group（ M22 stimulation for 24 h ＋ 15% serum of healthy rats）and JKN-administered group（ M22 stimulation for 24 h ＋ administration of 15% JKN for 24 h）. Autophagy was then observed using a transmission electron microscope,while autophagy-related LC3 and Beclin1 protein levels were detected by Western blot. Results The effect of M22 on the proliferation of FRTL-5 cells showed a significant doseeffect relationship; it was determined that cell proliferation activity was most obvious when 1 μg/mL M22 co-cultured FRTL-5 cells for 24 h. Compared with the normal group,the proliferation activity of FRTL-5 cells in the model group was significantly higher（ P〈0. 01） along with an increased release of c AMP in the cell supernatant（ P〈0. 01） and decreased expression of autophagy-related proteins（ P〈0. 01）. Compared with the model group,the proliferation of FRTL-5 cells was inhibited at medium and high doses JKN（ P〈0. 01）,while the r

关 键 词：甲亢宁胶囊 GRAVES病 M22 FRTL-5细胞 细胞自噬 自噬相关蛋白 

分 类 号：R285.5[医药卫生—中药学]