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 release of c AMP in the supernatant decreased（ P〈0. 05,P〈0. 01）; the group administered with the low dose did not show a statistically significant decrease（ P〈0. 05）,the expression of the aforementioned autophagy-related proteins increased in the JKN-administered group（ P〈0. 01）. Transmission electron microscopy showed that the number of autophagic corpuscles in the model group decreased as compared to that in the normal group,while the number of autophagic bodies in the JKNadministered group increased as compared to that in the model group. Conclusions M22 promotes the proliferation of FRTL-5 cells because of abnormal autophagy. Administration of JKN capsule can inhibit the proliferation of FRTL-5 cells by improving the capability of cells to undergo autophagy.
作者
魏军平
郑慧娟
李敏
王秋虹
魏璠
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)
出处
《中国中西医结合杂志》
CAS
CSCD
北大核心
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)
