17β-雌二醇对小鼠胸腺上皮细胞增殖和凋亡的影响

Effects of 17β-estradiol on the proliferation and apoptosis of murine thymic epithelial cells

为探讨17β-雌二醇对小鼠胸腺上皮细胞增殖和凋亡的影响及其分子机制，将小鼠胸腺上皮细胞系（MTEC1）细胞经不同浓度的17β-雌二醇处理24h后，采用CCK-8法检测细胞增殖活力的变化；流式细胞术检测细胞周期的变化；Annexin V—FITC／PI双染法检测细胞凋亡情况：DAPI染色法检测细胞核形态的变化；高通量测序技术获得差异基因表达谱并运用KEGG通路分析预测差异基因的作用通路；String基因互作分析差异基因的相互作用；实时荧光定量PCR验证p53通路相关基因的表达情况。结果显示，17β-雌二醇显著抑制MTEC1细胞的增殖活力，并呈浓度依赖性；使细胞周期出现G2／M期阻滞：能诱导MTEC1细胞凋亡，细胞核呈现不规则形态、核碎片、染色质凝聚和凋亡小体等典型的凋亡特征；KEGG通路分析显示，与细胞增殖和凋亡密切相关的p53信号通路呈极显著性差异：String基因互作分析显示，p53信号通路的相关基因互相作用，形成紧密联系的网络；p53信号通路的下游周期基因Cyclin B1表达下调，凋亡相关基因Trp53、Gadd45a、Fas、p21、Apaf1和Bax的表达水平显著上调。结果提示，雌激素可能通过p53信号通路对MTEC1细胞的增殖抑制、周期阻滞和细胞凋亡产生影响。

The effects and underlying molecular mechanism of 17β-estradiol on the proliferation and apoptosis of mouse thymic epithelial cells were investigated. In this study, the mouse thymic epithelial cell line 1（MTEC1） cells were treated with different concentrations of 17β-estradiol for 24 h. The cell viability was detected by means of the CCK-8 assay and the cell cycle was tested by flow cytometry. At the same time,the cell apoptosis was detected by Annexin V-FITC/PI staining. As well as,the nucleus division of MTECI cells was detected by DAPI staining. Moreover,the gene expression profile was determined by the high-throughput sequencing. And then,KEGG was applied to screen pathways and String was applied to analyze connection among different genes. In addition,some genes in p53 signaling pathway were verified by qRT-PCR. In result,the cell viability was inhibited by the treatment with 17β-estradiol in a dose-dependent manner,and flow cytometry analysis revealed that MTECI cell cycle was blocked in G2/M phase. Meanwhile,apoptotic cells were significantly increased after 17β-estradiol treatment. Similarly,the cell nucleus exhibited typical apoptotic morphology as characterized by cell nuclei shrinkage and chromatin condensation. Moreover,KEGG pathway analysis revealed that p53 signal- ing pathway was the significantly enriched pathway and String analysis presented that genes in p53 signaling pathway linked each other closely, qRT-PCR revealed that the expression of Cyclin B1 was significantly down-regulated,Trp53,Gadd45a,Fas,p21,Apafl and Bax were significantly up-regulated. Our results indicated that 17β-estradiol may play a key role in inhibiting MTECI proliferation,blocking cell cycle and inducing apoptosis through p53 signaling pathway.