【作者】 魏天南；

【导师】 胡建达；

【作者基本信息】 福建医科大学 ， 内科学， 2009， 硕士

【摘要】 目的①研究中药大黄素对人T淋巴细胞白血病Jurkat、Molt-4细胞株增殖、凋亡的影响。②观察大黄素对Jurkat、Molt-4细胞增殖、凋亡相关基因及蛋白表达水平的影响,探讨其作用机制。③观察大黄素对Jurkat、Molt-4细胞作用后PI3K/Akt信号转导通路中蛋白表达的变化。方法应用MTT法绘制细胞生长曲线、细胞克隆形成实验观察大黄素对Jurkat、Molt-4细胞增殖的影响; DNA片段化分析、Annexin V/PI双染流式细胞检测、末端缺口原位标记(TUNEL)法及DNA倍体分析检测大黄素对细胞凋亡的影响;蛋白印迹法(Western-Blot)检测c-myc、bcl-2、pro-与cleaved caspase-3、pro-caspase-8、pro-caspase-9、PARP蛋白以及PI3K/Akt信号转导通路中蛋白表达水平的变化。结果(1)细胞生长曲线结果显示大黄素能明显抑制Jurkat、Molt-4细胞增殖,48h半数抑制浓度(IC50)分别为20.7μmol·L-1和25.1μmol·L-1。DNA片段化、DNA倍体分析亚二倍体峰、TUNEL法凋亡细胞的检出及Annexin V/PI双染流式细胞检测等证实大黄素能有效诱导Jurkat细胞凋亡,细胞凋亡率在一定的药物浓度范围内与药物作用浓度和作用时间呈正相关。DNA片段化的检出同样表明大黄素可诱导Molt-4细胞凋亡。(2)80μmol·L-1大黄素作用Jurkat细胞不同时间和不同浓度大黄素作用Jurkat细胞后,c-myc、bcl-2、hTERT、pro-caspase-3、pro-caspase-8、procaspase-9蛋白表达下调,同时cleaved caspase-3和cleaved PARP蛋白的表达上调,并呈量效与时效关系。相同浓度大黄素作用Molt-4细胞后,bcl-2、c-myc、hTERT与procaspase-3蛋白表达下调。(3)大黄素可作用于Jurkat、Molt-4细胞Akt信号通路,下调Jurkat细胞中p-Akt、IκB-α、p-IκB-α、p-NF-κB、mTOR、p-mTOR、GSK-3β、p-GSK-3β、MAPK和p-MAPK的表达,而Akt、NF-κB表达变化不明显,Bad表达上调;大黄素还下调Molt-4细胞中Akt、p-AKT、m-TOR、p-mTOR蛋白表达。结论(1)大黄素能有效抑制Jurkat、Molt-4细胞增殖,诱导其凋亡,在一定浓度范围内呈剂量时间效应关系。大黄素可能是通过下调c-myc、bcl-2、hTERT及caspase家族的蛋白表达诱导细胞凋亡。(2) Akt信号通路参与了大黄素抑制Jurkat、Molt-4细胞增殖、诱导凋亡的过程。更多还原

【Abstract】 【Objective】To investigate the effects of Chinese traditional medicine Emodin on proliferation and apoptosis in T lymphocytic leukemic cell line Jurkat and Molt-4 and to explore the mechanisms of the effects by measuring the expression of proliferation and apoptosis related proteins as well as the expression of PI3K/AKT signaling pathway proteins.【Methods】Cell proliferation inhibition was detected by MTT assay. Cell apoptosis was measured by DNA ladder, Annexin V/PI double staining analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling(TUNEL) assay and cell cycle analysis. The expressions of apoptosis related proteins, caspase family members and PI3K/AKT signaling pathway proteins were determined by Western Blot.【Results】(1) Emodin inhibited proliferation in Jurkat and Molt-4 cells, with the IC50 in 48h at 20.7μmol·L-1 and 25.1μmol·L-1, respectively. Cell apoptosis was observed with both time and dose dependent manner in Jurkat cells and also observed in Molt-4 cells by the detection of DNA fragments. (2) In Jurkat cells, the expressions c-myc, hTERT and Bcl-2 were explored to be down-regulated after the treatment of Emodin in a time dependent manner. Caspase-3, PARP were both activated, while the expressions of procaspase-8 and 9 both decreased. In Molt-4 cells, c-myc, bcl-2 were down-regulated and caspase-3, PARP were both activated after the treatment of Emodin. (3)Emodin have effects on PI3K/AKT signaling pathway in both Jurkat and Molt-4 cells, with the down-regulation of p-Akt、IκB-α、p-IκB-α、 p-NF-κB、mTOR、p-mTOR、GSK-3β、p-GSK-3β、MAPK and p-MAPK in Jurkat cells and Akt、p-Akt、mTOR and p-mTOR in Molt-4 cells, while the expression of Akt and NF-κB showed no significant change and the expression of Bad in creased in Jurkat cells.【Conclusion】(1)Emodin could remarkbly inhibit proliferation and induce apoptosis in Jurkat and Molt-4 cells, and the down-regulation of bcl-2、c-myc、hTERT and caspase family members may have contributed to the apoptosis process.(2)PI-3K/Akt signaling pathway may have involved in the apoptosis of Jurkat and Molt-4 cells after treatment with Emodin.更多还原