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本文研究了金莲花总黄酮乙醇提取物(TFETC)诱导人HT-29结肠癌细胞凋亡的作用机制。MTT法测定TFETC对HT-29细胞增殖的影响,200μg/mL浓度TFETC对癌细胞的抑制率达到81%。通过4,6-diamidino-2-phenylindole(DAPI)法和流式细胞术检测到金莲花总黄酮乙醇提取物可以诱导癌细胞凋亡,200μg/mL浓度TFETC处理的癌细胞通过显微镜观察出现凋亡,亚G1期DNA含量达到28.9%。逆转录聚合酶链式反应(RT-PCR)法测定TFETC对HT-29细胞内Bcl-2,Bcl-xL,Bax,caspase-9,caspase-3和COX-2等基因表达的影响。TFETC在mRNA水准上下调抗凋亡基因Bcl-2和Bcl-xL,上调促凋亡基因Bax,caspase-9和caspase-3的表达。此外,TFETC还可抑制HT-29细胞内COX-2基因的表达,并呈剂量效应关系。本研究结果显示金莲花总黄酮乙醇提取物可以通过内源性线粒体途径诱导人HT-29结肠癌细胞的凋亡,同时金莲花总黄酮乙醇提取物还可通过下调COX-2基因的表达抑制HT-29细胞的增殖。
In this paper, we investigated the mechanism of TFETC-induced apoptosis in human HT-29 colon cancer cells. MTT assay TFETC HT-29 cell proliferation, 200μg / mL concentration of TFETC on the inhibition rate of cancer cells reached 81%. Apoptosis of cancer cells was detected by 4,6-diamidino-2-phenylindole (DAPI) method and flow cytometry, and the apoptosis of cancer cells treated with 200 μg / mL TFETC was observed by microscope , The sub-G1 DNA content reached 28.9%. The effect of TFETC on gene expression of Bcl-2, Bcl-xL, Bax, caspase-9, caspase-3 and COX-2 in HT-29 cells was determined by reverse transcription polymerase chain reaction (RT- TFETC down-regulated anti-apoptotic genes Bcl-2 and Bcl-xL at the mRNA level, and up-regulated the expression of pro-apoptotic genes Bax, caspase-9 and caspase-3. In addition, TFETC also inhibits COX-2 gene expression in HT-29 cells in a dose-dependent manner. The results of this study show that the ethanol extract of Trollius total flavonoids can induce apoptosis in human HT-29 colon cancer cells through the endogenous mitochondrial pathway. At the same time, the total flavonoids of Ethanol extract can also inhibit the expression of COX-2 gene by inhibiting the HT -29 cells.