mTOR介导小豆蔻明抗炎作用抑制卵巢癌细胞增殖的研究

发布时间：2018-04-20 04:16

  本文选题：小豆蔻明 + 炎症　； 参考：《福建医科大学》2014年硕士论文

【摘要】：目的 炎症与卵巢癌的生长、侵袭、血管生成等密切相关，靶向抗炎进而抑制肿瘤细胞增殖有望成为卵巢癌治疗的新途径。小豆蔻明（Cardamonin，CAR）具有抗炎抗肿瘤及抑制哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）活性的作用，但mTOR是否介导了CAR的抗炎作用，从而抑制卵巢癌细胞的增殖有待证实。本研究旨在明确CAR调节炎症抗肿瘤的作用及其相关靶标，为此类药物的研发提供实验依据。 方法 1细胞系 人卵巢癌细胞株SKOV3 2分组与给药 采用脂多糖（Lipopolysaccharide，LPS）诱导SKOV3卵巢癌细胞产生炎症，吡咯烷二硫代氨基甲酸酯（pyrrolidine dithiocarbamate，PDTC）阻断核转录因子-κB（nuclear factor-kappaB，NF-κB）介导的炎症途径。设立细胞对照组、LPS组、PDTC+LPS组，分别用RAP、不同剂量CAR处理，，各药物浓度为LPS（1μg/mL）、PDTC（100μM）、RAP（0.1μM）、CAR（100μM、30μM、10μM、3μM、1μM、0.1μM）。 3测定指标及方法 3.1显微镜观察细胞形态和密度 3.2MTT法测定细胞增殖 3.3PCR法检测炎症因子IL-6的mRNA表达 3.5Western Blot法检测NF-κB p65、mTOR、p-mTOR、S6K1及p-S6K1蛋白的表达 结果 1CAR可剂量依赖性抑制正常及LPS诱导的SKOV3细胞增殖； 2CAR可抑制正常及LPS诱导的SKOV3细胞IL-6mRNA和细胞核NF-κB p65蛋白表达；在正常SKOV3细胞中，低剂量CAR对IL-6mRNA表达抑制作用强于RAP和高剂量CAR（P0.01）；而在LPS预处理细胞中，RAP和高剂量CAR对IL-6的抑制作用强于低剂量CAR（P0.01）； 3LPS可激活mTOR和S6K1，显著增加p-mTOR、p-S6K1蛋白的表达（P0.01），在正常和LPS预处理细胞中，CAR均可抑制p-mTOR、p-S6K1的蛋白表达(P0.01)，CAR对二者总蛋白的表达无显著性影响； 4PDTC预作用可显著抑制LPS诱导的细胞核NF-κB p65蛋白表达（P0.01），而对LPS诱导的p-mTOR、p-S6K1无明显影响；同LPS预作用组比较，PDTC+LPS预作用后RAP和高剂量CAR对p-mTOR、p-S6K1蛋白表达的抑制作用减弱；同PDTC+LPS组比较，RAP和高剂量CAR对核NF-κB p65表达无明显抑制作用。 结论 CAR对正常及LPS诱导的SKOV3细胞增殖均有抑制作用，机制可能与CAR降低mTOR及下游S6K1的磷酸化从而影响NF-κB与IL-6的表达有关。
[Abstract]:Purpose Inflammation is closely related to the growth, invasion and angiogenesis of ovarian cancer. Cardamonine Carr) has anti-inflammatory and anti-tumor effects and inhibits the activity of mammalian rapamycin target protein target of rapamycin. However, whether mTOR mediates the anti-inflammatory effect of CAR and thus inhibits the proliferation of ovarian cancer cells remains to be confirmed. The aim of this study was to investigate the role of CAR in the regulation of inflammation and tumor and its related targets, and to provide experimental evidence for the development of these drugs. Method 1 cell line Human ovarian cancer cell line SKOV3 2 grouping and administration Lipopolysaccharide (LPS) was used to induce inflammation in SKOV3 ovarian cancer cells. Pyrrolidine dithiocarbamate- (PDTCc) blocked the inflammatory pathway mediated by nuclear transcription factor 魏 B(nuclear factor-kappa BNF- 魏 B. The control group was treated with CAR with different doses. The concentration of each drug was LPS(1 渭 g 路mL ~ (-1) ~ 100 渭 m ~ (-1) ~ (-1) ~ (-1) 渭 M ~ (-1) ~ (-1) 渭 m ~ (-1) ~ (-1) 渭 M ~ (-1) ~ (10) 渭 m ~ (-1) ~ (-1) 渭 M ~ (-1) ~ 10 渭 M ~ (-1) ~ (10 渭 m) ~ (-1) 渭 M ~ (-1) ~ (-1) 渭 M ~ (-1). 3 determination index and method 3.1 microscopic observation of cell morphology and density Determination of cell proliferation by 3.2MTT Detection of mRNA expression of inflammatory factor IL-6 by 3.3PCR Detection of the expression of p-mTORS6K1 and p-S6K1 protein in p65 mTORA of NF- 魏 B by 3.5Western Blot Result 1CAR inhibited the proliferation of normal and LPS induced SKOV3 cells in a dose-dependent manner. 2CAR inhibited the expression of IL-6mRNA and nuclear NF- 魏 B p65 protein in normal and LPS induced SKOV3 cells, and the expression of NF- 魏 B p65 protein in normal SKOV3 cells. The inhibitory effect of low dose CAR on IL-6mRNA expression was stronger than that of RAP and high dose carotene P0.01G, but the inhibitory effect of LPS pretreated cells on IL-6 was stronger than that of low dose carotene P0.01. 3LPS activated mTOR and S6K1, significantly increased the expression of p-mTOR-p-S6K1 protein. In normal and LPS pretreated cells, car could inhibit the expression of p-mTOR-p-S6K1 protein. The expression of p0. 01 protein in nuclear NF- 魏 B p65 induced by LPS was significantly inhibited by 4PDTC preconditioning, but the expression of p-S6K1 in p-mTORN induced by LPS was not significantly affected, and the inhibitory effect of RAP and high dose CAR on the expression of p-S6K1 protein in p-mTORB p65 protein was decreased compared with that in LPS pretreated group. Compared with PDTC LPS group, rap and high dose CAR had no obvious inhibitory effect on nuclear NF- 魏 B p65 expression. Conclusion Both normal and LPS induced proliferation of SKOV3 cells were inhibited by CAR. The mechanism may be related to the decrease of phosphorylation of mTOR and downstream S6K1 by CAR, which may affect the expression of NF- 魏 B and IL-6.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31


本文编号：1776202