齐墩果酸衍生物诱导人肝癌SMMC-7721细胞的凋亡机制

发布时间：2018-03-05 13:35

本文选题：齐墩果酸衍生物　切入点：人肝癌细胞系SMMC-7721　出处：《山西农业大学》2016年博士论文　论文类型：学位论文

【摘要】：研究发现五环三萜类化合物具有较好的抗肿瘤活性,齐墩果酸衍生物(Oleanolic Acid Derivatives, OAD)是一种五环三萜类化合物,本研究旨在探讨其对人肝癌细胞系SMMC-7721的作用及其机制。方法：(1)应用不同浓度的OAD作用于人肝癌细胞系SMMC-7721(以下简称SMMC-7721细胞)和人肝细胞HL-7702(以下简称HL-7702细胞),通过对MTT法对细胞生长曲线的测定、光学显微镜法对细胞病变的观察,研究OAD的细胞毒性作用。(2)通过光学显微镜法观察细胞膜通透性、线粒体膜电位(△Ψm)的荧光改变情况,应用分光光度计检测细胞内ATP的含量,酶联免疫法检测胞浆及线粒体内Cyt-C的含量,Western-blotting检测凋亡相关蛋白Bcl-2、Bax、pro-caspase-9、cleaved-caspase-9、pro-caspase-3、cleaved-caspase-3的蛋白表达水平,研究OAD诱导SMMC-7721细胞发生凋亡的机制。(3)采用qRT-PCR和Western-blotting技术检测不同浓度的OAD在不同时间点对SMMC-7721中Wnt/β-catenin信号通路中关键分子P-catenin、 c-myc和cyclin D1基因及蛋白水平的影响,Western-blotting技术进一步检测了β-catenin核转移水平以及β-catenin磷酸化水平,研究OAD对SMMC-7721细胞中WVnt/p-catenin信号通路的作用机制。结果：(1)OAD能引起SMMC-7721细胞皱缩、胞膜起泡以及凋亡小体的产生,15μM O AD在24h、48h、72h\ SMMC-7721细胞的最大抑制率分别是30.30%、63.00%、82.46%,对HL-7702的最大抑制率分别是37.20%、64.45%、84.68%,且呈时间和剂量依赖性。(2)7μM、9μM、11μM OAD作用于SMMC-7721细胞12h、24h、36h后,可引起SMMC-7721细胞膜磷脂酰丝氨酸(PS)外翻,经由Annexin V-FITC/PI双染后的SMMC-7721细胞膜绿色荧光及红色荧光均有所增加,且呈时间和剂量依赖性,即OAD可破坏SMMC-7721细胞膜的通透性,OAD作用于SMMC-7721细胞48h后抗凋亡蛋白Bcl-2被下调而促凋亡蛋白Bax被上调,并且促进了pro-caspase-9 和 pro-caspase-3的裂解,说明OAD促使SMMC-7721细胞发生了凋亡过程。(3)OAD处理SMMC-7721细胞后下调了细胞线粒体内ATP水平,当OAD浓度为11μM时,由细胞组的1648.46μmol/gprot降低为374.51 μmol/gprot,OAD还促进了线粒体内Cyt-C的释放,当OAD浓度为11μM时,线粒体Cyt-C的含量由细胞组的116.30nmol/L减少为40.15 nmol/L。与空白细胞组和DMSO组相比较,各浓度OAD药物处理组细胞中的红／绿荧光比例随着药物浓度的增加和时间的延长而减小。可见,OAD降低了SMMC-7721细胞线粒体膜电位(△ψm)。(4)OAD能下调SMMC-7721细胞Wnt/β-catenin信号通路关键分子β-catenin、c-myc和cyclin D1的基因及蛋白表达水平,并可抑制β-catenin的核转移和增加胞浆内β-catenin在Ser33/37/Thr41位点的磷酸化水平。结论：(1)OAD能抑制SMMC-7721细胞的增殖并引起细胞线粒体功能紊乱而诱导SMMC-7721细胞凋亡。(2)OAD通过抑制β-catenin的核转移以及增加β-catenin的磷酸化(Ser33/37/Thr41)水平来抑制SMMC-7721细胞中wnt/β-catenin信号通路的激活,从而诱导SMMC-7721细胞发生凋亡。综上所述,OAD有望成为一种新型的、有效的抑制肝癌细胞增殖、诱导凋亡的抗肝癌中药单体药物。
[Abstract]:The study found that three pentacyclic triterpenoids have good anti-tumor activity of oleanolic acid derivatives (Oleanolic, Acid Derivatives, OAD) is a three pentacyclic triterpenoids, the purpose of this study is to explore the SMMC-7721 on human hepatocellular carcinoma cell line and its working mechanism. Methods: (1) the effect of OAD with different concentrations on human hepatocellular carcinoma cells SMMC-7721 (hereinafter referred to as SMMC-7721 cells) and human liver cell HL-7702 (HL-7702 cell), based on the MTT method for the determination of growth curve of cells, optical microscope observation method of cell lesions, cytotoxic effect of OAD. (2) through the optical microscope to observe the permeability of cell membrane, mitochondrial membrane potential (delta only m) fluorescence changes, using spectrophotometer to detect the content of intracellular ATP content was measured by enzyme linked immunosorbent assay plasma and mitochondrial Cyt-C, Western-blotting detection of apoptosis related protein B Cl-2, Bax, pro-caspase-9, cleaved-caspase-9, pro-caspase-3, the expression level of cleaved-caspase-3 protein, the mechanism of OAD induced apoptosis of SMMC-7721 cells. (3) using qRT-PCR and Western-blotting to detect different concentrations of OAD at different time points of key molecules of Wnt/ beta -catenin signal pathway in SMMC-7721 P-catenin, effects of gene and protein level of c-myc and cyclin D1, Western-blotting technology to detect the beta -catenin nuclear translocation level and beta phosphorylation of -catenin, OAD on the WVnt/ mechanism of P-catenin signaling pathway in SMMC-7721 cells. Results: (1) OAD can induce SMMC-7721 cell shrinkage, cell membrane blebbing and apoptotic bodies, 15 M O AD in 24h, 48h, the maximum inhibitory rate of 72h SMMC-7721 cells were 30.30%, 63%, 82.46%, the maximum inhibition rate of HL-7702 were 37.20%, 64.45%, 84.68%, and Time and dose dependent manner. (2) 7 M, 9 M, 11 M OAD in SMMC-7721 cells 12h, 24h, 36h, SMMC-7721 can cause cell membrane phosphatidylserine (PS) increased valgus, via the SMMC-7721 cell membrane Annexin V-FITC/PI after staining with green fluorescence and red fluorescence were, dose and time dependent manner, OAD SMMC-7721 can destroy the cell membrane permeability, the effect of OAD on SMMC-7721 cells after 48h anti apoptotic protein Bcl-2 was down regulated and the pro apoptotic protein Bax was up-regulated, and promoted pro-caspase-9 and pro-caspase-3 OAD to make SMMC-7721 cleavage, apoptosis process. (3) OAD after treatment of SMMC-7721 cells with reduced ATP level of the mitochondria. When the OAD concentration is 11 M, reduced by 1648.46 mol/gprot cell group was 374.51 mol/gprot, OAD also promoted the mitochondrial release of Cyt-C, when the OAD concentration is 11 M, mitochondrial Cyt The content of -C by 116.30nmol/L cells were reduced to 40.15 nmol/L. compared with the blank cell group and DMSO group, the concentration of OAD treated red / green fluorescent cells in the proportion decreased with the increase of drug concentration and time. Therefore, OAD reduced SMMC-7721 cell mitochondrial membrane potential (Delta Psi m). (4) OAD can down regulate SMMC-7721 cell Wnt/ beta key molecules in -catenin signaling pathway beta -catenin gene and protein expression of c-myc and cyclin D1, and can inhibit the beta -catenin nuclear transfer and increase intracellular beta -catenin in phosphorylation of Ser33/37/Thr41 loci (1). Conclusion: OAD can inhibit SMMC-7721 cells the proliferation of cells and cause mitochondrial dysfunction and apoptosis in SMMC-7721 cells induced by OAD. (2) by inhibiting beta -catenin nuclear translocation and increased beta -catenin phosphorylation level (Ser33/37/Thr41) to inhibit SMMC-7721 cells in wnt/ beta The activation of -catenin signaling pathway can induce apoptosis of SMMC-7721 cells. In conclusion, OAD is expected to become a new type of anti hepatoma Chinese medicine monomer, which can effectively inhibit the proliferation and induce apoptosis of hepatoma cells.

【学位授予单位】：山西农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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