本文選題：齊墩果酸衍生物　切入點：人肝癌細(xì)胞系SMMC-7721　出處：《山西農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：研究發(fā)現(xiàn)五環(huán)三萜類化合物具有較好的抗腫瘤活性,齊墩果酸衍生物(Oleanolic Acid Derivatives, OAD)是一種五環(huán)三萜類化合物,本研究旨在探討其對人肝癌細(xì)胞系SMMC-7721的作用及其機制。方法：(1)應(yīng)用不同濃度的OAD作用于人肝癌細(xì)胞系SMMC-7721(以下簡稱SMMC-7721細(xì)胞)和人肝細(xì)胞HL-7702(以下簡稱HL-7702細(xì)胞),通過對MTT法對細(xì)胞生長曲線的測定、光學(xué)顯微鏡法對細(xì)胞病變的觀察,研究OAD的細(xì)胞毒性作用。(2)通過光學(xué)顯微鏡法觀察細(xì)胞膜通透性、線粒體膜電位(△Ψm)的熒光改變情況,應(yīng)用分光光度計檢測細(xì)胞內(nèi)ATP的含量,酶聯(lián)免疫法檢測胞漿及線粒體內(nèi)Cyt-C的含量,Western-blotting檢測凋亡相關(guān)蛋白Bcl-2、Bax、pro-caspase-9、cleaved-caspase-9、pro-caspase-3、cleaved-caspase-3的蛋白表達水平,研究OAD誘導(dǎo)SMMC-7721細(xì)胞發(fā)生凋亡的機制。(3)采用qRT-PCR和Western-blotting技術(shù)檢測不同濃度的OAD在不同時間點對SMMC-7721中Wnt/β-catenin信號通路中關(guān)鍵分子P-catenin、 c-myc和cyclin D1基因及蛋白水平的影響,Western-blotting技術(shù)進一步檢測了β-catenin核轉(zhuǎn)移水平以及β-catenin磷酸化水平,研究OAD對SMMC-7721細(xì)胞中WVnt/p-catenin信號通路的作用機制。結(jié)果：(1)OAD能引起SMMC-7721細(xì)胞皺縮、胞膜起泡以及凋亡小體的產(chǎn)生,15μM O AD在24h、48h、72h\ SMMC-7721細(xì)胞的最大抑制率分別是30.30%、63.00%、82.46%,對HL-7702的最大抑制率分別是37.20%、64.45%、84.68%,且呈時間和劑量依賴性。(2)7μM、9μM、11μM OAD作用于SMMC-7721細(xì)胞12h、24h、36h后,可引起SMMC-7721細(xì)胞膜磷脂酰絲氨酸(PS)外翻,經(jīng)由Annexin V-FITC/PI雙染后的SMMC-7721細(xì)胞膜綠色熒光及紅色熒光均有所增加,且呈時間和劑量依賴性,即OAD可破壞SMMC-7721細(xì)胞膜的通透性,OAD作用于SMMC-7721細(xì)胞48h后抗凋亡蛋白Bcl-2被下調(diào)而促凋亡蛋白Bax被上調(diào),并且促進了pro-caspase-9 和 pro-caspase-3的裂解,說明OAD促使SMMC-7721細(xì)胞發(fā)生了凋亡過程。(3)OAD處理SMMC-7721細(xì)胞后下調(diào)了細(xì)胞線粒體內(nèi)ATP水平,當(dāng)OAD濃度為11μM時,由細(xì)胞組的1648.46μmol/gprot降低為374.51 μmol/gprot,OAD還促進了線粒體內(nèi)Cyt-C的釋放,當(dāng)OAD濃度為11μM時,線粒體Cyt-C的含量由細(xì)胞組的116.30nmol/L減少為40.15 nmol/L。與空白細(xì)胞組和DMSO組相比較,各濃度OAD藥物處理組細(xì)胞中的紅／綠熒光比例隨著藥物濃度的增加和時間的延長而減小�？梢�,OAD降低了SMMC-7721細(xì)胞線粒體膜電位(△ψm)。(4)OAD能下調(diào)SMMC-7721細(xì)胞Wnt/β-catenin信號通路關(guān)鍵分子β-catenin、c-myc和cyclin D1的基因及蛋白表達水平,并可抑制β-catenin的核轉(zhuǎn)移和增加胞漿內(nèi)β-catenin在Ser33/37/Thr41位點的磷酸化水平。結(jié)論：(1)OAD能抑制SMMC-7721細(xì)胞的增殖并引起細(xì)胞線粒體功能紊亂而誘導(dǎo)SMMC-7721細(xì)胞凋亡。(2)OAD通過抑制β-catenin的核轉(zhuǎn)移以及增加β-catenin的磷酸化(Ser33/37/Thr41)水平來抑制SMMC-7721細(xì)胞中wnt/β-catenin信號通路的激活,從而誘導(dǎo)SMMC-7721細(xì)胞發(fā)生凋亡。綜上所述,OAD有望成為一種新型的、有效的抑制肝癌細(xì)胞增殖、誘導(dǎo)凋亡的抗肝癌中藥單體藥物。
[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.

【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R735.7

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