本文選題：姜黃素　切入點(diǎn)：L-FP(氟尿嘧啶　出處：《湖北中醫(yī)藥大學(xué)》2016年碩士論文

【摘要】：目的:姜黃素(curcumin,CUR)是中藥姜黃的主要活性成分,是一種脂溶性酚類色素,具有抗腫瘤、抗炎癥、抑制突變等多種作用。小劑量順鉑(DDP)與5-氟尿嘧啶(5-FU)聯(lián)合用藥(L-FP)是臨床上治療胃癌的常用化療方案。本文旨在探討姜黃素聯(lián)合L-FP對(duì)體外培養(yǎng)的胃癌MGC-803細(xì)胞的生長(zhǎng)、增殖、遷移和凋亡的影響及其機(jī)制,為臨床上聯(lián)合應(yīng)用姜黃素和L-FP治療胃癌提供理論基礎(chǔ)和實(shí)驗(yàn)依據(jù)。方法:采用細(xì)胞生物學(xué)和分子生物學(xué)的研究技術(shù)與方法,觀察姜黃素和/或不同劑量的L-FP對(duì)體外培養(yǎng)的胃癌MGC-803細(xì)胞的生長(zhǎng)、克隆形成、遷移、細(xì)胞周期、細(xì)胞凋亡的作用以及對(duì)相關(guān)調(diào)控蛋白Caspase-3、Caspase-8、Bax和Bcl-2的影響。主要觀察指標(biāo)及方法如下:1.MTT實(shí)驗(yàn):用姜黃素和/或不同劑量的L-FP分別處理96孔板培養(yǎng)的胃癌MGC-803細(xì)胞24h、48h和72h后,使用酶標(biāo)儀檢測(cè)每孔細(xì)胞的OD值,并計(jì)算細(xì)胞的生長(zhǎng)抑制率。2.平板克隆形成實(shí)驗(yàn):用姜黃素和/或不同劑量的L-FP處理6孔板培養(yǎng)的MGC-803細(xì)胞,24h后換不含藥物的培養(yǎng)液繼續(xù)培養(yǎng)10天,吉姆薩染色后計(jì)數(shù)并計(jì)算克隆形成率。3.Transwell遷移實(shí)驗(yàn):用含有姜黃素和/或不同劑量的L-FP的培養(yǎng)液培養(yǎng)Transwell小室中的胃癌MGC-803細(xì)胞,10h后,MTT方法檢測(cè)并計(jì)算遷移率。4.AO/EB雙染法:用姜黃素和/或不同劑量的L-FP處理6孔板培養(yǎng)的MGC-803細(xì)胞24h后,AO/EB雙染在熒光顯微鏡下拍照,觀察細(xì)胞形態(tài)、計(jì)數(shù)凋亡細(xì)胞數(shù)并計(jì)算凋亡率。5.流式細(xì)胞術(shù):姜黃素和/或不同劑量的L-FP處理6孔板培養(yǎng)的胃癌MGC-803細(xì)胞24h后,用PI單染方法檢測(cè)細(xì)胞周期;用Annexin V-FITC/PI雙染方法檢測(cè)細(xì)胞凋亡。6.酶標(biāo)儀比色法:用姜黃素和/或不同劑量的L-FP處理6孔板培養(yǎng)的 MGC-803細(xì)胞24h后,用酶標(biāo)儀比色法檢測(cè)Caspase-3和Caspase-8的活性,并計(jì)算其相對(duì)活性。7.Western blot方法:用姜黃素和/或不同劑量的L-FP處理6孔板培養(yǎng)的MGC-803細(xì)胞24h后,提取總蛋白,用Western blot法檢測(cè)Bax和Bcl-2的表達(dá)。結(jié)果:1.姜黃素聯(lián)合不同劑量的L-FP對(duì)體外培養(yǎng)的胃癌MGC-803細(xì)胞生長(zhǎng)的影響:用姜黃素和/或不同劑量的L-FP處理胃癌MGC-803細(xì)胞24h、48h和72h后,與對(duì)照組相比,各實(shí)驗(yàn)組均表現(xiàn)出了顯著的抑制生長(zhǎng)效應(yīng)(*P0.05或**P0.01或***P0.001);其中藥物處理24h和72h的抑制率,姜黃素聯(lián)合低劑量L-FP組與中劑量L-FP組相比和姜黃素聯(lián)合中劑量L-FP組與高劑量L-FP組相比,無(wú)顯著性差異(P0.05);而48h的抑制率,姜黃素聯(lián)合L-FP組顯著高于姜黃素或L-FP單用組(*P0.05)。2.姜黃素聯(lián)合不同劑量的L-FP對(duì)體外培養(yǎng)的胃癌MGC-803細(xì)胞克隆形成的影響:與對(duì)照組相比,各實(shí)驗(yàn)組細(xì)胞的克隆形成能力均顯著下降(*P0.05);并且姜黃素聯(lián)合L-FP組抑制率顯著高于姜黃素或L-FP單用組(*P0.05)。3.姜黃素聯(lián)合不同劑量的L-FP對(duì)體外培養(yǎng)的胃癌MGC-803細(xì)胞遷移的影響:與對(duì)照組相比,各實(shí)驗(yàn)組通過(guò)Transwell小室的細(xì)胞量明顯降低(*P0.05或**P0.01);其中姜黃素聯(lián)合L-FP組遷移率與姜黃素或L-FP單用組無(wú)顯著差異(P0.05)。4.姜黃素聯(lián)合不同劑量的L-FP對(duì)體外培養(yǎng)的胃癌MGC-803細(xì)胞周期的影響:與對(duì)照組相比,各實(shí)驗(yàn)組主要阻滯細(xì)胞于S期(*P0.05),而且姜黃素聯(lián)合L-FP組細(xì)胞在S期所占比例顯著高于姜黃素或L-FP單用組(*P0.05)。5.姜黃素聯(lián)合不同劑量的L-FP對(duì)體外培養(yǎng)的胃癌MGC-803細(xì)胞凋亡的影響:與對(duì)照組相比,各實(shí)驗(yàn)組均能顯著誘導(dǎo)MGC-803細(xì)胞發(fā)生凋亡(*P0.05),其中姜黃素聯(lián)合L-FP組的凋亡率顯著高于姜黃素或L-FP單用組(*P0.05),并且Annexin-V/PI雙染流式細(xì)胞儀檢測(cè)的各組細(xì)胞凋亡率與AO/EB雙染熒光顯微鏡檢測(cè)的各組細(xì)胞凋亡率沒有顯著差異(P0.05)。6.姜黃素聯(lián)合不同劑量的L-FP對(duì)體外培養(yǎng)的胃癌MGC-803細(xì)胞相關(guān)調(diào)控蛋白的影響:與對(duì)照組相比,各實(shí)驗(yàn)組細(xì)胞顯著增強(qiáng)了Bax的表達(dá),降低了Bcl-2的表達(dá),提高了Caspase-3和Caspase-8的活性(*P0.05或**P0.01);除了姜黃素聯(lián)合中劑量L-FP組的Bax相對(duì)表達(dá)量與高劑量L-FP單用組無(wú)顯著差異(P0.05),其余姜黃素聯(lián)合L-FP組的Bax相對(duì)表達(dá)量顯著高于姜黃素或L-FP單用組,Bcl-2相對(duì)表達(dá)量顯著低于姜黃素或L-FP單用組(*P0.05或**P0.01);姜黃素聯(lián)合低劑量L-FP組的Caspase-3和Caspase-8相對(duì)活性與中劑量L-FP單用組無(wú)顯著差異(P0.05),而姜黃素聯(lián)合中劑量L-FP組的Caspase-3和Caspase-8相對(duì)活性顯著高于高劑量L-FP單用組(*P0.05或**P0.01)。結(jié)論:1.姜黃素能增強(qiáng)L-FP對(duì)體外培養(yǎng)的胃癌MGC-803細(xì)胞的生長(zhǎng)抑制作用。2.姜黃素能增強(qiáng)L-FP對(duì)體外培養(yǎng)的胃癌MGC-803細(xì)胞克隆形成和遷移能力的抑制作用。3.姜黃素能增強(qiáng)L-FP對(duì)體外培養(yǎng)的胃癌MGC-803細(xì)胞周期中S期的阻滯作用。4.姜黃素能增強(qiáng)L-FP對(duì)體外培養(yǎng)的胃癌MGC-803細(xì)胞凋亡的誘導(dǎo)作用,其機(jī)制與抑制Bcl-2表達(dá)、促進(jìn)Bax表達(dá)和提高Caspase-3和Caspase-8活性有關(guān)。對(duì)于細(xì)胞凋亡的檢測(cè)而言,Annexin-V/PI雙染流式細(xì)胞儀檢測(cè)方法與AO/EB雙染熒光顯微鏡檢測(cè)方法的結(jié)果無(wú)顯著性差異。
[Abstract]:Objective: curcumin (curcumin, CUR) is the main active ingredient of turmeric, is a fat soluble phenolic pigment, anti-tumor, anti-inflammatory, inhibiting mutation and so on. The low dose of cisplatin (DDP) and 5- fluorouracil (5-FU) combination therapy (L-FP) is a commonly used chemotherapy treatment of gastric cancer. This paper aims to investigate the effect of curcumin and L-FP on cultured human gastric cancer MGC-803 cell growth, proliferation, migration and apoptosis effect and its mechanism, to provide theoretical and experimental basis for clinical application of curcumin combined with L-FP in the treatment of gastric cancer. Methods: the techniques and methods of using cell biology and molecular biology, to observe the effect of curcumin and / or different doses of L-FP on cultured human gastric cancer MGC-803 cell growth, colony formation, migration, cell cycle, apoptosis and protein related to the regulation of Caspase-3, Caspase-8, Bax and Bcl-2 The following effects. Main outcome measures and methods: 1.MTT experiment: treatment of gastric cancer cell line MGC-803 24h was cultured in 96 well plates respectively with curcumin and / or different doses of L-FP, 48h and 72h, using the ELISA OD was detected in each hole cells, and calculate the cell growth inhibition rate of.2. plate clone formation experiment: Curcumin and / or different doses of L-FP treatment 6 pore plate cultured MGC-803 cells after 24h culture medium not containing drugs to cultured for 10 days, after Giemsa staining counts and colony formation rate of.3.Transwell Migration Experiment: containing curcumin and / or different doses of L-FP cultured Transwell cell in gastric cancer MGC-803 cells, 10h, MTT method to detect and calculate the migration rate of.4.AO/EB double staining with curcumin and / or different doses of L-FP treatment of MGC-803 cells cultured in 24h 6 after AO/EB staining under fluorescence microscope and photographed, observe the cell Form, counting the number of apoptotic cells and apoptotic rate of.5. flow cytometry: Curcumin and / or different doses of L-FP treatment of MGC-803 gastric cancer cells 24h cultured in 6 well plates after stained with PI method to detect cell cycle; colorimetric method with Annexin V-FITC/PI double staining method to detect apoptosis.6. eliasa than by curcumin and / or different doses of L-FP treatment of MGC-803 cells cultured in 24h 6 after Caspase-3 and Caspase-8 detector colorimetric method with enzyme activity, and calculated the relative activity of.7.Western blot method: Curcumin and / or different doses of L-FP treatment of MGC-803 cells cultured in 24h 6 after extraction of total protein the expression of Bax and Bcl-2, with the detection of Western by blot. Results: 1. effects of curcumin combined with different doses of L-FP on the growth of in vitro cultured gastric cancer MGC-803 cells by curcumin and / or different doses of L-FP treatment of MGC-803 gastric cancer cells 24h, 48H And after 72h, compared with the control group, the experimental group showed significant growth inhibition effect (*P0.05 or **P0.01 or ***P0.001); inhibit drug treatment 24h and 72h was compared among them, dose L-FP group and high dose group L-FP curcumin combined with low dose of L-FP group compared with the medium dose L-FP group and curcumin in combination, no significant difference (P0.05); and the 48h inhibition rate of curcumin combined with L-FP group was significantly higher than that of curcumin or L-FP alone group (*P0.05) of.2. L-FP combined with different doses of curcumin on the cloning of gastric cancer MGC-803 cells cultured in vitro formation: compared with the control group, the experimental group cells clone forming ability were significantly decreased (*P0.05); and curcumin combined with inhibition rate of L-FP group was significantly higher than that of curcumin or L-FP alone group (*P0.05).3. L-FP of curcumin combined with different doses of the migration effect on in vitro cultured gastric cancer cells: MGC-803 and control group. Ratio of each experimental group by Transwell assay cells were significantly decreased (*P0.05 or **P0.01); the migration rate of curcumin combined with L-FP group and curcumin or L-FP alone group had no significant difference (P0.05) effect of.4. L-FP combined with different doses of curcumin in vitro cultured gastric cancer MGC-803 cell cycle: compared with the control group, the experiment the main group of cells were arrested in S phase (*P0.05), and curcumin combined with L-FP group the proportion of cells in S phase was significantly higher than that of curcumin or L-FP alone group (*P0.05) of.5. L-FP combined with different doses of curcumin on apoptosis of cultured human gastric cancer cell line MGC-803: compared with the control group, the experimental group were significantly induced apoptosis of MGC-803 cells (*P0.05), the apoptosis of curcumin combined with L-FP group was significantly higher than that of curcumin or L-FP alone group (*P0.05), and Annexin-V/PI double stained cells were detected by flow cytometry. The apoptosis rate With AO/EB double staining of apoptosis were detected by fluorescence microscopy was no significant difference (P0.05) effect of.6. L-FP combined with different doses of curcumin on cultured gastric cancer cells MGC-803 related regulatory protein: compared with the control group, the experimental group cells significantly enhanced the expression of Bax, reduce the expression of Bcl-2, increased by Caspase-3 and the activity of Caspase-8 (*P0.05 or **P0.01); L-FP group in addition to the dose of curcumin combined with Bax in the relative expression of high dose L-FP group had no significant difference (P0.05), the rest of curcumin combined with L-FP group Bax expression was significantly higher than that of curcumin or L-FP alone group, the relative expression of Bcl-2 was significantly lower than that of curcumin or L-FP the single group (*P0.05 or **P0.01); the relative activity of curcumin combined with low dose of L-FP group Caspase-3 and Caspase-8 and L-FP in single dose group had no significant difference (P0.05), and Jiang Huang combined with medium dose L-FP The relative activity of group Caspase-3 and Caspase-8 were significantly higher than that of the high dose L-FP group (*P0.05 or **P0.01). Conclusion: 1..3. curcumin can enhance the inhibitory effect of curcumin.2. inhibition of curcumin on cultured L-FP human gastric cancer cell line MGC-803 growth could enhance the L-FP on cloning of MGC-803 gastric cancer cells in vitro and migration ability to enhance L-FP can enhance the induction effect of L-FP on apoptosis of cultured human gastric cancer cell line MGC-803 to.4. inhibition in vitro of curcumin in gastric cancer MGC-803 cell cycle in S phase, the mechanism and inhibition of Bcl-2 expression, promote Bax expression and increase Caspase-3 and Caspase-8 activities. For the detection of apoptosis, there was no significant difference in Annexin-V/PI staining AO/EB method and flow cytometry staining with fluorescence microscope detection results.

【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.2

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