本文選題：白藜蘆醇　切入點：人肝癌細胞SMMC-7721　出處：《四川醫(yī)科大學》2015年碩士論文　論文類型：學位論文

【摘要】：目的:原發(fā)性肝癌是常見的消化系統(tǒng)惡性腫瘤之一,惡性程度較高,病灶容易轉移,治療效果差。白藜蘆醇(Resveratrol)是從虎杖、葡萄等植物中提取的一種多酚類物質,近年來研究顯示,白藜蘆醇具有抗腫瘤、保護血管神經(jīng)、抗炎、影響骨鈣水平等作用,是治療脂類代謝紊亂、炎癥和心臟疾病的有效成分。據(jù)文獻報道,白藜蘆醇具有抗肝癌的作用,但抗肝癌作用具體機制尚不清楚。誘導腫瘤細胞凋亡、抑制腫瘤細胞增殖是研究抗腫瘤藥物的常規(guī)途徑,本研究擬從體外角度,研究白藜蘆醇對人肝癌細胞SMMC-7721增殖及凋亡的影響,并初步探討其可能的作用機制。通過CCK-8法檢測白藜蘆醇影響人肝癌細胞SMMC-7721增殖的時效及量效關系,以確定白藜蘆醇發(fā)揮最大作用的濃度及時間范圍。應用Hoechst-33258染色及雙染流式細胞術(Annexin V/P)檢測凋亡細胞形態(tài)學及量的變化,Real time-PCR技術檢測白藜蘆醇干預前后人肝癌細胞SMMC-7721表達細胞色素P4501A1 m RNA(CYP1A1)的變化。蛋白印記分析(Western blot analysis)法檢測白藜蘆醇干預前后人肝癌細胞SMMC-7721表達細胞色素P4501A1蛋白的變化。方法:分別用濃度25μmol/L、50μmol/L、100μmol/L、200μmol/L的白藜蘆醇作用人肝癌細胞24h、48h、72h,CCK-8法檢測白藜蘆醇對人肝癌細胞SMMC-7721的增殖抑制作用。分別以無處理的人肝癌細胞SMMC-7721及相同劑量的DMSO為空白和隨機對照組,Annexin V/PI流式細胞術觀察100μmol/L的白藜蘆醇作用72h后對人肝癌細胞SMMC-7721凋亡率的影響,Hoechst-33258觀察100μmol/L白藜蘆醇作用72h后人肝癌細胞SMMC-7721中凋亡細胞形態(tài)學的變化。Real-time PCR分析100μmol/L的白藜蘆醇作用前后對人肝癌細胞SMMC-7721表達CYP1A1m RNA的影響。蛋白印記分析(Western blot analysis)法檢測100μmol/L的白藜蘆醇作用前后人肝癌細胞SMMC-7721中CYP1A1蛋白表達情況。結果:CCK-8檢測結果顯示,在體外培養(yǎng)狀態(tài)下,白藜蘆醇能顯著抑制人肝癌細胞SMMC-7721的增殖,在本實驗的濃度劑量范圍內,隨著白藜蘆醇濃度的不斷增大,抑制增殖的效果明顯增強,呈濃度依賴關系。同一濃度隨著作用時間的不斷延長,抑制增殖的效果明顯增強,呈時間依賴性。不同濃度白藜蘆醇(25μmol/L、50μmol/L、100μmol/L、200μmol/L)作用人肝癌細胞24h后增殖抑制率分別為(8.38±1.75%、15.40±3.32%、22.35±2.43%、15.57±1.73%),不同濃度白藜蘆醇(25μmol/L、50μmol/L、100μmol/L、200μmol/L)作用人肝癌細胞48h后增殖抑制率分別為(12.60±1.51%、20.70±3.21%、40.60±2.33%、51.67±3.70%),不同濃度白藜蘆醇(25μmol/L、50μmol/L、100μmol/L、200μmol/L)作用人肝癌細胞72h后增殖抑制率分別為(15.42±3.54%、24.57±3.81%、60.51±3.50%、65.51±2.41%),經(jīng)統(tǒng)計分析得出,不同濃度白藜蘆醇對人肝癌細胞SMMC-7721增殖抑制率不同(F=14.03,P=0.03),相同濃度白藜蘆醇在不同作用時間對人肝癌細胞SMMC-7721增殖抑制率也有明顯差異(F=15.04,P0.05),所以一定濃度白藜蘆醇作用一定時間范圍內,其對人肝癌細胞SMMC-7721的增殖抑制作用存在濃度及時間依賴性。從人肝癌細胞SMMC-7721在白藜蘆醇作用下的量效曲線上可以看出:100μmol/L白藜蘆醇作用72h后隨藥物濃度的升高抑制率斜率開始下降,說明單位時間內抑制效率開始下降,因此認為100μmol/L白藜蘆醇作用72h對人肝癌細胞SMMC-7721的抑制作用最為明顯。后續(xù)實驗以此作為作用截止點。Hoechst-33258染色結果顯示:100μmol/L的白藜蘆醇作用72h后人肝癌細胞SMMC-7721細胞核呈現(xiàn)明顯凋亡形態(tài),隨機對照組及空白組人肝癌細胞SMMC-7721細胞核無明顯變化。雙染色流式細胞儀檢測細胞凋亡率結果顯示:空白對照組、隨機對照組、100μmol/L白藜蘆醇實驗組的凋亡率分別為10.27%、12.31%、38.54%。RT-PCR結果:100μmol/L白藜蘆醇作用72h后細胞CYP1A1m RNA的表達出現(xiàn)顯著下降。進一步檢測發(fā)現(xiàn)CYP1A1蛋白的表達也呈現(xiàn)下降趨勢。結論:體外培養(yǎng)狀態(tài)下,白藜蘆醇能抑制人肝癌細胞SMMC-7721的增殖、促進其凋亡,其機制可能與白藜蘆醇在轉錄水平下調CYP1A1m RNA的表達、從而在翻譯水平減少CYP1A1的蛋白表達有關。
[Abstract]:Objective: primary hepatocellular carcinoma is one of the most common malignant tumor of digestive system, the higher the degree of malignancy, the lesion is easy to transfer and poor treatment effect. Resveratrol (Resveratrol) from Polygonum cuspidatum, an extraction of polyphenols from grape plants, recent studies have shown that resveratrol has anti-tumor anti-inflammatory, protect blood vessels and nerves. The effect of bone calcium levels, etc., is the treatment of lipid metabolism disorder, effective components of inflammation and heart disease. It is reported that resveratrol has anti hepatoma effect, but the anti cancer effects of the specific mechanism is still unclear. Induction of tumor cell apoptosis, inhibit the proliferation of tumor cells is the study of conventional ways of anticancer drugs, the in vitro study from the angle of effect of resveratrol on proliferation and apoptosis of human hepatocellular carcinoma cell line SMMC-7721, and to explore its possible mechanism. Through the detection of resveratrol in human hepatocellular carcinoma fine CCK-8 method. Time effect and dose effect relationship of SMMC-7721 cell proliferation, to determine resveratrol concentration and time play the biggest role. The scope of application of Hoechst-33258 staining and double staining flow cytometry (Annexin V/P) and the amount of morphological changes of apoptotic cells were detected, Real time-PCR detection of resveratrol in human hepatocellular carcinoma cells before intervention SMMC-7721 expression of cytochrome P4501A1 m RNA (CYP1A1). The changes of the Western blot analysis (Western blot analysis) method for the detection of resveratrol intervention before human hepatocellular carcinoma cell SMMC-7721 expression changes of cytochrome P4501A1 protein. Methods: with concentration of 25 mol/L, 50 mol/L, 100 mol/L, 200 mol/L of resveratrol in human hepatocellular carcinoma cells 24h 48h, 72h, and to investigate the inhibitory effect of resveratrol on proliferation of CCK-8 human hepatocellular carcinoma cell line SMMC-7721. In human hepatoma SMMC-7721 cells without treatment and the same dose of DMSO is empty and random on white Control group, resveratrol effect 72h Annexin V/PI flow cytometry in 100 mol/L on human hepatoma cell apoptosis rate of SMMC-7721, before and after treatment with resveratrol Hoechst-33258 changes of.Real-time PCR apoptosis 100 mol/L resveratrol 72h human hepatocellular carcinoma cells SMMC-7721 in the analysis of 100 mol/L the expression of CYP1A1m in human hepatoma cell line SMMC-7721 RNA. Western blot analysis (Western blot analysis) were detected before and after treatment with resveratrol 100 mol/L CYP1A1 protein expression in human hepatocellular carcinoma cell SMMC-7721. Results: CCK-8 test results showed that in vitro, resveratrol can significantly inhibit human hepatocellular carcinoma cell line SMMC-7721 the proliferation of concentration in the dose range of this experiment, with the increasing concentration of resveratrol, inhibited the proliferation effect significantly enhanced in a concentration dependent manner. With the same concentration 鐫，

本文編號：1587149