本文選題：白藜蘆醇苷 + 增殖��； 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：[研究背景和目的]白藜蘆醇苷(Polydatin,PD)是從寥科蓼屬虎杖(Polygonum Cuspidatum)的干燥根莖中提取的第4種單體,故又名虎杖結(jié)晶4號(hào),也稱為虎杖苷。同時(shí),在葡萄和葡萄酒中也存在大量的白藜蘆醇苷。近年來(lái),國(guó)內(nèi)外對(duì)白藜蘆醇苷的研究表明:白藜蘆醇苷在抗血小板聚集、抗血栓形成、加強(qiáng)心肌細(xì)胞收縮和舒張功能、改善休克之后重要組織器官的微循環(huán)等方面具有顯著效果。此外,還具有抗內(nèi)毒素休克性肺損傷、保護(hù)肝細(xì)胞、降血脂、抗脂質(zhì)過(guò)氧化、減輕子宮內(nèi)膜異位癥引起的慢性疼痛及改善結(jié)腸炎等作用。白藜蘆醇苷為白藜蘆醇的衍生物,之前有大量的臨床研究表明,白藜蘆醇具有良好的腫瘤預(yù)防和腫瘤治療的作用。然而,對(duì)于白藜蘆醇苷是否具有良好的腫瘤預(yù)防與治療作用的研究目前才剛剛起步。已有人初步觀察了白藜蘆醇苷對(duì)小鼠移植性肝癌、艾氏腹水癌的作用,發(fā)現(xiàn)可抑制腫瘤的生長(zhǎng)并引起G2/M期細(xì)胞周期阻滯。本文的目的在于探討白藜蘆醇苷對(duì)肝癌細(xì)胞在體內(nèi)外實(shí)驗(yàn)中是否具有增殖抑制作用,并進(jìn)一步認(rèn)識(shí)白藜蘆醇苷對(duì)人肝癌細(xì)胞凋亡及運(yùn)動(dòng)能力的影響,為之后的臨床研究提供更廣泛的實(shí)驗(yàn)依據(jù)。細(xì)胞凋亡是一種多發(fā)生于生理情況下細(xì)胞死亡現(xiàn)象,故稱為生理性死亡——凋亡(apoptosis),又稱程序性死亡(programmed cell death,PCD)。其重要的特點(diǎn)是:細(xì)胞腫脹溶解并釋放出裂解產(chǎn)物,使周圍組織發(fā)生炎癥反應(yīng)。1972年Kerr最先提出這一概念,細(xì)胞凋亡是多細(xì)胞生物體的一種重要的自穩(wěn)機(jī)制。它可以主動(dòng)地清除多余的有特異性、分化能力與機(jī)體不相適應(yīng)的以及已經(jīng)完成功能而又不再有用的細(xì)胞;清除有潛在危險(xiǎn)的細(xì)胞,如自身反應(yīng)性淋巴細(xì)胞及DNA損傷又得不到修復(fù)有癌變危險(xiǎn)的細(xì)胞等。故凋亡是調(diào)節(jié)生物體正常發(fā)育不可缺少的一種機(jī)制,此種調(diào)節(jié)一旦失敗會(huì)導(dǎo)致機(jī)體疾病,腫瘤的發(fā)生甚至死亡。腫瘤細(xì)胞屬于永生性細(xì)胞,腫瘤的發(fā)生不僅與細(xì)胞的異常增殖和分化有關(guān),也與細(xì)胞凋亡異常有關(guān)。[研究方法]本實(shí)驗(yàn)以人肝細(xì)胞癌細(xì)胞系HepG2為研究對(duì)象,通過(guò)采用2-(2-甲氧基-4-硝苯基)-3-(4-硝苯基)-5-(2,4-二磺基苯)-2H-四唑單鈉鹽(Cel Counting Kit簡(jiǎn)稱CCK試劑盒)試劑盒檢測(cè)不同濃度(12.5～2000μmol/L)和不同作用時(shí)間(24、48、72h)處理人肝癌細(xì)胞株的細(xì)胞抑制率;倒置顯微鏡和熒光顯微鏡觀察處理后的各組肝癌細(xì)胞株的細(xì)胞及細(xì)胞核形態(tài)的改變;流式細(xì)胞術(shù)檢測(cè)經(jīng)過(guò)不同濃度白藜蘆醇苷處理后,檢測(cè)分析各組細(xì)胞的凋亡率;通過(guò)粘附實(shí)驗(yàn)、運(yùn)動(dòng)實(shí)驗(yàn)和侵襲實(shí)驗(yàn)檢測(cè)白藜蘆醇苷對(duì)肝細(xì)胞癌細(xì)胞侵襲能力的影響,并比較其對(duì)不同細(xì)胞株作用的差別;實(shí)驗(yàn)數(shù)據(jù)以平均值士標(biāo)準(zhǔn)差(mean士SD)表示,采用析因設(shè)計(jì)的方差分析以及單因素方差分析,均數(shù)間多重比較采用LSD方法,方差不齊時(shí)采用Dunnet-t方法進(jìn)行;用SPSS19.0統(tǒng)計(jì)軟件對(duì)數(shù)據(jù)進(jìn)行處理。[實(shí)驗(yàn)結(jié)果]1.白藜蘆醇苷抑制肝癌細(xì)胞的生長(zhǎng)CCK-8實(shí)驗(yàn)結(jié)果表明:白藜蘆醇苷在12.5～200μM濃度,作用時(shí)間24～72h范圍內(nèi),處理肝癌細(xì)胞HepG2,以濃度和時(shí)間依賴方式抑制HepG2細(xì)胞增殖(P0.05),同時(shí),白藜蘆醇苷的濃度在12.5μmol/L以上時(shí),即可抑制肝癌細(xì)胞HepG2的增殖,濃度達(dá)到25μmol/L以上時(shí),可誘導(dǎo)HepG2細(xì)胞凋亡。2.白藜蘆醇苷誘導(dǎo)肝癌細(xì)胞凋亡流式細(xì)胞儀分析顯示:經(jīng)過(guò)不同濃度的白藜蘆醇苷處理肝癌細(xì)胞HepG2細(xì)胞后,在處理后24h后,對(duì)細(xì)胞進(jìn)行流式細(xì)胞儀檢測(cè),并與對(duì)照組對(duì)比,發(fā)現(xiàn)細(xì)胞凋亡比例隨著濃度的增加而明顯增多�？梢�,白藜蘆醇苷能誘導(dǎo)細(xì)胞發(fā)生凋亡,并呈濃度依賴性。3.白藜蘆醇苷改變肝癌細(xì)胞及細(xì)胞核形態(tài)倒置顯微鏡下觀察:形態(tài)變化24h組已可見較明顯差異。對(duì)照組細(xì)胞飽滿呈梭形或多邊形貼壁生長(zhǎng)良好,透光度好且細(xì)胞密度較大,僅有少量脫落漂浮于培養(yǎng)液的細(xì)胞。而實(shí)驗(yàn)組細(xì)胞生長(zhǎng)狀態(tài)明顯較對(duì)照組差,細(xì)胞皺縮,隨著濃度的增高,細(xì)胞存活越少,漂浮細(xì)胞增多。Hoechest33342熒光染色顯示:用不同濃度的白藜蘆醇苷處理肝癌細(xì)胞HepG2后,細(xì)胞及細(xì)胞核形態(tài)發(fā)生了明顯的變化,細(xì)胞體積縮小、變形,有凋亡小體形成,細(xì)胞核出現(xiàn)核固縮、核碎裂和核溶解,細(xì)胞處于凋亡階段,在形態(tài)上與正常的細(xì)胞核區(qū)別明顯,呈濃度時(shí)間依賴性。4.白藜蘆醇苷抑制肝癌細(xì)胞的侵襲能力通過(guò)運(yùn)動(dòng)實(shí)驗(yàn)和侵襲實(shí)驗(yàn)觀察表明:隨著白藜蘆醇苷的濃度和處理時(shí)間的增加,肝癌細(xì)胞HepG2侵襲和轉(zhuǎn)移能力明顯減弱,呈濃度時(shí)間依賴性。25μmol/L、50μmol/L的白藜蘆醇苷可抑制HepG2細(xì)胞(P0.01)的粘附力,對(duì)HepG2細(xì)胞(P0.01)的侵襲力也有抑制作用,運(yùn)動(dòng)能力無(wú)明顯變化。[結(jié)論]白藜蘆醇苷主要以濃度依賴和時(shí)間依賴的方式抑制肝癌細(xì)胞增殖,誘導(dǎo)肝癌細(xì)胞凋亡,并主要通過(guò)抑制肝癌細(xì)胞和FN的粘附及其對(duì)基底膜基質(zhì)的降解兩個(gè)環(huán)節(jié)來(lái)抑制肝癌細(xì)胞的侵襲能力,可降低肝癌細(xì)胞的侵襲和轉(zhuǎn)移能力。
[Abstract]:[research background and purpose] Polydatin (PD) is fourth monomers extracted from the dry rhizome of Polygonum Polygonum (Polygonum Cuspidatum). Therefore, it is also called polydatin No. 4, also called polydatin. At the same time, there are a lot of resveratrol in grape and wine. In recent years, the research table of resveratrol at home and abroad Resveratrol: resveratrol has significant effect on anti platelet aggregation, antithrombotic formation, strengthening myocardial cell contraction and diastolic function, and improving the microcirculation of important tissues and organs after shock. In addition, it also has anti endotoxic shock lung injury, protection of liver cells, lipid lowering, anti lipid peroxidation and alleviative endometriosis. The effects of chronic pain and ameliorate colitis. Resveratrol is a derivative of resveratrol. A large number of clinical studies have shown that resveratrol has a good role in cancer prevention and cancer treatment. However, the study on the role of resveratrol for cancer prevention and treatment has just started. Some people have preliminarily observed the effect of resveratrol on transplanted liver cancer in mice and Ehrlich ascites cancer, and found that it can inhibit the growth of tumor and cause cell cycle arrest in G2/M phase. The aim of this paper is to investigate whether resveratrol has inhibitory effect on hepatoma cells in vitro and in vivo, and further recognize resveratrol in human. The effect of apoptosis and movement ability of hepatoma cells provides a more extensive experimental basis for the subsequent clinical study. Apoptosis is a cell death phenomenon that occurs more in physiological conditions, so it is called apoptosis, also called programmed cell death (PCD). Its important feature is: cell swelling and dissolution This concept is first proposed in.1972 Kerr, which is an inflammatory reaction in the surrounding tissue. Apoptosis is an important self stabilizing mechanism of multicellular organisms. It can actively remove superfluous and specific cells that are not adapted to the body and have completed function but no longer useful. In addition to the potentially dangerous cells, such as their own reactive lymphocytes and DNA damage, they can not repair the cells that have the risk of canceration. Therefore, apoptosis is an indispensable mechanism for regulating the normal development of organisms. Once the failure of this regulation, the disease causes the body disease and the occurrence of the tumor to death. The tumor cells belong to the immortal cells and the tumor. The occurrence is not only related to abnormal proliferation and differentiation of cells, but also related to abnormal apoptosis. [research methods] this experiment is based on human hepatocellular carcinoma cell line HepG2, by using 2- (2- methoxy -4- nitrophenyl) -3- (4- nitrophenyl) -5- (2,4- two sulfonyl benzene) -2H- four azole monosodium salt (Cel Counting Kit) kit The cell inhibition rate of human hepatocellular carcinoma cell lines treated with different concentration (12.5 ~ 2000 mol/L) and different action time (24,48,72h) was detected. The changes of cell and nuclear morphology of hepatoma cell lines after treatment were observed by inverted microscope and fluorescence microscope. Flow cytometry was used for detection and analysis after the treatment of resveratrol with different concentrations. The effect of resveratrol on the invasiveness of hepatocellular carcinoma cells was detected by adhesion test, exercise test and invasion test, and the difference of the effect of resveratrol on the different cell lines was compared. The experimental data were expressed by the mean standard deviation (mean, SD), and the variance analysis of factorial design and the analysis of single factor variance were used. LSD method was used in multiple comparison and Dunnet-t method was used when the variance was not homogeneous. The data were processed with SPSS19.0 statistical software. [experimental results showed that resveratrol inhibited the growth of hepatoma cells by CCK-8 experimental results. The results showed that resveratrol was in the concentration of 12.5 to 200 mu M, and the action time was 24 ~ 72h, and the liver cancer cell HepG2 was treated. Concentration and time dependent manner inhibited HepG2 cell proliferation (P0.05). At the same time, when the concentration of resveratrol was above 12.5 u mol/L, the proliferation of HepG2 cells could be inhibited. When the concentration was above 25 mu mol/L, the apoptosis of HepG2 cells apoptosis.2. resveratrol induced hepatoma cells apoptosis flow cytometer analysis showed that after different concentrations After treating hepatoma cell HepG2 cells with resveratrol, after treated with 24h, the cell was detected by flow cytometry, and compared with the control group, it was found that the percentage of apoptosis increased with the increase of concentration. It was found that resveratrol could induce apoptosis and the concentration dependent.3. resveratrol changed the liver cancer cells. The morphological changes in the 24h group showed that the cells in the control group were full of spindle or polygonal adherence, with good transmittance and large cell density, and only a small amount of cells that were floating in the culture medium, and the cell growth of the experimental group was significantly worse than that of the control group. The higher the concentration, the less the cell survival, the.Hoechest33342 fluorescent staining of the floating cells showed that the cell and nuclear morphology changed obviously after the treatment of the liver cancer cell HepG2 with different concentrations of resveratrol. The cell size, the deformation, the apoptosis and the nuclear condensation, the nucleus fragmentation and nuclear disintegration, the nucleus disintegration, the cell At the stage of apoptosis, there was a distinct difference in morphology from normal nuclei. The inhibitory effect of resveratrol on the invasiveness of hepatoma cells was observed by a concentration of time dependent.4.. The results showed that with the increase of the concentration and treatment time of resveratrol, the ability of invasion and metastasis of hepatoma cell HepG2 weakened obviously and showed a concentration. Time dependent.25 mu mol/L, 50 micron mol/L of resveratrol can inhibit the adhesion of HepG2 cells (P0.01), inhibit the invasion of HepG2 cells (P0.01) and exercise no obvious changes. [Conclusion] resveratrol mainly inhibits the proliferation of hepatoma cells in a concentration dependent and time dependent manner, and induces apoptosis of hepatoma cells. The invasion and metastasis ability of hepatoma cells can be reduced by inhibiting the adhesion of hepatoma cells and FN and the degradation of the basal membrane matrix by two links.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張利萍;馬會(huì)杰;布會(huì)敏;王妹玲;李茜;齊釗;張翼;;白藜蘆醇甙減輕缺血/再灌注誘發(fā)的大鼠心肌細(xì)胞凋亡(英文)[J];生理學(xué)報(bào);2009年04期

2 ;Anticancer activity of resveratrol on implanted human primary gastric carcinoma cells in nude mice[J];World Journal of Gastroenterology;2005年02期

3 舒仕瑜,王興勇,凌智瑜,盧仲毅;Effect of polydatin on phospholipase A_2 in lung tissues in rats with endotoxic shock[J];Chinese Journal of Traumatology;2004年04期

4 田京偉,楊建雄,傅風(fēng)華,蔣王林,王振華,王超云;白藜蘆醇苷對(duì)羥自由基所致大鼠腦線粒體氧化損傷的保護(hù)作用[J];中國(guó)藥理學(xué)通報(bào);2003年11期

5 ;Antitumor and immunomodulatory activity of resveratrol on experimentally implanted tumor of H22 in Balb/c mice[J];World Journal of Gastroenterology;2003年07期

6 王佾先,亢壽海,張琴芬,徐榮華;白藜蘆醇苷的抗癌作用及對(duì)癌細(xì)胞周期的影響[J];浙江中西醫(yī)結(jié)合雜志;2003年05期

7 舒仕瑜;白藜蘆醇苷生物活性及藥理作用[J];兒科藥學(xué);2002年01期

8 莫志賢,邵紅霞;白藜蘆醇苷體外對(duì)過(guò)氧化氫導(dǎo)致小鼠肝細(xì)胞損傷的保護(hù)作用[J];中國(guó)藥理學(xué)通報(bào);2000年05期

9 張佩文,余傳林,王耀忠,駱蘇芳,孫莉莎,李銳松;3,4′,5-三羥基芪-3-β-單-D-葡萄糖苷對(duì)血管內(nèi)皮前列環(huán)素的影響與血小板聚集的關(guān)系(英文)[J];Acta Pharmacologica Sinica;1995年03期

10 王躍忠,駱蘇芳,張佩文,余傳林;3,4′,5-三羥基芪-3-β-單-D-葡萄糖苷減輕動(dòng)脈內(nèi)皮損傷性血栓形成的作用(英文)[J];Acta Pharmacologica Sinica;1995年02期

相關(guān)博士學(xué)位論文 前1條

1 張玉松;虎杖苷抗腫瘤作用及機(jī)制研究[D];蘇州大學(xué);2013年

，

本文編號(hào)：1785631