白藜芦醇苷对人肝癌细胞HepG2细胞株凋亡的促进作用的实验研究

发布时间：2018-04-22 04:31

本文选题：白藜芦醇苷 + 增殖　；参考：《南方医科大学》2017年硕士论文

【摘要】：[研究背景和目的]白藜芦醇苷(Polydatin,PD)是从寥科蓼属虎杖(Polygonum Cuspidatum)的干燥根茎中提取的第4种单体,故又名虎杖结晶4号,也称为虎杖苷。同时,在葡萄和葡萄酒中也存在大量的白藜芦醇苷。近年来,国内外对白藜芦醇苷的研究表明:白藜芦醇苷在抗血小板聚集、抗血栓形成、加强心肌细胞收缩和舒张功能、改善休克之后重要组织器官的微循环等方面具有显著效果。此外,还具有抗内毒素休克性肺损伤、保护肝细胞、降血脂、抗脂质过氧化、减轻子宫内膜异位症引起的慢性疼痛及改善结肠炎等作用。白藜芦醇苷为白藜芦醇的衍生物,之前有大量的临床研究表明,白藜芦醇具有良好的肿瘤预防和肿瘤治疗的作用。然而,对于白藜芦醇苷是否具有良好的肿瘤预防与治疗作用的研究目前才刚刚起步。已有人初步观察了白藜芦醇苷对小鼠移植性肝癌、艾氏腹水癌的作用,发现可抑制肿瘤的生长并引起G2/M期细胞周期阻滞。本文的目的在于探讨白藜芦醇苷对肝癌细胞在体内外实验中是否具有增殖抑制作用,并进一步认识白藜芦醇苷对人肝癌细胞凋亡及运动能力的影响,为之后的临床研究提供更广泛的实验依据。细胞凋亡是一种多发生于生理情况下细胞死亡现象,故称为生理性死亡——凋亡(apoptosis),又称程序性死亡(programmed cell death,PCD)。其重要的特点是:细胞肿胀溶解并释放出裂解产物,使周围组织发生炎症反应。1972年Kerr最先提出这一概念,细胞凋亡是多细胞生物体的一种重要的自稳机制。它可以主动地清除多余的有特异性、分化能力与机体不相适应的以及已经完成功能而又不再有用的细胞;清除有潜在危险的细胞,如自身反应性淋巴细胞及DNA损伤又得不到修复有癌变危险的细胞等。故凋亡是调节生物体正常发育不可缺少的一种机制,此种调节一旦失败会导致机体疾病,肿瘤的发生甚至死亡。肿瘤细胞属于永生性细胞,肿瘤的发生不仅与细胞的异常增殖和分化有关,也与细胞凋亡异常有关。[研究方法]本实验以人肝细胞癌细胞系HepG2为研究对象,通过采用2-(2-甲氧基-4-硝苯基)-3-(4-硝苯基)-5-(2,4-二磺基苯)-2H-四唑单钠盐(Cel Counting Kit简称CCK试剂盒)试剂盒检测不同浓度(12.5～2000μmol/L)和不同作用时间(24、48、72h)处理人肝癌细胞株的细胞抑制率;倒置显微镜和荧光显微镜观察处理后的各组肝癌细胞株的细胞及细胞核形态的改变;流式细胞术检测经过不同浓度白藜芦醇苷处理后,检测分析各组细胞的凋亡率;通过粘附实验、运动实验和侵袭实验检测白藜芦醇苷对肝细胞癌细胞侵袭能力的影响,并比较其对不同细胞株作用的差别;实验数据以平均值士标准差(mean士SD)表示,采用析因设计的方差分析以及单因素方差分析,均数间多重比较采用LSD方法,方差不齐时采用Dunnet-t方法进行;用SPSS19.0统计软件对数据进行处理。[实验结果]1.白藜芦醇苷抑制肝癌细胞的生长CCK-8实验结果表明:白藜芦醇苷在12.5～200μM浓度,作用时间24～72h范围内,处理肝癌细胞HepG2,以浓度和时间依赖方式抑制HepG2细胞增殖(P0.05),同时,白藜芦醇苷的浓度在12.5μmol/L以上时,即可抑制肝癌细胞HepG2的增殖,浓度达到25μmol/L以上时,可诱导HepG2细胞凋亡。2.白藜芦醇苷诱导肝癌细胞凋亡流式细胞仪分析显示:经过不同浓度的白藜芦醇苷处理肝癌细胞HepG2细胞后,在处理后24h后,对细胞进行流式细胞仪检测,并与对照组对比,发现细胞凋亡比例随着浓度的增加而明显增多。可见,白藜芦醇苷能诱导细胞发生凋亡,并呈浓度依赖性。3.白藜芦醇苷改变肝癌细胞及细胞核形态倒置显微镜下观察:形态变化24h组已可见较明显差异。对照组细胞饱满呈梭形或多边形贴壁生长良好,透光度好且细胞密度较大,仅有少量脱落漂浮于培养液的细胞。而实验组细胞生长状态明显较对照组差,细胞皱缩,随着浓度的增高,细胞存活越少,漂浮细胞增多。Hoechest33342荧光染色显示:用不同浓度的白藜芦醇苷处理肝癌细胞HepG2后,细胞及细胞核形态发生了明显的变化,细胞体积缩小、变形,有凋亡小体形成,细胞核出现核固缩、核碎裂和核溶解,细胞处于凋亡阶段,在形态上与正常的细胞核区别明显,呈浓度时间依赖性。4.白藜芦醇苷抑制肝癌细胞的侵袭能力通过运动实验和侵袭实验观察表明:随着白藜芦醇苷的浓度和处理时间的增加,肝癌细胞HepG2侵袭和转移能力明显减弱,呈浓度时间依赖性。25μmol/L、50μmol/L的白藜芦醇苷可抑制HepG2细胞(P0.01)的粘附力,对HepG2细胞(P0.01)的侵袭力也有抑制作用,运动能力无明显变化。[结论]白藜芦醇苷主要以浓度依赖和时间依赖的方式抑制肝癌细胞增殖,诱导肝癌细胞凋亡,并主要通过抑制肝癌细胞和FN的粘附及其对基底膜基质的降解两个环节来抑制肝癌细胞的侵袭能力,可降低肝癌细胞的侵袭和转移能力。
[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.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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