【作者】 孙晓娟；

【导师】 王留兴；

【作者基本信息】 郑州大学 ， 肿瘤学， 2012， 硕士

【摘要】 背景和目的在恶性肿瘤的高发病率和高死亡率的今天,恶性肿瘤的防治和治疗已经成为学者们所面临的难点和重点。恶性肿瘤的治疗已经经历了一个漫长的过程。外科手术从广泛的切除治疗,到组织和功能的保存治疗,已经发展到目前的预防性手术治疗、微创治疗、个体化治疗；内科治疗从无特异性的细胞毒药物治疗,对不同阶段的癌细胞的大剂量药物杀伤治疗,已经发展到靶向治疗、生物治疗等；放射治疗也发展为联合治疗,对正常的组织损伤减小,更好的提高疗效。肿瘤一旦发现,多数已经为中晚期,而对于中晚期的肿瘤已经不能手术治疗,多采用化学治疗。临床上长期化学治疗后,发现化疗药物有耐药性,对患者有较多的毒副作用,且长期应用费用较高,这些都使患者的生活质量大大降低。近几年对天然药物的开发和应用已经成为研究的热点。中药在治疗肿瘤方面主要体现其扶正祛邪的观点,来抑制肿瘤细胞的生长活性和作用于细胞周期诱导凋亡。中药与西药相比,来源广泛、价格低、副作用小,可通过中药单药或者复方合剂来进行研究,提取其抗肿瘤成分,获得新的抗肿瘤药物,为治疗肿瘤开辟新的途径。黄精、巴戟天、白芷为传统的中药,提取其抗肿瘤的主要成分来进行研究。本研究选择人食管癌Eca-109细胞株、人胃癌HGC-27细胞株、人直肠癌HCT-8细胞株,进行体外抗肿瘤实验,分别将不同浓度和不同组合的中药作用于三种细胞,进行细胞生物活性的检测、药物作用后细胞形态变化和对细胞周期的影响,通过对细胞生物活性的检测、细胞形态的变化和细胞周期的影响,为后续对实体转移瘤的体内实验研究提供进一步的理论依据。方法1药物的制备根据参考文献中药物的提取方法来进行各药物抗肿瘤主要成分的提取。三种中药的抗肿瘤成分主要为黄静多糖、巴戟天水提液、白芷异欧前胡素。实验中将各主要成分溶解并稀释为实验所需的药物浓度。药物的浓度=中药重量/培养液体积。2细胞培养Eca-109、HGC-27、HCT-8细胞用含10%灭活胎牛血清的RPMI-1640培养基,在37℃、5%CO2的细胞培养箱中常规培养。24h-48h换液,48h-72h传代。3细胞抑制率的测定取Eca-109、HGC-27、HCT-8对数生长期细胞,均以5×104/m1细胞浓度接种于96孔板,每孔接种200μL,培养36h后,设对照组(未加药)和实验组,每组设6个平行孔,置于37℃、5%CO2培养箱中常规培养48h,采用噻唑蓝(MTT)还原法来检测各实验组细胞的生长抑制率。实验重复3次(n=18)。4观察细胞形态学采用倒置显微镜观察各药物作用于Eca-109细胞48小时后的形态学的变化。5流式细胞仪检测细胞周期变化。6统计学分析采用统计学软件SPSS17.0分析,采用单因素方差分析及χ2检验进行分析,以a=0.05为检验水准。结果1黄精、巴戟天、白芷对三种肿瘤细胞的生长抑制作用1.1对Eca-109细胞的生长抑制作用黄精浓度达到20mg/ml时,抑制率可达到56.69%,巴戟天浓度达到20mg/ml时,抑制率可达到61.66%,白芷浓度达到4mg/ml时,抑制率可达到24.27%,小剂量的三药合用时,抑制率可达到88.31%。1.2对HGC-27细胞的生长抑制作用黄精多糖浓度达到20mg/ml时,抑制率可达到51.65%,巴戟天水提液浓度达到20mg/ml时,抑制率可达到56.96%,白芷浓度达到4mg/ml时,抑制率可达到23.84%,小剂量三药合用时,抑制率可达到87.96%。1.3对HCT-8细胞的生长抑制作用黄精多糖浓度达到20mg/ml时,抑制率可达到58.19%,巴戟天水提液浓度达到20mg/ml时,抑制率可达到60.19%,白芷浓度达到4mg/ml时,抑制率可达到28.27%,小剂量三药合用时,抑制率可达到87.70%。2空白对照组：Eca-109细胞形态为多边形,且贴壁生长,生长状态良好。黄精组(1mg/ml)和白芷组(0.5mg/ml)：大部分的贴壁细胞失去了正常形态,细胞形态变为圆形并且折光性较差。三联组(黄精4mg/ml+巴戟天4mg/ml+白芷0.5mg/ml)：成团的细胞互相分离,细胞形态缩小且凝聚,细胞膜变得皱缩,核染色质深染且固缩,聚拢在核膜的边缘,可多见空泡变形。3体外培养的Eca-109细胞、HGC-27细胞、HCT-8细胞经过三种单药分别作用和联合作用后,各个实验组的细胞S期百分比率明显的升高,GO/G1期细胞的百分比率明显的下降；细胞被阻滞于S期,抑制细胞凋亡。结论1黄精、巴戟天、白芷三种中药均能明显抑制Eca-109细胞,HGC-27细胞,HCT-8细胞的生长活性；且小剂量药物组合连用时,更明显的抑制了细胞生长的活性。2黄精、巴戟天、白芷三种中药将Eca-109细胞,HGC-27细胞,HCT-8细胞阻滞于S期,且三药组合作用时细胞阻滞于S期,可诱导细胞凋亡。3本实验结果表明,黄精、巴戟天、白芷三种中药单用及组合应用时均有抗肿瘤的作用,机制可能为作用于细胞周期将细胞阻滞于S期和诱导细胞凋亡。本实验为进一步的后期研究和临床应用提供了科学的理论依据。更多还原

【Abstract】 Background and purposeThe prevention and treatment of malignant tumor have become difficulty and key the scholars face as a result of the high incidence and mortality of cancer. The treatment of malignant tumor has experienced a long process. Surgical resection from extensive to the organization and function of the conservative therapy, have been developed to the current preventive surgery, minimally invasive treatment and individual therapy. Medical treatment has been developed from the nonspecific cytotoxic drugs therapy, the different stages of the cancer cells of high-dose drug killer treatment, to targeting therapy, biotherapy, etc. Accompanied with other therapies, radiotherapy can be less likely to damage the healthy tissue and improve the curative effect. When the tumor was found, most of them were already the middle-late stage. The middle-late tumor patients who were medically inoperable were generally offered chemical treatment. The patients of the long-term chemotherapy were found to have drug resistance, more side effects, higher cost and lower the quality of life. The development and application of the nature medicines has become the research focus these years. The anti-tumor mechanism of traditional Chinese medicine may inhibit the growth of tumor cells and induce apoptosis through affecting the different phase of tumor cell generation cycle, which is the viewpoint of strengthening body resistance and eliminating evil. Compared with western medicine, traditional Chinese medicine has extensive sources, low price and slight side effects. Traditional Chinese medicine can be researched through single drug or compound mixture, withdrawing its anti-tumor components, accessing to new anticancer drugs, and opening new avenues for the treatment of tumors.Rhizoma polygonati, Morinda officinalis, radix angelicae dahuricae are the traditional Chinese medicine, being extracted the main component for the anti-tumor research. This experiment chooses human esophageal carcinoma Eca-109cell, human lung cancer HGC-27cell, human colon cancer HCT-8cell as experimental objects.In this study, deal with three types of cells with different concentrations and different combinations of Chinese herb extracts in vitro, then detect cell biological activity, changes in cell morphology and cell cycle. These experiments will provide further theoretical basis for the experimental study of follow-up entity metastases.Methods1Drug preparation According to the literature, the drug in the extraction method to extract anti-tumor components of crude extracting from all kinds of medicine。 Three kinds of Chinese traditional medicine antitumor composition mainly for Rhizoma polygonati polysaccharide, morinda officinalis how liquid, radix angelicae dahuricae isoimperatorin. Major composition dissolved in the experiment for different drug concentration. Drug concentration=Traditional Chinese medicine weight/medium volume.2Cell culture Eca-109, HGC-27, HCT-8cells used to contain10%inactivated fetal bovine serum RPMI-1640medium, at37℃and routinely cultured in5%CO2incubator.24to48hour for liquid,48-72hour passage.3The determination of the inhibition rates Take the Eca-109, HGC-27, HCT-8cells in the logarithmic growth phase,5x104/ml concentration of cells inoculated in96-well plates, each well inoculated with200μL, cultured36h, set in the control group (no dosing)and experimental groups, each group of six parallel hole, at37℃ and routinely cultured in5%CO2incubator, after48h, thiazolyl blue (MTT) reduction method to determine the growth inhibitory rate of cells of each experimental group. The experiment was repeated three times (n=18).4Watch cell morphology After drug action in Eca-109cells48hours, observed the morphological changes using an inverted microscope.5Flow cytometry to detect cell cycle6Statistical analysis statistical software SPSS17.0analysis, single factor analysis of variance test with a=0.05for the inspection standards.Results1Inhibitory effect of Rhizoma polygonati,morinda officinalis how,radix angelicae dahuricae on the three tumor cells.1.1Eca-109cell growth of inhibition when Rhizoma polygonati concentration was20mg/ml, the inhibition rate reached56.69%, when Morinda officinalis how concentration was20mg/ml, the inhibition rate reached61.66%,when radix angelicae dahuricae concentration was4mg/ml, the inhibition rate of24.27%,when three drugs combined, the highest inhibition rate of88.31%.1.2HGC-27cell growth of inhibition when Rhizoma polygonati concentration was20mg/ml, the inhibition rate reached51.65%, when Morinda officinalis how concentration was20mg/ml, the inhibition rate reached56.96%,when radix angelicae dahuricae concentration was4mg/ml, the inhibition rate of23.84%,when three drugs combined, the highest inhibition rate of87.96%.1.3HCT-8cell growth of inhibition when Rhizoma polygonati concentration was20mg/ml, the inhibition rate reached58.19%, when Morinda officinalis how concentration was20mg/ml, the inhibition rate reached60.19%,when radix angelicae dahuricae concentration was4mg/ml, the inhibition rate of28.27%,when three drugs combined, the highest inhibition rate of87.70%.2Blank control group:Eca-109cell morphology have a polygon, and the adherent growth, growth in good condition. Rhizoma polygonati (lmg/ml) and radix angelicae dahuricae (0.5mg/ml):Most of the adherent cells lose their normal morphology, cell shape into a round and less refraction. Triple set group (the Rhizoma polygonati4mg/ml+the Morinda officinalis how4mg/ml+the radix angelicae dahuricae0.5mg/ml):into a group of cells separated from each other, cohesion and narrow cells, cell membrane becomes wrinkled, nuclear chromatin deeply stained pyknotic gathered in the nuclear the edge of the membrane, may be more common in the vacuole deformation.3、Eca-109cells, HGC-27cells and HCT-8cells were cultured in vitro,by different single-agent and combination therapy, the percentage rate of cells in S phase of the experimental group significantly increased,and in G0/G1phase cells have the percentage rate significant decrease; cells were blocked in S phase and inhibition of apoptosis.Conclusions1Rhizoma polygonati, Morinda officinalis, radix angelicae dahuricae three kinds of traditional Chinese medicine can significantly inhibit Eca-109cells, HGC-27cells and HCT-8cell growth activity. And a combination of drugs used in conjunction, a more pronounced inhibition of cell growth activity.2Rhizoma polygonati, Morinda officinalis, radix angelicae dahuricae three kinds of traditional Chinese medicine let Eca-109cells, the HGC-27cells and HCT-8cells arrest in S phase and the role of three-drug combination can induce apoptosis.3The results suggest that, Rhizoma polygonati, Morinda officinalis, radix angelicae dahuricae all have antitumor activity when used alone and the combination of three kinds of traditional Chinese medicine, the mechanism may be the role of the cell cycle cells were arrested in S phase and induction of apoptosis. This experiment provides a scientific and theoretical basis for further post-research and clinical applications.更多还原