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沙滩马鞭草提取物对HeLa细胞的生长抑制及诱导凋亡作用的实验研究

Inhibitive Effect and Mechanism of Verbena Littoralis H.B.K. on HeLa Cells from Cervical Cancer

【作者】 吴佳梅

【导师】 方瑾;

【作者基本信息】 中国医科大学 , 细胞生物学, 2010, 硕士

【摘要】 恶性肿瘤是目前危害人体生命和健康的严重疾病之一,据世界卫生组织(WHO)统计资料表明,全世界癌症每年发病约1000万人,死亡约700万人,已成为人类仅次于心血管病的第二杀手。因此探索有效的肿瘤治疗方法成为必然。在恶性肿瘤的治疗中,药物治疗占有重要地位。在合成的化学类抗肿瘤药物中绝大多数是以天然抗肿瘤活性成分为先导的化合物。因而,从天然植物成分中,筛选能够引起细胞凋亡,抑制细胞生长的药物,将是今后抗肿瘤治疗最理想的途径。沙滩马鞭草是原产于巴拉圭的多年生药用植物。目前有关沙滩马鞭草药理活性的研究主要集中在对神经生长因子(NGF)的增强作用方面,还未见沙滩马鞭草及其提取物有抗肿瘤活性的报道。本研究对从沙滩马鞭草中提取的16个单一成分的抗肿瘤活性进行了初步筛查,并对其中抗肿瘤活性较强的16#成分的抗肿瘤机制进行了初步探讨。宫颈癌是常见妇科恶性肿瘤之一,发病率和死亡率在女性恶性肿瘤中居第二位,仅次于乳腺癌。为此,我们选择了HeLa细胞(人宫颈癌细胞)为研究对象,观察沙滩马鞭草提取物对其生长的抑制作用,并从细胞凋亡方面来进一步探讨沙滩马鞭草提取物诱导细胞凋亡的机理。材料与方法一、细胞培养:将HeLa细胞接种于含10%小牛血清的RPMI 1640培养液中,置于37℃,5%的CO2培养箱中,2~3d传代1次,取对数生长期细胞用于实验。二、沙滩马鞭草提取物的制备单体化合物编号1-16,由沈阳药科大学提供,从沙滩马鞭草Verbena littoralisH.B.K.的甲醇提取物中利用硅胶吸附柱色谱、Sephadex LH-20柱色谱和半制备型HPLC等方法分离得到,通过理化常数和光谱数据分析(EI-MS、1HNMR、13C NMR、HMQC和HMBC)鉴定其结构。每个单体化合物经DMSO溶解制成储备液,4℃避光保存。三、MTT法检测沙滩马鞭草提取物对肿瘤细胞的生长抑制作用1、沙滩马鞭草16种提取物抗肿瘤活性的筛查采用MTT的方法,选用大肠癌细胞系Clone A对沙滩马鞭草16种提取物的生物学活性进行筛查。2、16#化合物对不同肿瘤细胞系的生长抑制作用采用MTT的方法,用筛选到的有生物学活性的化合物对不同肿瘤细胞系进行作用,观察其对不同肿瘤细胞的生长抑制作用是否存在选择性。四、16#化合物对HeLa细胞的生长抑制和诱导凋亡作用1、16#化合物对HeLa细胞系的生长抑制作用我们以生长抑制作用最为明显的HeLa细胞作为研究对象,采用不同浓度的16#化合物作用HeLa细胞24h后,观察其生长抑制作用。2、16#化合物对HeLa细胞作用后的细胞形态变化观察不同浓度的16#化合物作用HeLa细胞24h,相差显微镜下对形态进行观察;不同浓度的16#化合物作用HeLa细胞24h,荧光显微镜下观察其形态变化。3、流式细胞术检测16#化合物对HeLa细胞的诱导凋亡作用不同浓度的16#化合物作用HeLa细胞24h,采用Annexin-v/PI凋亡检测试剂盒进行凋亡率的测定。4、凝胶电泳检测16#化合物对HeLa细胞作用的DNA片段化分别用10、20μmol的16#化合物作用HeLa细胞24h,收集细胞、提取全基因组DNA,经2%的琼脂糖凝胶电泳检测其基因组DNA变化。实验结果一、沙滩马鞭草提取物的筛选与生物活性的鉴定1、沙滩马鞭草16种提取物抗肿瘤活性的筛查沙滩马鞭草16种提取物中的第16#化合物对Clone A细胞的生长抑制作用最为显著,说明16#化合物是抗肿瘤作用的有效成分。2、16#化合物对不同肿瘤细胞系的生长抑制作用采用MTT的方法,用筛选到的有生物学活性的16#化合物对不同肿瘤细胞系进行作用,发现16#化合物对不同的肿瘤细胞均具有生长抑制作用。二、16#化合物对HeLa细胞的生长抑制和诱导凋亡作用1、16#化合物对HeLa细胞系的生长抑制作用16#化合物对HeLa细胞的生长具有明显的抑制作用。且随药物浓度的增加,HeLa细胞的生长抑制率也逐渐增高。2、16#化合物作用宫颈癌HeLa细胞后的形态学变化不同浓度的16#化合物作用HeLa细胞24h后,相差显微镜下对其形态进行观察可见:细胞变圆、体积变小、细胞间隔稍增大,并可见凋亡小体。不同浓度的16#化合物作用HeLa细胞24h后,DAPI染色后荧光显微镜观察可见明显的凋亡小体。3、流式细胞术检测16#化合物对HeLa细胞的诱导凋亡作用采用Annexin-v/PI双染标定后。经流式细胞仪检测可见,16#化合物可以诱导HeLa细胞凋亡,并且具有浓度依赖性。4、凝胶电泳检测16#化合物对HeLa细胞作用的DNA片段化16#化合物作用HeLa细胞24h,收集细胞、提取全基因组DNA,经2%的琼脂糖凝胶电泳凝胶成像后,在20μmol时可见明显的凋亡梯带。结论本实验结果表明,沙滩马鞭草琨类提取物16#化合物对HeLa细胞的生长有明显的抑制作用,其作用是由诱导HeLa细胞凋亡引起的。

【Abstract】 At present, The malignant tumor is one serious diseases harms of human body life and health, According to World Health Organization (WHO) statistical data indicated, cancer is taken approximately 10,000,000 people and death approximately 7,000,000 people every year in the world, it’s have become the second murderer to be only inferior the cardiovascular disease. Therefore explores the effective tumor method of treatment to become inevitably.In malignant tumor treatment, the medicine treatment holds the important status. In the synthesis chemistry class anti-tumor medicine the overwhelming majority is take the natural anti-tumor active constituent as forerunner’s compound. Thus, from the natural medicine, screening can cause the cell to perish weakly, the suppression cell grows the medicine, will be the next anti-tumor treats the most ideal way.The Verbena littoralis H.B.K. is a perennial medicinal plant that produces originally in Paraguay. At present,the research mainly concentrates in the Verbena littoralis H.B.K. to the nerve growth factor (NGF) enhancement function aspect, the Verbena littoralis H.B.K. and the extraction has the anti-tumor active report has not seen.This research will especially pay close attention to the anti-tumor activity of Verbena littoralis H.B.K. and its induce apoptosis mechanism.Cervical cancer is one of common gynecology tumors, incidence rate and the mortality rate occupy second in the feminine malignant tumor, is only inferior to breast cancer. Therefore, we have chosen the HeLa cell (human cervix uterus cancer cell) as the object of study, observes the Verbena littoralis H.B.K. extraction to its growth inhibitory action, and perishes from the cell the aspect further to discuss the mechanism weakly which the Verbena littoralis H.B.K. extraction induction cell perishes weakly.Materials and Methods1. Identify of biological activity and screening of the Verbena littoralis H.B.K. extraction(1) Screening of the different compound of the Verbena littoralisH.B.K. extractionUses the method of MTT, Selects colon cancer cell line Clone A to screening biological activity of the 16 kind of compound of the Verbena littoralis H.B.K. extraction(2) Screening of the 16# compound to different tumor cell lineof theVerbena littoralis H.B.K.The anti-proliferation effect of Verbena littoralis H.B.K. on Clone A, HeLa, HT-29, HL-60, CCL-187 cells was measured with MTT. Observes it whether has the selectivity to the different tumor cell growth inhibitory action2. Cell growth inhibitory action of the 16# compound to Cervical cancer HeLa cellWe take the HeLa cell as the object of study,because of the grow inhibitory action most obvious of the HeLa cell. HeLa cells were exposed to different concentration of Verbena littoralis H.B.K. extraction 16# compound for 24h, Observes its growth inhibitory action3. The Morphological change of HeLa cells were exposed to Verbena littoralis H.B.K. extraction 16# compound(1) Phase contrast microscope HeLa cells were exposed to different concentration of Verbena littoralis H.B.K. extraction 16# compound for 24h, Observes its Morphological change on the Phase contrast microscope.(2) Fluorescence microscopeHeLa cells were exposed to different concentration of Verbena littoralis H.B.K. extraction 16# compound for 24h, Observes its Morphological change on the Fluorescence microscope.4. Genome team DNA change of HeLa cells were exposed to Verbena littoralis H.B.K. extraction 16# compoundAfter treating different concentration of Verbena littoralis H.B.K. extraction 16# compound for 24h.Collecting all the HeLa cells, extracting DNA in conventional methods, using Agarose gel electrophoresis, UV-light camera observation.5.Detection of apoptosis rate of HeLa cells were exposed to Verbena littoralis H.B.K. extraction 16# compoundAdding different concentration of Verbena littoralis H.B.K. extraction 16# compound for 24h, incubate for 24h. Collecting all the HeLa cells, detect apoptosis rate by FCMResults1. Identify of biological activity and screening of the Verbena littoralis H.B.K. extraction(1) Screening of the different compound of the Verbena littoralis H.B.K.The 16# compound cell growth inhibitory action is most remarkable to Clone A, explained the 16# compound is the anti-tumor function effective component.(2) The growth inhibitory action to different tumor cell line of 16# compound Uses MTT the method, the different tumor cell line were exposed to 16# compound of Verbena littoralis H.B.K. extraction, discovered the 16# compound has the growth inhibitory action to the different tumor cell.2.Cell growth inhibitory action of the 16# compound to Cervical cancer HeLa cellThe proliferation of HeLa cells significantly inhibited by Verbena littoralis H.B.K. with a dose dependant manner. When the concentration of Verbena littoralis H.B.K. extraction thing 16# compound was increased, the growth inhibition rate increased gradually.3.The Morphological change of HeLa cells were exposed to Verbena littoralis H.B.K. extraction 16# compoundAfter HeLa cells were exposed to different concentration of Verbena littoralis H.B.K. extraction thing 16# compound for 24h, the apoptotic cell were found with Fluorogenic microscope and phase contrast microscopy, the cell changes circle, the volume changes small, the nuclear envelope was largely disrupted and apoptotic cell shoeed.4.Genome team DNA change of HeLa cells were exposed to Verbena littoralisH.B.K. extraction 16# compoundThe HeLa cells were exposed to 16# compound for 24h,collection cell, withdraws entire genome team DNA, Agarose gel electrophoresis demonstration 20μmol after the 16# compound affects the HeLa cell to appear obvious DNA Ladder.5.Detection of apoptosis rate of HeLa cells were exposed to Verbena littoralis H.B.K. extraction 16# compoundAfter HeLa cells treated by different concentration of Verbena littoralis H.B.K. extraction thing 16# compound for 24h, the induction of apoptosis took on a dose dependent manner. Apoptotic rate of HeLa cells reached 21.82% with 25μmol Verbena littoralis H.B.K. extraction thing 16# compound for 24h.ConclusionThe results of our study showed that Verbena littoralis H.B.K. extraction thing 16# compound inhibit the growth of HeLa cells through the apoptosis of HeLa cells induced with Verbena littoralis H.B.K. extraction thing 16# compound.

【关键词】 沙滩马鞭草HeLa细胞凋亡肿瘤
【Key words】 Verbena littoralis HB.K.HeLaApoptosistumor
  • 【分类号】R285.5
  • 【被引频次】3
  • 【下载频次】144
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