自拟方抑癌汤对人肺癌NCI-H446细胞增殖抑制作用的体外实验研究

【作者】 杜进军；

【导师】 葛正行；

【作者基本信息】 贵阳中医学院 ， 中西医结合临床， 2007， 硕士

【摘要】 目的:本课题应用细胞培养技术,通过自拟方抑癌汤对人肺癌NCI-H446细胞株进行干扰研究,探讨其增殖抑制作用。旨在从苗药中药组方中找到抑制肺癌细胞增殖的新方法,为治疗肺癌提供一种新的治疗思路。方法:将NCI-H446细胞进行培养,绘制NCI-H446细胞生长曲线,取处在对数生长期的NCI-H446细胞进行实验。首先确定抑癌汤的作用浓度范围,将抑癌汤分为1g/ml、100mg/ml、10 mg/ml、1 mg/ml、100ug/ml、10 ug/ml、1 ug/ml、0.1ug/ml共8个浓度梯度,对照组为只加等量的细胞培养液,总共9组。每一组各设4个复孔,使用MTT法测定细胞的存活率。取细胞的存活率为50%时的药物浓度范围做为抑癌汤的给药参考浓度。再将抑癌汤在20ug/ml、40ug/ml、60ug/ml、80ug/ml、100ug/ml五个浓度和在24小时、48小时、72小时这三个观测时间点上与空白组(培养基对照组)进行比较观察。最后取抑癌汤60ug/ml的浓度做为抑癌汤的给药浓度,与西药组;抑癌汤加西药组(60ug/ml浓度组并加用西药)和以只加培养液的空白对照组,共四个大组(抑癌汤组、西药组、抑癌汤加西药组、空白组),每一组各设4个复孔,进行对照研究,使用MTT法测定细胞的抑制率,以上实验均重复三次。结果:1:绘制细胞生长曲线,通过倒置显微镜下进行细胞计数,得到细胞倍增时间为26小时。2:抑癌汤对NCI-H446细胞毒实验得到细胞存活率50%时的药物浓度范围在1-100ug/ml之间,根据该浓度范围可以为细胞增殖抑制实验提供给药药物参考浓度。3:细胞增殖抑制实验:抑癌汤在20ug/ml、40ug/ml、60ug/ml、80ug/ml、100ug/ml浓度和在24小时、48小时、72小时这三个观测时间点与空白组(培养基对照组)比较均有统计学差异。抑癌汤组三次实验经过统计分析得到:P=0.492,即抑癌汤组三次重复实验无统计学差异,说明实验具有可重复性。结果显示不同浓度抑癌汤作用48h对NCI-H446细胞的增殖具有明显的抑制作用,随着作用时间的延长抑制作用逐渐减弱,且随着给药剂量的增大,抑制作用增强。抑癌汤在24小时、48小时、72小时的抑制率分别达到53.47%、54.47%和10.45%。4:四个给药组之间的比较:抑癌汤组选用的中间浓度(60ug/ml)。抑癌汤加西药组与抑癌汤组;抑癌汤加西药组与西药组;抑癌汤组与西药组;抑癌汤组与空白组;抑癌汤加西药组与空白组;西药组和空白组的细胞抑制率有统计学差异。通过给药组间均数两两比较:抑癌汤加西药组优于抑癌汤组;抑癌汤加西药组优于西药组;西药组优于抑癌汤组;抑癌汤组优于空白组;抑癌汤加西药组优于空白组;西药组优于空白组并有统计学差异。结论:1:人小细胞肺癌NCI-H446细胞倍增时间为26小时,增殖快。2:结果显示不同浓度抑癌汤作用48h对NCI-H446细胞的增殖具有明显的抑制作用,随着作用时间的延长抑制作用逐渐减弱。3:抑癌汤对NCI-H446细胞随着给药剂量的增大,抑制作用增强。4:抑癌汤加西药组优于抑癌汤组;抑癌汤加西药组优于西药组;西药组优于抑癌汤组。抑癌汤在体外对人小细胞肺癌NCI-H446细胞具有抑制作用,通过理气、活血止痛、燥湿祛痰、清热解毒的方法抑制癌细胞增殖。具有潜在药用价值,值得进一步深入研究。更多还原

【Abstract】 Objective: The topic cell culture technology, Since the adoption of the proposed side suppressor soup on human lung cancer cell line NCI-H446 interference, explore its proliferation. From seedling to the Chinese Medicines board prescription drugs found lung cancer cell proliferation inhibition new method for the treatment of lung cancer with a new treatment ideas.Methods: NCI-H446 cells were cultured in plotting NCI-H446 cells growth curve, from the log at the NCI-H446 cells for experiments. First determine the role of tumor suppressor soup concentration of tumor suppressor soup into 1g/ml,100mg/ml.10mg/ml,1mg/ml,100ug/ml,10ug/ml, 1ug/ml, 0.1ug/ml a total of eight concentration gradient, the control group only increase contour cell culture media, a total of nine. Each group set up four Minute hole, MTT assay was used to measure cell survival. Cells from the 50% survival rate for the drug concentration as a tumor suppressor soup administrationb reference concentration. In further unrest 20ug/ml,40ug/ml,60ug/ml,80ug/ml suppressor, 100ug/ml 5 concentration and 24 hours, 48 hours, This 72-hour observation time three point blank (media control group) were compared observation. For the final soup 60ug/ml suppressor as the concentration of tumor suppressor soup administration concentration, and the Western group. suppressor Tonga medicine group (60ug/ml concentrations and increases with western medicine) and the only culture fluid of the control group. A total of four larger groups (suppressor of group, medicine group, the tumor suppressor Tonga medicine group, the control group), a group set up four of the Minute hole, Comparative study, the MTT assay was used to measure the cell growth rate, the above experiments were repeated three times.Results:1 : Drawing cell growth curve inverted microscope for cell counting, cell doubling time was 26 hours. 2 : NCI-H446 tumor suppressor soup of cytotoxic cell experiments at the 50% survival rate of drug concentration in the range of 1-10 0ug/ml between, according to the concentration range for the inhibition of cell proliferation with reference to the concentration of drug administration. 3 : inhibition of cell proliferation : the tumor suppressor 20ug/ml,40ug/ml,60ug/ml soup, 80ug/ml,100ug/ml concentration and 24 hours, 48 hours, This 72-hour observation time three point blank Group (media control group) were compared statistically significant difference. Suppressor soup three experimental groups after statistical analysis : P=0.492. suppressor soup that is repeated three experimental groups no significant difference on experiments with repeatability. The results showed that different concentrations of tumor suppressor role of soup for 48 hours NCI-H446 cells was inhibited, With the duration of effect gradually weakened, and with the dosage increased, the inhibition. Suppressor soup in 24 hours, 48 hours, 72 hours of inhibition rate reached 53.47%, respectively, 54.47% and 10.45%.4 : four administration of comparisons between groups : Group selected tumor suppressor soup in the middle concentration (60ug/ml). Suppressor Tonga medicine group and tumor suppressor soup; Suppressor Tonga western medicine and western medicine; Suppressor soup and western medicine; suppressor soup group and control group; suppressor Tonga medicine group and control group; Western and blank cells inhibition rate was statistically different. Through the administration of several groups were compared on February 2 : suppressor Tonga medicine group than suppressor soup; Suppressor Tonga better than Western medicine group; WM group than suppressor soup; suppressor soup is better than the blank group; suppressor Tonga medicine group is better than the blank group; Western medicine is better than the blank group and the significant difference.Conclusion: 1 : human small cell lung cancer NCI-H446 cells doubling time of 26 hours, rapid proliferation. 2 : The results showed that different concentrations of tumor suppressor role of soup for 48 hours NCI-H446 cells was inhibited, With the duration of effect gradually diminish. 3 : soup of NCI-H446 tumor suppressor cells with the dosage increased, the inhibition. 4 : Tonga medicine group suppressor tumor suppressor than soup; Suppressor Tonga better than Western medicine group; WM group than suppressor Soup. Suppressor unrest in vitro human small cell lung cancer NCI-H446 cells were inhibited by Shugan Jimmy gas, Huoxuezhitong, and removing expectorant, Qingrejiedu inhibition of cell proliferation. Have potential medicinal value, it is worth further study.更多还原