【作者】 任靖；

【导师】 王敏伟； 孙立新；

【作者基本信息】 沈阳药科大学 ， 药理学， 2005， 硕士

【摘要】 本文通过对白英乙醇提取物、乙酸乙酯部位、正丁醇部位体外对人肝癌BEL-7402、人胃腺癌SGC-7901及人宫颈癌HeLa细胞等肿瘤细胞的增殖抑制作用及体内对小鼠移植性肝癌H₂₂和肉瘤S₁₈₀肿瘤生长抑制作用的研究确定白英抗肿瘤作用主要有效部位，并对其体外诱导人宫颈癌HeLa细胞死亡机制进行初步研究。选取体外培养的人肝癌BEL-7402、人胃腺癌SGC-7901和人宫颈癌HeLa细胞为模型，以半数抑制浓度(IC₅₀)为指标，采用MTT法初步筛选白英抗肿瘤作用主要有效部位；体内以小鼠移植性肿瘤肝癌H₂₂和肉瘤S₁₈₀为模型，以抑瘤率为指标，进一步确定白英抗肿瘤作用的主要有效部位；以体外培养的人宫颈癌HeLa细胞为模型，采用形态学荧光染色、LDH活力测定、流式细胞术分析等方法对白英乙酸乙酯部位的抗肿瘤作用机制进行初步研究。体外实验结果表明白英不同提取部位对体外培养的BEL-7402、SGC-7901和HeLa细胞均具有显著的细胞增殖抑制活性，且对细胞生长的抑制作用呈现显著的浓度、时间依赖关系，作用48h后对BEL-7402、SGC-7901和HeLa细胞的IC₅₀值分别为：乙醇提取物：287.4±5.8μg·mL^-1、176.1±5.2μg·mL^-1和297.5±7.0μg·mL^-1；乙酸乙酯部位：106.2±5.8μg·mL^-1、65.4±6.0μg·mL^-1和33.7±4.9μg·mL^-1；正丁醇部位：180.2±6.3μg·mL^-1、114.9±4.9μg·mL^-1和59.3±4.8μg·mL^-1。体内实验表明白英不同提取部位对小鼠移植性肉瘤S₁₈₀均具有显著的抗肿瘤活性，其中乙醇提取物高剂量组和乙酸乙酯部位高剂量组对肉瘤S₁₈₀及肝癌H₂₂的生长抑制率均达到40％以上；正丁醇部位对小鼠移植性肉瘤S₁₈₀的抑制作用显著而对肝癌H₂₂的抑制作用不显著。50μg·mL^-1白英乙酸乙酯部位处理的HeLa细胞经Hoechst 33258形态学荧光染色出现明显的凋亡小体；LDH活力分析实验表明，白英乙酸乙酯部位处理HeLa细胞诱导细胞死亡的过程中有一定的凋亡细胞的分布，合适剂量的白英乙酸乙酯部位诱导细胞凋亡；对其采用流式细胞术分析，适当剂量或作用时间后出现凋亡特征峰；随药物浓度增大或作用时间延长，乙酸乙酯部位处理的HeLa细胞各时相的细胞百分含量发生显著的变化，G₀／G₁期细胞百分含量呈显著上升趋势。由实验结果可见，白英乙酸乙酯部位是白英抗肿瘤作用主要有效部位；白英乙酸乙酯部位体外可诱导HeLa细胞凋亡，且对HeLa细胞呈现显著的细胞周期抑制作用，可将细胞周期阻滞在G₀／G₁期。更多还原

【Abstract】 The in vitro and vivo antitumor activity of some fractions of Solarium lyratum was studied in this dissertation.The inhibitory effect of some fractions on proliferation of human hepatocarcinoma cell lines BEL-7402, human gastric carcinoma cell lines SGC-7901 and human cervix epithelial cell lines HeLa were measured by MTT colorimetric assay in vitro. The mouse tumor model was used to investigate the effect of some fractions on tumor growth. The mechanism of antitumor effects was studied preliminarily by morphology analysis, lactate dehydrogenase（LDH） activity-based assay and flow cytometry（FCM） in HeLa cells.The studies demonstrate that some fractions inhibited proliferation of several tumor cell lines, including BEL-7402 cells(IC₅₀.ethanol extracts: 287.40±5.84μg·mL^-1, IC₅₀. Etoac fractions: 106.23±5.79μg·mL^-1 and IC₅₀. n-BuOH fractions: 180.22±6.32μg·mL^-1), SGC-7901 Cells(IC₅₀. ethanol extracts: 176.14±5.18μg·mL^-1, IC₅₀. EtoAC fractions: 65.44±6.01μg·mL^-1 and IC₅₀.n-BuOH fractions: 114.89±4.89μg·mL^-1) and HeLa cells(IC₅₀, ethanoi extracts: 297.45±6.95μg·mL^-1, IC₅₀. Etoac fractions: 33.66±4.87μg·mL^-1 and IC₅₀. n-BuOH fractions: 59.31±4.78μg·mL^-1). When the concentration of some fractions varied from low to high, they were able to inhibit the proliferation of tumor cells in a concentration-dependent manner in vitro. At high doses of ethanol extracts and EtoAC fractions, the tumor inhibition rates on mice bearing sarcoma 180(S₁₈₀) and hepatocarcinoma 22(H₂₂) were both over 40%. N-BuOH fractions could significantly inhibit the tumor growth of S₁₈₀. But there was no influence of n-BuOH fractions on the growth of H₂₂.In EtoAC fractions-treated HeLa cells for 12 h, apoptotic bodies were formed, nuclear damage was observed by Hoechst 33258 staining. Treatment with various concentrations of EtoAC fractions for 12 h, HeLa cells underwent apoptosis as measured by LDH activity-based assay. At the same time, apoptosis was confirmed by the appearance of apoptotic peak on FCM in HeLa cells. FCM results showed that HeLa cells treated with EtoAC fractions were remarkably arrested at G₀／G₁ phase in a time and concentration-dependent manner.Main active fractions were confirmed as EtoAC fractions. The possible mechanism of antitumor effects of EtoAC fractions maybe associate with inducing apoptosis and inducing G₀/G₁ phase arrest.更多还原