【作者】 朱丽丽；

【导师】 李惠芬；

【作者基本信息】 天津医科大学 ， 药理学， 2006， 硕士

【摘要】 目的:以传统抗癌中药重楼为研究对象,优选最佳中试提取工艺,分离、纯化、精制得到抗肿瘤活性单体PCB5-9与PCB4—5,探讨重楼皂苷诱导人低分化胃腺癌SGC—7901细胞凋亡的作用机制,以期发现药物作用新靶点,为寻求治疗肿瘤的新方法奠定基础。方法:（1）应用正交试验设计,以薯蓣皂苷元得率为考察指标,筛选重楼总皂苷的最佳提取工艺条件。以乙醇为提取溶剂提取得浸膏,浸膏水溶液依次经石油醚、乙酸乙酯、正丁醇萃取得重楼总皂苷,然后分别经大孔吸附树脂柱层析、硅胶柱层析、反相ODS柱层析、RP-HPLC制备色谱制备得重楼皂苷单体成分。（2）采用MTT比色法研究中药重楼皂苷单体对肿瘤细胞株K562、SGC—7901体外增殖反应的抑制作用,确定其对体外培养的K562、SGC-7901细胞的作用剂量。（3）重楼皂苷单体经5（6）-TAMRA-5-maleimide荧光试剂标记后,MTT法观察重楼皂苷、荧光标记的重楼皂苷及游离荧光对SGC-7901细胞的抗肿瘤作用,并采用荧光显微镜评价该荧光标记药物的细胞染色情况。（4）琼脂糖凝胶电泳法观察重楼皂苷诱导肿瘤细胞SGC—7901凋亡的作用。（5）应用激光共聚焦显微术,以钙离子荧光探针Fluo-3/AM标记SGC-7901细胞内钙离子,观察不同浓度的PCB5-9（83-93）作用于癌细胞10min后,药物对细胞内钙离子浓度动态变化的影响,并观察药物作用于细胞24h后,细胞内荧光强度的变化。结果:（1）重楼皂苷的最佳提取工艺条件为乙醇浓度为70%,提取时间为5h,料液比为8倍,在此条件下从中提取,分离出重楼皂苷PCB 5-9（83-93）与PCB4-5（39-48）,经进一步的分离纯化,经HPLC归一化方法鉴定其纯度分别为97.63%和91.80%,结构有待进一步的鉴定。（2）体外实验结果表明PCB 5-9、PCB4-5对肿瘤细胞株K562、SGC—7901均有抑制作用,其抑制率与时间、剂量呈依赖性关系。其中,PCB 5-9对K562、SGC—7901细胞的最高抑制率分别为88.54%和75.08%;72h的IC₅₀分别为6.55μg/mL与20.48μg/mL;PCB4-5最高抑制率为88.58%和75.68%;72h的IC₅₀分别为5.69μg/mL和4.83μg/mL。（3）荧光标记的重楼皂苷体外实验结果表明,标记后的重楼皂苷对SGC-7901细胞也具有抑制作用,浓度为0.03mmol/l时,抑制率为52.28%;游离荧光对肿瘤细胞没有抑制作用,荧光显微镜下观察标记的药物作用细胞2.5h后,细胞内出现红色荧光。（4）琼脂糖凝胶电泳结果显示从PCB 5-9（83-93）处理24h的SGC-7901细胞中提取的DNA,出现较典型的梯状电泳条带,且随着药物浓度的升高,条带亮度增加,10μg/mL药物组和阳性药顺铂组条带亮度最高。（5）激光共聚焦显微观察结果表明不同浓度药物孵育SGC-7901细胞10min后,LSCM动态观察细胞内的钙离子,发现没有显著变化,而药物作用24h后,细胞内钙离子浓度显著升高,且[Ca²⁺]i随着药物浓度的增加而升高。结论:正交设计优化的重楼皂苷提取工艺条件可放大,适用于工业提取;重楼皂苷对体外培养的人白血病细胞K562和人低分化胃腺癌细胞株SGC-7901的生长有抑制作用,且呈明显的剂量与时间依赖性关系,并诱导SGC-7901细胞凋亡,细胞内Ca²⁺浓度升高,提示药物可能通过钙和钙调蛋白途径诱导细胞凋亡;标记的重楼皂苷对SGC-7901具有抑制作用,且能透过细胞膜进入活细胞。更多还原

【Abstract】 Objective:We selected Rhizoma Paridis as research object to optimize extraction process by mass trial,separated and purificated two anti-tumor active components PCB5-9 and PCB4—5 respectively,to investigate the mechanisms of saponins-induced apoptosis on SGC-7901 cells,in order to find new drug action target and establish theory foundation of new method of treating human poorly differentiated gastric adenocarcinoma.Methods:（1） We applied orthogonal design to optimize the extraction process for the saponins from Rhizoma Paridis.We used ethyl alcohol as extracting solvent to gain Chinese medical concrete,used petroleum ether,ethyl acetate and n-Butanol to extract saponins from it;and used macoropore resin,gelica gel,ODS,RP-HPLC to separate saponins.（2） Applied MTT assay to detect the inhibition effect of saponins from Rhizoma Paridis against human erythroleukemia cell line K562 and human poorly differentiated gastric adenocarcinoma cell line SGC-7901 culturing in vitro, and determined effective dose and median inhibition concentration(IC₅₀) of saponins to SGC-7901 cells and K562 cell after being treated with it.（3） Saponins from Rhizoma Paridis were marked by fluorescent reagent 5（6） -TAMRA-5-maleimide,applied MTT assay to detect the inhibition effect of saponins from Rhizoma Paridis、saponins marked by TAMRA and TAMRA against SGC-7901culturing in vitro,and to investigated cell-stained process of the fluorescent labeling saponins by fluorescence microscope.（4） Investigated the mechanisms of saponins-induced apoptosis on SGC-7901 cells by the way of DNA gel electroplhoresis.（5） The concentration of Ca²⁺ intra-cellular([Ca²⁺]i) was measured by the laser scanning confocal microscope（LSCM）.We treated SGC-7901 with different concentration of saponins for 24hours,used the fluorescent probe Ca²⁺ to mark the intra-cellular Ca²⁺ to observe its statical effect on concentration of Ca²⁺.We marked the intra-cellular Ca²⁺,then gave SGC-7901 with different concentration of saponins for 10 minutes to observe its dynamic effect on concentration of Ca² immediately.Results:（1） The optimum contraction conditions of saponins from Rhizoma Paridis were as follows:70%ethyl alcohol,5hours,8 times dosage liquor ratio.According to this condition,we abstracted and separated saponins PCB 5-9（83-93） and PCB4-5 （39-48）,their purification were 97.63%and 91.80%respectively by HPLC.Their structures needed further verification.（2） Studies in vitro indicated that two compositions could inhibit growth of K562、SGC—7901 and their inhibition rate had dose-time depended relationship.The highest inhibition rate of PCB 5-9（83-93） on K562、SGC—7901 were 88.54%and 75.08%respectively,IC₅₀ at 72h 6.55μg/mL and 20.48μg/mL respectively;PCB 4-5（39-48） respectively 88.58%and 75.68%,IC₅₀ at 72h 5.69μg/mL、4.83μg/mL respectively.（3） Studies of fluorescent labeling saponins on SGC-7901 in vitro showed fluorescent labeling saponins could inhibit growth of SGC-7901,when the concentration was 0.03mmol/L,its inhibition rate was 52.28%.5（6）-TAMRA-5- maleimide had no inhibition action on SGC-7901, red fluorescence appeared in cell by the way of fluorescence microscope after SGC-7901 had been treated by fluorescent labeling saponins for 2.5h.（4） Agarose gel electrophoresis showed typical DNA ladder strip after cell SGC-7901 had been treated with PCB 5-9（83-93） for 24h,and brightness of strip increased along with the concentration of it raises.（5） The concentration of Ca²⁺ intra-cellular([Ca²⁺]i) measured by LSCM after different concentration of saponins treated SGC-7901 for 24h showed significant variation（P＜0.05） compared with control;[Ca²⁺]i had no change when saponins treated SGC-7901 for 10min.Conclusion:The optimum contraction conditions of saponins from Rhizoma Paridis by the way of orthogonal experiment design could be applied in industry.Saponins could inhibit the proliferation of the SGC-7901 and K562,and induce cell SGC-7901 apoptosis.The increased concentration of Ca²⁺ suggested calcium-calmodulin pathway might be one of it’s mechanism.Fluorescent labeling saponins had inhibition action on SGC-7901,and it could get into cells successfully.更多还原