【作者】 杨军；

【导师】 丁敏；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2005， 硕士

【摘要】 目的:复方斑蝥胶囊是根据卫生部颁布的标准生产的纯中药胶囊制剂,临床上用于治疗多种癌症,以原发性肝癌为主,显示了较好的疗效,作用机制在国内外还未见报道。血清药理学方法是研究中药复方的有效手段。因此,本课题拟运用血清药理学方法观察复方斑蝥胶囊对人肝癌细胞 SMMC-7721 的增殖和细胞周期、细胞凋亡以及凋亡相关蛋白的影响,探讨其作用机制。 方法:1、采用 MTT 法确定制备含药血清的条件1.1.确定最佳采血时间: 取新西兰大耳白兔 3 只,用 20 ml PBS(pH 7.4)将临床等效剂量复方斑蝥胶囊内容物制成混悬液灌胃给药,每日一次,连续三日,末次给药后 1、2、3、4、5 h 采血,分离血清。以不同采血时间的含药血清对人肝癌细胞 SMMC-7721 进行 MTT 试验,确定最佳采血时间。1.2.确定最佳给药剂量: 取新西兰大耳白兔 12 只,分为 1、2、3 倍临床等效剂量组、对照组,每组 3 只。20 ml PBS(pH 7.4)将复方斑蝥胶囊内容物按相应剂量制成混悬液,灌胃给药,每日一次,连续三日,末次给药后于最佳采血时间点采血,分离血清。以不同剂量组的含药血清对人肝癌细胞SMMC-7721 进行 MTT 试验,确定含药血清最佳给药剂量。2、采用 MTT 法,连续 5 天,观察含药血清对人肝癌细胞 SMMC-7721 生更多还原

【Abstract】 OBJECTIVE:Fufang Banmao Capsule is a Chinese traditional medicinemade according to the standard of Ministry of Health. It is used to curemulti-cancer especially liver cancer, it’s curative effect is good, but it’smechanism hasn’t been reported. Serum pharmacological method is a goodapproach to study Chinese traditional medicine. So, we are going toobserve effects of proliferation, cell cycle, apoptosis and apoptosis relatedprotein of Fufang Banmao Capsule on human hepatocellular carcinomaSMMC-7721 cells and investigate it’s mechanism.METHODS:1.MTT assay was used to confirm the condition of preparing drug serum.1.1.Confirm the best time of collecting blood: Content of Fufang Banmao Capsule in equivalent dose were made intosuspension with 20ml PBS and given it to three New Zealand rabbitsintragastrically, once a day and continue for three days.Collected bloodafter the last administration at 1h, 2h, 3h, 4h, 5h and separated the serumsample. Proceed to MTT assay with each drug serum and calculateinhibiting rate to confirm the best time of collecting blood.1.2.Confirm the best dose of collecting blood: Twelve New Zealand rabbits were grouped under 1, 2, 3 equivalentdose and control,three in each group.Content of Fufang Banmao Capsule ineach dose were made into suspension with 20ml PBS and given it to therabbits intragastrically, once a day and continue for three days. Collectedblood after the last administration at the best time and separated the serumsample. Proceed to MTT assay with each drug serum of different dose andcalculate inhibiting rate to confirm the best dose of administration ofcollecting blood.2.Observed effect of the drug serum on the proliferation in humanhepatocellular carcinoma SMMC-7721 cells by MTT assay and continuefor five days.3.The drug serum incubated with human hepatocellular carcinomaSMMC-7721 cells for 72h.3.1.Cell cycle and the apoptosis index were determined by flow cytometry.3.2.The morphological and ultra structural changes were observed bytransmission electron microscope.3.3.The effect of Fufang Banmao Capsule on hepatocellular carcinomaprotein expression of Bcl-2 and Bax were detected byimmunocytochemistry.RESULTS:1.The drug serum of each time could significantly inhibit the proliferationof SMMC-7721 cells and the highest inhibting rate was 3h(p<0.01). Thedrug serum of each dose could significantly inhibit the proliferation ofSMMC-7721 cells dose-dependently and the highest inhibting rate was thethree times dose(p<0.01).2.The SMMC-7721 growth curves showed the drug serum could inhibitproliferation of the SMMC-7721 cells time-dependently.3.Flow cytometry analysis showed that the drug serum treated cellsunderwent a G0/G1 arrest, with the fraction of cells in G1 phase increasingto 85.6% and the fraction of cells in S phase decreasing to 14.4% and thefraction of cells in G2/M phase decreasing to zero. There was a apoptosispeak and the rate of apoptosis is 10.4%. There were no significantlydifference between cells control and serum control. In controls nucleolus isbig and nucleolinus is evidence, euchromatin is abundance and organelle isless, there are short microvillus on the membrane. In drug serum-treatedcells there is a typical apoptosis hepatocarcinoma cell, we could findheterochromatin gather ground and enrichment and nuclear fragmentation.Drug serum-treated protein express of Bcl-2 decreasing(P<0.01)and proteinexpress of Bax has no obvious change(P>0.05).CONCLUSION:1.Fufang Banmao Capsule could significantly inhibit the proliferation ofSMMC-7721 cells time/dose-dependently.2.Fufang Banmao Capsule could disturb cell cycle progression and induceapoptosis.Possible mechanism may be down-regulation of the expression ofBcl-2.更多还原