【作者】 马秀英；

【导师】 周黎明；

【作者基本信息】 四川大学 ， 药理学， 2007， 硕士

【摘要】 目的：观察埃克特注射液对人肝癌细胞SMMC-7721及小鼠H₂₂肝癌移植性肿瘤的抑制作用，并探讨以紫草萘醌类化合物为主要成分，制备而成的埃克特注射液的抗肿瘤作用机制。方法：用MTT法分析0.1，0.2，0.4，0.8，1.6，3.2，6.4，12.8，25.6mg·L^-1的埃克特注射液对人肝癌细胞SMMC-7721的增殖抑制作用，并计算半数抑制浓度IC₅₀；以1mg·L^-1，2mg·L^-1，4mg·L^-1埃克特注射液，0·05 mg·L^-1阿霉素为阳性对照作用于SMMC-7721细胞，绘制作用7天生长曲线；建立小鼠H₂₂肝癌移植性实体瘤模型，随机分为阴性对照组、环磷酰胺组、埃克特注射液高、中、低剂量组及助溶剂对照组，除环磷酰胺全程仅给药一次，其余各组隔天给药一次，共6次，通过绘制瘤块生长曲线及计算肿瘤抑制百分率评价体内抑瘤效果，瘤组织固定行免疫组化，半定量检测凋亡相关蛋白Bcl-2、Bax及Caspase-3的表达情况；Giemsa及Hoechst 33258染色观察不同剂量埃克特注射液作用SMMC-7721细胞48h后细胞形态的变化，观察有无凋亡特征性变化；流式细胞术检测不同剂量埃克特注射液作用SMMC-7721细胞48h后细胞凋亡率及细胞周期变化，并检测Bax及Bcl-2蛋白的表达情况；RT-PCR检测埃克特注射液不同剂量组作用SMMC-7721细胞24h后，bcl-2 mRNA及bax mRNA的表达情况。结果：（1）MTT结果显示埃克特注射液对SMMC-7721细胞具有显著抑制作用，呈明显量效关系，作用48小时的IC₅₀为2.325 mg·L^-1；生长曲线实验显示埃克特注射液对SMMC-7721细胞的抑制作用呈明显时效和量效关系；（2）体内实验表明，腹腔注射0.5 mg·kg^-1，1 mg·kg^-1，2 mg·kg^-1埃克特注射液6次，对小鼠H₂₂肝癌移植性肿瘤有一定的抑制作用，抑制率分别为34.37％（P＜0.05），42.36％（P＜0.05），57.99％（P＜0.01），呈剂量依赖性；生长曲线也显示给药各剂量组实体瘤体积与阴性对照相比增长减缓，且呈一定的时效及量效关系。（3）Giemsa染色后在光镜下可见埃克特注射液给药组多数细胞出现形态改变，胞体变小，胞质浓缩拉长，呈拉丝状，核质比增大，核膜完整，核固缩，染色质不均一或边集的典型凋亡特征；Hoechst 33258染色后给药组细胞核出现细胞核固缩，染色质不均一或边集呈致密强荧光，出现凋亡小体等明显的凋亡形态学变化；流式细胞术检测显示埃克特注射液作用SMMC-7721细胞48h后，随着剂量的增大凋亡率升高，1mg·L^-1，2mg·L^-1，4mg·L^-1剂量组细胞凋亡率分别为11.73％，20.57％，27.13％，中高剂量组均检测到明显亚G₁凋亡峰；给药组S期细胞明显降低，G₂／M期细胞明显增多，细胞周期阻滞于G₂／M期，且药物浓度越高，此趋势越明显；药物处理后Bcl-2及Bax蛋白的表达均下调，且Bcl-2／Bax值降低；RT-PCR检测结果显示给药组bcl-2 mRNA及bax mRNA表达水平均出现下调，bcl-2／bax比值降低；体内实验瘤组织免疫组化结果显示给药组Bcl-2蛋白表达下调，Bax及Caspase-3蛋白表达均出现上调。结论：埃克特注射液对人肝癌SMMC-7721细胞具有明显体外抗增殖作用，对小鼠移植性肿瘤H₂₂也呈现了明显的抑制作用。其效应机制可能与阻滞细胞周期于G₂／M期，下调bcl-2 mRNA及Bcl-2蛋白表达，降低Bcl-2／Bax比值，上调Caspase-3蛋白表达，诱导肿瘤细胞凋亡，抑制肿瘤细胞增殖有关。更多还原

【Abstract】 Objective : To investigate the inhibitory effect of Aikete injection （AKT） on human hepatic cancer cell line SMMC-7721 and Hepatic cancer H₂₂ . To study the mechanism of anti-hepatocarcinoma of Aikete injection which is mainly composed of Naphthoquinones derivatives.Methods: MTT assay and growth curve assay were used to assess the inhibitory effect of Aikete injection on proliferation of SMMC-7721 cells. SMMC-7721 cells were treated with AKT at the dose 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6mg·L^-1 in MTT assay to analyze the inhibitory effect on proliferation of SMMC-7721 cells and calculated IC₅₀ after treated for 48 hours; After treated with AKT at the dose 1, 2, 4mg·L^-1, 0.05 mg·L^-1 Adriamycin （ADM） as positive control for 7 days, growth curve of SMMC-7721 cells was completed; Hepatocarcinoma H₂₂ model in mice was performed to study anti-hepatocarcinoma activity of AKT in vivo which were injected by intraperitoneal injection for 6 times, cyclophosphamide （CTX） as positive control by intraperitoneal injection just for once. After 11 days, the weight of tumor、spleen and thymus were observed to calculate the percentage of inhibition on tumor, and complete the growth curve of tumor by measuring length and width of tumor. Apoptosis-related protein （Caspase-3, Bcl-2 and Bax） were checked by immunohistochemical staining （IHC） . Its apotosis-inducing effect was approved by morphological observation by Giemsa staining and Hoechst 33258 fluorescent staining. The expressions of Bax and Bcl-2 protein, apoptosis rates and cell cycle were analyzed by flow cytometry （FCM）. The expression of bax mRNA and bcl-2 mRNA were analyzed by RT-PCR.Results: After treated with AKT for 48 hours, MTT assay showed AKT caninhibit the proliferation of SMMC-7721 cells and had obvious dose-effect relationship. IC₅₀ of AKT to SMMC-7721 cells was 2.325mg·L^-1. The dose-effect and time-effect relationship were described in growth curve. The experiment in vivo showed AKT of 0.5, 1, 2mg·kg^-1 had significant inhibitory effect on tumor and dose-related effect on mice with hepatocarcinoma, the inhibitory rate of tumor ranged from 34.37%（P<0.05） to 57.99 %（P<0.01）. Cells shrinkage, nuclear pyknosis, chromatin condensation which were the characteristics of cell apoptosis were observed by Hoechst 33258 and Giemsa staining. FCM showed Cells in S phase decreased, cells in G₂/M phase increased, the cell cycle was arrested in G2/M phase. In groups of 2mg·L^-1 and 4mg·L^-1, the sub-G₁ peak was detected, the sub-G₁ peaks at the dose 4mg·L^-1 was obvious. The percentage of apoptosis increased. The expression of Bcl-2 and Bax protein was down-regulated, while the index of Bcl-2/Bax was decreased in the AKT group. RT-PCR showed that AKT could down-regulate the expression of bcl-2 mRNA and bax mRNA, and decreased the index of bcl-2 mRNA/bax mRNA. It was found that AKT could down-regulate the expression of Bcl-2 protein and up-regulate the expression of Bax and Caspase-3 protein in Hepatocarcinoma H22. Conclusion: AKT could inhibit proliferation of SMMC-7721 cells in vitro and the growth of Hepatocarcinoma H₂₂ in vivo. It may induce Hepatocarcinoma cells apoptosis through arresting cell cycle in G₂/M phase, down-regulating the expression of bcl-2 mRNA and Bcl-2 protein, decreasing the index of Bcl-2/Bax, and up-regulating the expression of Caspase-3 protein.更多还原