光叶番荔枝中二萜类化合物抑制肝癌细胞株SMMC-7721增殖及机制的研究

【作者】 夏国豪；

【导师】 章永红； 潘良熹；

【作者基本信息】 南京中医药大学 ， 肿瘤内科， 2003， 硕士

【摘要】 目的 研究光叶番荔枝中二萜类化合物药A（-）-kaur-16-en-19-oic acid、药B（-）-kauran-19-al-17-oic acid对人肝癌细胞株SMMC-7721的抑制作用及其机理。 方法 以MTT法检测SMMC-7721细胞经药A、药B作用后的活力，用荧光显微镜、透射电镜、扫描电镜观察其形态变化，用流式细胞仪分析细胞周期的变化及计算凋亡率，用免疫细胞化学染色的方法观察细胞凋亡调控基因的表达情况。 结果5umol／L以上浓度的药A和10umol／L以上浓度的药B对SMMC-7721细胞有较强的抑制作用，以25umol／L的药A作用最强，抑制率高达81.05％，浓度增加和减少其抑制率均下降，48小时IC₅₀为19.5umol/L，药B对SMMC-7721细胞的抑制作用与浓度呈线性相关，72小时IC₅₀为47.5umol／L，两化合物的抑制作用均呈时间依赖性，25umol／L浓度的药A作用于SMMC-7721细胞48小时，在流式细胞仪上可见亚G₀／G₁峰，药A、药B使细胞周期阻滞于G₀-G₁期，细胞凋亡率分别为43.31％、24.95％；经25umol／L浓度的药A作用后，在荧光显微镜、透射电镜、扫描电镜下观察到细胞凋亡的特征形态改变，如细胞体积缩小，胞膜皱缩，表面微绒毛消失，核固缩、沿核膜分布等，用免疫细胞化学染色的方法观察到Bcl-2基因表达降低，bax表达增加。 结论 光叶番荔枝中二萜类化合物药A、药B对人肝癌细胞株SMMC-7721有显著的抑制作用，诱导SMMC-7721癌细胞凋亡，抑制Bcl-2基因的表达，增加bax基因的表达。更多还原

【Abstract】 OBJECTIVE To study the inhibitory effects of diterpenoid compound ( drug A.drug B) isolated from Annona glabra Linn on the proliferotion of SMMC-7721 cells lines and its mechanism .METHODS SMMC-7721 cells were treated with compound drug A and drug B in various concentrations for various days,then observed and analysed by MTT assay ,Flurescent microscopy,Transmission electron microscopy (TEM), Scanning electron microscopy(SEM),Flow cytometry(FCM),Agarose gel electrophoresis and Immunohisto-chemical staining.RESULTS In vitro studies,by means of MTT assaysjt was found the more then 5umol/L drug A and the more then 10umol/L drug B could significantly inhibit the growth of SMMC-7721,the inhibitory effect of 25umol/L drug A was the most power and its 72h inhibition rate was 81.05%. After 48h of treatment with 25umol/L drug A and drug B, "Sub-G0/G1"peaks was recorded in DNA histogram of FCM . It was also showed by FCM that the cell cycle was arrested in G0/G1 phase and the apoptotic rate were43.31%and 24.95% respectively. After 72h of treatment with 25umol/L drug A under the inspection of Flurescent microscopy , SEM and TEM, most of SMMC-7721 cells displayed the typically morphological features of apoptosis, including cell shrinkage,condensation and fragmentation of nuclear chromation,etc. It was observed by Immunohistochemical staining that drug A downregulated expression of bcl-2 and upregulated expression of bax genes .CONCLUSION Diterpenoid compound (drug A,drug B) isolated from Annona glabra Linn inhibite the proliferotion of SMMC-7721 cells lines and induced SMMC-7721 cells apoptosis with downregulation of bcl-2 gene expression and upregulation of bax genes expression.更多还原