斑蝥素酸镁对人肝癌细胞SMMC-7721及其裸鼠皮下移植瘤的影响
Effect of magnesium cantharidate on hepatocellular carcinoma SMMC-7721 cells and subcutaneoushepatocellular SMMC-7721 carcinoma transplantation tumors in nude mice
晏 容1, 3** 刘 云2,3 朱欣婷1, 3 易小飞1 刘 流1 李晓飞1, 3
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DOI:10.7679/j.issn.2095-1353.2015.054
作者单位:1. 遵义医学院基础医学院,遵义 563003;2. 遵义医学院医学与生物学研究中心,遵义 563003;3. 贵州省普通高等学校特色药物肿瘤防治特色重点实验室,遵义 563003
中文关键词:斑蝥素酸镁,人肝癌细胞SMMC-7721,裸鼠,抗肿瘤,细胞凋亡
英文关键词:magnesium cantharidate, hepatocellular carcinoma cells SMMC-7721. nude mice, anti-tumor; cell apoptosis
中文摘要:
【目的】 探讨斑蝥素酸镁对人肝癌细胞SMMC-7721及其裸鼠皮下移植瘤的影响。【方法】 1、采用磺酰罗丹明染色法(SRB法)检测不同浓度斑蝥素酸镁在体外对人肝癌细胞SMMC-7721增殖的抑制作用;2、流式细胞术检测斑蝥素酸镁对人肝癌细胞SMMC-7721细胞周期和细胞凋亡的影响;3、Hoechst33342染色观察斑蝥素酸镁对人肝癌细胞SMMC-7721细胞形态的影响;4、透射电镜观察斑蝥素酸镁作用后人肝癌细胞SMMC-7721超微结构的变化;5、建立人肝癌细胞SMMC-7721裸鼠皮下移植瘤模型,实验组每只裸鼠瘤周注射斑蝥素酸镁6.26×10-5 mmol,对照组给予相同容积的无菌生理盐水瘤周注射,计算抑瘤率;6、原位末端标记染色(TUNEL)法检测人肝癌裸鼠皮下移植瘤组织细胞凋亡情况。【结果】 1、斑蝥素酸镁对人肝癌细胞SMMC-7721有比较明显的抑制作用,抑制率随药物浓度的增加而升高,呈剂量效应关系,其半数抑制浓度(IC50)为1.79 mmol/L;2、流式细胞检测结果显示:人肝癌细胞SMMC-7721在斑蝥素酸镁的作用下,G0/ G1期细胞减少,G2/M期细胞增加,细胞出现G2/M期阻滞;细胞凋亡率随斑蝥素酸镁浓度加大而逐渐增加;3、Hoechst33342染色镜下显示:斑蝥素酸镁作用后人肝癌细胞SMMC-7721出现凋亡细胞形态特征;4、透射电镜观察:斑蝥素酸镁作用后人肝癌细胞SMMC-7721出现细胞核异形、染色质聚集成团、边集,见凋亡小体;5、斑蝥素酸镁组肿瘤体积、重量显著小于生理盐水组(P﹤0.05),抑瘤率为49%;6、TUNEL法提示斑蝥素酸镁组移植瘤组织细胞凋亡率显著高于生理盐水组(χ2=92.609,P﹦0.000)。【结论】 斑蝥素酸镁对人肝癌细胞SMMC-7721在体内外均有抑制增殖作用,并可以诱导肿瘤细胞凋亡,其凋亡的发生与细胞分裂期阻滞有关。
英文摘要:[Objectives] To investigate the effect of magnesium cantharidate on hepatocellular carcinoma SMMC-7721 cells and subcutaneous SMMC-7721 transplantation tumors in nude mice. [Methods] 1. The sulforhodamine B(SRB) assay was employed to evaluate the effect of magnesium cantharidate on the inhibition of hepatocellular carcinoma cells in vitro. 2. Flow cytometry(FCM)was used to measure the effect of magnesium cantharidate on cell-cycle arrest and cell apoptosis of hepatocellular carcinoma cells. 3. Hoechst 33342 staining was used to observe the effect of magnesium cantharidate on morphological changes in hepatocellular carcinoma cells. 4. Transmission electron microscopy (TEM) was used to observe the effect of magnesium cantharidate on the ultra-structure of hepatocellular carcinoma cells. 5. A model system using subcutaneous transplantation tumors composed of hepatocellular carcinoma cells was set up in nude mice. The experimental group was injected with magnesium cantharidate (6.26×10-5 mmol) around the tumor and the control group was given the same volume of sterile saline. The inhibition rates of the tumor in the two groups were calculated and compared. 6. Apoptosis in the experimental group was observed with the TUNEL staining method. [Results] 1. The results show that magnesium cantharidate had an obvious inhibitory effect on the proliferation of hepatocellular carcinoma cells, and that this increased gradually with dosage. The IC50 value was 1.79 μmol/L.2.FCM at which point the cell-cycle of SMMC-7721 changed. The ratio of cultured cells declined in the G0/G1 phase, whereas cells increased dramatically in the G2/M phase. The cell-cycle arrested in the G2/M phase. Cell apoptosis increased gradually with concentration of magnesium cantharidate. 3. Hoechst 33342 staining showed that hepatocellular carcinoma cells displayed morphological features typical of apoptotic cells. 4. TEM revealed nuclear abnormities, including concentrations of chromatin on the nuclear edge and the formation of apoptotic bodies. 5. The volume and weight of tumors in the magnesium cantharidate group was significantly lower than in the sterile saline control group (P <0.05) and the inhibition rate was 49%. 6. The TUNEL assay indicated that the apoptosis rate in the magnesium cantharidate group was significantly higher than in the sterile saline group(χ2=92.609, P=0.000). [Conclusion] Magnesium cantharidate can inhibit the proliferation of SMMC-7721 hepatocellular carcinoma cells both in vitro and in vivo, and induce the apoptosis of cancer cells. Apoptosis may be associated with the arrest of cell division.