摘要
目的观察苦参碱对肝癌干细胞增殖及侵袭转移能力的影响，探讨苦参碱对肝癌干细胞的作用机制。方法应用无血清肿瘤干细胞培养基，悬浮培养人肝癌细胞株SMMC-7721形成克隆，收集后裸鼠皮下接种并予以顺铂筛选，取耐药细胞继续悬浮培养获得干细胞克隆。采用不同浓度苦参碱体外作用72h后，噻唑蓝（MTI＇）比色法观察肝癌干细胞的增殖能力变化，荧光定量聚合酶链反应（PCR）、Westernblot法检测侵袭转移相关基因细胞黏附调节基因（CAR）、E-钙黏蛋白（E—cadhenn）、层粘连蛋白（Laminin）、纤连蛋白（Fibronectin）表达水平的变化。结果在肿瘤干细胞培养基和顺铂的双重筛选下，SMMC-7721细胞能形成悬浮生长的干细胞克隆。5、50、100、200mg／L苦参碱体外作用72h后，可明显抑制肝癌干细胞的增殖，抑制率分别为（3．0±0．3）％、（26．9±0．5）％、（55．1±0．6）％、（77．4±0．8）％。荧光定量PCR结果显示，50mg／L苦参碱可诱导肿瘤干细胞CAR、E-cadherin、Laminin、Fibronectin基因表达上调至未处理组的15．2、8．5、4．9和6．3倍，差异有统计学意义，与Western blot的结果-致。结论- 定浓度的苦参碱可明显抑制肝癌干细胞的体外增殖，并抑制其侵袭转移能力。
Objective To investigate the effect of matrine on proliferation, invasion and metastasis of hepatocellular carcinoma （HCC） stem cells in vitroand the action mcchanims. Methods SMMC-7721 cells were cuhured in tumor stem cell specific medium to form sphere, harvested, inoculated subcutaneous-ly into the nude mice, and treated with different concentrations of cisplatin. The drug resistant ceils were resuspended and purified. Methyl thiazol tetrazolium （MTT） test was performed to detect the inhibitory effects of matrine against tumor proliferation. Real-time polymerase chain reaction （real-time PCR） and Western blotting were used to detect the changes of CAR, E-cadherin, Laminin and Fibronectin. Results Through the double filter of TSC medium and cisplatin-resistance, SMMC-7721 stem cells could form spheres in TSC medium. The inhibition ratio of HCC stem cells was （3.0 ± 0. 3 ） %, （26. 9 ± 0. 5 ） %, （55. 1 ± 0. 6）%, and （77. 4 ± 0. 8 ）%, respectively in the presence of matrine （5, 50, 100,200 mg/L） for 72 h in vitro. The real-time PCR revealed that 50 mg/L matrine increased the expression ofcell adhe-sion regulates gene （ CAR）, E-cadherin, Laminin and Fibronectin up to 15.2, 8. 5, 4. 9 and 6. 3 times re-spectively, which was consistent to the result of Western blotting. Conclusion Appropriate concentration of matrine could significantly inhibite the proliferation and invasion of HCC stem cells.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2013年第1期61-63,共3页
Chinese Journal of Experimental Surgery
基金
浙江省中医药科技计划资助项目（2010ZA111）
宁波市自然科学基金资助项目（2010A610054）
关键词
癌
肝细胞
肿瘤干细胞
苦参碱
增殖
转移
Carcinoma,hepatocellular
Tumor stem cell
Matrine
Proliferation
Metastasis
