【作者】 王玉美；

【导师】 杨雁；

【作者基本信息】 安徽医科大学 ， 药理学， 2007， 硕士

【摘要】 目的：探讨黄芪提取物（CAE）抗肝癌的作用机制。方法：利用MTT方法建立小牛血清（NBS）、转化生长因子-β₁（TGF-β₁）体外刺激HepG2细胞增殖模型，研究CEA(5，10，20，40，80mg·L^-1)五个剂量对NBS、TGF-β₁诱导的HepG2细胞增殖的影响；采用免疫沉淀和Western-blot方法检测CAE对TGF-β₁诱导的HepG2细胞内Smad2C磷酸化的影响；Western-blot方法检测3种MAPK抑制因子对TGF-β₁诱导的Smad2C、Smad2L、Smad3C、Smad3L磷酸化的影响；采用浸润小室镜下计数细胞的方法，检测CAE(40mg·L^-1)对TGF-β₁诱导的HepG2细胞移行的影响。结果：1．TGF-β₁(9pM·L^-1)、12．5％NBS作用0．5、1h时无明显促进HepG2细胞增殖作用，但作用2，4，8，16，24h均明显促进HepG2细胞增殖，CAE(5～80 mg·L^-1)呈浓度依赖型地抑制HepG2细胞增殖。2．CAE(40 mg·L^-1)明显抑制TGF-β₁(200 pM·L^-1，12 h)诱导的HepG2细胞移行。3．TGF-β₁(9 pM·L^-1)作用1 h，明显促进Smad2C、Smad2L、Smad3C、Smad3L磷酸化，CAE高中低三个剂量呈浓度依赖性地抑制由TGF-β₁诱导的Smad2C磷酸化。4．c-Jun氨基末端激酶（JNK）抑制因子(1，3，10μM·L^-1)呈浓度依赖性地抑制Samd2C、Smad3L的磷酸化，JNK抑制因子(1，3，10μM·L^-1)有抑制Smad3C磷酸化的趋势，对Smad2L磷酸化无明显影响；p38抑制因子(1，3，10μM·L^-1)呈浓度依赖性地抑制Smad2C、Smad3L磷酸化，p38抑制因子(1，3，10μM·L^-1)有抑制Smad3C磷酸化的趋势，对Smad2L磷酸化无明显影响；细胞外信号调节激酶（ERK）抑制因子(1，3，10μM·L^-1)有抑制Smad2L磷酸化的趋势；对Smad2C、Smad3L、Smad3C磷酸化无明显影响。结论：1．CAE可抑制HepG2细胞增殖与移行，提示，CAE可能具有抑制肝癌细胞生长与浸润的作用；2．CAE高、中、低三个剂量可呈浓度依赖性地抑制由TGF-β₁诱导的Smad2C磷酸化，提示，CAE可能经由TGF-β／Smad信号转导通路从而发挥抗肝癌的作用；3．JNK的抑制因子和p38的抑制因子明显抑制TGF-β₁诱导的Smad2C、Smad3L的磷酸化，提示，TGF-β可能通过活化JNK和p38信号途径促进Smad3L磷酸化，参与了肝肿瘤形成过程。更多还原

【Abstract】 Objective To study the mechanism of Anti-hepatoma action of Compound Astragalus Extract（CAE）. Methods The HepG2 cells were stimulated by TGF-Pi or serum and treated with CAE(5～80 mg·L^-1), the proliferation of the cells were analyzed by MTT. The effect of CAE on expression level of pSmad2C and the effects of MAPK inhibitors on expression levels of pSmad2C, pSmad2L, pSmad3C, pSmad3L were investigated respectively by immunoprecipitation and/or Western blotting method in HepG2 cells induceded by TGF-β₁. The effect of CAE on invasive capacity was determined by using transwells, stained by hematoxylin and counted the infiltrating cells in HepG2 cells induced by TGF-β₁. Result 1. The proliferation of the cells was not remarkably stimulated by TGF-β₁ （9 pM） or serum （12.5%） for 0.5 and 1h, but significantly for 2, 4, 8, 16, 24 h, and the elevated proliferation was inhibited by CAE(5～80 mg·L^-1) treatment in a dose-dependent manner. 2. The invasive capacity was decreased by CAE(40 mg·L^-1) treatment in HepG2 cells induced by TGF-β₁ (200 pM·L^-1,12 h). 3. The phosphorylations of Smad2C, Smad2L, Smad3C, and Smad3L were induced by 9 pM·L^-1 TGF-β₁ at 1 h in the HepG2 cells, and the phosphorylation of Smad2C was inhibited by CAE (20～80 mg·L^-1) in a dose-dependent manner. 4. The phosphorylations of Smad2C and Smad3L in HepG2 cells induced by TGF-pi (9 pM·L^-1, 1h) were inhibited by inhibitor for JNK (1, 3, 10μM·L^-1) in a dose-dependent manner, the phosphorylation of Smad3C in HepG2 cells induced by TGF-β₁ (9 pM·L^-1, 1h) was slightly inhibited by inhibitor for JNK (1, 3, 10μM·L^-1),but the phosphorylation of Smad2L in HepG2 cells induced by TGF-β₁(9 pM·L^-1, 1 h)was not affected by inhibitor for JNK (1,3, 10μM·L^-1). The phosphorylations of Smad2C and Smad3L in HepG2 cells induced by TGF-pi (9 pM·L^-1, 1 h) were inhibited by inhibitor for p38(l, 3, 10 μM·L^-1) in a dose-dependent manner, the phosphorylation of Smad3C in HepG2 cells induced by TGF-β₁ (9 pM·L^-1, 1h) was slightly inhibited by inhibitor for p38(l, 3, 10μM·L^-1), but the phosphorylationof Smad2L in HepG2 cells induced by TGF-β₁ (9 pM·L^-1, 1 h) was not affected by inhibitor for p38(l, 3,10μM·L^-1). The phosphorylation of Smad2L in HepG2 cells induced by TGF-β₁ (9 pM·L^-1, 1h) was slightly inhibited by inhibitor for inhibitor for ERK (1, 3, 10 uM·L^-1), but the phosphorylations of Smad2C, Smad3C, Smad3L were not affected by inhibitor for ERK (1, 3, 10μM·L^-1). Conclusion 1. CAE could inhibit proliferation and invasive capacity of HepG2 cells; these suggest that CAE may inhibit growth and invasion of hepatoma cells. 2. CAE inhibit phosphorylation of Smad2C in HepG2 cells induced by TGF-β₁ in a dose-dependent manner, these suggest that CAE may inhibit liver cancer pathogenesis through TGF-β/Smad signaling pathway. 3. Inhibitors for p38 and JNK could inhibit respectively phosphorylation of Smad2C and Smad3L in HepG2 cells induced by TGF-β₁, these suggest that TGF-β₁ might induce the phosphorylation of Smad2C/3L via activated JNK and p38 and then involve in liver cancer pathogenesis.更多还原