OBJECTIVE: To evaluate the anti-hepatoma effect of Calmodulin antagonist 0 - 4-ethoxyl-butyl-Berbamine (EBB), one of the berbamine derivatives. METHODS: Monotetrazolium (MTT) method was used to analyze the effect of EBB on the proliferation and growth inhibition effect. Of a hepatoma cell line in vitro. A mouse hepatoma model was induced by injection of hepatoma cells (H22) in the abdominal cavity. The effect of EBB on survival at different concentrations as well as in combination with 5-FU were investigated in vivo. Flow cytometry analysis, dot blot hybridization, western blot, immunochemistry, enzyme-linked lectin assay (ELISA), trifluoperazine (TFP) and electron microscopic observation were used to study the effect of EBB on cell cycle process, P53 mRNA and protein levels, calmodulin content and ultrastractural changes of hepatoma cells. RESULTS: EBB exerts a very strong inhibitory effect on human hepatoma cell line 7402 and mouse hepatoma cell line H22 in vitro. The IC(50) value of EBB for the two cell lines are 3.312 microg/ml and 1.167 microg/ml, respectively. The sensitivity of H22 cells to 5-FU can be markedly enhanced: The IC(50) dosage of 5-Fu can be decreased from 0.75 microg/ml down to 0.15 microg/ml, when jointly administered with nontoxic dosages of EBB (IC(10)). In vivo, EBB can prolong the lifespan of mice with ascites H22 to more than three months. 64% of mice survived, while all animals in the control group died by the 18th day. When EBB (5 mg x kg(-1) x d(-1)) is jointly used with 5-FU (25 mg x ml(-1) x d(-1)), 73% of mice with ascites H22 survived, much higher than 27% in the 5-FU treated group. EBB can enhance the anti-hepatoma ability of 5-Fu treatment. EBB mechanism against hepatoma: P53 expression in the EBB treated group is substantially higher than that in the control group. EBB increased the translation of P53. As a calmodulin antagonist, EBB decreases amount of the CaM in hepatoma cells and blocked the hepatoma cell proliferation cycle at the G(2)M phase. Before the G(0)/G(1) phase, a
目的利用基因工程技术获得重组的人促血液血管生成素(HAPO),以探讨其对骨髓细胞的作用。方法提取人胎肝总RNA,利用RT-PCR、克隆HAPO cDNA插入表达载体pET32c,使之在大肠杆菌BL21(DE3)中表达;用DEAESepharose Fast Flow阴离子交换柱、Ni-Chelating Sepharose Fast Flow亲和柱以及SP Sepharose Fast Flow阳离子交换柱分离纯化,肠激酶酶切,液体培养小鼠骨髓细胞。结果经IPTG诱导HAPO实现了在大肠杆菌中的可溶性表达,经一系列层析获得融合的rh-HAPO,肠激酶酶切除去N-端融合部分后,获得高纯度的rh-HAPO。液体培养小鼠骨髓实验发现rh-HAPO可促进CD34+细胞和flk-1细胞的增殖。结论重组蛋白rh-HAPO可促进造血干/祖细胞的增殖。
