以人工饲养的北星二号蚯蚓为材料,通过对14种进口和国产离子交换树脂和 DEAE-纤维素的筛选试验和比较研究,确定了蚯蚓纤溶酶的分离纯化的较佳工艺:组织匀浆或磷酸缓冲液保温提取→Amberlite IRA 904或 DEAE-纤维素离子交换柱层析分离→S...以人工饲养的北星二号蚯蚓为材料,通过对14种进口和国产离子交换树脂和 DEAE-纤维素的筛选试验和比较研究,确定了蚯蚓纤溶酶的分离纯化的较佳工艺:组织匀浆或磷酸缓冲液保温提取→Amberlite IRA 904或 DEAE-纤维素离子交换柱层析分离→Sephadex G 100或 Sephadtx G 75凝胶层析纯化→真空冷冻干燥得蚯蚓纤溶酶冻干粉。该工艺所需设备简单,操作方便,适合中。展开更多
Background The earthworm fibrinolytic enzyme (EFE) is a complex protein enzyme that is widely distributed in the earthworm's digestive cavity. Possessing strong protein hydrolysis activity, EFE not only has a direc...Background The earthworm fibrinolytic enzyme (EFE) is a complex protein enzyme that is widely distributed in the earthworm's digestive cavity. Possessing strong protein hydrolysis activity, EFE not only has a direct effect on fibrin, but also can activate plasminogen. Its therapeutic and preventative effects on thrombosis-related disease have been confirmed clinically. Recently, there has been increased interest in the anti-tumor activity of EFE. In this study, the anti-tumor activity of EFE, isolated from Eisenia foetida, on human hepatoma cells was evaluated in vitro and in vivo. The potential mechanisms involved were also studied. Methods In vitro experiments were performed in four human hepatoma cell lines: HLE, Huh7, PLC/PRF/5 and HepG2. After treatment with EFE in various concentrations, the inhibition of the rate of cell proliferation was measured. For the in vivo studies, tumor-bearing models xenografted with Huh7 cells were developed in nude mice, and then the mice were fed with EFE once a day for 4 weeks, and the control group received only saline. An inhibitory effect on tumor growth was observed. Also, apoptosis was observed with flow cytometric assay and fluorescent dye staining with acridine orange and ethidium bromide (AO/EB). The expression of matrix metalloproteinase 2 (MMP-2) were detected by Western blotting assay. Results After treatment with various concentrations of EFE, the proliferation of all hepatoma cell lines was suppressed to varying degrees in vitro. The IC50 for HLE, Huh7, PLC/PCF/5 and HepG2 were 2.11, 5.87, 25.29 and 17.30 uku/ml, respectively. After administration of EFE orally for 4 weeks, the growth of tumor xenograft of Huh7 cells in nude mice was significantly inhibited in vivo. The tumor inhibitory rates in the EFE 500 uku/(kg·d) and 1000 uku/(kg·d) groups were 46.08% (compared with control group, P=0.026) and 57.52% (compared with control group, P=0.002) respectively. Meanwhile, the average weight of body, spleen or thymus did not show any remarkable differences among the various groups. The population in sub-G1 stage was more in the EFE treated groups than in the control group according to flow cytometric assay. After treatment with EFE 0, 5, 10 uku/ml for 72 hours, the apoptotic rates were 3.5%, 10.9% and 12.3% in HLE cells, and 5.0%, 24.7% and 34.5% in Huh7 cells respectively. Under fluorescent staining with AO/EB, the apoptotic morphological changes could be detected more significantly in the EFE treated groups than in the untreated groups. After treatment with EFE in doses of 0, 5, 10 uku/ml for 72 hours, the apoptotic rates were 3.02%, 8.76%, 10.54% in HLE cells, and 3.95%, 18.27%, 30.89% in Huh7 cells respectively. The apoptosis-inducing effects of EFE occurred in a dose dependent manner. Western blotting assay showed that, after treatment with EFE, the secretions of MMP-2 were significantly inhibited in HLE and Huh7 cells. Conclusions EFE showed significant anti-tumor activity in hepatoma cells both in vitro and in vivo, which may be because EFE could induce apoptosis of hepatoma cells and inhibit the expression of MMP-2. This suggests that EFE has a potential role in the treatment of hepatoma.展开更多
文摘以人工饲养的北星二号蚯蚓为材料,通过对14种进口和国产离子交换树脂和 DEAE-纤维素的筛选试验和比较研究,确定了蚯蚓纤溶酶的分离纯化的较佳工艺:组织匀浆或磷酸缓冲液保温提取→Amberlite IRA 904或 DEAE-纤维素离子交换柱层析分离→Sephadex G 100或 Sephadtx G 75凝胶层析纯化→真空冷冻干燥得蚯蚓纤溶酶冻干粉。该工艺所需设备简单,操作方便,适合中。
文摘Background The earthworm fibrinolytic enzyme (EFE) is a complex protein enzyme that is widely distributed in the earthworm's digestive cavity. Possessing strong protein hydrolysis activity, EFE not only has a direct effect on fibrin, but also can activate plasminogen. Its therapeutic and preventative effects on thrombosis-related disease have been confirmed clinically. Recently, there has been increased interest in the anti-tumor activity of EFE. In this study, the anti-tumor activity of EFE, isolated from Eisenia foetida, on human hepatoma cells was evaluated in vitro and in vivo. The potential mechanisms involved were also studied. Methods In vitro experiments were performed in four human hepatoma cell lines: HLE, Huh7, PLC/PRF/5 and HepG2. After treatment with EFE in various concentrations, the inhibition of the rate of cell proliferation was measured. For the in vivo studies, tumor-bearing models xenografted with Huh7 cells were developed in nude mice, and then the mice were fed with EFE once a day for 4 weeks, and the control group received only saline. An inhibitory effect on tumor growth was observed. Also, apoptosis was observed with flow cytometric assay and fluorescent dye staining with acridine orange and ethidium bromide (AO/EB). The expression of matrix metalloproteinase 2 (MMP-2) were detected by Western blotting assay. Results After treatment with various concentrations of EFE, the proliferation of all hepatoma cell lines was suppressed to varying degrees in vitro. The IC50 for HLE, Huh7, PLC/PCF/5 and HepG2 were 2.11, 5.87, 25.29 and 17.30 uku/ml, respectively. After administration of EFE orally for 4 weeks, the growth of tumor xenograft of Huh7 cells in nude mice was significantly inhibited in vivo. The tumor inhibitory rates in the EFE 500 uku/(kg·d) and 1000 uku/(kg·d) groups were 46.08% (compared with control group, P=0.026) and 57.52% (compared with control group, P=0.002) respectively. Meanwhile, the average weight of body, spleen or thymus did not show any remarkable differences among the various groups. The population in sub-G1 stage was more in the EFE treated groups than in the control group according to flow cytometric assay. After treatment with EFE 0, 5, 10 uku/ml for 72 hours, the apoptotic rates were 3.5%, 10.9% and 12.3% in HLE cells, and 5.0%, 24.7% and 34.5% in Huh7 cells respectively. Under fluorescent staining with AO/EB, the apoptotic morphological changes could be detected more significantly in the EFE treated groups than in the untreated groups. After treatment with EFE in doses of 0, 5, 10 uku/ml for 72 hours, the apoptotic rates were 3.02%, 8.76%, 10.54% in HLE cells, and 3.95%, 18.27%, 30.89% in Huh7 cells respectively. The apoptosis-inducing effects of EFE occurred in a dose dependent manner. Western blotting assay showed that, after treatment with EFE, the secretions of MMP-2 were significantly inhibited in HLE and Huh7 cells. Conclusions EFE showed significant anti-tumor activity in hepatoma cells both in vitro and in vivo, which may be because EFE could induce apoptosis of hepatoma cells and inhibit the expression of MMP-2. This suggests that EFE has a potential role in the treatment of hepatoma.