目的探讨arresten抑制人胃癌细胞SGC-7901裸鼠移植瘤生长的作用。方法构建包含arresten cDNA的分泌型真核表达质粒pcDNA3.1(+)-ss-arresten,并以脂质体法将重组质粒转染至SGC-7901细胞。蛋白印迹法(W estern b lot)检测SGC-7901是否分...目的探讨arresten抑制人胃癌细胞SGC-7901裸鼠移植瘤生长的作用。方法构建包含arresten cDNA的分泌型真核表达质粒pcDNA3.1(+)-ss-arresten,并以脂质体法将重组质粒转染至SGC-7901细胞。蛋白印迹法(W estern b lot)检测SGC-7901是否分泌表达arresten,同时,通过生长曲线绘制及生长周期测定的方法了解上述转基因措施本身对SGC-7901细胞的体外生物学特性的影响。将转基因的SGC-7901细胞株接种至裸鼠皮下,使其在接种局部表达arresten,观察肿瘤生长情况。结果成功构建了分泌型真核表达载体pcDNA3.1(+)-ss-arresten,并获得了可分泌表达arresten的人胃癌SGC-7901细胞株。质粒转染对SGC-7901细胞增殖特性无影响。转基因SGC-7901细胞的裸鼠移植瘤生长缓慢。结论arresten抑制人胃癌SGC-7901移植瘤的生长,其途径可能是通过抑制其滋养血管的生长而实现。展开更多
BACKGROUND: The proliferation and metastasis of cancers depend on angiogenesis. This property provides the feasibility for the treatment of cancer by inhibition of angiogenesis, and many angiogenic inhibitors have bee...BACKGROUND: The proliferation and metastasis of cancers depend on angiogenesis. This property provides the feasibility for the treatment of cancer by inhibition of angiogenesis, and many angiogenic inhibitors have been demonstrated to effectively inhibit angiogenesis and consequently the growth of solid cancer. As for the newly identified angiogenesis inhibitor, arresten, some studies have found its high activity on restrainting tumor vessel. This study was to assess the anti-angiogenic activity of arresten. METHODS: The arresten gene was obtained from a healthy puerpera's placenta tissue by the reverse transcriptase-polymerase chain reaction (RT-PCR) method, and molecular cloning to prokaryotic expression plasmid pBV220 by recombination strategy. The prokaryotic expression plasmid pBV220/arr was identified by restriction enzyme digestion and sequenced. The pBV220/arr was transformed into E. coli JM109, DH5α, BL21 and BL21 (DE3) by the CaCl_2 transformation method. The arresten expression level was detected by SDS-PAGE. The expressed product was purlfled, re-naturalized and detected for its biological activity of inhibiting the angiogenesis of chorioallantoic membrane (CAM). RESULTS: The arresten gene was cloned and pBV220/arr was constructed. The arresten expression level of protein was highly increased after pBV220/arr was transformed into E. coli BL21 (DE3). SDS-PAGE showed that the expressed arresten proteins were mainly inclusion bodies and had a molecular weight of 26 kDa. The expressed arresten protein showed evident biological activities. CONCLUSIONS: The successful construction of recombinant plasmid pBV220/arr and the effective expression in E. coil have laid a foundation for further study of its anti-angiogenic function and may pave the way for future antitumor application.展开更多
To express recombinant arresten in Escherichia coli (E.Coli) and investigate its biological activity, prokaryotic expression vector of human arresten gene was constructed by gene engineering. Human arresten gene was a...To express recombinant arresten in Escherichia coli (E.Coli) and investigate its biological activity, prokaryotic expression vector of human arresten gene was constructed by gene engineering. Human arresten gene was amplified from recombinant plasmid pGEMArr by polymerase chain reaction (PCR), and inserted into prokaryotic expression vector pRSET containing T7 promoter. Restriction analysis and DNA sequencing verified that the arresten gene was correctly cloned into the expression vector. The recombinant plasmid pRSETAt was subsequently transformed into E.coli BL21 (DE3), and the target gene was expressed under induction of IPTG. SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 29 kD (1 kD=0.992 1 ku) amounted to 29 % of the total bacterial proteins. After purification and renaturation, the recombinant protein could significantly suppress the proliferation of human umbilical vein endothelial cells (HUVECs). These results suggested that the expression of a biologically active form of human arresten in the pRSET expression system laid a foundation for further study on the mechanistic insight into arresten action on angiogenesis and the development of powerful anti-cancer drugs.展开更多
The eukaryotic expression of human arresten gene and its effect on the proliferation of in vitro cultured vascular smooth cells （VSMCs） in vitro were investigated. COS-7 cells were transfected with recombinant eukar...The eukaryotic expression of human arresten gene and its effect on the proliferation of in vitro cultured vascular smooth cells （VSMCs） in vitro were investigated. COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome. Forty-eight h after transfection, reverse transcription-polymerase chain reaction （RT-PCR） was used to detect the expression of arresten mRNA in the cells, while Western blot assay was applied to detect the expression of arresten protein in concentrated supernatant. Primary VSMCs from thoracic aorta of male Sprague-Dawley rats were cultured using the tissue explant method, and identified by immunohistochemical staining with a smooth muscle-specific anti-α- actin monoclonal antibody before serial subcuhivation. VSMCs were then co-cultured with the concentrated supernatant and their proliferation was detected using Cell Counting Kit-8 （CCK-8） in vitro. The results showed that RT-PCR revealed that the genome of arresten-transfected cells contained a 449 bp specific fragment of arresten gene, suggesting the successful transfection. Success- ful protein expression in supernatants was confirmed by Western blot. CCK-8 assay showed that the proliferation of VSMCs were inhibited significantly by arresten protein as compared with control cells （F=40. 154, P〈0.01）. It was concluded that arresten protein expressed in eukaryotic cells can inhibit proliferation of VSMCs effectively in vitro, which would provide possibility to the animal experiments.展开更多
文摘目的探讨arresten抑制人胃癌细胞SGC-7901裸鼠移植瘤生长的作用。方法构建包含arresten cDNA的分泌型真核表达质粒pcDNA3.1(+)-ss-arresten,并以脂质体法将重组质粒转染至SGC-7901细胞。蛋白印迹法(W estern b lot)检测SGC-7901是否分泌表达arresten,同时,通过生长曲线绘制及生长周期测定的方法了解上述转基因措施本身对SGC-7901细胞的体外生物学特性的影响。将转基因的SGC-7901细胞株接种至裸鼠皮下,使其在接种局部表达arresten,观察肿瘤生长情况。结果成功构建了分泌型真核表达载体pcDNA3.1(+)-ss-arresten,并获得了可分泌表达arresten的人胃癌SGC-7901细胞株。质粒转染对SGC-7901细胞增殖特性无影响。转基因SGC-7901细胞的裸鼠移植瘤生长缓慢。结论arresten抑制人胃癌SGC-7901移植瘤的生长,其途径可能是通过抑制其滋养血管的生长而实现。
基金This work was supported by a grant from Science and Technology Fund of Shanxi Province, China (No. 042082).
文摘BACKGROUND: The proliferation and metastasis of cancers depend on angiogenesis. This property provides the feasibility for the treatment of cancer by inhibition of angiogenesis, and many angiogenic inhibitors have been demonstrated to effectively inhibit angiogenesis and consequently the growth of solid cancer. As for the newly identified angiogenesis inhibitor, arresten, some studies have found its high activity on restrainting tumor vessel. This study was to assess the anti-angiogenic activity of arresten. METHODS: The arresten gene was obtained from a healthy puerpera's placenta tissue by the reverse transcriptase-polymerase chain reaction (RT-PCR) method, and molecular cloning to prokaryotic expression plasmid pBV220 by recombination strategy. The prokaryotic expression plasmid pBV220/arr was identified by restriction enzyme digestion and sequenced. The pBV220/arr was transformed into E. coli JM109, DH5α, BL21 and BL21 (DE3) by the CaCl_2 transformation method. The arresten expression level was detected by SDS-PAGE. The expressed product was purlfled, re-naturalized and detected for its biological activity of inhibiting the angiogenesis of chorioallantoic membrane (CAM). RESULTS: The arresten gene was cloned and pBV220/arr was constructed. The arresten expression level of protein was highly increased after pBV220/arr was transformed into E. coli BL21 (DE3). SDS-PAGE showed that the expressed arresten proteins were mainly inclusion bodies and had a molecular weight of 26 kDa. The expressed arresten protein showed evident biological activities. CONCLUSIONS: The successful construction of recombinant plasmid pBV220/arr and the effective expression in E. coil have laid a foundation for further study of its anti-angiogenic function and may pave the way for future antitumor application.
基金This project was supported by grants from the National Natural Science Foundation of China ( No.30271242,30371396).
文摘To express recombinant arresten in Escherichia coli (E.Coli) and investigate its biological activity, prokaryotic expression vector of human arresten gene was constructed by gene engineering. Human arresten gene was amplified from recombinant plasmid pGEMArr by polymerase chain reaction (PCR), and inserted into prokaryotic expression vector pRSET containing T7 promoter. Restriction analysis and DNA sequencing verified that the arresten gene was correctly cloned into the expression vector. The recombinant plasmid pRSETAt was subsequently transformed into E.coli BL21 (DE3), and the target gene was expressed under induction of IPTG. SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 29 kD (1 kD=0.992 1 ku) amounted to 29 % of the total bacterial proteins. After purification and renaturation, the recombinant protein could significantly suppress the proliferation of human umbilical vein endothelial cells (HUVECs). These results suggested that the expression of a biologically active form of human arresten in the pRSET expression system laid a foundation for further study on the mechanistic insight into arresten action on angiogenesis and the development of powerful anti-cancer drugs.
基金This project was supported by a grant from National Natu-ral Sciences Foundation of China ( No . 30371396 ,30271242)
文摘The eukaryotic expression of human arresten gene and its effect on the proliferation of in vitro cultured vascular smooth cells （VSMCs） in vitro were investigated. COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome. Forty-eight h after transfection, reverse transcription-polymerase chain reaction （RT-PCR） was used to detect the expression of arresten mRNA in the cells, while Western blot assay was applied to detect the expression of arresten protein in concentrated supernatant. Primary VSMCs from thoracic aorta of male Sprague-Dawley rats were cultured using the tissue explant method, and identified by immunohistochemical staining with a smooth muscle-specific anti-α- actin monoclonal antibody before serial subcuhivation. VSMCs were then co-cultured with the concentrated supernatant and their proliferation was detected using Cell Counting Kit-8 （CCK-8） in vitro. The results showed that RT-PCR revealed that the genome of arresten-transfected cells contained a 449 bp specific fragment of arresten gene, suggesting the successful transfection. Success- ful protein expression in supernatants was confirmed by Western blot. CCK-8 assay showed that the proliferation of VSMCs were inhibited significantly by arresten protein as compared with control cells （F=40. 154, P〈0.01）. It was concluded that arresten protein expressed in eukaryotic cells can inhibit proliferation of VSMCs effectively in vitro, which would provide possibility to the animal experiments.
