目的构建pcD N A3.1(+)/G STP1真核表达载体并将其稳定转染到肺鳞癌细胞N C I-H 520中,建立稳定转染的N C I-H 520细胞系,为后续研究奠定基础。方法以pD N R-LIB-G STP1为模板,用PC R扩增G STP1 cD N A的编码区,并将扩增的cD N A片段与...目的构建pcD N A3.1(+)/G STP1真核表达载体并将其稳定转染到肺鳞癌细胞N C I-H 520中,建立稳定转染的N C I-H 520细胞系,为后续研究奠定基础。方法以pD N R-LIB-G STP1为模板,用PC R扩增G STP1 cD N A的编码区,并将扩增的cD N A片段与载体连接后亚克隆到真核表达载体pcD N A3.1(+)中。重组子经酶切分析及测序鉴定后,用脂质体转染技术将其导入到人肺鳞癌细胞系N C I-H 520,经G 418筛选并建立稳定的转染细胞株,应用W estern blotting检测转染前后该细胞株G STP1基因在蛋白水平的表达。结果 pcD N A3.1(+)/G STP1经酶切鉴定及D N A测序证实序列完全正确,真核表达载体构建成功;经W estern blotting检测,重组质粒转染株中G STP1基因的蛋白表达水平明显高于对照组,证实G STP1基因已稳定转染到N C I-H 520细胞中并得到表达。结论成功构建了pcD N A3.1(+)/G STP1真核表达质粒,建立了稳定表达G STP1的N C I-H 520细胞系,为进一步研究G STP1的生物学功能奠定了基础。展开更多
Annexin A5 is a Ca2?-dependent phospholipidbinding protein and protein kinase C inhibitory protein. It has a potential role in cellular signal transduction, inflammation, growth and differentiation. In this study, we ...Annexin A5 is a Ca2?-dependent phospholipidbinding protein and protein kinase C inhibitory protein. It has a potential role in cellular signal transduction, inflammation, growth and differentiation. In this study, we evaluated the expression of this protein in lung tumor tissues and subsequently established a NCI-H520 cell line that stably expresses the wild-type ANXA5 gene to determine the effects of annexin A5 upregulation on the cell morphology, proliferation and metastasis potential in vitro.The effects of annexin A5 on NCI-H520 cells were tested by crystal violet staining, CCK-8 assay, scratch wound assay, and Transwell assay. The expressions of Akt,PCNA, vimentin, and E-cadherin were examined by Western blot assay. In this study, we demonstrated that annexin A5 is expressed at lower levels in tumor tissues compared with normal tissues. Additionally, the upregulation of this protein may inhibit the proliferation, migration, and invasion abilities of NCI-H520 cells in vitro. The transfected cells were arrested in the G1/S phase of the cell cycle, and the expression levels of Akt, PCNA and Vimentin were downregulated, while E-cadherin was upregulated.展开更多
文摘目的构建pcD N A3.1(+)/G STP1真核表达载体并将其稳定转染到肺鳞癌细胞N C I-H 520中,建立稳定转染的N C I-H 520细胞系,为后续研究奠定基础。方法以pD N R-LIB-G STP1为模板,用PC R扩增G STP1 cD N A的编码区,并将扩增的cD N A片段与载体连接后亚克隆到真核表达载体pcD N A3.1(+)中。重组子经酶切分析及测序鉴定后,用脂质体转染技术将其导入到人肺鳞癌细胞系N C I-H 520,经G 418筛选并建立稳定的转染细胞株,应用W estern blotting检测转染前后该细胞株G STP1基因在蛋白水平的表达。结果 pcD N A3.1(+)/G STP1经酶切鉴定及D N A测序证实序列完全正确,真核表达载体构建成功;经W estern blotting检测,重组质粒转染株中G STP1基因的蛋白表达水平明显高于对照组,证实G STP1基因已稳定转染到N C I-H 520细胞中并得到表达。结论成功构建了pcD N A3.1(+)/G STP1真核表达质粒,建立了稳定表达G STP1的N C I-H 520细胞系,为进一步研究G STP1的生物学功能奠定了基础。
基金supported by the National Basic Research Program of China (2012CB967003)National Natural Science Foundation of China (81272225)+2 种基金the Research Fund for the Docoral Program of Higher Education of China (20132106110006)the Science Foundation of Liaoning Province Education Administration (L2013350)Dalian Science Foundation (2011E12SF036).
文摘Annexin A5 is a Ca2?-dependent phospholipidbinding protein and protein kinase C inhibitory protein. It has a potential role in cellular signal transduction, inflammation, growth and differentiation. In this study, we evaluated the expression of this protein in lung tumor tissues and subsequently established a NCI-H520 cell line that stably expresses the wild-type ANXA5 gene to determine the effects of annexin A5 upregulation on the cell morphology, proliferation and metastasis potential in vitro.The effects of annexin A5 on NCI-H520 cells were tested by crystal violet staining, CCK-8 assay, scratch wound assay, and Transwell assay. The expressions of Akt,PCNA, vimentin, and E-cadherin were examined by Western blot assay. In this study, we demonstrated that annexin A5 is expressed at lower levels in tumor tissues compared with normal tissues. Additionally, the upregulation of this protein may inhibit the proliferation, migration, and invasion abilities of NCI-H520 cells in vitro. The transfected cells were arrested in the G1/S phase of the cell cycle, and the expression levels of Akt, PCNA and Vimentin were downregulated, while E-cadherin was upregulated.
