摘要
目的 :建立CROC - 1基因表达被阻断的FL -CROC - 1-细胞系 ,研究CROC - 1基因在细胞生长中的作用。方法 :运用反义技术将新近发现的人泛素缀合酶样蛋白基因CROC - 1的适当长度cDNA片断克隆到本室改建的真核细胞表达载体pMAMneo -amp-中 ,经限制性内切酶图谱分析筛选出反向插入的表达CROC - 1反义RNA的重组质粒。将此反义表达重组质粒 (pMAM -antiCROC - 1)用改良的磷酸钙法转染人羊膜FL细胞并用含G418的培养基筛选 ,测定G418抗性的FL -CROC - 1-细胞系的生长速度。结果 :建立的FL-CROC - 1-细胞系在地塞米松诱导下CROC - 1基因表达被反义抑制后 ,其生长速度较对照FL细胞及转染了载体pMAMneo -amp-的FL细胞 (FL -MAMneo)的明显要慢 (P <0 0 5 )。结论 :本研究成功地建立了CROC -1基因表达被阻断的FL -CROC - 1-细胞系 ,并提示CROC - 1基因编码的泛素缀合酶样蛋白在细胞生长中起正调控作用。
AIM:To establish FL- CROC -1 - cell line in which CROC -1 gene expression was blocked and study the role of CROC -1 gene in cell growth. METHODS:The appropriate length cDNA fragment of the recently identified human gene CROC -1 which encodes ubiquitin-conjugating enzyme like protein(Ubc-like protein) was cloned into the reconstructed eukaryotic expression vector pMAMneo-amp - by antisense strategy. The recombinant plasmid which can express CROC -1 antisense RNA was selected by restriction enzyme map analysis. The antisense expression recombinant plasmid pMAM-anti CROC -1 was then transfected into human amnion FL cells by a modified calcium phosphate-mediated transfection procedure and selected with MEM medium containing 400 μg/mL geneticin. Finally ,the growth rate of the G418 resistant FL- CROC -1 - cell line was determined. RESULTS:When the antisense inhibition of CROC -1 gene expression was induced by dexamethasone, the growth rate of the FL- CROC -1 - cell line was obviously slower as compared with that of the control FL cell and FL-MAMneo cell which was established by transfection of plasmid pMAMneo-amp - ( P< 0.05). CONCLUSIONS:FL- CROC -1 - cell line was successfully established in this study and the result of FL- CROC -1 - cell growth suppression suggests that CROC -1 gene encoded Ubc-like protein plays a positive regulation role in cell growth.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2000年第7期577-580,共4页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助! (No .39830 2 10 )
