人干细胞因子受体c-Kit稳定表达细胞株的构建  

Establishment of Engineered Cells with Stably Expressing the Human Stem Cell Factor Receptor c-Kit

作　　者：熊安琪[1] 陈松森[1] 狄旭[1] 刘长征[1] 姚杰[1] 

机构地区：[1]中国医学科学院基础医学研究所,中国协和医科大学基础医学院,北京100005

出　　处：《中国生物化学与分子生物学报》2001年第4期478-482,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家"九五"攻关项目资助 (No.96 CO2 0 1 10 1)&&

摘　　要：干细胞因子 (SCF)是一种重要的造血因子 ,其受体c Kit具有酪氨酸激酶活性 .SCF c Kit介导的细胞内信号转导在造血、肥大细胞的生成及其功能、以及生殖细胞和黑色素细胞的发育中起着关键的作用 .通过构建人c kitcDNA的pcDNA3.1真核表达载体 ,转染不表达人c kit的小鼠髓系祖细胞FDC P1.经Zeocin抗性筛选 ,PCR、RT PCR、Western印迹分析、流式细胞仪分析等方法检测到人c kit基因的稳定整合和表达 ,并分布于细胞表面 ,证明获得了稳定表达人c Kit受体的细胞株 .用MTT法检测重组细胞株增殖特性 ,表明重组人SCF可刺激其增殖 .为进一步研究人cStem cell factor(SCF)is a cytokine critical for normal hematopoiesis.The receptor for SCF is c\|Kit, a tyrosine kinase.The signal transduction mediated by SCF/c\|Kit plays a critical role in hematopoiesis,in mast cell production,and in the development of germ cells and melanocytes.Human c\|kit cDNA was constructed into a mammalian expression vector pcDNA3.1,then transfected into a murine myeloid progenitor FDC\|P1 which does not express c\|kit. The transformants were selected by Zeocin and detected by PCR,RT\|PCR,Western blot and flow cytometric analysis.The results indicate that c\|kit gene is stably integrated and expressed on the surface of FDC\|P1 cells and stable cell lines which continuously express human c\|kit gene were obtained.The proliferation tests using MTT method showed that the recombinant cell lines proliferated after the stimulation of recombinant human SCF.The results implied that cell lines obtained may be useful for the further research on both the intercellular signal transduction mediated by human c\|Kit receptor and the determination of the biological activity of the recombinant human SCF.

关 键 词：人干细胞因子受体c-Kit 转染 FDC-P1细胞株 表达 增殖 

分 类 号：Q786[生物学—分子生物学]