摘要
目的:了解优势表达鞘氨醇激酶-1(SphK-1)对慢性粒细胞白血病细胞系K562细胞生物学特性的影响。方法:使用pLXSN、pLXSN-SphK-1的重组逆转录病毒液感染K562细胞并用终浓度1 000 ng/m l的G418筛选感染pLXSN和pLXSN-SphK-1基因的K562细胞;取对数生长期的K562-pLXSN和K562-SphK细胞提取蛋白收集胞浆蛋白,应用BCA-200蛋白检测试剂盒进行蛋白定量。根据蛋白定量取蛋白含量相同的蛋白提取上清行γ3-2P-ATP掺入法检测K562-pLXSN和K562-SphK细胞SphK-1活性;并应用MTT法检测K562-pLXSN和K562-SphK细胞的增殖情况及对不同剂量格列卫的敏感性。结果:K562细胞转染SphK-1基因比转染空载体后的细胞SphK-1酶活性显著增高;K562-pLXSN细胞和K562-SphK细胞培养24 h、48和72 h后细胞增殖无显著性差异;当格列卫浓度为0.1和0.25μmol/L时,K562-PLXSN和K562-SphK细胞培养72 h后的细胞存活率无明显差异;当格列卫浓度为0.5至10μmol/L时,K562-SphK细胞存活率显著低于K562-PLXSN细胞(P<0.05)。结论:优势表达SphK-1基因对K562细胞自然增殖无显著影响,但增加了细胞对浓度高于0.5μmol/L格列卫的敏感性。
Objective：To understand the impact on the biology of K562 cell by the dominant expression of sphingosine kinase-1, Methods： K562 cells were separately infected with pLXSN and pLXSN-SphK recombinant retrovirus supernatant, and then selected for 1 000 ng/ml G418 resistance. The protein of well-growthing K562-pLXSN and K562-SphK cells were collected, and the intracellular SphK-1 activities were assayed. The proliferation status and the sensitivity to Gleevec of K562-SphK cells and K562-pLXSN cells were evaluated with MTT. Results： The activity of SphK-1 in K562-SphK cells was significantly higher than that in K562-pLXSN cells. There was no markedly difference in the proliferation status between K562-SphK cells and K562-pLXSN cells. K562-SphK cells were more sensitive to Gleevec than K562-pLXSN cells after Gleevec concentration at 0.5-10 μmol/L（ P 〈 0.05 ）. Conclusions： Dominant expression of SphK-1 in K562 cells didnt affect cell proliferation, but increased the sensitivity to higher concentration of Gleevec.
出处
《军医进修学院学报》
CAS
北大核心
2007年第1期20-22,共3页
Academic Journal of Pla Postgraduate Medical School
