联合诱导法对K562细胞红系分化及其相关基因和膜蛋白表达的影响  

Co-induce of K562 cells into erythroid differentiation and expression of gene and membrane protein on cells

作　　者：黄前川[1] 李津婴[1] 周虹[1] 许燕群[1] 黄正霞[1] 陈莉[1] 

机构地区：[1]第二军医大学长海医院血液科,上海200433

出　　处：《临床检验杂志》2009年第1期10-13,共4页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然科学基金资助项目(编号:30570460)

摘　　要：目的优化K562细胞体外红系诱导分化条件,分析红系相关基因和膜蛋白的表达,探讨K562细胞的分化潜能。方法采用红系诱导联合培养法,优化丁酸钠、促红细胞生成素、氯化高铁血红素、枸橼酸铁等成分组合和剂量组合。鉴定指标选用红系血红素合成酶(eALAS)mRNA和粒系bcr/ablmRNA表达丰度分析、细胞形态学观察、联苯胺染色阳性率计数。红系分化进一步鉴定指标选用Real-timePCR检测10种红系分化相关基因的表达;流式细胞术测定红系细胞膜标志物。结果K562细胞诱导120h后,联苯胺染色阳性率达100%;α、β收缩蛋白(spectrinα、β)、带3蛋白(band3)、eALAS、整合素相关蛋白(CD47)、RhD血型抗原(RhD)、锚蛋白(ankyrin)、红细胞膜带4.2蛋白(EPB4.2)的mRNA水平为对照组的8.05、14.58、22.87、14.52、26.53、3.48、2.71、1.94倍,而bcr/ablmRNA水平为对照组的39%;红系膜蛋白CD47、band3、RhD水平明显增高(P<0.01),血型糖蛋白A(GPA)无明显差异。结论优化组合体外红系诱导分化法可使K562细胞稳定向红系分化,并且细胞状态良好,易于进行红系统疾病研究;CD47、band3、RhD这3种膜蛋白可以作为早期红系分化的敏感指标。Objective To optimize the inducing conditions for differentiation of K562 cells into erythroid lineage in vitro, and assess the potentiality of erythroid differentiation of K562 cells. Methods The expressions of relevant gene and membrane protein were examined. K562 ceils were co-cultured with sodium butyrate, erythropoietin, hemin and ferric citrate at optimized concentrations. The morphology of the cells was observed under microscope. The hemoglobin content in K562 cells was monitored by benzidine staining. The expression of bcr/abl and eALAS mRNA represented the abundance of granulocytic and erythroid lineage respectively. The expression of 10 ery- throid differentiation-related genes and certain erythroid membrane markers were determined by real-time PCR and flow cytometry. Re- sults The percentage of benzidine-positive cells induced under the optimized conditions reached 100% following the inducement of 120 hours. The mRNA levels of speetrin alpha, spectrin beta, band 3, eALAS, CD47, RhD, ankyrin, EPB 4.2 determined by realtime- PCR increased up to 8.05, 14.58, 22.87, 14.52, 26.53, 3.48, 2.71 and 1.94-fold respectively compared to the control group, while the mRNA level of bcr/abl was 0.39-fold reduction. The expression of 3 red cell membrane markers （ CD47, band3 and RhD） in the induced cells was much higher than that in the controls （P 〈 0.01 ）. No difference of glycophorin A expression was found between the 2 groups. Conclusions K562 cells could stably differentiate into erythroid lineage by optimized co-induce with multi-factors, and the cells were in desirable conditions and suitable for research on the disorders involved red blood cells. RhD, CD47 and band 3 may be sensitive erythroid markers in early phase.

关 键 词：K562细胞 红系诱导分化 基因和膜蛋白表达 

分 类 号：Q78[生物学—分子生物学]