联合培养法优化K562细胞红系诱导分化及红系相关基因和膜蛋白表达分析

Optimization of multi-factors co-induce of K562 cell into erythroid differentiation and analysis of gene and membrane protein expression of erythroid cell

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，优化诱导试剂丁酸钠、红细胞生成素、氯化高铁血红素等成分组合和剂量组合。诱导效率鉴定指标选用红系ALAS 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Realtime-PCR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。结果优化红系诱导组合联合培养K562细胞120h后，联苯胺染色计数阳性细胞可达1。0％。Realtime-PCR检测Spectrinα、Spectrinβ、band3、eALAS、CD47、RhD、EPB4．2的mRNA水平分别为对照组的8．05、14．58、22．87、14．52、26．53、3．48、1．94倍，而bcr／ablmRNA水平为对照组的0．39倍。流式细胞术测定红系膜蛋白CD47，band3、RhD水平明显增高（P〈0．01），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，良好细胞状态利于转外源基因操作，进行红系统疾病研究。RhD比GPA更早在红系细胞表达，可作为早期红系特异标记。

Objective Optimization of the conditions to induce K562 cell into erythroid linage, and assessment of K562 cell erythroid differentiation potentiality by examining the expression of relevant gene and membrane protein. Method K562 cells were co-cultured with sodium butyrate, erythropoiehuman and hemin at the optimal concentrations. The morphology of the cells was observed by microscope and the hemo- globin content of K562 cells was monitored by benzidine staining. The abundance of P210 bcr/abl and eALAS mRNA, respectively representting granulocytic and erythroid lineages were used to evaluate the op- timized induction. Further explore was performed by Realtime-PCR and flow cytometry to test 10 kinds of erythroid differentiation-related gene expression as well as some of erythroid membrane markers. Result The percentage of benzidine positive cells induced by the optimized condition was up to 100% at 120 hours. Realtime-PCR showed the level of Spectrinα, Spectrinβ, band3, eALAS, CD47, RhD, EPB4.2 was 14.52, 22.87, 26.53, 8.05, 14.58, 2.71, 3.48 fold respectively compared to the control group,while that of the bcr/abl was 0.39 fold. The expression of three red cell membrane markers （CD47, band3 and RhD） were much higher than those of the controls （P〈0.01） and the expression of GPA was negative. Conclusion K562 cells could stably differentiate into erythroid linage by the optimized culturation of multi-factors co-in duce. And all the cells were in good condition suitable for transgenic experiment in research on red blood cell disorders. Our findings also demonstrated that RhD expression was earlier than GPA at the stage of erythoid maturity so could be a reliably specific early-erythroid marker.