CAJ | 학술논문

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/abl mRNA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120 h后,联苯胺染色计数阳性细胞可达100%.Realtime-PCR检测Spectrina、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ber/abl mRNA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.
목적 우화K562세포체외홍계유도분화조건,분석홍계상관기인화막단백적표체,탐토K562세포적분화잠능.방법 채용홍계유도연합배양법,우화유도시제정산납、홍세포생성소、록화고철혈홍소등성분조합화제량조합.유도효솔감정지표선용홍계ALAS mRNA화립계bcr/abl mRNA표체봉도분석、세포형태학관찰、련분알염색양성솔계수.홍계분화진일보감정지표선용Realtime-PCR검측10충홍계분화상관기인적표체;류식세포술측정홍계세포막표지물.결과 우화홍계유도조합연합배양K562세포120 h후,련분알염색계수양성세포가체100%.Realtime-PCR검측Spectrina、Spectrinβ、band3、eALAS、CD47、RhD、EPB4.2적mRNA수평분별위대조조적8.05、14.58、22.87、14.52、26.53、3.48、1.94배,이ber/abl mRNA수평위대조조적0.39배.류식세포술측정홍계막단백CD47,band3、RhD수평명현증고(P<0.01),GPA무명현차이.결론우화조합체외홍계유도분화법가사K562세포은정향홍계분화,량호세포상태리우전외원기인조작,진행홍계통질병연구.RhD비GPA경조재홍계세포표체,가작위조기홍계특이표기.
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 hemoglobin 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 optimized induction.Further explore was performed by Reahime-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.Reahime-PCR showed the level of Spectrina,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 eontrol 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<O.01)and the expression of GPA was negative.Conclusion K562 cells could stably differentiate into erythroid linage by the optimized cuhuration of multi-factors CO-induce.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.