人类ERMAP-siRNA对Ara-C诱导K562细胞向红细胞系分化过程的影响

Effects of Human ERMAP-siRNA on Erythroid Differentiation of K562 Cells Induced by Ara-C

为探讨人类ERMAP基因在红细胞分化发育过程中的作用,本研究设计ERMAP-dsDNA,制备ERMAP-shRNA表达质粒,并建立稳定表达ERMAP-shRNA的K562细胞系(即ERMAP-shRNA/K562细胞系),观察Ara-C诱导ERMAP-shRNA/K562细胞向红细胞系分化过程中,细胞形态、联苯胺染色细胞阳性率和细胞表面标记等的变化,同时以FQ-PCR检测K562细胞人类ERMAP基因表达量的变化。结果发现:经Ara-C诱导72小时,ERMAP-shRNA/K562细胞与对照组比较,体积较大,胞浆量较少,着色大部分呈深蓝色或蓝紫色,部分细胞仍可见1-2个核仁;联苯胺染色阳性率由1.17%增加至2.04%(p<0.05),但仍低于Ara-C诱导K562组(p<0.05);CD36-/CD235a+细胞比例从8.83%增至11.28%,CD36+/CD235a+细胞比例从1.23%增至2.64%,CD36+/CD235a-细胞比例从0.59%增至1.47%,均明显低于Ara-C诱导K562细胞组;与此同时,ERMAP-shRNA/K562细胞ERMAP基因的表达量从诱导前的2.52×10-3缓慢增加至诱导72小时后的4.53×10-3,明显低于K562细胞组。结论:ERMAP-shRNA可抑制Ara-C诱导K562细胞向红系分化的过程,这进一步提示人类ERMAP基因与红细胞分化发育过程有关。

In order to investigate the potential role of human ERMAP gene in erythropoiesis, the ERMAP-dsDNA was designed, ERMAP-shRNA expressing plasmids was constructed, and ERMAP-shRNA/K562 cell was established. Cell morphology, biphenylamine staining, expression of cell surface antigens as well as quantitative level of human ERMAP gene were observed during K562 cells differentiating toward erythroid lineage induced by Ara-C. The results showed that at 72 hours after Ara-C treatment, ERMAP-shRNA/K562 cell size became large with increasing cytoplasm content. The percentage of biphenylamine positive cells increased from 1.17% to 2.04% （p 〈 0.05 ）, but still lower than that in group K562 ＋ Ara-C. The percentage of CD36^-/CD235a^＋ increased from 8.83% to 11.28%, CD36^＋/ CD235a^＋ increased from 1.23% to 2.64%, and CD36^＋/CD235a^- increased from 0.59% to 1.47% respectively, which were all lower than that in group K562 ＋ Ara-C at either time point. At the same time, the level of ERMAP expression increased slowly from 2.52×10^-3 to 4.53×10^-3, which was also significantly lower than that of group K562 ＋ Ara-C. It is concuded that the ERMAP-sbRNA inhibits the Ara-C-induced erythroid differentiation of K562 cells, which further suggests that there is relationship between hERMAP and erythroid differentiation and development.