Pathological analysis of ribosomal protein S19 deficient Diamond-Blackfan anemia

为了分析RPS 19突变型Diamond-Blackfan贫血（DBA）的病理机制，我们构建了12种1点突变型RPS 19表达载体。当突变基因转染K562细胞时，12个载体中有11个不能产生突变的RPS 19蛋白。由于蛋白体抑制剂MG 132挽救了蛋白质表达，因此蛋白质降解是由蛋白体消化引起的。这些结果表明，RPS 19突变的DBA患者被认为缺乏RPS 19。当针对RPS 19基因的siRNA抑制K562细胞中RPS 19基因的表达时，K562细胞停止增殖而不发生凋亡。结果显示，转染后的细胞p21、p57表达增加，Rb蛋白磷酸化，Ki 67/Hoechst 33342染色显示细胞进入G 0期阻滞，并将含有针对RPS 19的siRNA序列的慢病毒载体转染人CD 34阳性细胞。转导细胞出现G 0期阻滞，红系祖细胞部分严重缺陷（CD 34 + CD 71 highCD 45 RA-），表明RPS 19突变型DBA是由RPS 19蛋白表达水平引起的，其主要机制是造血干细胞或早期红系祖细胞发生G 0期阻滞。

In order to analyze the pathology of the RPS19 mutated Diamond-Blackfan anemia (DBA), we have developed the 12 kinds of one point mutated RPS 19 expression vectors. When the mutated genes were transfected in K562 cells, 11 out of 12 vectors could not produce the mutated RPS19 protein. Since the proteosome inhibitor, MG 132 rescued the protein expression, the protein extinctions were caused by digestion in the proteosome. From these results showed that RPS 19 mutated DBA patients are thought to lack of RPS19.When the expression of RPS19 genes were repressed by siRNA against RPS19 gene in K562 cells, they stopped proliferating without apoptosis. In these cells, the expression of p21 and p57 were increased, and Rb were phospholyrated, and Ki 67/Hoechst 33342 staining showed that they went into the G0 arrest.Furthermore, human CD34 positive cells were transduced by the lentiviral vector that contained the siRNA sequence against RPS19. The transduced cells showed G0 arrest, and the severe defect of the erythroid progenitor fraction (CD34+CD71highCD45RA-).Together with these results, RPS19 mutated DBA are caused by the expression level of RPS19 protein, and the main mechanism of this disease is G0 arrest in the hematopoietic stem cells or early erythroid progenitors.