目的探讨益髓生血颗粒含药血清对K562细胞向红系分化的影响。方法用SD大鼠制备益髓生血颗粒含药血清,分高、中、低和对照组,各组含药血清以20%终浓度添加到1640培养基中,并对K562细胞进行干预培养。采用联苯胺染色法观察各组血清干预K562细胞后,对蓝染细胞进行计数,计算细胞蓝染阳性率;采用实时荧光定量聚合酶链反应(real time polymerase chain reaction,RT-PCR)方法观察益髓生血颗粒干预K562细胞后珠蛋白基因mRNA表达情况。结果联苯胺染色结果显示高、中、低、对照组与空白组比较均能够显著提高K562细胞蓝染率(P<0.0001);与对照组比较,益髓生血颗粒含药血清高、中剂量组K562细胞蓝染数量提高(P<0.001)。RT-PCR结果显示益髓生血颗粒含药血清,高剂量组与对照组比较,能够促进α和γ-珠蛋白基因表达(P<0.05)。结论益髓生血颗粒含药血清能够促进K562细胞向红系分化。
Objective: To examine the clinical effects of Yisui Shengxue Granules(益髓生血颗粒) in the treatment of β-thalassemia and explore its mechanism on DNA methylation levels. Methods: A randomized placebo-controlled double-blinded trial was conducted. Forty patients with β-thalassemia were recruited and distributed randomly by envelope method into an experimental group and a control group, 20 patients in each group. The patients were given Yisui Shengxue Granules in the experimental group and placebo in the control group(12 g/bag, 3 times a day) during a 3-month intervention. Before and after 1, 2, and 3 months of treatment, peripheral intravenous blood was sampled, and blood parameters such as hemoglobin(Hb), red blood cells(RBCs), reticulocytes(Ret), and fetal hemoglobin(HbF) were analyzed. Mononuclear cells from 5 patients, who showed an obvious treatment effect, were isolated by density gradient centrifugation. DNA methylation was analyzed using an Affymetrix USA GeneChip Human Promoter 1.0 Array and Input-promoter 1.0. Results: Compared with pre-treatment, there was an obvious increase in Hb and RBCs counts after 1, 2, and 3 months in the experiment group(P<0.01 or P<0.05). Meanwhile, HbF increased from the 2 nd to the 3 rd month(P<0.05). In the control group, Hb and RBCs showed no obvioas change. After 3-month treatment, DNA methylation results from 5 patients revealed that there were 24 hypomethylated genes and 3,685 hypermethylated genes compared with pre-treatment. Genes of insulin-like growth factor 1 receptor(IGF1 R) and Janus kinase 3(JAK3) revealed the most relations with other genes(degree: 21) and genes of 1-phosphatidylinositol-4, 5-bisphosphate phosphodiesterase gamma 2(PLCG2) and mitogen-activated protein kinase 10(MAPK10) showed a stronger intermediary role(betweenness centrality=0.04). Conclusions: JAK3 and MAPK10 are two key genes in bone marrow and the lymphatic system, and JAK3 is likely to be related to hematopoietic cytokines in the process of early hematopoiesis.(Registration No. NCT
