尿多酸肽对原代红系细胞γ-珠蛋白表达的体外研究

In vitro study of urinary polypeptide on expression ofγ-globin in primary erythrocytes

目的:探讨尿多酸肽(CDA-Ⅱ)对体外培养原代红系细胞γ-珠蛋白基因和蛋白表达的影响,为CDA-Ⅱ对β-地中海贫血的治疗提供新思路。方法:以不同浓度CDA-Ⅱ(0 mg/mL、1 mg/mL、2 mg/mL、3 mg/mL、4 mg/mL)作用原代红系细胞24 h、48 h和72 h,采用实时荧光定量聚合酶链反应(RT-PCR)和Cell counting kit-8(CCK-8)法分别检测γ-珠蛋白基因的表达及细胞增殖-毒性情况;以3 mg/mL CDA-Ⅱ为实验组、200 nmol/L地西他滨和100μmol/L羟基脲为阳性对照组以及等体积PBS+DMSO为阴性对照组作用细胞后,采用RT-PCR检测γ-珠蛋白、DNA甲基转移酶1(DNMT1)和FOXO3a mRNA表达的变化;Western Blot(WB)检测γ-珠蛋白表达的情况。结果:RT-PCR结果表明,3 mg/mL CDA-Ⅱ作用细胞48 h和72h后上调γ-珠蛋白基因的表达作用明显(P<0.05),分别为空白对照组(0 mg/mL)的2.5倍和2.68倍;CCK-8结果显示细胞存活率存在时间和浓度依赖性下降,24 h、48 h和72 h的半数抑制浓度(IC50)分别为(5.28±0.67)mg/mL、(3.89±0.63)mg/mL、(2.35±0.20)mg/mL;与阴性对照组比较,CDA-Ⅱ组与地西他滨、羟基脲阳性对照组既可以降低DNMT1基因的表达(P<0.05)、升高FOXO3a基因的表达,同时可以上调γ-珠蛋白基因和蛋白的表达(P<0.01)。结论:CDA-Ⅱ对红系细胞γ-珠蛋白基因和蛋白的高表达可能与DNMT1和FOXO3a有关。

Objective: To investigate the effect of urinary polypeptide(CDA-Ⅱ)on the expression of γ-globin gene and protein in primary erythrocytes cells in vitro,and to provide a new concept for the treatment of β-thalassemia by CDA-Ⅱ.Methods: Primary erythroid cells were treated with different concentrations of CDA-Ⅱ(0 mg/mL,1 mg/mL,2 mg/mL,3 mg/mL,4 mg/mL)for 24 h,48 h,and 72 h,the expression of γ-globin gene and cell proliferation-toxicity were detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and Cell counting kit-8(CCK-8),respectively.The γ-globin DNA methyltransferase1(DNMT1)and FOXO3 a mRNA expression were detected by RT-qPCR after cells treated with 3 mg/mL of CDA-Ⅱ(CDA-Ⅱ experimental group),200 nmol/L of decitabine and 100 μmol/L of hydroxyurea(two positive control group),and equal volume PBS+DMSO(negative control group).The expression of γ-globin was detected by Western blot(WB).Results: The results of RT-qPCR showed that the expression of γ-globin gene was significantly up-regulated after treated with 3 mg/mL CDA-Ⅱ for 48 h and 72 h(P<0.05),which was 2.5 and 2.68 times higher than that of the blank control group(0 mg/mL CDA-Ⅱ),respectively.CCK-8 results showed that the cell survival rate was decreased in a time-dependent and concentration-dependent manner.The 50%inhibitory concentration(IC50)at 24 h,48 h,and 72 h were(5.28±0.67)mg/mL,(3.89±0.63)mg/mL,and(2.35 ± 0.20)mg/mL,respectively.Compared with the negative control group,CDA-Ⅱ experimental group,decitabine and hydroxyurea positive control group could decrease the expression of DNMT1 gene(P<0.05),increase the expression of FOXO3 a gene,and up-regulate the expression of γ-globin gene and protein(P<0.01).Conclusion: The effect of in primary erythroid cells CDA-Ⅱ on the high expression of γ-globin gene and protein in erythroid cells may be related to DNMT1 and FOXO3 a.