Zhang J, Zhang X, Chen P
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, CAMS & PUMC, Beijing 100005.
Chin Med Sci J. 2001 Dec;16(4):187-93.
To examine the effect of isobutyramide synthesized in our laboratory on human and murine globin gene expression and to test cell toxicity of the drug.
MEL cells were transfected with the recombinant construct muLCRAgammapsibetadeltabeta and the stable transformants were cultured in the medium with different concentrations of isobutyramide. The experimental mice and rabbit were injected with different doses of isobutyramide. The globin mRNAs were analyzed by RNase protection assay. The hematological toxicity and electrolyte toxicity of the drug were tested.
An inducible and dose-dependent expression of the human gamma-, beta- and mouse alpha-globin gene was observed in the transfected MEL cells. The induction of the human gamma-globin gene is significant stronger than that of the beta-globin gene. With 2.5 approximately 5 mmol/L isobutyramide, the induction of the human gamma-globin gene is even more effective than that of mouse alpha-globin gene. After a 15-day injection under the doses of 500 approximately 900 mg x kg(-1) x d(-1), the level of the mouse embryonic epsilon(y)-globin mRNA could be significantly induced up to 3 approximately 4 fold of that of uninjected controls. The changes of hemoglobin(Hb), RBC, hematocrit(HCT), WBC, derived from mice injected with different doses of isobutyramide at the interval of 24 hours for 2 approximately 4 weeks, were generally within the normal range. In rabbits injected with isobutyramide in the same regiment for 2 weeks, the concentration of blood K+, Na+, Cl- and CO2 were all within normal range and serum ionic osmotic pressure remained stable as well.
Our results suggested that isobutyramide is a weak inducer of cell differentiation, but it can selectively activate transcription of human gamma-globin gene at a certain degree, and it can act on early stages of erythroid progenitor differentiation in adult mice and activate transcription of embryonic epsilon(y)-globin gene and have no hematological toxicity. Our results have further proved the potential value of isobutyramide in treatment of beta-thalassemia and sickle cell disease.
研究本实验室合成的异丁酰胺对人及小鼠珠蛋白基因表达的影响，并检测该药物的细胞毒性。
用重组构建体muLCRAgammapsibetadeltabeta转染MEL细胞，稳定转染子在含不同浓度异丁酰胺的培养基中培养。给实验小鼠和兔子注射不同剂量的异丁酰胺。通过核糖核酸酶保护试验分析珠蛋白mRNA。检测药物的血液学毒性和电解质毒性。
在转染的MEL细胞中观察到人类γ-、β-和小鼠α-珠蛋白基因的诱导性和剂量依赖性表达。人类γ-珠蛋白基因的诱导明显强于β-珠蛋白基因。在2.5至5 mmol/L异丁酰胺作用下，人类γ-珠蛋白基因的诱导甚至比小鼠α-珠蛋白基因更有效。在500至900 mg·kg⁻¹·d⁻¹剂量下注射15天后，小鼠胚胎ε(y)-珠蛋白mRNA水平可显著诱导至未注射对照组的3至4倍。在2至4周内每隔24小时给注射不同剂量异丁酰胺的小鼠检测血红蛋白（Hb）、红细胞（RBC）、血细胞比容（HCT）、白细胞（WBC）的变化，总体在正常范围内。在以相同方案给兔子注射异丁酰胺2周后，血液中K⁺、Na⁺、Cl⁻和CO₂浓度均在正常范围内且血清离子渗透压也保持稳定。
我们的结果表明异丁酰胺是一种弱的细胞分化诱导剂，但它能在一定程度上选择性激活人类γ-珠蛋白基因转录，它可作用于成年小鼠红系祖细胞分化的早期阶段并激活胚胎ε(y)-珠蛋白基因转录且无血液学毒性。我们的结果进一步证明了异丁酰胺在治疗β地中海贫血和镰状细胞病方面的潜在价值。
