Ationu A, Humphries A, Layton D M
Department of Haematological Medicine, King's College School of Medicine and Dentistry, London SE5 9RS, UK.
Int J Mol Med. 1999 Jan;3(1):21-4. doi: 10.3892/ijmm.3.1.21.
The metabolic defect of triosephosphate isomerase (TPI) deficiency is reversible in deficient lymphoblastoid cells when cultured in the presence of human K562 erythroleukemia cells or plasma as exogenous source of functional enzyme. However, plasma contains a variety of undefined biological response modifiers whose effects on TPI gene expression are unknown. In the present study, TPI deficient lymphoblastoid cells were cultured in serum-free medium for 24 h (controls) and stimulated with fresh frozen plasma (FFP) at final concentrations of 20, 40, and 60% for 9 h. Changes in TPI mRNA expression were monitored by slot and Northern blot hybridisations using a specific human TPI cDNA probe. For equivalent loading of total RNA, TPI mRNA expression in FFP-treated lymphoblastoid cells exceeded that for controls by on average 20-fold. Additional studies with the transcription inhibitor, actinomycin D, revealed a rapid degradation of TPI mRNA in controls compared to FFP-treated cells, indicating that the stability of the TPI transcript was affected by plasma. These data suggest that functional or regulatory elements within the TPI gene promoter can be modulated by biological response modifiers. An understanding of the transcriptional control of TPI may provide useful insights into the development of gene therapy strategies for TPI deficiency.
当在存在人K562红白血病细胞或血浆作为功能性酶的外源来源的情况下进行培养时，磷酸丙糖异构酶（TPI）缺乏症的代谢缺陷在缺乏TPI的淋巴母细胞样细胞中是可逆的。然而，血浆含有多种未定义的生物反应调节剂，其对TPI基因表达的影响尚不清楚。在本研究中，将缺乏TPI的淋巴母细胞样细胞在无血清培养基中培养24小时（作为对照），并用终浓度为20%、40%和60%的新鲜冷冻血浆（FFP）刺激9小时。使用特异性人TPI cDNA探针通过狭缝杂交和Northern印迹杂交监测TPI mRNA表达的变化。为了使总RNA等量上样，FFP处理的淋巴母细胞样细胞中TPI mRNA表达平均比对照高出20倍。使用转录抑制剂放线菌素D的进一步研究表明，与FFP处理的细胞相比，对照中TPI mRNA迅速降解，这表明TPI转录本的稳定性受血浆影响。这些数据表明，TPI基因启动子内的功能元件或调控元件可被生物反应调节剂调节。对TPI转录控制的理解可能为TPI缺乏症的基因治疗策略的开发提供有用的见解。
