Ye Zhang-Qun, Kong De-Bo, Chen Zhi-Qiang, Yao Lin-Fang, Guo Hui, Yu Xiao, Liu Guan-Lin, Yang Wei-Min
Department of Urology, Tongji Hospital, Tongji Medical School, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China.
Int J Mol Med. 2007 Oct;20(4):521-6.
Hyperoxaluria can lead to multiple pathologic conditions such as recurrent urolithiasis, oxalosis, nephrocalcinosis and even renal failure, but there is no known oxalate-degrading pathway in the human body, and current therapeutic options for patients with hyperoxaluria are limited. Oxalyl-CoA decarboxylase and formyl-CoA transferase are the key enzymes in the oxalate catabolism of Oxalobacter formigenes which dwell in the intestine of vertebrates and have an important symbiotic relationship with their hosts. The aim of this study was to insert the oxalate-degrading enzyme genes into human embryo kidney (HEK) 293 cells and to evaluate if the oxalate-degrading enzymes could be expressed in these cells and keep their enzyme activity. We present here the cloning of the two genes from O. formigenes and their subsequent expression in HEK293 cells. The results showed that the expression of oxalyl-CoA decarboxylase and formyl-CoA transferase was confirmed by RT-PCR and Western blotting, and the proteins were located in the cytoplasm of transfected cells. Furthermore, the transfected cells were capable to a certain degree of degrading oxalate. These findings suggest that the transfer of oxalate-degrading enzyme genes into human cells is possibly a potential candidate for the gene therapy of hyperoxaluria.
高草酸尿症可导致多种病理状况，如复发性尿路结石、草酸沉着症、肾钙质沉着症甚至肾衰竭，但人体中尚无已知的草酸降解途径，目前针对高草酸尿症患者的治疗选择有限。草酰辅酶A脱羧酶和甲酰辅酶A转移酶是栖居于脊椎动物肠道内且与其宿主具有重要共生关系的产甲酸草酸杆菌草酸分解代谢中的关键酶。本研究的目的是将草酸降解酶基因导入人胚肾（HEK）293细胞，并评估草酸降解酶是否能在这些细胞中表达并保持其酶活性。我们在此展示了从产甲酸草酸杆菌中克隆这两个基因及其随后在HEK293细胞中的表达。结果显示，通过逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法（Western blotting）证实了草酰辅酶A脱羧酶和甲酰辅酶A转移酶的表达，且这些蛋白质位于转染细胞的细胞质中。此外，转染细胞在一定程度上能够降解草酸。这些发现表明，将草酸降解酶基因转入人体细胞可能是高草酸尿症基因治疗的一个潜在候选方法。
