中草酸钙尿石患者尿凝血酶原F1分子异常及其机制研究

Urinary prothrombin fragment 1（UPTF1）is an important component of calcium oxalate crystal surface binding proteins（CSBP）. It was manifested there was the correlation between UPTF1 and urolithiasis in vitro, but the role of UPTF1 on urolithiasis in vivo is ambiguous. Using DEAE-Sepharose CL-6B to isolate prothrombin, and using a seeded crystallization technique, it was observed that CSBP and UPTF1 of normal subjects could apparently inhibit calcium oxalate crystal growth. CSBP of stone former showed lower inhibitory activity than that of normal subjects. Using enzyme-linked immunoadsordent assay, it was observed that the combining ability with calcium oxalate crystal of UPTF1 in calcium oxalate stone formers was significantly weaker than that in normal subjects. When protein-bound Gla was determined in the crystal matrix and urine of normal subjects and calcium oxalate stone formers by high performance liquid chromatography, it was found that protein-bound Gla in the crystal matrix and urine of normal subjects and calcium oxalate stone formers were significantly less than that of normal subjects. it was concluded that fully carboxylated Gla-bound proteins are inhibitory factors of calcium oxalate calculus. Using reverse transcriptase-polymerase chain reaction and immunohistochemical techniques, UPTF1 was visible as cytoplasmic staining in the epithelial cells of the TALH and the distal convoluted tubule and kidney tissue showed clear evidence of prothrombin mRNA expression, it is concluded that UPTF1 is synthesized and excreted in kidney. When the activity of carboxylase was detected by carboxylation reaction in vitro(calcium oxalate stone formers and mice) , it was observed that the activity of vitamin K dependent carboxylase was higher in the group of experimental urolithiasis in kidney, it was verified that the vitamin K dependent carboxylase may play a role in the pathology of urolithiasis. When the gene mutations were determined by single conformation polymorphism(SSCP) and DNA sequencing, gene mutations was not observed ofγ- carboxyglutamic acid domain of prothrombin in patients with calcium oxalate calculus, it was verified that the formation of calcium oxalate calculus is not related to the genetic mutation of γ- carboxyglutamic acid domain of prothrombin. It was found that the carboxylation defection of UPTF1 play an important role in the pathologenesis of calcium oxalate calculus, and the activity reduction of vitamin K dependent carboxylase result in carboxylation defection of UPTF1. It provided a theory foundation to explore pathogenesis of calcium oxalate calculus for next step. Our investigation on UPTF1 belongs to the part of research on CSBP, and represents the latest research direction, and establishes a new pathway for the research on etiological factor of urolithiasis in our country.

有研究显示某些尿蛋白分异常与尿石形成有关。本研究拟先通过鉴定草酸钙尿石患者尿凝血酶原片段落(UPTF1)分子特征及抑制草酸钙晶体生长和聚集的活性变化以了解UPTF1分子异常情况；再研究凝血酶原基因突变和UPTF1分子中谷氨酸的羧基化机制以探讨UPTF1分子异常折机制。这有助于阐明尿石症发病机制、为尿石症的基因诊断及临床治疗提供理论依据。.