Studies on forensic applications of human ribonucleases

虽然人体体液中含有多种核酸酶，但这些酶在法医学领域还没有得到应用。因此，本研究旨在使这些酶在这一领域有更广泛的应用。(1)从人尿液中分离纯化了脱氧核糖核酸酶I和脱氧核糖核酸酶II两种脱氧核糖核酸酶，并对其进行了详细鉴定。通过等电聚焦和免疫印迹相结合的技术或新设计的酶谱方法，可以在人的尿液或血清中检测到至少10种不同的表型。遗传研究表明，DNase I在一个常染色体座位上由4个共显性等位基因控制，其基因频率分布良好。因此，人类DNase I基因多态性被认为是一种有效的法医个体化工具。其次，我们发现尿液和白细胞DNA酶II活性水平存在遗传多态性：日本研究人群可以分为两种截然不同的类型，高活性和低活性。来自家庭研究，DNaseII。低活性类型为隐性等位基因所致。(2)成功地从人红细胞、脾和肾中分离纯化并鉴定了不同的糖核酸酶(RNasea)。这些核糖核酸酶的酶学和免疫学性质非常相似，但它们的碳水化合物组成有很大的差异。人类核糖核酸酶的器官或组织特异性可能是由于它们的碳水化合物部分的差异。(3)采用等电聚焦电泳法和特异性抗体免疫印迹技术，发现凝血和纤溶系统中的一个因子--人蛋白C抑制物存在遗传多态性。

Although there are many nucleolytic enzymes in human body fluids, these enzymes have not been used in the field of forensic sciences at all. So, this study was designed in order to make a wider variety of applications of these enzymes in this field. (1) Two types of deoxyribonuclease, DNases I and II, were purified separately from human urine, and then characterized in detail. By means of the combined technique of isoelectric focusing and immunoblotting or the newly devised zymogram method, at least ten distinct phenotypes could be detected in human urinary or serum DNaseI. From the genetic surveys, it has been known that DNase I was controlled by four codominant alleles at a single autosomal locus with the favorable distribution of its gene frequencies. Therefore, the genetic polymorphism of human DNase I was found to be useful as an effective tool for forensic individualization. Next, we discovered the existence of genetic polymorphism in the activity levels of urinary and leukocytic DNase IIs : the Japanese study population could be classified into two distinct types, high-activity and low-activity. From the family study, DNaseII. was found to be controlled by two alleles at a single autosomal locus the low activity type was due to an recessive allele. (2) We succeeded in purification and characterization of different sibonucleases (RNasea) from human erythrocytes, spleen and kidney. The enzymological and immunological properties of the RNases were very similar but there was a wide difference among their carbohydrate compositions. The organ- or tissue-specificities of human RNases may be due to the differences in their carbohydrate moieties. (3) We discovered the existence of genetic polymorphism in human protein C inhibitor, a factor in the system of blood coagulation and fibrinolysis, using isoelectric focusing electrophoresis and immunoblotting with specific antibodies.