Preparation of homotrimer-gels composed of type I collagen α1 chain for novel biomaterials as drug-delivery.

生物材料给药系统是药理学领域的重要研究课题。从牛骨和皮肤中获得的胶原蛋白是用于此目的的最有用的载体生物材料之一，其通过在体温下形成凝胶包埋药物和作为疫苗中的稳定剂。近年来，研究认为致病性朊蛋白的污染来源于牛骨，明胶过敏是由牛Ⅰ型胶原α2链引起的。为开发无致敏性、无病原体的胶原蛋白，本研究采用重组技术制备了由人I型胶原α1链{α1（I）_3}组成的新型同源三聚体凝胶，并利用胶原蛋白凝胶进行药物控释，重点研究了一种新型药物释放系统（DDS）。用体外翻译法制备了由{α1（I）_3}组成的同源三聚体凝胶。将克隆的人α1链cDNA（胶原结构域，3.2kbp）分别插入pIVEX 2.3d和pIVEX 1.3WG载体中，构建了人α1链的真核表达载体。由于两种表达系统表达的重组α1链蛋白产量都很低，而且都是以不溶性部分回收，因此需要寻找更有效的表达系统。在使用内皮抑制素作为抗血管生成因子的药物给药研究中，内皮抑制素的全身给药在类风湿性关节炎动物模型中具有关节炎抑制作用。在对荷瘤小鼠的另一项给药研究中，注射包埋有omithine脱羧酶（ODC）的反义寡核苷酸的去端肽胶原，其已知是肿瘤生长的调节剂，显示小鼠中的肿瘤消退。提示胶原凝胶包埋药物是一种有效的新型给药系统。

The drug-delivery systems using biomaterials are important research subjects in the field of pharmacology. The collagen obtained from bovine bone and skin, has been one of the most useful carrier biomaterials for this purpose by entrapping drugs in gel formation at body temperature and for a stabilizer in vaccine. Recently, it is suggested that contamination of pathogenic prion protein would be derived from bovine bone and that gelatin allergy was caused by bovine α2 chain of type I collagen. To develop nonallergenic and pathogen-free collagen, we investigated on preparation of novel homotrimer-gel composed of human type I collagen α1 chains {α1(I)_3} in recombinant technology, and the controlled release of drugs using the collagen-gel, focusing on a new drug delivery system (DDS) in this research project. Preparation of homotrimer-gel composed of {α1(I)_3} was examined using in vitro translation methods. The cloned human α1 chain cDNA (collagenous domain, 3.2 kbp) was inserted into vectors which were used pIVEX 2.3d for bacterial expression system and pIVEX 1.3WG for wheat germ expression system. Since recombinant α1 chain protein by both expression systems showed very low yield and recovered in insoluble fraction, it needed to search more effective expression system. In drug administration study using endostatin as antiangiogenic factor, the systemic administration of endostatin had an arthritis inhibiting effect in rheumatoid arthritis animal model. In the other administration study for tumor bearing mice, the injections of atelocollagen entrapped of antisence-oligonucleotides of omithine decarboxylase (ODC) which is known to be regulator of tumor growth, showed regression of tumor in the mice. These results suggested that collagen-gel entrapped of drugs was thought to be effective new DDS.