重组人透明质酸酶药效学研究

发布时间：2018-05-16 10:53

本文选题：透明质酸 + 透明质酸酶　；参考：《中国人民解放军军事医学科学院》2017年硕士论文

【摘要】：透明质酸(HA)又名玻尿酸,是由N-乙酰氨基葡萄糖与D-葡萄糖醛酸为双糖单位聚合而成的糖胺聚糖,其双糖单位中的N-乙酰氨基葡萄糖与D-葡萄糖醛酸以β-1,3糖苷键相连,双糖单位之间则以β-1,4糖苷键进行聚合。由于双糖单位聚合度的差异,导致透明质酸的分子量分布较为宽泛,天然的HA主要以高分子透明质酸(HMW HA,Mr2×106Da)方式存在。分子量是决定HA生物学功能的重要参数。细胞外基质的主要成分是HMW HA,其高粘弹性是细胞外基质得以抵抗外界压缩力的根源,依赖于高粘弹性,HMW HA也用于眼科手术。低分子量透明质酸(LMW HA,1×105 DaMr2×106 Da)具有良好的保湿性和润滑性,广泛用于化妆品;研究发现,超低分子量透明质酸(VLMW HA,Mr1×105 Da)具有促血管生成作用、促创伤愈合作用、免疫调节活性和抗肿瘤活性,在机体愈合、免疫增强和肿瘤治疗等领域有重要的潜在应用价值。因此我们建立了高纯度低分子量HA的酶切制备法:依赖r Hu PH20酶解特性,利用GPC法测定酶解产物分子量,建立酶解产物分子量变化与酶解时间的动力学模型,调控酶解反应,获取特定分子量的HA以满足不同的临床需求,同时也为进一步探究体内酶解反应行为和功能研究提供有效和重要的线索。透明质酸酶(HAase)是专一性催化降解HA的酶的统称,部分HAase也可以在一定程度上降解软骨素和硫酸软骨素。人源性HAase主要有三种:HAase 1、HAase 2和PH20,其中PH20是降解人体内HA的主要酶类。本实验室建立了大规模重组人透明质酸酶(r Hu PH20)的生产流程,并设定一系列的质量控制标准以确保制备高纯度的r Hu PH20,为后续药效学研究奠定了坚实的基础。皮下注射是经典的微量体积注射方式,注射液必须经由皮肤间质进入到作用靶点,而这种由胶原蛋白、弹性蛋白、纤维连接蛋白和透明质酸等结构性大分子组成的皮肤间质,具有复杂三维立体结构,限制了皮下注射药物的扩散速率和输液量。其中HA是皮肤间质复杂立体结构的“基架”,同时HA的亲水性促使水分子嵌入到网状结构内部,构建成网状实体弹性结构,阻碍了流体在皮肤间质中流动,阻碍了注射液的扩散吸收。本实验以裸鼠为研究对象,以台盼蓝模拟联用药物作为可视化的示踪剂,r Hu PH20显著提高皮下注射给药的扩散速率;以新西兰兔为实验对象,皮下注射大体量生理盐水制造皮下水肿模型,造模前预注r Hu PH20,在不产生组织损伤条件下,显著性提高皮下注射输液量;造模后注射r Hu PH20,局部皮下水肿逐渐减小,20分钟后水肿消失,表明r Hu PH20可以有效治疗局部皮下水肿;以SD大鼠为研究对象,r Hu PH20与NGF联用滴鼻给药可促进NGF入脑效率,显著增加NGF经鼻入脑速率和在各脑组织中的分布,对创伤性脑损伤和神经中枢性疾病的治疗具有重大潜在应用价值。皮肤间质中HA的半衰期只有15-20小时,意味着注射部位的皮肤间质在24小时内会恢复损伤,不产生任何组织变化和炎症表征。r Hu PH20的体内的半衰期只有10分钟,极短的半衰期意味着几乎不会引起组织损伤和炎症反应。同时r Hu PH20是人源性透明质酸酶,具有底物专一性,对联用药物和组织中其它蛋白无作用,r Hu PH20良好的兼容性确保了临床用药的安全性。rHu PH20作为扩散剂辅助皮下注射是一种新型的“大体量皮下给药方式”,相比于静脉注射,具有技术要求低、操作简单、成本低廉和输液效率高等优势,可替代静脉注射成为慢性疾病患者自助型注射类药物给药方式,例如糖尿病患者注射胰岛素;替代不便于静脉注射环境和条件的大体量注射方式,例如皮肤烧伤患者的输液;为一类因溶解度、生物利用度和稳定性等因素限制而不能进行静脉注射的药物提供新的给药途径。r Hu PH20与NGF联用滴鼻给药途径是一种新的NGF给药方式,显著增加NGF经鼻入脑速率和在各脑组织中的分布,为神经中枢靶向给药提供快速、高效和便捷的给药方式。
[Abstract]:Hyaluronic acid (HA), also known as hyaluronic acid, is a glycosaminoglycan from N- acetylglucosamine and D- glucuronic acid as a disaccharide unit. The N- acetylglucosamine in the double sugar unit is linked to the D- glucuronic acid with the beta -1,3 glycoside bond, and the disaccharide unit is polymerized with the beta -1,4 glycoside bond. Due to the difference of the degree of polymerization of the disaccharide units The distribution of the molecular weight of hyaluronic acid is more extensive, and the natural HA is mainly in the way of high molecular hyaluronic acid (HMW HA, Mr2 x 106Da). Molecular weight is an important parameter to determine the biological function of HA. The main component of the extracellular matrix is HMW HA, and its high viscoelasticity is the root of the extracellular matrix to resist the external compression, and is dependent on the height of the extracellular matrix. Viscoelasticity, HMW HA is also used in ophthalmological surgery. Low molecular weight hyaluronic acid (LMW HA, 1 * 105 DaMr2 * 106 Da) has good moisturizing and lubricity and is widely used in cosmetics. The study found that ultra low molecular weight hyaluronic acid (VLMW HA, Mr1 * 105 Da) has angiogenesis, wound healing, immunomodulatory activity and antitumor activity. It has important potential application value in the fields of body healing, immune enhancement and tumor treatment. Therefore, we have established a high purity and low molecular weight HA enzyme cutting preparation method: dependent on the properties of R Hu PH20 enzyme hydrolysis, the molecular weight of the hydrolysate by GPC method, the kinetic model of the molecular weight change and the enzymolysis time of the hydrolysate, and the enzymatic hydrolysis reaction To obtain specific molecular weights of HA to meet different clinical needs, and to provide an effective and important clue to further explore the behavior and function of enzyme reaction in vivo. Hyaluronidase (HAase) is the general name for the enzyme that catalyzes the degradation of HA, and part of HAase can also degrade chondroitin and chondroitin sulfate at a certain degree. There are three main types of sex HAase: HAase 1, HAase 2 and PH20, in which PH20 is the main enzyme to degrade HA in human body. The production process of large-scale recombinant human hyaluronidase (R Hu PH20) is established in this laboratory, and a series of quality control standards are set up to ensure the preparation of high purity R Hu PH20, which lays a solid foundation for subsequent pharmacodynamic research. Subcutaneous injection is a classic micro volume injection. The injection must enter the target target via the skin stroma, and the skin interstitial, consisting of structural macromolecules such as collagen, elastin, fibronectin and hyaluronic acid, has a complex three-dimensional structure, limiting the diffusion rate and loss of subcutaneous injection of drugs. HA is the "base" of the complex three-dimensional structure of the skin. At the same time, the hydrophilicity of the HA causes water molecules to be embedded into the network structure, which is constructed into a reticular solid elastic structure, which hinders the flow of fluid in the skin interstitial and hinders the diffusion and absorption of the injection. As a visual tracer, R Hu PH20 significantly improved the diffusion rate of subcutaneous injection; a New Zealand rabbit was taken as the experimental object, subcutaneous injection of large body volume of saline to make subcutaneous edema model, pre injection of R Hu PH20, and under the condition of no tissue injury, significant subcutaneous injection infusion volume; R Hu PH20, local injection after modeling, and partial injection of R Hu. Hypodermic edema gradually decreased and edema disappeared after 20 minutes, indicating that R Hu PH20 could effectively treat local subcutaneous edema; SD rats were used as the research object. R Hu PH20 and NGF combined with nasal drip could promote the efficiency of NGF entry to the brain, significantly increase the rate of nasal entry into the brain and the distribution of R in the brain tissues, and to the traumatic brain injury and nerve central disease. The treatment has a great potential application value. The half-life of HA in the skin is only 15-20 hours, which means that the skin interstitium at the injection site will recover within 24 hours, without any tissue change and inflammation, the half-life of the body of the.R Hu PH20 is only 10 minutes, and the short half-life means that tissue damage is almost impossible to cause. Inflammatory reaction. Meanwhile, R Hu PH20 is a human hyaluronidase, which has substrate specificity, has no effect on other drugs and other proteins in the tissue. The good compatibility of R Hu PH20 ensures the safety of clinical medication.RHu PH20 as a diffusion agent assisted subcutaneous injection is a new type of "large volume subcutaneous administration", compared with intravenous infusion. It has the advantages of low technical requirements, simple operation, low cost and high infusion efficiency. It can replace intravenous injection as a self-service injection drug for patients with chronic diseases, such as insulin injection for diabetics, instead of a large volume injection which is inconvenient for intravenous injection environment and conditions, such as infusion of skin burn patients. For a class of drugs which are limited by solubility, bioavailability and stability and cannot be injected intravenously, a new route of administration of.R Hu PH20 and NGF is a new way of NGF administration, which significantly increases the rate of NGF through the nose and the distribution of the brain in all brain tissues, and provides a rapid delivery for the target drug delivery of the nerve center. Efficient and convenient way of drug delivery.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

【参考文献】