本文選題：透明質(zhì)酸 + 透明質(zhì)酸酶�。� 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年碩士論文

【摘要】：透明質(zhì)酸(HA)又名玻尿酸,是由N-乙酰氨基葡萄糖與D-葡萄糖醛酸為雙糖單位聚合而成的糖胺聚糖,其雙糖單位中的N-乙酰氨基葡萄糖與D-葡萄糖醛酸以β-1,3糖苷鍵相連,雙糖單位之間則以β-1,4糖苷鍵進行聚合。由于雙糖單位聚合度的差異,導(dǎo)致透明質(zhì)酸的分子量分布較為寬泛,天然的HA主要以高分子透明質(zhì)酸(HMW HA,Mr2×106Da)方式存在。分子量是決定HA生物學(xué)功能的重要參數(shù)。細胞外基質(zhì)的主要成分是HMW HA,其高粘彈性是細胞外基質(zhì)得以抵抗外界壓縮力的根源,依賴于高粘彈性,HMW HA也用于眼科手術(shù)。低分子量透明質(zhì)酸(LMW HA,1×105 DaMr2×106 Da)具有良好的保濕性和潤滑性,廣泛用于化妝品;研究發(fā)現(xiàn),超低分子量透明質(zhì)酸(VLMW HA,Mr1×105 Da)具有促血管生成作用、促創(chuàng)傷愈合作用、免疫調(diào)節(jié)活性和抗腫瘤活性,在機體愈合、免疫增強和腫瘤治療等領(lǐng)域有重要的潛在應(yīng)用價值。因此我們建立了高純度低分子量HA的酶切制備法:依賴r Hu PH20酶解特性,利用GPC法測定酶解產(chǎn)物分子量,建立酶解產(chǎn)物分子量變化與酶解時間的動力學(xué)模型,調(diào)控酶解反應(yīng),獲取特定分子量的HA以滿足不同的臨床需求,同時也為進一步探究體內(nèi)酶解反應(yīng)行為和功能研究提供有效和重要的線索。透明質(zhì)酸酶(HAase)是專一性催化降解HA的酶的統(tǒng)稱,部分HAase也可以在一定程度上降解軟骨素和硫酸軟骨素。人源性HAase主要有三種:HAase 1、HAase 2和PH20,其中PH20是降解人體內(nèi)HA的主要酶類。本實驗室建立了大規(guī)模重組人透明質(zhì)酸酶(r Hu PH20)的生產(chǎn)流程,并設(shè)定一系列的質(zhì)量控制標準以確保制備高純度的r Hu PH20,為后續(xù)藥效學(xué)研究奠定了堅實的基礎(chǔ)。皮下注射是經(jīng)典的微量體積注射方式,注射液必須經(jīng)由皮膚間質(zhì)進入到作用靶點,而這種由膠原蛋白、彈性蛋白、纖維連接蛋白和透明質(zhì)酸等結(jié)構(gòu)性大分子組成的皮膚間質(zhì),具有復(fù)雜三維立體結(jié)構(gòu),限制了皮下注射藥物的擴散速率和輸液量。其中HA是皮膚間質(zhì)復(fù)雜立體結(jié)構(gòu)的“基架”,同時HA的親水性促使水分子嵌入到網(wǎng)狀結(jié)構(gòu)內(nèi)部,構(gòu)建成網(wǎng)狀實體彈性結(jié)構(gòu),阻礙了流體在皮膚間質(zhì)中流動,阻礙了注射液的擴散吸收。本實驗以裸鼠為研究對象,以臺盼藍模擬聯(lián)用藥物作為可視化的示蹤劑,r Hu PH20顯著提高皮下注射給藥的擴散速率;以新西蘭兔為實驗對象,皮下注射大體量生理鹽水制造皮下水腫模型,造模前預(yù)注r Hu PH20,在不產(chǎn)生組織損傷條件下,顯著性提高皮下注射輸液量;造模后注射r Hu PH20,局部皮下水腫逐漸減小,20分鐘后水腫消失,表明r Hu PH20可以有效治療局部皮下水腫;以SD大鼠為研究對象,r Hu PH20與NGF聯(lián)用滴鼻給藥可促進NGF入腦效率,顯著增加NGF經(jīng)鼻入腦速率和在各腦組織中的分布,對創(chuàng)傷性腦損傷和神經(jīng)中樞性疾病的治療具有重大潛在應(yīng)用價值。皮膚間質(zhì)中HA的半衰期只有15-20小時,意味著注射部位的皮膚間質(zhì)在24小時內(nèi)會恢復(fù)損傷,不產(chǎn)生任何組織變化和炎癥表征。r Hu PH20的體內(nèi)的半衰期只有10分鐘,極短的半衰期意味著幾乎不會引起組織損傷和炎癥反應(yīng)。同時r Hu PH20是人源性透明質(zhì)酸酶,具有底物專一性,對聯(lián)用藥物和組織中其它蛋白無作用,r Hu PH20良好的兼容性確保了臨床用藥的安全性。rHu PH20作為擴散劑輔助皮下注射是一種新型的“大體量皮下給藥方式”,相比于靜脈注射,具有技術(shù)要求低、操作簡單、成本低廉和輸液效率高等優(yōu)勢,可替代靜脈注射成為慢性疾病患者自助型注射類藥物給藥方式,例如糖尿病患者注射胰島素;替代不便于靜脈注射環(huán)境和條件的大體量注射方式,例如皮膚燒傷患者的輸液;為一類因溶解度、生物利用度和穩(wěn)定性等因素限制而不能進行靜脈注射的藥物提供新的給藥途徑。r Hu PH20與NGF聯(lián)用滴鼻給藥途徑是一種新的NGF給藥方式,顯著增加NGF經(jīng)鼻入腦速率和在各腦組織中的分布,為神經(jīng)中樞靶向給藥提供快速、高效和便捷的給藥方式。
[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.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R96

【參考文獻】

相關(guān)期刊論文 前1條

1 呂鵬;張金龍;宋小紅;侯利華;陳薇;;利用SEC-HPLC法測定重組人透明質(zhì)酸酶的純度[J];生物技術(shù)通訊;2016年03期

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本文編號：1896549