负载PTHrP1-34可降解PLGA缓释微球的可控释放及细胞生物学研究

发布时间：2018-04-13 21:38

本文选题：甲状旁腺相关肽 + 聚乙交酯-丙交酯　；参考：《吉林大学》2014年硕士论文

【摘要】：研究背景及目的：甲状旁腺激素（Parathyroid Hormone, PTH）是具有促进成骨细胞形成及加强成骨细胞矿化作用的治疗骨质疏松症药物。而甲状旁腺相关肽（PTHrP）是一种与PTH（1～34）在基因编码、分子结构、受体构成和信号传导方面有许多相同或相似之处的多肽类物质，具有与PTH相似的生物学活性。目前很多研究将PTHrP用于增加种植体周围骨密度，提高种植体早期稳定性方面，以减少骨愈合时间。口腔种植体早期稳定性的关键因素在于牙槽骨的质量和剩余牙槽骨的体积，受系统性骨质疏松的影响。然而PTHrP不同的给药方式对骨组织的影响不同，间歇的、低剂量的给药能促进成骨，增加骨量；持续的、大剂量的PTHrP会引起破骨细胞活化，抑制成骨细胞功能。另外，PTHrP存在着半衰期时间短、容易变性、靶向性差且价钱高昂等问题，因此研制控制其释放的载体，提高其生物利用率是有效的解决以上缺陷的途径。聚乙交酯-丙交酯(PLGA)是一种生物相容性良好的生物可降解高分子材料，具有良好的可降解性、可吸收性，而且可作为药物载体以控制药物释放的速度。因此研制以PLGA作为药物控释载体负载PTHrP，可控制药物释放速度，从而达到长时间缓释的目的。方法：采用W1/O/W2挥发法制备实验所需的包载PTHrP1-34的PLGA微球。通过SEM、核磁、红外等观察PLGA载药微球结构；利用紫外分光光度法在280nm 考察载药微球的体外释放特性。并通过体细胞毒性评价，碱性磷酸酶测定等检测负载PTHrP1-34的PLGA微球对小鼠前骨细胞的分化、细胞活性及凋亡效应的影响。结果： 1、PLGA重复单元中乳酸和羟基乙酸单元的比约为85:15，，分子量Mn=85000，PDI=1.26。通过观察载药微球具有良好的球形结构，平均粒径约为8μm。包载PTHrP1-34的PLGA微球的载药率和包封率分别为0.84%和72.3%。包载PTHrP1-34的PLGA微球能实现长达25day的持续释放。 2、PTHrP1-34载药微球的细胞学评价：间歇性使PTHrP1-34作用于MC3T3-E1时，浓度为1×10-9mol/L的PTHrP1-34对MC3T3-E1增殖及ALP活性促进效应最大，与文献报道略有差异；包载了PTHrP1-34的PLGA缓释微球间歇性作用于MC3T3-E1时，相比于空白对照组和纯药组，总浓度在1×10-9mol/L时具有明显的促进MC3T3-E1细胞增殖以及增加ALP活性的作用（P0.05）。结论： 1、包载PTHrP1-34的PLGA微球的持续释放可以达到25day，能较好地实现PTHrP1-34在生理温度下的长期持续释放。 2、最适浓度10-9mol/L的PTHrP1-34间歇性给药时可促进MC3T3-E1增殖及增加ALP活性。 3、包载了PTHrP1-34的PLGA缓释微球作用于MC3T3-E1时，可以明显促进MC3T3-E1的增殖与分化，提示包载了PTHrP1-34的PLGA缓释微球，在口腔种植领域的应用具有较好的应用前景，为临床解决种植体周围骨量不足等问题提供了新的可能的解决方案。
[Abstract]:Research background and purpose:
Parathyroid hormone (Parathyroid, Hormone, PTH) is promote osteoblast formation and enhance osteoblast mineralization in the treatment of osteoporosis drugs. And parathyroid hormone related peptide (PTHrP) is a kind of PTH (1~34) in the gene encoding, molecular structure, a polypeptide of many of the same or similar structure and receptor signal transduction, with biological activity similar to that of PTH. At present a lot of research will be PTHrP to increase the bone density around the implant, improve early implant stability, to reduce the time of bone healing.
The key factor lies in the stability of early dental implant alveolar bone quality and alveolar bone volume, affected by systemic osteoporosis. However, PTHrP administered different effects on bone tissue intermittent, low doses of the drug can promote bone formation, increase bone mass; sustained, high dose PTHrP induced osteoclast activation, inhibition of osteoblast function. In addition, PTHrP has a short half-life, easy degeneration, targeting the poor and high price, so the development of the release of vector control, improve their bioavailability is the effective way to solve the above defects. Polyglycollide - C poly (PLGA) is a kind of biodegradable polymer materials, has good biodegradability and absorbability, and can be used as drug carrier to control the rate of drug release. Therefore the development of PLGA as drug controlled release The carrier load PTHrP can control the rate of drug release so as to achieve a long time release.
Method:
The PLGA microspheres containing PTHrP1-34 were prepared by W1/O/W2 evaporation method. The structure of PLGA drug loaded microspheres was observed by SEM, NMR and IR, and 280nm was detected by ultraviolet spectrophotometry.
The release characteristics of drug loaded microspheres were investigated. The effects of PTHrP1-34 loaded PLGA microspheres on the differentiation, cell viability and apoptosis of mouse bone marrow cells were detected by somatic cytotoxicity and alkaline phosphatase assay.
Result:
1, lactic acid and glycolic acid unit PLGA repeat unit was 85:15 and the ratio of molecular weight of Mn=85000 and PDI=1.26. through the observation of microspheres with spherical structure good sustained release, the average particle size is about the drug loading rate of 8 M. PLGA microspheres loaded PTHrP1-34 and encapsulation rate were 0.84% PLGA microspheres and 72.3%. package PTHrP1-34 can achieve up to 25day.
2, the cytological evaluation of PTHrP1-34 loaded microspheres:
The effect of PTHrP1-34 on intermittent MC3T3-E1, concentration of 1 * 10-9mol/L PTHrP1-34 on MC3T3-E1 proliferation and ALP activity of promoting effect, slightly with the reported difference; PTHrP1-34 loaded PLGA microspheres intermittent effect on MC3T3-E1 when compared to the blank control group and pure drug group, the total concentration significantly stimulated the proliferation of MC3T3-E1 cells along with the increase of ALP activity in 1 * 10-9mol/L (P0.05).
Conclusion:
1, the continuous release of PLGA microspheres loaded with PTHrP1-34 can reach 25day, which can achieve a good long-term sustained release of PTHrP1-34 at physiological temperature.
2, PTHrP1-34 intermittent administration of the optimum concentration of 10-9mol/L can promote the proliferation of MC3T3-E1 and increase the activity of ALP.
3, PTHrP1-34 loaded PLGA microspheres on MC3T3-E1, can significantly promote the proliferation and differentiation of MC3T3-E1, suggesting that PLGA sustained-release microspheres containing PTHrP1-34, has a good prospect of application in the field of oral implantology, to solve the growing problem of clinical bone defects around the body provides a new possible solution.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R783.6

【参考文献】