纳米靶向FSH受体卵巢早衰大鼠模型建立的相关研究

发布时间：2017-12-31 12:06

本文关键词：纳米靶向FSH受体卵巢早衰大鼠模型建立的相关研究　出处：《复旦大学》2012年硕士论文　论文类型：学位论文

【摘要】：卵巢早衰(premature ovarian failure, POF)是指女性40周岁之前发生的卵巢功能衰竭,以雌激素缺乏(E225pg/mL)及促性腺激素,包括卵泡刺激素(Follicle Stimulating Hormone, FSH)和黄体生成素(Luteinizing Hormone, LH)水平升高(FSH40IU/L,LH30IU/L)为特征,可发生在月经初潮之前或之后,表现为继发性闭经、月经稀发或绝经、不孕。POF是引起妇女不孕和内分泌紊乱的常见原因,女性30岁之前的发病率为0.1%,40岁之前的发病率为1%。虽然POF的发病率日趋增高,但其发病机制仍不甚明了。为了深入研究POF的发生及进展规律,探寻安全、有效的治疗方法,我们需要建立一种符合病因学同时又理想、可靠的动物模型。靶向治疗具有很强的目的性,可以显著减少药物对其它组织的毒副作用。本课题利用FSH多肽片段作为靶向头基,联合纳米粒载体,探索构建主动靶向大鼠FSH受体的纳米系统,为进一步建立更加有效的FSH受体功能抑制型POF动物模型的提供理论基础。第一部分根据大鼠FSHβ亚基第32-52氨基酸序列成功制备了rFSHβ32-52多肽片段,并将Maleimide-PE-PLA和MPEG-PLA按质量比1：9混合,采用复乳/溶媒蒸发法制备了载香豆素-6的NP、rFSHβ32-NP,粒径分别为100和120nm左右,Zeta电位值为-14mV左右。NP表面经多肽修饰后粒径有一定程度的增加,电位值无明显的变化。为了验证rFSH32-NP的靶向性,第二部分采用香豆素-6荧光标记的NP及rFSHβ32-NP对细胞进行靶向内吞能力的定性及定量检验,评价了rFSHβ32-52对大鼠卵巢颗粒细胞的靶向能力。相较于FSHR阴性的SKOV3细胞,FSHR阳性的CaOV3和大鼠卵巢颗粒细胞对rFSHβ32-NP的摄取率显著增加,并且呈现出一定的时间和浓度依赖性。说明利用rFSHβ32-52多肽片段作为靶向头基的纳米粒对大鼠卵巢颗粒细胞具有主动靶向的作用。本研究构建的rFSHβ32-NP给药系统,实现了对大鼠卵巢颗粒细胞靶向给药的目的,为进一步创建规范统一化的POF动物模型提供了理论和实验依据,具有一定的参考价值。
[Abstract]:Premature ovarian failure (premature ovarian failure, POF) refers to the ovarian failure in women 40 years of age before, with the lack of estrogen (E225pg/mL) and gonadotropins, follicle stimulating hormone (Follicle Stimulating, Hormone, FSH) and luteinizing hormone (Luteinizing, Hormone, LH) levels (FSH40IU/L, LH30IU/L) as the characteristic, after can occur in or before menarche, as secondary amenorrhea, oligomenorrhea or menopause, infertility.POF are a common cause of infertility in women and endocrine disorders, the incidence of women before the age of 30 was 0.1%, the incidence rate of 1%. before the age of 40, although the incidence of POF is increasing, but its pathogenesis is still not very clear. For the occurrence and progress of law, in-depth study of POF to explore the safe, effective treatment, we need to establish an accord with the etiology and ideal animal model of targeted therapy has reliable. A strong purpose, can significantly reduce the side effects on other tissues drugs. The issue of the use of FSH polypeptides as targeting ligand, combined with nanoparticles, explore the construction of targeted nano system of the rat FSH receptor, FSH receptor function to further establish a more effective inhibition of the POF animal model theory the foundation.
The first part according to the 32-52 amino acid sequence of rat FSH were successfully synthesized rFSH beta subunit beta 32-52 peptide fragments, and Maleimide-PE-PLA and MPEG-PLA at the mass ratio of 1:9 mixture, using double emulsion / solvent evaporation method to prepare loading coumarin -6 NP, rFSH beta 32-NP, the particle size was about 100 and 120nm respectively, Zeta potential the value is about -14mV.NP after the surface modification with FSHP diameter increased to a certain extent, the potential value of no significant changes.
In order to verify the rFSH32-NP target, the second part of the coumarin fluorescent -6 markers NP and rFSH beta 32-NP qualitative and quantitative test of the ability to swallow inward target cells, and evaluate the rFSH beta 32-52 on rat ovarian granulosa cell targeting ability. Compared to FSHR negative SKOV3 cells of rFSH beta 32-NP uptake ovarian granulosa cells CaOV3 and FSHR positive rate in rats increased significantly, and showed a time and concentration dependent manner. The use of rFSH beta peptide fragments of 32-52 nanoparticles as a targeting ligand has the effect of active targeting on rat ovarian granulosa cells.
The rFSH beta 32-NP delivery system has achieved the goal of targeting the ovarian granulosa cells in rats. It provides a theoretical and experimental basis for further establishing standardized and unified POF animal models, and has a certain reference value.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R711.75;R-332

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