生长因子缓释微球修复压力性尿失禁鼠功能及结构的研究
发布时间：2018-09-13 21:46
【摘要】：背景胰岛素样生长因子-1可用于治疗经阴道分娩的压力性尿失禁病人损伤的尿道外括约肌。新的药物控制释放系统—海藻酸钙-多聚鸟氨酸-明胶微球可用于包裹并保持生长因子的活性,实现活性药物缓慢释放,持续发挥治疗作用。目的1.制备IGF-1缓释微球并检测其理化性质,分析微球的缓释效果;2.构建产伤动物模型,通过功能学试验以及组织学检测验证IGF-1缓释微球对其尿控功能及结构的恢复作用。方法1通过乳化交联法制备可缓释IGF-1的海藻酸钙-多聚鸟氨酸-明胶微球。在光镜下观察微球的形态并进行粒径分析,对微球的常温体外孵育的稳定性、体外机械强度、生物安全性进行检测分析,后通过ELISA试剂盒计算微球中IGF-1的包封率、载药率及释放特性。2使用阴道扩张来模拟产伤模型。将动物分为四个组,分别是假手术组、生理盐水组、空载微球组和IGF-1微球组,按分组进行处理。手术一周后进行漏尿点压试验和尿道外括约肌肌电图监测。功能检测后处死,取出尿道组织,处理后切片进行染色,观察尿道及周围组织显微形态结构改变。结果1微球的制备、理化性质及缓释效果:经乳化交联法可得到大小均一且稳定的微球,平均直径为(91.72±3.51)μm;微球体外孵育实验证明微球在等渗透压生理盐水中形态稳定,并无膨胀或破裂;微球体外机械强度测试中,震荡摇晃60小时后,微球的完整率仍为98.4±0.3%,表现出良好的稳定性;三个不同浓度(100%、50%、20%)的微球浸出液通过相对增值率RGR计算其细胞毒性分级均为0到1级,证明微球没有细胞生物毒性;微球释放IGF-1有“突释”的特点,累计36小时微球中约90%的IGF-1生长因子被释放,其后12天内的释放曲线呈现缓慢微量的线性增加趋势;缓释微球包裹IGF-1的载药量为:606.52ng/g,包封率为45.3%。2 IGF-1缓释微球注射治疗女性压力性尿失禁模型鼠的作用及机制探讨:手术一周后对SD鼠进行漏尿点压试验,假手术组的漏尿点压为44.4 ± 3.4 cmH2O,而生理盐水组与空载微球组的漏尿点压分别为23.9± 1.3 cmH2O和21.7± 0.8 cmH20,与假手术组相比显著降低(p0.05),而IGF-1微球组的漏尿点压为28.4 ± 1.2 cmH2O,比空载微球组有显著性地提高(p0.05);Masson's Trichrome染色可发现阴道扩张损伤后的尿道外括约肌有广泛的断裂,肌肉排列紊乱,组织间有胶原纤维沉积,经过了 IGF-1微球治疗后可观察到肌肉的层数比生理盐水组和空载微球组有明显的恢复,而且胶原纤维的沉积也更少,尿道外括约肌的形态结构也完整;对于假手术组,CD31阳性的毛细血管密度在生理盐水组和空载微球组明显下降,而经过IGF-1微球治疗的大鼠的尿道组织具有更高密度的毛细血管密度,与生理盐水组和空载微球组比较有统计学差异(p0.05)。结论使用乳化交联法可构建大小均一、形态稳定、有相当机械强度和无生物毒性的海藻酸钙-多聚鸟氨酸-明胶微球,微球对IGF-1的载药率和释放特性均符合针对压力性尿失禁治疗需求的生长因子缓释系统的设计要求;而使用IGF-1缓释微球治疗压力性尿失禁大鼠能有效促进受损的尿道括约肌的损伤后恢复以及血管再生,能明显提高大鼠的尿控功能以及改善组织结构。IGF-1缓释微球有望作为一种新型的治疗压力性尿失禁的方法。
[Abstract]:BACKGROUND Insulin-like growth factor-1 (IGF-1) can be used to treat urethral external sphincter injury in patients with stress urinary incontinence during vaginal delivery. 1. Preparation of sustained-release microspheres of IGF-1 and its physicochemical properties were tested to analyze the sustained-release effect of the microspheres; 2. Establishment of an animal model of birth injury and verification of the recovery of urinary control function and structure of the sustained-release microspheres of IGF-1 by functional test and histological examination. Methods 1. The sustained-release of IGF-1 was prepared by emulsifying and crosslinking method. Microspheres. The morphology of microspheres was observed under light microscope and the size of microspheres was analyzed. The stability, mechanical strength and biological safety of microspheres incubated at room temperature in vitro were tested and analyzed. The encapsulation efficiency, drug loading rate and release characteristics of IGF-1 in microspheres were calculated by ELISA kit. One week after operation, leak point pressure test and external urethral sphincter electromyography (ESEMG) were performed. The urethral tissues were removed and stained. Results 1. Preparation, physicochemical properties and sustained-release effect of the microspheres: The microspheres with uniform size and stable average diameter (91.72 65507 The complete rate of the microspheres was 98.4 6550 The release curve of long-acting factor showed a slow linear increasing trend within 12 days after the release of long-acting factor; the drug loading of sustained-release microspheres encapsulated with IGF-1 was 606.52 ng/g, and the encapsulation rate was 45.3%. 2 The effect and mechanism of sustained-release microspheres on female stress urinary incontinence model rats: leak point pressure test and artificial hand test were carried out one week after operation. The leak point pressure of the operation group was 44.4 (+ 3.4) cmH2O, while that of the saline group and the empty microsphere group were 23.9 (+ 1.3) cmH2O and 21.7 (+ 0.8) cmH20, which were significantly lower than that of the sham operation group (p0.05), while that of the IGF-1 microsphere group was 28.4 (+ 1.2) cmH2O, which was significantly higher than that of the empty microsphere group (p0.05). Staining revealed extensive rupture of the external urethral sphincter after vaginal dilatation injury, disorder of muscle arrangement and deposition of collagen fibers between tissues. In sham operation group, the capillary density of CD31 positive group decreased significantly in normal saline group and no-loaded microsphere group, while the urethral tissue of rats treated with IGF-1 microsphere had higher density of capillary density, which was significantly different from that of normal saline group and no-loaded microsphere group (p0.05). Conclusion Emulsion intercourse was used. The microspheres with uniform size, stable morphology, considerable mechanical strength and non-toxicity could be constructed. The drug loading rate and release characteristics of the microspheres met the design requirements of growth factor sustained-release system for stress urinary incontinence treatment. The sustained-release microspheres of IGF-1 were used to treat stress urine. Incontinence rats can effectively promote the recovery of injured urethral sphincter and vascular regeneration, significantly improve the urinary control function and improve the tissue structure of rats. IGF-1 sustained-release microspheres are expected to be a new treatment for stress urinary incontinence.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R694.54
，
本文编号：2241832
[Abstract]:BACKGROUND Insulin-like growth factor-1 (IGF-1) can be used to treat urethral external sphincter injury in patients with stress urinary incontinence during vaginal delivery. 1. Preparation of sustained-release microspheres of IGF-1 and its physicochemical properties were tested to analyze the sustained-release effect of the microspheres; 2. Establishment of an animal model of birth injury and verification of the recovery of urinary control function and structure of the sustained-release microspheres of IGF-1 by functional test and histological examination. Methods 1. The sustained-release of IGF-1 was prepared by emulsifying and crosslinking method. Microspheres. The morphology of microspheres was observed under light microscope and the size of microspheres was analyzed. The stability, mechanical strength and biological safety of microspheres incubated at room temperature in vitro were tested and analyzed. The encapsulation efficiency, drug loading rate and release characteristics of IGF-1 in microspheres were calculated by ELISA kit. One week after operation, leak point pressure test and external urethral sphincter electromyography (ESEMG) were performed. The urethral tissues were removed and stained. Results 1. Preparation, physicochemical properties and sustained-release effect of the microspheres: The microspheres with uniform size and stable average diameter (91.72 65507 The complete rate of the microspheres was 98.4 6550 The release curve of long-acting factor showed a slow linear increasing trend within 12 days after the release of long-acting factor; the drug loading of sustained-release microspheres encapsulated with IGF-1 was 606.52 ng/g, and the encapsulation rate was 45.3%. 2 The effect and mechanism of sustained-release microspheres on female stress urinary incontinence model rats: leak point pressure test and artificial hand test were carried out one week after operation. The leak point pressure of the operation group was 44.4 (+ 3.4) cmH2O, while that of the saline group and the empty microsphere group were 23.9 (+ 1.3) cmH2O and 21.7 (+ 0.8) cmH20, which were significantly lower than that of the sham operation group (p0.05), while that of the IGF-1 microsphere group was 28.4 (+ 1.2) cmH2O, which was significantly higher than that of the empty microsphere group (p0.05). Staining revealed extensive rupture of the external urethral sphincter after vaginal dilatation injury, disorder of muscle arrangement and deposition of collagen fibers between tissues. In sham operation group, the capillary density of CD31 positive group decreased significantly in normal saline group and no-loaded microsphere group, while the urethral tissue of rats treated with IGF-1 microsphere had higher density of capillary density, which was significantly different from that of normal saline group and no-loaded microsphere group (p0.05). Conclusion Emulsion intercourse was used. The microspheres with uniform size, stable morphology, considerable mechanical strength and non-toxicity could be constructed. The drug loading rate and release characteristics of the microspheres met the design requirements of growth factor sustained-release system for stress urinary incontinence treatment. The sustained-release microspheres of IGF-1 were used to treat stress urine. Incontinence rats can effectively promote the recovery of injured urethral sphincter and vascular regeneration, significantly improve the urinary control function and improve the tissue structure of rats. IGF-1 sustained-release microspheres are expected to be a new treatment for stress urinary incontinence.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R694.54
，
本文编号：2241832
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