壳聚糖—磷酸甘油—庆大霉素凝胶系统经圆窗龛局部给药的实验研究

发布时间：2018-03-22 19:36

本文选题：壳聚糖-磷酸甘油凝胶　切入点：庆大霉素　出处：《山东大学》2012年博士论文　论文类型：学位论文

【摘要】：第一部分壳聚糖-磷酸甘油-庆大霉素凝胶圆窗龛局部给药治疗梅尼埃病的有效性及安全性研究研究目的：目前利用庆大霉素(GT)耳毒性,治疗梅尼埃病眩晕,得到越来越多的耳科医生的关注。庆大霉素内耳局部给药用于治疗梅尼埃病,操作简单,疗效确切。由于给药浓度及剂量的不同,不同研究表明,庆大霉素除了发挥对前庭毛细胞的破坏作用外,其对内耳毛细胞亦有不同程度的损伤。该研究应用壳聚糖-磷酸甘油(CGP)凝胶携带庆大霉素,通过圆窗龛给药的方式,可控及缓释地将庆大霉素释放进入内耳,与传统鼓室内注射庆大霉素相比,评估其治疗梅尼埃病的安全性及有效性。实验方法：听力及前庭功能正常的C57BL/6J小鼠(6-8周)24只,随机分成3组,A组为经鼓膜穿刺隔日注射庆大霉素硫酸盐(6mg/ml,10ul)组；B组为经鼓膜穿刺隔2日注射庆大霉素硫酸盐(6mg/ml,10ul)组；C组为应用CGP凝胶缓释系统经圆窗龛释放庆大霉素(300mg/ml,0.2ul)组。A、B组小鼠均接受2周重复药物注射,C组小鼠仅接受1次圆窗龛给药。听力学评估：在注射前及注射后14天,采应用脑干诱发电位(ABR)评估小鼠的听力变化情况；前庭功能评估：在注射前及注射后的14天内,每天进行垂直转鼓实验,评估小鼠的前庭功能变化情况。实验结果： ABR检测示：在注射后的第14天,A组小鼠ABR阈值在16kHz、24kHz、32kHz、40kHz均比用药前明显提高,差异有显著统计学意义(p0.05)；B组小鼠在注射后第14天,仅有40kHzABR阈值较注射前显著提高,差异有统计学意义(p=0.030,p0.05),其余频率,ABR阈值均未见明显提高,差异无统计学意义(p0.05)；C组小鼠各频率ABR阈值较注射前均无显著提高,差异无统计学意义(p0.05)。垂直转鼓实验示： A组小鼠在术后第10天的保持时间比术前明显降低(p=0.031,p0.05),差异有统计学意义,而在术后第14天的保持时间与术前相比无明显差异(p=0.105,p0.05),无统计学意义；B组小鼠在术后所有时间点均与术前数据无显著性差异(p0.05)；C组小鼠的平均保持时间在术后第6天、第10天与术后第14天均明显低于术前(p值分别为0.041,0.034,0.006；p值均0.05)。C组小鼠的平均保持时间在术后14天明显低于A组(p=0.019；p0.05)和B组(p=0.016；p0.05)。实验结论：壳聚糖-磷酸甘油(CGP)凝胶缓释系统携带庆大霉素进入内耳,可以造成持续而稳定的前庭功能下降,而不引起显著的听觉功能下降,较之传统的鼓室注射治疗,其是一种安全而有效的内耳缓释给药系统,有良好的临床应用前景。第二部分壳聚糖-磷酸甘油-庆大霉素凝胶经圆窗龛给药后庆大霉素在内耳毛细胞的分布研究目的：目前利用庆大霉素耳毒性,治疗梅尼埃病眩晕,得到越来越多的耳科医生的关注。庆大霉素内耳局部给药用于治疗梅尼埃病,操作简单,疗效确切。但是由于直接注射庆大霉素至中耳后,不能准确地控制与圆窗膜接触的药量,而且部分药液通过咽鼓管排除中耳腔,或被中耳腔粘膜吸收,因此导致进入内耳的庆大霉素的剂量有很大波动性,因此也产生了很大的疗效差异。为克服现有方法的局限,建立一种安全有效并且方便的给药模式,使其能够在单次应用后,即可持续缓慢地释放庆大霉素至内耳,我们建立了壳聚糖-磷酸甘油凝胶系统应用于庆大霉素内耳局部给药。前期研究通过与传统鼓室内注射的对照,已经证明壳聚糖-磷酸甘油-庆大霉素凝胶在小鼠圆窗龛局部注射后,缓释庆大霉素至内耳的能力,及其对耳蜗、前庭系统功能的影响。但是对于此缓释凝胶携带庆大霉素进入内耳后,庆大霉素在内耳的分布情况,我们尚不清楚,因此,此实验的目的是研究庆大霉素在该缓释凝胶系统的缓释作用下,在内耳毛细胞中如何分布的,以及如何影响内耳毛细胞的形态及功能的。实验方法：将硫酸庆大霉素(gentamicin, GT)和德州红(texas red, TR)混合后置于37。搅拌过夜使其结合形成庆大霉素德州红螯合物(gentamicin-texas red, GTTR),然后将GTTR与壳聚糖-磷酸甘油凝胶(CGP)混合形成壳聚糖-磷酸甘油-庆大霉素凝胶(CGP-GTTR-hydrogel)。通过耳后入路暴露小鼠左侧耳蜗圆窗龛,取0.5ul该凝胶注入圆窗龛内,让其左耳向上平卧半小时待胶体凝固。分别于凝胶注射后1天及7天,采取心脏灌注4%多聚甲醛的方式处死小鼠,快速将其耳蜗取下,蜗内灌注固定液,固定2小时后,解剖其基底膜、椭圆囊斑及球囊斑,用Phalloidin-Alexa-488进行染色,进行铺片,使用Leica激光共聚焦显微镜观察分析,Phalloidin与GTTR使用红绿双色荧光通道进行扫描观察,选择一定的区域进行荧光半定量分析,测定得到的平均像素强度即为荧光强度。观察庆大霉素在内耳的分布规律及毛细胞的形态。实验结果：在前庭系统中,经圆窗龛局部注射壳聚糖-磷酸甘油-庆大霉素凝胶1天后,红色颗粒状GTTR荧光主要位于球囊斑上的毛细胞胞质内,其荧光强度在注射后1天和7天分别是14.13±4.09,4.69±1.76,注射后7天时的荧光强度明显强于注射后1天时的荧光强度,差异有统计学意义(p0.05)。另外,注射后1天时表皮板表面的纤毛束明显多于注射后7天时的纤毛束。在耳蜗系统中,经圆窗龛局部注射壳聚糖-磷酸甘油-庆大霉素凝胶1天后,可观察到红色颗粒状GTTR荧光主要位于耳蜗基底膜的底转及中转,其荧光强度分别为51.59±6.42,20.64±4.14,底转强度明显强于中转荧光强度(p0.01),差异有统计学意义。同样地,在注射凝胶7天后,红色颗粒状GTTR荧光仍然位于耳蜗基底膜的底转及中转,其荧光强度分别为43.01±3.61,12.17±2.43,底转强度明显强于中转荧光强度(p0.01),差异有统计学意义。在凝胶注射后的1天和7天,耳蜗的顶转均未见到红色颗粒状荧光。同时,比较耳蜗底转在注射后1天和7天的荧光强度可见,注射后1天的荧光强度明显强于注射后7天,差异有统计学意义(p0.05)；同样比较耳蜗中转在注射后1天和7天的荧光强度发现同样的规律,注射后1天的荧光强度明显强于注射后7天,差异有统计学意义(p0.05)。另外,在注射后7天,耳蜗底转部分外毛细胞缺失,但是在注射后1天和7天,在耳蜗的顶转及中转均未见毛细胞的缺失。研究结论：该研究探讨了庆大霉素通过该凝胶缓释系统释放进入内耳后,庆大霉素在内耳的分布有两个基本规律,第一是时间依赖性,即给药时间越长,药物在耳蜗毛细胞中的浓度越低；第二是底转-顶转的浓度梯度变化规律,即越靠近圆窗龛的部位如耳蜗底转及前庭,药物的浓度越高。利用该凝胶系统通过圆窗龛释放庆大霉素进入内耳,可以较好的保存低频及中频听力,同时可以破坏耳蜗的前庭功能及底转毛细胞的部分功能。因此,此种给药系统可以应用于临床上已经存在高频听力损失的梅尼埃病人的治疗中,为临床上难治性梅尼埃病的治疗提出了一种全新的治疗方案。
[Abstract]:The first part
The chitosan glycerol phosphate gel - gentamicin round window niche topical treatment of Meniere's disease study of the efficacy and safety of
The purpose of the study:
The use of gentamicin (GT) ototoxicity, treatment of vertigo in Meniere's disease, get more and more attention. Aurist gentamicin administered for the treatment of inner ear Meniere's disease, simple operation, definite curative effect. Due to the different concentration and dose, different studies have shown that, in addition to play the gentamicin damage to vestibular hair cells. It also has a different degree of damage to the hair cells of the inner ear. The research and application of chitosan glycerophosphate (CGP) gel with gentamicin, through the round window niche the mode of administration, and the controlled release of gentamicin released into the inner ear, compared with the traditional intratympanic gentamicin injection, to assess the safety of the treatment of Meniere's disease and effective.
Experiment method:
Hearing and vestibular function in normal C57BL/6J mice (6-8 weeks) 24 rats were randomly divided into 3 groups, group A underwent tympanic membrane puncture after injection gentamycin sulfate (6mg/ml, 10ul) group; group B underwent tympanic membrane puncture every 2 days injection of gentamicin sulfate (6mg/ml, 10ul) group; group C with CGP gel release through the round window niche release of gentamicin (300mg/ml, 0.2ul).A group, B group of mice received 2 weeks of repeated injections of the drug, the mice in group C received only 1 times in the round window niche administration.: audiological assessment before injection and 14 days after injection, application of brainstem auditory evoked potential (ABR) changes in hearing assessment in mice; vestibular function assessment: before injection and after injection 14 days, vertical drum test every day, changes in the evaluation of vestibular function in mice.
The experimental results:
ABR showed: in Fourteenth days after injection, the mice in group A ABR in 16kHz 24kHz, 32kHz threshold, 40kHz, were higher than before treatment significantly improved, the difference was statistically significant (P0.05); B group of mice fourteenth days after the injection, only 40kHzABR threshold significantly increased before injection, the difference was statistically significant (p=0.030 P0.05, ABR), the rest of the frequency, thresholds were not improved obviously, the difference was not statistically significant (P0.05); C group of mice ABR threshold of each frequency than before the injection were not significantly improved, the difference was not statistically significant (P0.05).
Vertical drum experiment: A group of mice on the tenth postoperative day retention time is significantly lower than that before surgery (p=0.031, P0.05), the difference was statistically significant, and after fourteenth days of retention time compared with preoperative had no significant difference (p=0.105, P0.05), no statistical significance; B mice in operation after all the time points were compared with preoperative data showed no significant difference (P0.05); C group of mice maintained an average time on the sixth day after operation, tenth days and fourteenth days after operation were significantly lower than the preoperative (P = 0.041,0.034,0.006; P 0.05) on the 14 day after operation was significantly lower than A group average retention time of mice in.C group (p=0.019; P0.05) and B group (p=0.016; P0.05).
The experimental conclusion:
Chitosan glycerophosphate (CGP) gel delivery system with gentamicin into the inner ear, can cause persistent vestibular function decline, without causing the auditory function decreased significantly, compared with the traditional intratympanic injection therapy, which is a safe and effective ear drug delivery system has good prospect of clinical application.
The second part
The chitosan glycerol phosphate gentamicin gel through the round window niche for the distribution of gentamicin in the inner hair cells after administration
The purpose of the study:
The use of gentamicin ototoxicity, treatment of vertigo in Meniere's disease, get more and more attention. Aurist gentamicin administered for the treatment of inner ear Meniere's disease, simple operation, definite curative effect. But because of the direct injection of gentamicin to the middle ear, can accurately control the charge and the round window membrane contact, and part of the liquid through the eustachian tube out of the middle ear cavity or is the middle ear cavity mucosal absorption, thus resulting in the inner ear of gentamicin dose have great volatility, therefore produce a very different effect. In order to overcome the limitation of existing methods to establish a safe and effective and convenient mode of administration, so that it can be in a single application, you can continue to slowly release of gentamicin the inner ear, we established the application of chitosan glycerophosphate gel system in the inner ear of gentamicin topically. Preliminary studies comparing with the traditional drum The control of indoor injection, has proved that chitosan glycerophosphate gel - gentamicin in mice after local injection of round window niche, the ability to release gentamicin to the inner ear, and its effect on cochlear, vestibular function. But for the sustained-release gel carrying gentamicin into the inner ear, the distribution of gentamicin in the inner ear, we do not know. Therefore, the objective of this experiment is to study the effect of gentamicin in sustained-release sustained-release gel system, how the distribution in inner ear hair cells, and how to influence the morphology and function of inner ear hair cells.
Experiment method:
Gentamycin sulfate (gentamicin, GT) and Dezhou red (Texas Red, TR) mixed in 37. stirred overnight the combination of the formation of gentamicin Dezhou red chelate (gentamicin-texas red, GTTR), and then GTTR and chitosan glycerol phosphate gel (CGP) mixed into chitosan glycerophosphate gel (gentamicin CGP-GTTR-hydrogel). Through the posterior approach in mice exposed to the left ear cochlea round window niche, 0.5ul of the gel into the round window niche, let the left ear to supine to half an hour respectively. Solidification colloidal gel injection after 1 days and 7 days, take heart perfusion of 4% paraformaldehyde the mice were killed quickly the cochlea removed, cochlear perfusion fixative, fixed after 2 hours, the anatomy of the basal membrane, utricle and saccule, using Phalloidin-Alexa-488 staining of flatmount using Leica laser confocal microscope observation and analysis, Phalloidin and GTTR Both red and green fluorescence channel were observed, some regional fluorescence semi quantitative analysis, measuring the average pixel intensity is obtained. The fluorescence intensity observed in the distribution of gentamicin and hair cells of the inner ear morphology.
The experimental results:
In the vestibular system, the round window niche of local injection of gentamicin chitosan glycerol phosphate gel after 1 days, the red fluorescence of GTTR was mainly located in the granular saccule on hair cells in the cytoplasm, the fluorescence intensity in 1 days after injection and 7 days were 14.13 + 4.09,4.69 + 1.76, the fluorescence intensity at 7 days after injection the fluorescence intensity was stronger than the 1 days after the injection, the difference was statistically significant (P0.05). In addition, 1 days after injection of epidermal surface of stereociliary bundles significantly more than the injection of stereociliary bundles after 7 days.
In the cochlear system, the round window niche of local injection of gentamicin chitosan glycerol phosphate gel for 1 days, observed the red granular GTTR fluorescence mainly located in the basilar membrane of the cochlea basal turn and transfer, the fluorescence intensity was 51.59 + 6.42,20.64 + 4.14, the base is stronger than the transfer fluorescence intensity (P0.01). The difference was statistically significant. Similarly, in 7 days after the injection of gel, red granular GTTR fluorescence is still in the basilar membrane of the cochlea basal turn and transfer, the fluorescence intensity was 43.01 + 3.61,12.17 + 2.43, the base is stronger than the transfer fluorescence intensity (P0.01), there was significant difference in the gel after injection of 1. And 7 days, the cochlea apical turn were not seen red granular fluorescence. At the same time, compared with the basal turn of the cochlea after injection in fluorescence intensity of 1 and 7 days after injection of visible, fluorescence intensity of 1 days was higher than that in the 7 days after injection, the difference was statistically The significance (P0.05); also in the cochlea after injection of fluorescent intensity transfer 1 days and 7 days and found the same rules, 1 days after injection of fluorescence intensity was stronger than the 7 days after injection, the difference was statistically significant (P0.05). In addition, in 7 days after injection, cochleostomy part of outer hair cell loss however, in 1 days after injection and 7 days, in the absence of the top and the cochlear hair cells were not found in transit.
The conclusion of the study:
The study of gentamicin through the gel delivery system released into the inner ear after gentamicin has two basic rules of distribution in the inner ear, the first is the time dependence of the delivery time is longer, the drug concentration in the cochlear hair cells in the lower; second is the bottom to top concentration gradient changes turn, i.e. the more close to the round window niche sites such as the basal turn of the cochlea and vestibular, the higher the concentration of drugs into the inner ear through the round window niche. The release of gentamicin using the gel system, keeping the low frequency and middle frequency hearing can be better, some functions of hair cells and can turn the court before the destruction of the cochlear function and bottom. Therefore, this kind of treatment to the system can be applied to clinical medicine already exists on the high frequency hearing loss in patients with Meniere's, put forward a new therapeutic scheme for clinical treatment of intractable Meniere's disease.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R764.3

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