本文選題：殼聚糖-磷酸甘油凝膠　切入點(diǎn)：慶大霉素　出處：《山東大學(xué)》2012年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分 殼聚糖-磷酸甘油-慶大霉素凝膠圓窗龕局部給藥治療梅尼埃病的有效性及安全性研究 研究目的： 目前利用慶大霉素(GT)耳毒性,治療梅尼埃病眩暈,得到越來越多的耳科醫(yī)生的關(guān)注。慶大霉素內(nèi)耳局部給藥用于治療梅尼埃病,操作簡(jiǎn)單,療效確切。由于給藥濃度及劑量的不同,不同研究表明,慶大霉素除了發(fā)揮對(duì)前庭毛細(xì)胞的破壞作用外,其對(duì)內(nèi)耳毛細(xì)胞亦有不同程度的損傷。該研究應(yīng)用殼聚糖-磷酸甘油(CGP)凝膠攜帶慶大霉素,通過圓窗龕給藥的方式,可控及緩釋地將慶大霉素釋放進(jìn)入內(nèi)耳,與傳統(tǒng)鼓室內(nèi)注射慶大霉素相比,評(píng)估其治療梅尼埃病的安全性及有效性。 實(shí)驗(yàn)方法： 聽力及前庭功能正常的C57BL/6J小鼠(6-8周)24只,隨機(jī)分成3組,A組為經(jīng)鼓膜穿刺隔日注射慶大霉素硫酸鹽(6mg/ml,10ul)組；B組為經(jīng)鼓膜穿刺隔2日注射慶大霉素硫酸鹽(6mg/ml,10ul)組；C組為應(yīng)用CGP凝膠緩釋系統(tǒng)經(jīng)圓窗龕釋放慶大霉素(300mg/ml,0.2ul)組。A、B組小鼠均接受2周重復(fù)藥物注射,C組小鼠僅接受1次圓窗龕給藥。聽力學(xué)評(píng)估：在注射前及注射后14天,采應(yīng)用腦干誘發(fā)電位(ABR)評(píng)估小鼠的聽力變化情況；前庭功能評(píng)估：在注射前及注射后的14天內(nèi),每天進(jìn)行垂直轉(zhuǎn)鼓實(shí)驗(yàn),評(píng)估小鼠的前庭功能變化情況。 實(shí)驗(yàn)結(jié)果： ABR檢測(cè)示：在注射后的第14天,A組小鼠ABR閾值在16kHz、24kHz、32kHz、40kHz均比用藥前明顯提高,差異有顯著統(tǒng)計(jì)學(xué)意義(p0.05)；B組小鼠在注射后第14天,僅有40kHzABR閾值較注射前顯著提高,差異有統(tǒng)計(jì)學(xué)意義(p=0.030,p0.05),其余頻率,ABR閾值均未見明顯提高,差異無統(tǒng)計(jì)學(xué)意義(p0.05)；C組小鼠各頻率ABR閾值較注射前均無顯著提高,差異無統(tǒng)計(jì)學(xué)意義(p0.05)。 垂直轉(zhuǎn)鼓實(shí)驗(yàn)示： A組小鼠在術(shù)后第10天的保持時(shí)間比術(shù)前明顯降低(p=0.031,p0.05),差異有統(tǒng)計(jì)學(xué)意義,而在術(shù)后第14天的保持時(shí)間與術(shù)前相比無明顯差異(p=0.105,p0.05),無統(tǒng)計(jì)學(xué)意義；B組小鼠在術(shù)后所有時(shí)間點(diǎn)均與術(shù)前數(shù)據(jù)無顯著性差異(p0.05)；C組小鼠的平均保持時(shí)間在術(shù)后第6天、第10天與術(shù)后第14天均明顯低于術(shù)前(p值分別為0.041,0.034,0.006；p值均0.05)。C組小鼠的平均保持時(shí)間在術(shù)后14天明顯低于A組(p=0.019；p0.05)和B組(p=0.016；p0.05)。 實(shí)驗(yàn)結(jié)論： 殼聚糖-磷酸甘油(CGP)凝膠緩釋系統(tǒng)攜帶慶大霉素進(jìn)入內(nèi)耳,可以造成持續(xù)而穩(wěn)定的前庭功能下降,而不引起顯著的聽覺功能下降,較之傳統(tǒng)的鼓室注射治療,其是一種安全而有效的內(nèi)耳緩釋給藥系統(tǒng),有良好的臨床應(yīng)用前景。 第二部分 殼聚糖-磷酸甘油-慶大霉素凝膠經(jīng)圓窗龕給藥后慶大霉素在內(nèi)耳毛細(xì)胞的分布 研究目的： 目前利用慶大霉素耳毒性,治療梅尼埃病眩暈,得到越來越多的耳科醫(yī)生的關(guān)注。慶大霉素內(nèi)耳局部給藥用于治療梅尼埃病,操作簡(jiǎn)單,療效確切。但是由于直接注射慶大霉素至中耳后,不能準(zhǔn)確地控制與圓窗膜接觸的藥量,而且部分藥液通過咽鼓管排除中耳腔,或被中耳腔粘膜吸收,因此導(dǎo)致進(jìn)入內(nèi)耳的慶大霉素的劑量有很大波動(dòng)性,因此也產(chǎn)生了很大的療效差異。為克服現(xiàn)有方法的局限,建立一種安全有效并且方便的給藥模式,使其能夠在單次應(yīng)用后,即可持續(xù)緩慢地釋放慶大霉素至內(nèi)耳,我們建立了殼聚糖-磷酸甘油凝膠系統(tǒng)應(yīng)用于慶大霉素內(nèi)耳局部給藥。前期研究通過與傳統(tǒng)鼓室內(nèi)注射的對(duì)照,已經(jīng)證明殼聚糖-磷酸甘油-慶大霉素凝膠在小鼠圓窗龕局部注射后,緩釋慶大霉素至內(nèi)耳的能力,及其對(duì)耳蝸、前庭系統(tǒng)功能的影響。但是對(duì)于此緩釋凝膠攜帶慶大霉素進(jìn)入內(nèi)耳后,慶大霉素在內(nèi)耳的分布情況,我們尚不清楚,因此,此實(shí)驗(yàn)的目的是研究慶大霉素在該緩釋凝膠系統(tǒng)的緩釋作用下,在內(nèi)耳毛細(xì)胞中如何分布的,以及如何影響內(nèi)耳毛細(xì)胞的形態(tài)及功能的。 實(shí)驗(yàn)方法： 將硫酸慶大霉素(gentamicin, GT)和德州紅(texas red, TR)混合后置于37。攪拌過夜使其結(jié)合形成慶大霉素德州紅螯合物(gentamicin-texas red, GTTR),然后將GTTR與殼聚糖-磷酸甘油凝膠(CGP)混合形成殼聚糖-磷酸甘油-慶大霉素凝膠(CGP-GTTR-hydrogel)。通過耳后入路暴露小鼠左側(cè)耳蝸圓窗龕,取0.5ul該凝膠注入圓窗龕內(nèi),讓其左耳向上平臥半小時(shí)待膠體凝固。分別于凝膠注射后1天及7天,采取心臟灌注4%多聚甲醛的方式處死小鼠,快速將其耳蝸取下,蝸內(nèi)灌注固定液,固定2小時(shí)后,解剖其基底膜、橢圓囊斑及球囊斑,用Phalloidin-Alexa-488進(jìn)行染色,進(jìn)行鋪片,使用Leica激光共聚焦顯微鏡觀察分析,Phalloidin與GTTR使用紅綠雙色熒光通道進(jìn)行掃描觀察,選擇一定的區(qū)域進(jìn)行熒光半定量分析,測(cè)定得到的平均像素強(qiáng)度即為熒光強(qiáng)度。觀察慶大霉素在內(nèi)耳的分布規(guī)律及毛細(xì)胞的形態(tài)。 實(shí)驗(yàn)結(jié)果： 在前庭系統(tǒng)中,經(jīng)圓窗龕局部注射殼聚糖-磷酸甘油-慶大霉素凝膠1天后,紅色顆粒狀GTTR熒光主要位于球囊斑上的毛細(xì)胞胞質(zhì)內(nèi),其熒光強(qiáng)度在注射后1天和7天分別是14.13±4.09,4.69±1.76,注射后7天時(shí)的熒光強(qiáng)度明顯強(qiáng)于注射后1天時(shí)的熒光強(qiáng)度,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。另外,注射后1天時(shí)表皮板表面的纖毛束明顯多于注射后7天時(shí)的纖毛束。 在耳蝸系統(tǒng)中,經(jīng)圓窗龕局部注射殼聚糖-磷酸甘油-慶大霉素凝膠1天后,可觀察到紅色顆粒狀GTTR熒光主要位于耳蝸基底膜的底轉(zhuǎn)及中轉(zhuǎn),其熒光強(qiáng)度分別為51.59±6.42,20.64±4.14,底轉(zhuǎn)強(qiáng)度明顯強(qiáng)于中轉(zhuǎn)熒光強(qiáng)度(p0.01),差異有統(tǒng)計(jì)學(xué)意義。同樣地,在注射凝膠7天后,紅色顆粒狀GTTR熒光仍然位于耳蝸基底膜的底轉(zhuǎn)及中轉(zhuǎn),其熒光強(qiáng)度分別為43.01±3.61,12.17±2.43,底轉(zhuǎn)強(qiáng)度明顯強(qiáng)于中轉(zhuǎn)熒光強(qiáng)度(p0.01),差異有統(tǒng)計(jì)學(xué)意義。在凝膠注射后的1天和7天,耳蝸的頂轉(zhuǎn)均未見到紅色顆粒狀熒光。同時(shí),比較耳蝸底轉(zhuǎn)在注射后1天和7天的熒光強(qiáng)度可見,注射后1天的熒光強(qiáng)度明顯強(qiáng)于注射后7天,差異有統(tǒng)計(jì)學(xué)意義(p0.05)；同樣比較耳蝸中轉(zhuǎn)在注射后1天和7天的熒光強(qiáng)度發(fā)現(xiàn)同樣的規(guī)律,注射后1天的熒光強(qiáng)度明顯強(qiáng)于注射后7天,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。另外,在注射后7天,耳蝸底轉(zhuǎn)部分外毛細(xì)胞缺失,但是在注射后1天和7天,在耳蝸的頂轉(zhuǎn)及中轉(zhuǎn)均未見毛細(xì)胞的缺失。 研究結(jié)論： 該研究探討了慶大霉素通過該凝膠緩釋系統(tǒng)釋放進(jìn)入內(nèi)耳后,慶大霉素在內(nèi)耳的分布有兩個(gè)基本規(guī)律,第一是時(shí)間依賴性,即給藥時(shí)間越長(zhǎng),藥物在耳蝸毛細(xì)胞中的濃度越低；第二是底轉(zhuǎn)-頂轉(zhuǎn)的濃度梯度變化規(guī)律,即越靠近圓窗龕的部位如耳蝸底轉(zhuǎn)及前庭,藥物的濃度越高。利用該凝膠系統(tǒng)通過圓窗龕釋放慶大霉素進(jìn)入內(nèi)耳,可以較好的保存低頻及中頻聽力,同時(shí)可以破壞耳蝸的前庭功能及底轉(zhuǎn)毛細(xì)胞的部分功能。因此,此種給藥系統(tǒng)可以應(yīng)用于臨床上已經(jīng)存在高頻聽力損失的梅尼埃病人的治療中,為臨床上難治性梅尼埃病的治療提出了一種全新的治療方案。
[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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R764.3

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