本文選題：氣管狹窄 + 西羅莫司　； 參考：《南方醫(yī)科大學(xué)》2017年博士論文

【摘要】：氣管狹窄是呼吸危重癥醫(yī)學(xué)科常見的重癥疾病之一。置入氣管支架后可迅速解除呼吸困難癥狀,是目前臨床上治療氣管狹窄的重要手段之一。隨著支架適應(yīng)癥逐漸拓寬,發(fā)現(xiàn)支架置入后會(huì)出現(xiàn)支架內(nèi)再狹窄、咳嗽、感染、出血等各種并發(fā)癥,嚴(yán)重阻礙氣管支架的臨床廣泛應(yīng)用。支架內(nèi)再狹窄的機(jī)制與成纖維細(xì)胞的異常激活引起肉芽組織增生有關(guān),所以抑制成纖維細(xì)胞活性減少肉芽組織增生成為治療氣管狹窄的研究重點(diǎn)。抗增殖藥物西羅莫司不僅有抗感染、抗增殖、抗排斥的作用,研究發(fā)現(xiàn)還可抑制肉芽組織生長,是治療氣管狹窄比較有前景的藥物之一。但全身應(yīng)用抗增殖藥物會(huì)出現(xiàn)細(xì)胞毒性反應(yīng)、水鈉潴留等副作用。因此,通過局部應(yīng)用抗增殖藥物使藥物特異性直接作用于靶向病灶,抑制氣管成纖維細(xì)胞的過度增殖,直接發(fā)揮藥物治療效果,又避免藥物全身應(yīng)用對全身其他器官的影響,是解決該氣管狹窄的一個(gè)研究方向。為此,是否可以設(shè)計(jì)一種特殊的支架,既可以有足夠的支撐力保持管腔通暢,又可以局部持續(xù)釋放藥物抑制支架置入后的肉芽組織增生,防止支架內(nèi)再狹窄,從而拓展氣管支架在臨床上的應(yīng)用呢?本課題以鎳鈦合金支架為平臺(tái),以PLGA為載藥材料,西羅莫司為抗增殖藥物,利用浸涂法制備西羅莫司氣管支架。評估自制西羅莫司涂層支架的結(jié)構(gòu)及性能;在體外環(huán)境下,評估西羅莫司涂層支架體外藥物釋放過程,并在動(dòng)物上初步探索西羅莫司涂層支架的有效性及安全性,為改進(jìn)支架設(shè)計(jì)提供實(shí)驗(yàn)依據(jù)。目的1.西羅莫司涂層氣管支架的制備方法;2.西羅莫司涂層氣管支架體外藥物釋放過程;3.西羅莫司氣管涂層支架在動(dòng)物模型上應(yīng)用的有效性及安全性。方法1.西羅莫司氣管支架的制備方法用超聲波及有機(jī)溶劑將裸鎳鈦合金支架清洗干凈。在避光條件下,按不同西羅莫司/PLGA質(zhì)量比配置浸涂液,采用浸涂法制備藥物涂層支架。用掃描型電鏡觀察支架表面及涂層結(jié)構(gòu)的形態(tài)。2.西羅莫司氣管支架體外藥物釋放過程將制作好的藥物涂層支架置入含磷酸鹽緩沖液的試管中完全浸泡,于37℃恒溫同時(shí)在75轉(zhuǎn)/分不斷攪拌的培養(yǎng)搖床箱中48小時(shí),收集溶出介質(zhì)用于藥物濃度測定,并用新鮮磷酸鹽緩沖液完全取代,重復(fù)上述過程直至42天結(jié)束。用酶放大免疫測定法,測定磷酸鹽緩沖液中西羅莫司的含量。3.西羅莫司氣管支架在動(dòng)物模型上應(yīng)用的安全性及有效性研究將16只成年新西蘭白兔隨機(jī)分為實(shí)驗(yàn)組(n=8)和對照組(n=8),橫向切開兔氣管1/2周徑,實(shí)驗(yàn)組置入自制西羅莫司涂層支架后閉合氣管,對照組同等條件下置入裸金屬支架。分別于術(shù)后第2、4周使用超細(xì)氣管鏡觀察支架及支架內(nèi)肉芽組織生長情況,并根據(jù)肉芽組織阻塞氣道的程度估算氣管狹窄程度。術(shù)后第4周處死所有動(dòng)物,取氣管大體標(biāo)本,HE染色后進(jìn)行組織學(xué)觀察。結(jié)果1.掃描電鏡觀察清洗過的裸支架,支架表面干凈無雜質(zhì),提示超聲波及有機(jī)溶劑法可用于清洗支架;電鏡掃描藥物涂層支架顯示支架表面光滑,藥膜均勻光滑地附著在支架上,支架涂層厚度約4-5um,浸涂法可成功制備西羅莫司涂層氣管支架。2.西羅莫司/PLGA質(zhì)量比為1:10的支架上攜帶的藥物量最多,繼續(xù)增加西羅莫司/PLGA量(1:5)卻不能提高支架的載藥量,提示支架涂層的載藥量存在飽和效應(yīng)。氣管涂層支架平均載藥量為80.07±2.26ug,處于西羅莫司安全治療窗內(nèi),不會(huì)產(chǎn)生藥物相關(guān)毒副作用。連續(xù)6周的體外藥物濃度測定發(fā)現(xiàn),西羅莫司氣管支架的藥物釋放在前10天內(nèi)呈快速釋放過程,此后緊跟一個(gè)緩慢釋放過程。支架攜帶的藥物大約有50%左右在2周時(shí)藥物被釋放,28天時(shí)大約近70%藥物被釋放,而42天時(shí)大約支架上80%的藥物被釋放。3.實(shí)驗(yàn)過程中實(shí)驗(yàn)組及對照組各有1只動(dòng)物死亡,余實(shí)驗(yàn)動(dòng)物均存活至實(shí)驗(yàn)終點(diǎn),且均出現(xiàn)不同程度的氣管狹窄。超細(xì)氣管鏡觀察顯示,術(shù)后2周,對照組動(dòng)物氣管內(nèi)支架區(qū)輕度肉芽組織生長,部分動(dòng)物可見大量黏液潴留。實(shí)驗(yàn)組未見明顯肉芽組織生長,部分動(dòng)物可見較多黏液潴留。術(shù)后4周,對照組動(dòng)物氣管內(nèi)支架區(qū)出現(xiàn)輕至中度肉芽組織增生,個(gè)例出現(xiàn)氣管嚴(yán)重堵塞,肉芽組織增生部位尤以支架上緣及下緣為著,氣管切開處未見狹窄。實(shí)驗(yàn)組動(dòng)物氣管內(nèi)支架區(qū)輕度肉芽組織增生,增生部位主要位于支架上緣。實(shí)驗(yàn)過程中未發(fā)現(xiàn)支架移位、斷裂等現(xiàn)象。取支架內(nèi)肉芽組織增生部位及氣管切開縫合處的氣管大體標(biāo)本進(jìn)行組織學(xué)觀察,結(jié)果表明實(shí)驗(yàn)組(812.95±235.89)肉芽組織厚度(um)小于對照組(1577.53±507.97)(t=3.612,P=0.004);實(shí)驗(yàn)組(平均秩次4.93)炎癥反應(yīng)程度輕于對照組(平均秩次10.07)(Mann-WhitneyU=6.500,P=0.014),且炎癥反應(yīng)越強(qiáng),肉芽組織增生越明顯(相關(guān)系數(shù)r=0.809,P=0.000)。結(jié)論1.超聲波及有機(jī)溶劑法可用于清潔支架;浸涂法可成功制作符合實(shí)驗(yàn)要求的西羅莫司氣管支架模型;2.西羅莫司/PLGA的最佳藥物/涂層比例為1:10,且支架涂層的載藥量存在飽和效應(yīng);西羅莫司涂層支架體外藥物釋放是先快速釋放,后緩慢釋放的過程;西羅莫司涂層支架攜帶的藥物量在安全治療窗內(nèi),是一個(gè)較安全的新型氣管藥物涂層支架;3.支架覆蓋區(qū)域的氣管組織炎癥反應(yīng)程度與肉芽組織增生存在相關(guān)性;自制的西羅莫司氣管藥物支架可以有效抑制支架內(nèi)肉芽組織形成,減輕局部炎癥反應(yīng),有潛在的臨床應(yīng)用價(jià)值,可為改進(jìn)支架設(shè)計(jì)提供實(shí)驗(yàn)室依據(jù)。
[Abstract]:Tracheal stenosis is one of the most common respiratory and critical care medicine, a severe disease. Tracheal stent implantation can rapidly relieve the symptoms of dyspnea, is one of the important means of treatment of tracheal stenosis in clinic at present. With the support of indications gradually widened and found that after stenting will infection in stent restenosis, cough, bleeding and other complications. Clinical application of tracheal stent. Hinder stent restenosis mechanism and abnormal activation of fibroblasts by granulation tissue, so inhibit fibroblast activity to reduce the increasing emphasis on the formation of granulation tissue for the treatment of tracheal stenosis. Anti proliferative drug sirolimus not only anti infection, anti proliferation, anti rejection effect. The study found that can inhibit the growth of granulation tissue, is one of the more promising drugs for the treatment of tracheal stenosis. But the systemic application of antiproliferative drugs will be fine Cell toxicity, sodium retention and other side effects. Therefore, the local application of anti proliferative drugs to drug specific direct effects on target lesions, inhibit the proliferation of fibroblasts in the trachea, directly play the effect of the drugs, but also to avoid the general drug application to other organs of the body, is a research to solve the trachea the direction of stenosis. Therefore, whether we can design a special bracket, can have enough supporting force to maintain patency of the lumen, and can inhibit the local sustained release of the drug after stent implantation of granulation tissue, prevent stent restenosis, thus expanding tracheal stent in clinical application? The nickel titanium alloy stent platform, using PLGA as a drug carrier material, sirolimus as anti proliferative drugs, prepared by dip coating method using sirolimus stents. Assessment of the structure and performance of homemade sirolimus eluting stent in; In vitro, evaluation of sirolimus eluting stent in vitro drug release process, effectiveness and safety in animal and explore the sirolimus eluting stent, to provide the experimental basis for improved scaffold design. Methods to prepare 1. sirolimus coated tracheal stent; 2. West Romo our tracheal stent in vitro drug release coating process; effectiveness and safety 3. application of sirolimus eluting stent in tracheal animal model. A method for preparing 1. sirolimus tracheal stent with ultrasound and organic solvent will bare nickel titanium alloy stent clean. In the dark condition, according to the different /PLGA mass ratio of sirolimus coated liquid, prepared by dip coating method. Drug eluting stent stent observation the surface coating structure and morphology of.2. sirolimus tracheal stent in vitro drug release process of the drug eluting stent prepared by scanning electron microscope device Completely immersed into phosphate buffer containing the test tube, at constant temperature of 37 DEG C at 75 RPM stirring culture shaker box for 48 hours, medium for drug concentration determination of dissolution and collection, with fresh phosphate buffer was replaced completely, the process is repeated until the end of 42 days. The immunoassay using enzyme amplification the safety and effectiveness of.3. determination of sirolimus sirolimus tracheal stent in the phosphate buffer used in animal models of 16 adult New Zealand white rabbits were randomly divided into experimental group (n=8) and control group (n=8), transverse incision of rabbit tracheal diameter 1/2, the experimental group was made of sirolimus eluting stents after closed tracheal. The control group bare metal stent implantation under the same conditions. Respectively in ultrafine bronchoscope to observe the growth of granulation tissue and stent stent used in 2,4 weeks after surgery, and according to the degree of obstruction of the airway granulation tissue To estimate the degree of tracheal stenosis. All animal were sacrificed at fourth weeks after operation, the tracheal specimen HE histological observation after staining. Results 1. bare stents SEM cleaned, stent surface clean and free of impurities, suggesting that ultrasonic and organic solvent method can be used for cleaning the stent; scanning electron microscope show that the stent drug-eluting stent smooth surface. Smooth membrane attached to the stent, stent coating thickness of about 4-5um, the dip coating method can be successfully prepared sirolimus coated tracheal stent.2. /PLGA quality than most sirolimus drug carrying for 1:10 on the support, continue to increase the amount of /PLGA of sirolimus (1:5) can improve the loading capacity of the scaffold, drug loading prompt stent coating there is a saturated effect. The trachea coated stent the average drug loading was 80.07 + 2.26ug, a sirolimus safe therapeutic window, does not produce side for 6 consecutive weeks of drugs. The determination of drug concentration in vitro, drug sirolimus tracheal stent release in 10 days before a quick release process, then followed by a slow release process. There are about 50% in 2 weeks when the drug is released drug stents carry 28 days, nearly 70% of drug was released on the 42 day the drug about support the 80% is the release of.3. during the experiment, the experimental group and control group each with 1 animal deaths, more than the experimental animal survived to the experiment end point, and there are different degrees of tracheal stenosis. Ultrafine bronchoscope observation showed, after 2 weeks, the control group animal tracheal stent mild growth of granulation tissue, visible part of animal a large number of mucus retention. The experimental group had no obvious growth of granulation tissue, part animal visible mucus retention. After 4 weeks, the control group animal tracheal stent area of mild to moderate hyperplasia of granulation tissue, a gas Guan Yanzhong Jam, especially in parts of granulation tissue hyperplasia and inferior edge of stent for tracheotomy, no stenosis stent. Mild hyperplasia of granulation tissue in experimental animal group in the trachea, hyperplasia of parts were mainly located in the edge of stent stent migration was found. During the experiment, the fracture phenomenon. Gross specimens of granulation tissue supporting frame the hyperplastic part and tracheal cutdown at tracheal histological observation, the results show that the experimental group (812.95 + 235.89) granulation tissue thickness (UM) than the control group (1577.53 + 507.97) (t=3.612, P=0.004); experimental group (mean rank 4.93) the degree of inflammatory reaction than that in the control group (mean rank 10.07) (Mann-WhitneyU=6.500, P=0.014), and the inflammatory response is stronger, the more obvious hyperplasia of granulation tissue (correlation coefficient r=0.809, P=0.000). Conclusion: 1. ultrasonic and organic solvent method can be used to clean the stent can be successfully established; meet the test requirements of the San Siro dipping The cumulativeincidence tracheal stent model; optimal drug / /PLGA ratio of 2. sirolimus coating at 1:10, and stent coating loading saturation effect; the sirolimus eluting stent in vitro drug release is the first release, after the process of slow release; the amount of drug sirolimus eluting stent carrying in safe therapeutic window, is a relatively safe model airway drug eluting stent; the correlation between 3. support coverage area of the tracheal tissue and the degree of inflammatory reaction and hyperplasia of granulation tissue; sirolimus eluting stent can effectively inhibit the homemade granulation tissue formation, reduce the inflammatory response and has potential clinical application value, can provide the basis for the improvement of laboratory support design.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R56

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