西罗莫司涂层气管支架的制备及应用研究

发布时间：2018-04-16 15:24

本文选题：气管狭窄 + 西罗莫司　；参考：《南方医科大学》2017年博士论文

【摘要】：气管狭窄是呼吸危重症医学科常见的重症疾病之一。置入气管支架后可迅速解除呼吸困难症状,是目前临床上治疗气管狭窄的重要手段之一。随着支架适应症逐渐拓宽,发现支架置入后会出现支架内再狭窄、咳嗽、感染、出血等各种并发症,严重阻碍气管支架的临床广泛应用。支架内再狭窄的机制与成纤维细胞的异常激活引起肉芽组织增生有关,所以抑制成纤维细胞活性减少肉芽组织增生成为治疗气管狭窄的研究重点。抗增殖药物西罗莫司不仅有抗感染、抗增殖、抗排斥的作用,研究发现还可抑制肉芽组织生长,是治疗气管狭窄比较有前景的药物之一。但全身应用抗增殖药物会出现细胞毒性反应、水钠潴留等副作用。因此,通过局部应用抗增殖药物使药物特异性直接作用于靶向病灶,抑制气管成纤维细胞的过度增殖,直接发挥药物治疗效果,又避免药物全身应用对全身其他器官的影响,是解决该气管狭窄的一个研究方向。为此,是否可以设计一种特殊的支架,既可以有足够的支撑力保持管腔通畅,又可以局部持续释放药物抑制支架置入后的肉芽组织增生,防止支架内再狭窄,从而拓展气管支架在临床上的应用呢?本课题以镍钛合金支架为平台,以PLGA为载药材料,西罗莫司为抗增殖药物,利用浸涂法制备西罗莫司气管支架。评估自制西罗莫司涂层支架的结构及性能;在体外环境下,评估西罗莫司涂层支架体外药物释放过程,并在动物上初步探索西罗莫司涂层支架的有效性及安全性,为改进支架设计提供实验依据。目的1.西罗莫司涂层气管支架的制备方法;2.西罗莫司涂层气管支架体外药物释放过程;3.西罗莫司气管涂层支架在动物模型上应用的有效性及安全性。方法1.西罗莫司气管支架的制备方法用超声波及有机溶剂将裸镍钛合金支架清洗干净。在避光条件下,按不同西罗莫司/PLGA质量比配置浸涂液,采用浸涂法制备药物涂层支架。用扫描型电镜观察支架表面及涂层结构的形态。2.西罗莫司气管支架体外药物释放过程将制作好的药物涂层支架置入含磷酸盐缓冲液的试管中完全浸泡,于37℃恒温同时在75转/分不断搅拌的培养摇床箱中48小时,收集溶出介质用于药物浓度测定,并用新鲜磷酸盐缓冲液完全取代,重复上述过程直至42天结束。用酶放大免疫测定法,测定磷酸盐缓冲液中西罗莫司的含量。3.西罗莫司气管支架在动物模型上应用的安全性及有效性研究将16只成年新西兰白兔随机分为实验组(n=8)和对照组(n=8),横向切开兔气管1/2周径,实验组置入自制西罗莫司涂层支架后闭合气管,对照组同等条件下置入裸金属支架。分别于术后第2、4周使用超细气管镜观察支架及支架内肉芽组织生长情况,并根据肉芽组织阻塞气道的程度估算气管狭窄程度。术后第4周处死所有动物,取气管大体标本,HE染色后进行组织学观察。结果1.扫描电镜观察清洗过的裸支架,支架表面干净无杂质,提示超声波及有机溶剂法可用于清洗支架;电镜扫描药物涂层支架显示支架表面光滑,药膜均匀光滑地附着在支架上,支架涂层厚度约4-5um,浸涂法可成功制备西罗莫司涂层气管支架。2.西罗莫司/PLGA质量比为1:10的支架上携带的药物量最多,继续增加西罗莫司/PLGA量(1:5)却不能提高支架的载药量,提示支架涂层的载药量存在饱和效应。气管涂层支架平均载药量为80.07±2.26ug,处于西罗莫司安全治疗窗内,不会产生药物相关毒副作用。连续6周的体外药物浓度测定发现,西罗莫司气管支架的药物释放在前10天内呈快速释放过程,此后紧跟一个缓慢释放过程。支架携带的药物大约有50%左右在2周时药物被释放,28天时大约近70%药物被释放,而42天时大约支架上80%的药物被释放。3.实验过程中实验组及对照组各有1只动物死亡,余实验动物均存活至实验终点,且均出现不同程度的气管狭窄。超细气管镜观察显示,术后2周,对照组动物气管内支架区轻度肉芽组织生长,部分动物可见大量黏液潴留。实验组未见明显肉芽组织生长,部分动物可见较多黏液潴留。术后4周,对照组动物气管内支架区出现轻至中度肉芽组织增生,个例出现气管严重堵塞,肉芽组织增生部位尤以支架上缘及下缘为著,气管切开处未见狭窄。实验组动物气管内支架区轻度肉芽组织增生,增生部位主要位于支架上缘。实验过程中未发现支架移位、断裂等现象。取支架内肉芽组织增生部位及气管切开缝合处的气管大体标本进行组织学观察,结果表明实验组(812.95±235.89)肉芽组织厚度(um)小于对照组(1577.53±507.97)(t=3.612,P=0.004);实验组(平均秩次4.93)炎症反应程度轻于对照组(平均秩次10.07)(Mann-WhitneyU=6.500,P=0.014),且炎症反应越强,肉芽组织增生越明显(相关系数r=0.809,P=0.000)。结论1.超声波及有机溶剂法可用于清洁支架;浸涂法可成功制作符合实验要求的西罗莫司气管支架模型;2.西罗莫司/PLGA的最佳药物/涂层比例为1:10,且支架涂层的载药量存在饱和效应;西罗莫司涂层支架体外药物释放是先快速释放,后缓慢释放的过程;西罗莫司涂层支架携带的药物量在安全治疗窗内,是一个较安全的新型气管药物涂层支架;3.支架覆盖区域的气管组织炎症反应程度与肉芽组织增生存在相关性;自制的西罗莫司气管药物支架可以有效抑制支架内肉芽组织形成,减轻局部炎症反应,有潜在的临床应用价值,可为改进支架设计提供实验室依据。
[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.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R56

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