缓释载药聚乳酸凝胶预防气管纤维组织增生的实验研究

发布时间：2018-04-02 13:12

本文选题：丝裂霉素　切入点：地塞米松磷酸钠　出处：《南方医科大学》2012年硕士论文

【摘要】：研究背景喉气管狭窄(laryngo tracheal stenosis,LTS)最常见的病因是机械性外伤和医源性损伤,机械性外伤包括喉气管外伤后的软骨骨折或者气道挫裂和移位变形,医源性损伤包括气管插管、气管切开、喉部放疗及喉部手术等因素。喉气管狭窄以声门下狭窄(SGS,subglottic stenosis)和气管狭窄(TS,tracheal stenosis)最常见,目前治疗方法包括手术治疗、激光治疗、支架扩张及药物治疗等。常用的手术方法包括气管重建术、环状软骨切除术、激光手术包括C02激光、Nd:YAG激光等方法。对于预防喉气管狭窄手术后再狭窄及喉气管恶性肿瘤阻塞需要支架的支撑,但是目前可行的支架都不能顺应气道的解剖结构而导致气道的损伤,对喉气管粘膜刺激引发的咳嗽,支架移位、脱落,支架上下端部位肉芽的生长及有可能拔管后再次发生喉气管狄窄等一些常见并发症尚未得到满意的解决。对于良性的喉气管狭窄,半年至1年后拔出支撑管后还有因纤维组织增生而再次狭窄的可能,肉芽组织的形成是喉狭窄支架置入最常见的并发症。近年来丝裂霉素C(mitomycin C,MMC)抗肿瘤、抗瘢痕形成、减少肉芽组织增生和防止组织粘连的作用得到了公认,被广泛应用于临床相关治疗中。 MMC最早发现于1956年,是从头状链霉菌培养液中分离提取的一种广谱抗肿瘤抗后素。作为一种广谱抗肿瘤药物,MMC被用作系统化疗药,应用于乳腺、肺、胰腺及结肠直肠癌的治疗中。MMC具有抗肿瘤和抑制增殖的双重作用,作为一种烷化剂,其主要作用机制是与DNA的双螺旋形成交联,破坏DNA的结构和功能,抑制增殖期DNA的复制,对增殖期中的细胞均有杀伤作用,同时也作用于静止期的细胞,因而可用来阻止手术区的血管再生和瘢痕形成。研究表明,MMC在体内和体外均能抑制成纤维细胞增生,从而减少各种细胞外基质成分合成。MMC可使体外培养的成纤维细胞中cyclin D1的表达下调,caspase-3的表达上调。cyclin D1是细胞周期中G1期的关键调控蛋白,其下调将阻止细胞由G1期进入S期,从而抑制细胞增殖。caspase-3被称为死亡蛋白酶,其上调将激活凋亡级联反应,使细胞进入不可逆的凋亡过程,诱导细胞凋亡。MMC可能通过上述途径抑制细胞增殖,诱导细胞凋亡,使成纤维细胞减少,预防瘢痕产生。在预防声带术后的粘连及气管手术后的狭窄中,丝裂霉素可能也通过这些途径发挥作用。局部应用丝裂霉素联合气道置入支架治疗是一种有前景的方法。在耳鼻咽喉头颈外科中,丝裂霉素C对预防喉粘连和鼻腔粘连有一定疗效,但所有研究均是一次性用药,把含有丝裂霉素C的棉片在创面敷贴4-5分钟,不能完全解决粘连问题。国外报道用聚乳酸聚乙醇酸(Poly L-lactide-co-glycolide, PLGA)纳米微粒可以作为动物喉部的缓慢释药载体,该实验中将载有德克萨斯红葡聚糖、肝细胞生长因子及牛血清白蛋白的PLGA纳米微粒注射到小鼠声带,体外实验中载有这三种不同物质的PLGA均可释放12d-14d天以上,红葡聚糖在体内持续释放达1周,肝细胞生长因子在体外仍可以减少TGF-β诱导3T3型成纤维细胞中胶原的生长,说明了PLGA载药纳米微粒可以长期释放药物并且可以保持药物的生物活性。聚乳酸凝胶为可注射用生物降解性材料,其生物组织相容性好。聚乳酸凝胶在冷藏条件下为液态,常温下为胶体状,可携带药物,在喉气管疾病应用方而,可作为良好的注射用药物缓释载体。温敏型聚乳酸凝胶注射到体内后,在体温作用下原位凝固,可将其作为药物释放载体,把所需剂量的药物与聚乳酸凝胶混合后注射到患病部位,可迅速形成凝胶。这种原位形成的水凝胶可以和周围组织牢固的结合,可控制在特定部位形成凝胶,在病灶处精确地释放包裹的药物,有效地控制了凝胶中药物的释放范围,减轻全身的不良反应。地塞米松磷酸钠(Dexamethasone sodium phosphate, DSP)为糖皮质激素类用药,具有明显抗炎作用,其对各种原因(物理、化学、生物、免疫等)引起炎症都有很强的抗炎作用,减轻炎症早期的渗出、水肿、毛细血管扩张、白细胞浸润及吞噬反应。在炎症后期可抑制毛细血管和纤维母细胞的增生,延缓肉芽组织生成,防止粘连及瘢痕形成。其价格便宜,局部用药不良反应少,可抑制纤维组织增生,本实验将聚乳酸凝胶作为MMC、DSP的缓释载体并用于喉气管外壁纤维组织增生的动物模型,研究其对气管纤维组织增生的抑制作用,为未来研究PLGA支架涂层药物缓释载体对喉气管狭窄的治疗提供实验基础。目的探讨载MMC与DSP的聚乳酸凝胶在体内外的缓释情况及对气管纤维组织增生的抑制作用。方法在体外缓释实验中,将聚乳酸凝胶加入双蒸水,于4℃冰箱中放置24h以上,直至完全溶解得到澄明溶液,搅拌下加入MMC、DSP搅拌10min。37℃水浴放置5min,制成原位凝胶。取1mg/ml的MMC聚乳酸凝胶1ml及2mg/ml的DSP聚乳酸凝胶1ml分别置于10mlPH为7.4PBS中,在37℃恒温摇床中,以120r/min的速度持续摇荡。分别于1、4、6、8、12h,1、3、5、7、14、21、28、35d取出样本200μl并加入等量的新鲜PBS溶液。各样本通过0.45μm微孔滤膜过滤,测定释放介质中MMC及DSP的含量,药物的累积浓度通过紫外分光光度法测定。通过观察各时间点留取的浸出液分别测出在365nm及242nm处的紫外分光光度值,实验重复3次。在体内缓释试验中,通过喉气管外壁损伤建立纤维组织增生的动物模型。将42只新西兰大白兔按照完全随机的方法分成7组后进行气管外壁刮伤,将0.2ml载有不同的药物剂量的聚乳酸凝胶与明胶海绵混匀后置于损伤后的气管外壁,并用半圆形的硅胶管固定。实验组包括载MMC组3组(0.1mg,0.21ng,0.4mg).载药DSP组3组(1mg,2mg,3mg)和空白对照组1组。分别于术后1、3、7、10、14、21d从O.4mgMMC组耳缘静脉抽血1ml,用质谱方法测量动物体内药物释放浓度。术后1、7、14d从耳缘静脉抽血1.5ml测定血常规观察白细胞计数。术后4周通过空气栓塞处死动物后取气管外壁标本,标本经过福尔马林固定,石蜡切片,HE染色,光学显微镜下观察纤维组织增生厚度。统计学方法：实验数据通过SPSS13.0统计软件进行分析,采用单因素方差分析(One-Way ANOVA),组间比较采用LSD(Least-significant Difference),P0.05表示差异有统计学意义。结果药物体外实验发现1mgMMC缓释时间可达35天以上,2mgDSP缓释时间可达28天以上,DSP早期释放相对较快,6hDSP累积释放浓度达(31.51±2.31)%,28d时累积释放浓度达(92.01±1.76)%,以后释放逐渐缓慢,逐渐趋于平衡。MMC在21d之前释放较缓慢,累积释放浓度达(32.05±1.81)%,35d时累积释放浓度达(84.28±3.06)%。在动物体内两种药物凝胶在术后4周尚未完全降解,各组气管外壁纤维组织增生厚度如下：空白组：0.82±0.29mm；0.1mgMMC组：0.65±0.16mm； O.2mgMMC组：O.49±0.24mm；0.4mgMMC组：O.17±0.09mm；1mgDSP组：0-31±O.33mm；2mgDSP组：0.18±0.13mm:3mgDSP组：0.14±0.13mm。通过One-Way ANOVA LSD检验,两种药物各剂量组(除0.1mgMMC组外)与对照组比较,纤维组织增生厚度差异具有统计学意义(F=9.482,P=0.000),说明药物(除0.1mgMMC组外)对气管壁纤维组织增生有明显抑制作用；在MMC凝胶不同剂量组中,0.4mgMMC组与其他剂量组纤维组织增生厚度差异均有统计学意义(F=11.973,P=0.001),0.4mgMMC纤维组织增生的平均厚度最小,说明0.4mg MMC为动物体内抑制纤维组织增生效果最好的最低剂量；在DSP不同剂量组中,总体差异无显著统计学意义(F=0.988,P=0.395),1mgDSP为动物体内最大程度抑制气管瘢痕形成的合适剂量；0.4mg MMC与DSP各组间抑制纤维组织增生的程度差异均无统计学意义(F=0.893,P=0.462)。其中0.4mgMMC组,从第1-21天动物体内MMC血浆中平均浓度为(0.16±0.12)ng/ml,在第7天释放浓度达到高峰,第10天以后药物释放逐渐趋于平衡。术后1、7、14d抽血观察白细胞计数,白细胞计数分别为(9.03±1.57)×109/L、(9.85±1.63)×109/L、(10.17±3.11)×109/L,通过One-Way ANOVA LSD检验发现3组白细胞计数差别无显著性统计学意义(P0.05),说明载有MMC的聚乳酸凝胶对动物的骨髓无明显抑制作用。结论 MMC及DSP聚乳酸凝胶在动物体内及体外释放时间均可长达4周以上。在新西兰大白兔体内MMC抑制纤维增生的最合适剂量为0.4mg,DSP最合适剂量为1mg、MMC、DSP聚乳酸凝胶均可作为预防纤维组织增生的临床用药。本研究结果为未来研究生物可降解聚乳酸聚乙醇酸(Poly L-lactide-co-glycolide, PLGA)涂层药物缓释载体支架对喉气管狭窄的治疗提供实验基础。
[Abstract]:Research background
Laryngotracheal stenosis (laryngo tracheal stenosis, LTS) is the most common cause of mechanical trauma and iatrogenic injury, including mechanical traumatic laryngotracheal injuries after cartilage fracture or airway laceration and displacement, iatrogenic injury including tracheal intubation, incision of trachea, larynx and laryngeal surgery radiotherapy and other factors. The throat in order to tracheal stenosis subglottic stenosis (SGS, subglottic, stenosis) and tracheal stenosis (TS, tracheal stenosis) is the most common current treatment methods include surgery, laser therapy, stent expansion and drug therapy. Surgery commonly used methods include tracheal reconstruction, cricoidectomy, laser surgery including C02 laser, Nd:YAG laser method for the prevention of laryngotracheal stenosis and restenosis after surgery of laryngeal and tracheal malignant tumor obstruction need support, but at present the feasible bracket can not adapt to the anatomic structure of airway and lead to airway The damage of cough, laryngeal and tracheal mucosa irritation caused by stent migration, fall off, the lower part of the bracket granulation growth, may occur again after extubation, laryngeal and tracheal narrow common complications has not been satisfactorily solved. For laryngotracheal benign stricture, half a year to 1 years after pulling out and supporting tube because of the hyperplasia of fibrous tissue and re stenosis may, the formation of granulation tissue is the most common complication of laryngeal stenosis stent implantation. In recent years, mitomycin C (mitomycin C MMC) anti tumor, anti scar formation, reduce the proliferation of granulation tissue and prevent tissue adhesion has been recognized and has been widely applied in clinical treatment.
MMC was first discovered in 1956, is from Streptomyces caespitosus cultivating a broad-spectrum extraction liquid anti tumor and anti vasopressin. As a broad-spectrum anticancer drug, MMC was used as the system used in chemotherapy, breast, lung, pancreatic and colorectal cancer.MMC treatment has double antitumor effect and inhibited the proliferation of and as an alkylating agent, its main mechanism is the double helix and the formation of DNA crosslinking, damage to the structure and function of DNA, inhibit the proliferation of DNA replication, killing of proliferating cells in the important role, but also in quiescent cells, and thus can be used to prevent angiogenesis and scar the formation. The results show that MMC can inhibit the proliferation of fibroblasts in vivo and in vitro, thus reducing the various components of extracellular matrix synthesis of.MMC can make the in vitro expression of fibroblast in cyclin D1 down regulated the expression of Caspase-3. .cyclin D1 is the cell cycle in G1 phase of the key regulatory protein, its downregulation will prevent cells from G1 phase to S phase, thus inhibiting the proliferation of.Caspase-3 cells is called death protease, its increase will activate the apoptotic cascade, the cells into the irreversible process of apoptosis, cell apoptosis induced by.MMC may inhibit cell proliferation by the pathway, induce cell apoptosis, fibroblasts decreased, prevent scar stenosis. In tracheal surgery to prevent adhesions and vocal cords postoperatively, MMC can through these pathways play a role.
The treatment of topical mitomycin combined airway stent implantation is a promising approach. In otorhinolaryngology head and neck surgery, mitomycin C for the prevention of laryngeal adhesion and adhesion of nasal cavity has certain curative effect, but all of them is a one-time medication, the cotton sheet containing mitomycin C in wound dressing for 4-5 minutes, can not completely solve the adhesion the problem with the foreign reports. PLGA (Poly L-lactide-co-glycolide PLGA) nanoparticles can be used as animal throat slow release carrier, the experiment with Texas red dextran, hepatocyte growth factor and bovine serum albumin PLGA nanoparticles injected into mice carrying the vocal cords, the in vitro experiments of three different substances PLGA can release more than 12d-14d days, red dextran in vivo sustained release up to 1 weeks, the hepatocyte growth factor in vitro could reduce the fibroblast induced 3T3 TGF- beta The growth of collagen in cells shows that PLGA nanoparticles can release drugs for a long time and keep the biological activity of the drug.
Polylactic acid gel as biodegradable material for injection and its biocompatibility. The polylactic acid gel under refrigerated conditions for liquid at room temperature for colloid, can carry drugs applied in laryngeal tracheal diseases, can be used as a good drug carrier injection. Wen Min type polylactic acid gel was injected into the in vivo, at body temperature in situ solidification, can be used as drug delivery carrier, the desired drug dosage and polylactic acid gel mixture was injected into the diseased part, can quickly form a gel. The in situ formed hydrogels can be firmly combined with the surrounding tissue, can control the formation of gel at a specific location, accurate drug the release of the parcel in lesions and effectively control the extent of drug delivery in the gel, reduce systemic adverse reactions.
Dexamethasone sodium phosphate (Dexamethasone sodium, phosphate, DSP) for glucocorticoid medication has significant anti-inflammatory effects, for various reasons (physical, chemical, biological, immunological etc.) cause inflammation have potent anti-inflammatory effects, reduce inflammatory exudation, early edema, capillary dilatation, leukocyte infiltration and phagocytosis. In the late inflammation can inhibit the proliferation of capillaries and fibroblasts, delaying the generation of granulation tissue, preventing adhesion and scar formation. It is cheap, less adverse reactions and local drug, can inhibit the proliferation of fibrous tissue, the polylactic acid gel as a carrier of DSP MMC, and the animal model for laryngotracheal wall fibrous tissue hyperplasia the study on the inhibition of tracheal fibroplasia, for the future research of PLGA stent coating drug carrier to provide the experimental basis for the treatment of laryngotracheal stenosis.
objective
To investigate the sustained release of polylactic acid gel containing MMC and DSP in vivo and in vitro and the inhibition of tracheal fibrous tissue proliferation.
Method
In vitro release experiment, the polylactic acid gel with double distilled water, placed in the refrigerator at the temperature of 4 24h or more, until completely dissolved by a clear solution, stirring MMC, DSP 10min.37 5min placed C water bath stirring, made of in situ gel. The 1mg/ml MMC 1ml and 2mg/ ml polylactic acid gel DSP polylactic acid gel 1ml 10mlPH were placed in 7.4PBS, 37 in constant temperature shaker, with the speed of 120r/min continuous swing. In 1,4,6,8,12h, the fresh PBS solution 1,3,5,7,14,21,28,35d take a sample of 200 L and adding the same amount of each sample. By 0.45 m microporous membrane filtration, determination of the release of MMC and DSP in the medium, the cumulative drug concentration by UV spectrophotometry. The leaching solution at each time point for the measured UV in 365nm and 242nm spectrophotometry, the experiment was repeated 3 times.
In vivo release experiment, through the establishment of animal model of laryngeal and tracheal wall injury of fibrous tissue hyperplasia. 42 New Zealand white rabbits were randomly divided into 7 groups after the method of tracheal wall scraping 0.2ml containing different doses of polylactic acid gel and gelatin sponge after mixing in the injury of tracheal wall. The silicone tube and fixed with semicircular. The experimental group including MMC group 3 groups (0.1mg, 0.21ng, 0.4mg). The drug loaded DSP 3 groups (1mg, 2mg, 3mg) and blank control group. 1 groups respectively after 1,3,7,10,14,21d O.4mgMMC group from ear vein blood 1ml, release concentration by using mass spectrometry the measurement of animal drugs. Postoperative 1,7,14d were determined from the ear vein blood 1.5ml to observe blood white cell count at 4 weeks after operation by air embolism death animal after tracheal wall specimens were fixed in formalin, paraffin section, HE staining, optical display The thickness of fibrous tissue was observed under microscopes.
Statistical methods: the experimental data were analyzed by SPSS13.0 statistical software, and the single factor analysis of variance (One-Way ANOVA) was used. The comparison between groups was LSD (Least-significant Difference), P0.05 showed that the difference was statistically significant.
Result
In vitro experiments showed that the 1mgMMC release time up to 35 days, 2mgDSP release time up to 28 days, DSP early release is relatively fast, 6hDSP cumulative release concentration of (31.51 + 2.31)%, 28d cumulative release concentration of (92.01 + 1.76)%, after gradually slow, tending to balance.MMC before 21d slow release, the cumulative release concentration of (32.05 + 1.81)%, 35d cumulative release concentration of (84.28 + 3.06)%.
In two kinds of animal drug gel in 4 weeks after the operation has not been completely degraded, tracheal wall thickness of fibrous tissue hyperplasia were as follows: blank group: 0.82 + 0.29mm; group 0.1mgMMC: 0.65 + 0.16mm; group O.2mgMMC: O.49 + 0.24mm; group 0.4mgMMC: O.17 + 0.09mm; 1mgDSP group: 0-31 + O.33mm; group 2mgDSP: 0.18 group: 0.14 + 0.13mm:3mgDSP + 0.13mm. by One-Way ANOVA LSD test, two kinds of drugs in each dose group (except group 0.1mgMMC) compared with the control group, with statistically significant difference between the thickness of fibrous tissue hyperplasia (F=9.482, P=0.000), indicating that the drug (except 0.1mgMMC group) had significant inhibitory effects on proliferation of fibrous tissue in the tracheal wall; different doses of MMC gel group, 0.4mgMMC group and other groups of hyperplasia of fibrous tissue thickness differences were statistically significant (F=11.973, P=0.001), the average thickness of fibrous tissue proliferation in 0.4mgMMC's 0.4mg MMC. To inhibit the proliferation of fibrous tissue in vivo animal effect best in the lowest dose; different doses of DSP group, no significant difference (F=0.988, P=0.395), the appropriate dose of 1mgDSP was the animal maximum suppression of trachea scar formation; 0.4mg MMC and DSP were inhibited the proliferation of fibrous tissues showed no significant difference (the degree of F=0.893, P=0.462). In 0.4mgMMC group, from the 1-21 day average concentration of MMC in plasma for animal in vivo (0.16 + 0.12) ng/ml, on the seventh day release concentration reached the peak, tenth days after the drug release has gradually come to balance. Observe the white blood cell count blood 1,7,14d after operation, white blood cell count (9.03 + 1.57 respectively.) * 109/L (9.85 + 1.63) * 109/L (10.17 + 3.11) * 109/L One-Way ANOVA LSD, through the inspection found 3 groups of white blood cell count no significant difference statistically significant (P0.05), shows that MMC loaded polylactic acid gel There is no obvious inhibitory effect on the bone marrow of animals.
MMC and DSP polylactic acid gel in animal in vivo and in vitro release time can be up to more than 4 weeks. The most suitable dose of inhibitory fiber hyperplasia in New Zealand rabbits in MMC is 0.4mg, DSP is the most suitable dose of 1mg, MMC, DSP polylactic acid gel can be used as prevention of fibrous tissue hyperplasia clinical treatment. The results of this study for the future study of biodegradable polylactic acid polyglycolic acid (Poly, L-lactide-co-glycolide, PLGA) coating drug release carrier to provide the experimental basis of treatment of laryngotracheal stenosis.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R767.1

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