磁性温敏凝胶作为卡介苗载体在膀胱肿瘤灌注治疗中的应用研究

发布时间：2018-04-10 21:37

本文选题：卡介苗 + 膀胱灌注　；参考：《山东大学》2014年博士论文

【摘要】：研究背景膀胱癌是泌尿系统最常见的恶性肿瘤之一,发病率较高。膀胱癌包括尿路上皮癌、鳞状细胞癌和腺细胞癌,其中尿路上皮癌最为常见,虽然理论上经尿道膀胱肿瘤电切术可完全切除非肌层浸润性膀胱癌,但术后复发率可高达67%。因此中国、欧洲及美国的泌尿外科指南建议所有非肌层浸润性膀胱癌患者术后均应进行辅助性膀胱灌注治疗。卡介苗(Bacillus Calmette-Guerin, BCG)是膀胱灌注常用的一种生物制剂,为一种减毒活菌,具有一定的抗原性、致敏性和残余毒性。经过多年的实践,BCG己被公认为最有效的预防膀胱肿瘤复发的免疫制剂,适用于表浅的、非肌层浸润性膀胱肿瘤的术后灌注治疗。 BCG膀胱灌注虽然疗效确切,但其不良反应发生率较高,限制了其临床应用。且有研究报道,30～50%的膀胱肿瘤病人BCG灌注失败,因无法耐受副作用、对BCG治疗反应差、肿瘤进展或复发等,最终停用BCG灌注治疗。增加BCG的灌注剂量是增强BCG治疗反应的最直接方法,但矛盾的是BCG灌注的剂量越大,虽然抗肿瘤效果明显,但副作用随之增加,如何提高BCG的疗效同时降低其不良反应,成为研究的热点。由于人周期性的排尿,药物灌注后很快随尿液排出体外,其与膀胱粘膜的作用时间较短,通常只有1～2小时。药物膀胱内灌注后,在较短的时间段内,病人对膀胱内药物的反应个体差异较大,部分病人甚至没有反应。膀胱内药物灌注的效果主要与膀胱内药物浓度及持续时间相关,而非药物灌注剂量。因此,BCG灌注后在膀胱内的持续作用时间对治疗效果至关重要。基于壳聚糖(CS)及p-甘油磷酸钠(β-glycerophosphate, GP)的温敏凝胶是一种生物材料,在室温条件下,CS和GP的混合溶液呈液态,加热至37℃体温条件下,即转变为CS/GP凝胶。CS凝胶在药物载体、细胞包裹及组织工程等方面得到广泛应用,是优良且极具发展前途的医用缓释体系。因此我们设想,CS/GP温敏凝胶可以装载BCG进行膀胱灌注,以期延长BCG在膀胱内的滞留时间,提高BCG的疗效。 CS/GP温敏凝胶载体进入膀胱后依然会随尿液排出体外。我们设想利用磁性颗粒及外加磁场防止凝胶载体随尿液排出。Fe304磁性纳米粒(Fe3O4magnetic nanoparticle, Fe3O4-MNP)是一种纳米级铁氧体,具有磁响应性好,在液体中可均匀分散,且在生物体内可被降解,无毒副作用等特性,作为磁性材料在生物领域中得到广泛应用。因此我们将Fe3O4-MNP包装进入CS/GP温敏凝胶内,在外加磁场条件下,利用Fe3O4-MNP所提供的磁牵引力,实现凝胶载体在膀胱内的贴壁粘附。研究目的本课题中我们以CS/GP凝胶为基质,设计了一种搭载BCG、Fe3O4-MNP的载体复合物(Fe3O4-BCG-CS/GP),在常温条件下为液体,进入体内后可转变为凝胶,用以进行BCG的膀胱灌注,以期延长BCG在膀胱内的作用时间,并最终增强BCG的抗膀胱肿瘤作用。方法 1. Fe3O4-BCG-CS/GP凝胶载体系统的构建、表征检测及体内应用可行性评估 1.1Fe3O4-BCG-CS/GP载体复合物的制备根据已有文献报道及我们的前期实验,制备方法如下： 1)将脱乙酰度为95%的CS溶于0.1mol/L的稀盐酸中； 2)室温下持续充分搅拌2小时； 3)将CS溶液中不溶性颗粒过滤； 4)将p-GP粉末溶于蒸馏水中； 5)将CS溶液及β-GP溶液在4℃C冰箱中冷藏10分钟； 6)持续搅拌的状态下将p-GP溶液逐滴加入CS溶液中,直至形成均一的溶液； 7)然后将一定量的BCG及Fe3O4-MNP加入混合溶液中,采用机械搅拌、超声分散的方法混合均匀； 1.2CS/GP溶液及Fe3O4-BCG-CS/GP复合物溶液的体外凝胶化时间测定不同浓度配比的CS/GP溶液及Fe3O4-BCG-CS/GP载体复合物凝胶化时间的测定按照如下经典方法进行：将溶液加入离心管中；37℃水浴锅中加热；按照一定的时间间隔,从水浴锅中取出离心管,倒置观察；倒置30s样品不流动,即可认为凝胶化完成；记录不同CS和GP配比的CS/GP溶液凝胶化时间,Fe3O4-BCG-CS/GP载体复合物溶液凝胶化时间同理测定。 1.3大鼠体内凝胶化时间测定及冰冻切片检查利用大鼠测定体内凝胶化时间。灌注前,进行腹腔注射麻醉,取3Fr硬膜外导管自制大鼠导尿管。用空针抽出大鼠膀胱内尿液。将0.1ml Fe3O4-BCG-CS/GP载体复合物溶液注入大鼠膀胱内。根据步骤2中测定的凝胶化时间,处死大鼠,取出膀胱,观察凝胶化完成情况。然后对大鼠膀胱进行冰冻切片检查,观察复合物凝胶后的形态。 1.4数码显微镜及扫描电镜检查我们采用数码显微镜及扫描电镜来观察Fe3O4-BCG-CS/GP复合物凝胶的表面形态。Fe3O4-BCG-CS/GP复合物溶液在37℃水浴中完成凝胶化。取样品少量冷冻干燥。放置于数码显微镜下观察摄片。喷镀贵金属银,增强导电性,处理完成后在扫描电镜下观察复合物凝胶的表面形态。 1.5Fe3O4-BCG-CS/GP复合物凝胶大鼠膀胱内滞留时间测定雌性’Wistar大鼠36只用以测定Fe3O4-BCG-CS/GP复合物凝胶在膀胱内的滞留时间。大鼠膀胱灌注前2小时禁饮。麻醉后进行膀胱灌注,所有大鼠处于4kG外加磁场中饲养。灌注后每4小时处死大鼠3只；取膀胱标本立即进行冰冻切片检查,HE染色观察膀胱内残留凝胶形态,抗酸染色及复染HE染色观察凝胶中剩余BCG的形态。 2. Fe3O4-BCG-CS/GP凝胶载体系统抗膀胱肿瘤作用的研究 2.1实验设计本部分实验使用24只雌性Wistar大鼠。根据以往成熟的实验设计,采用BBN诱发大鼠膀胱肿瘤原位模型后进行抗肿瘤实验。BBN加入每日饮水中,浓度为0.05%,共喂养8周。10周后,24只大鼠根据灌注药物的不同随机分为4组：第一组(Group1):为对照组,给予0.1ml的PBS灌注；第二组(Group2):给予1mg/0.1ml的BCG进行膀胱灌注；第三组(Group3):CS/GP溶液灌注组,每次灌注0.1ml；第四组(Group4):给予Fe3O4-BCG-CS/GP复合物溶液0.1ml,含1mg BCG。四组大鼠每周灌注一次,共6次。实验的1-8周为肿瘤诱导期,11～16周为治疗期。四组大鼠均饲养于强度为4kG的磁场环境中。 2.2抗肿瘤效果验证 20周后麻醉下处死所有大鼠,收集大鼠膀胱组织观察。详细记录每只大鼠的肿瘤数目及每个肿瘤的体积,计算每组大鼠的肿瘤发生率及每只大鼠的平均肿瘤体积。直径超过0.5mm的肿物拟定为肿瘤,列入统计中。肿瘤体积按如下公式计算： V (mm3)=1/2XaXb2(a是肿瘤的最大直径,b为肿瘤的最短直径)。 2.3尿液细胞因子分析膀胱灌注后,将4组大鼠转移至代谢笼内收集尿液。第一次膀胱灌注后24小时收集尿液,每日收集1次,共6次。按实验计划,在处死大鼠前收集尿液1次。每次收集的时间约2小时。收集尿液的离心管内含蛋白酶抑制剂,收集时放置于冰块上。收集完成后离心、储存于-80℃冰箱中待检。本实验采用双抗体夹心ELISA法来检测大鼠尿液中IL-2和IFN-γ的浓度,描绘出其变化曲线。通过计算曲线下面积(area under the curve, AUC)来估算大鼠接受单次膀胱灌注后尿液中细胞因子分泌的总量,据此评估大鼠膀胱内免疫反应的强弱。 2.4免疫组化检查大鼠膀胱灌注BCG后引起膀胱内CD4+的淋巴细胞浸润,其在BCG抗膀胱肿瘤的作用机制中起重要作用。处死所有大鼠后,收集大鼠膀胱组织进行免疫组化检查。我们采用CD4抗体免疫组化染色的方法检测膀胱粘膜中浸润的CD4+淋巴细胞。 2.5统计分析所有数据读取等检测步骤重复进行三遍,指标进行统计学处理,采用Prism5统计软件进行分析,计量数值以x±s表示,根据需要选用配对资料的t检验或单因素方差分析进行统计分析,选用双侧检验。P0.05为差异有统计学意义。结果 1. Fe3O4-BCG-CS/GP凝胶载体系统的构建、表征检测及体内应用可行性评估实验所制备的CS/GP溶液无色半透明状,性质均一,在体外加热至37℃C的条件下,CS/GP溶液成功完成凝胶化,凝胶化后溶液失去流动性。倾倒离心管,凝胶固定不流动。Fe3O4-BCG-CS/GP复合物成功完成凝胶化,样品呈灰黑色,性质均一。实验发现,CS及GP的浓度皆可影响凝胶化时间。由于复合物凝胶中Fe3O4-MNP的存在,凝胶保留了一定的磁响应性。经冰冻切片、HE染色后观察,Fe3O4-BCG-CS/GP载体复合物亦在大鼠膀胱内完成凝胶化。由于CS为有机物,冰冻切片法较好地保留了复合物凝胶在膀胱内的形态,显示Fe3O4-BCG-CS/GP复合物凝胶载体呈网格状的微结构,大小、直径不一。数码显微镜及电镜下可观察到复合物凝胶的表面同样呈现大小不同的网格状结构,与冰冻切片所示结构相似。在磁场作用下,复合物凝胶在膀胱内贴附到膀胱壁表面。随着复合物凝胶在膀胱内作用时间的延长,其在粘膜表面的降解增多、随尿液排出的损耗增大以及尿液的溶蚀作用,其浓度和密度也逐渐降低,凝胶网格溶胀扩大,网格壁变薄。经过48小时,我们仍可在膀胱内检测到少量凝胶存留,因此我们可以认为,磁性凝胶作为BCG载体,可以在外加磁场的作用下,在膀胱内滞留时间长达48个小时。 2. Fe3O4-BCG-CS/GP凝胶载体系统抗膀胱肿瘤作用的研究在抗大鼠膀胱肿瘤的研究中,大鼠灌注Fe3O4-BCG-CS/GP复合物后,所有24只大鼠均按计划完成20周的实验过程。在第20周末,处死大鼠,取大鼠膀胱组织进行检查。Fe3O4-BCG-CS/GP复合物与普通BCG都显示出明显抗肿瘤诱导作用。Fe3O4-BCG-CS/GP复合物组大鼠的平均肿瘤体积较BCG组大鼠明显减小,但在肿瘤数目方面的差异没有统计学意义。Fe3O4-BCG-CS/GP复合物灌注后大鼠尿液中IL-2及IFN-γ浓度较普通BCG组明显升高,且分泌总量较大。通过CD4染色,Fe3O4-BCG-CS/GP复合物灌注的大鼠膀胱粘膜下有更多CD4+淋巴细胞浸润。结论本实验中成功制备了以CS/GP温敏凝胶为载体、以Fe3O4-MNP为磁性牵引物、搭载BCG的Fe3O4-BCG-CS/GP凝胶载体系统,经验证可在体内、体外成功完成凝胶化,保留了较好的磁响应性。凝胶载体呈网格状,BCG等搭载物在凝胶载体中相对独立,利于BCG的释放。Fe3O4-BCG-CS/GP凝胶载体系统在外加磁场作用下,可在大鼠膀胱内延长BCG的作用时间达48小时；动物实验显示,与普通BCG相比,同等剂量条件下Fe3O4-BCG-CS/GP凝胶载体所搭载的BCG可刺激膀胱分泌更多细胞因子,募集大量炎性细胞浸润,在膀胱内产生更强的粘膜免疫反应,其抗膀胱肿瘤作用优于普通BCG。我们认为以CS/GP温敏凝胶为载体搭载Fe3O4-MNP、BCG后构建的Fe3O4-BCG-CS/GP凝胶载体系统,增强了BCG的免疫活性及抗肿瘤效应,为BCG的应用提供了一种新的思路,有望成为用于膀胱癌术后灌注预防膀胱癌复发的更好的免疫制剂。
[Abstract]:Research background
Bladder cancer is one of the most common malignant tumor in urinary system, high incidence of bladder cancer. Including urothelial carcinoma, squamous cell carcinoma and adenocarcinoma, including urothelial carcinoma is the most common, although the theory of transurethral resection of bladder tumor complete resection of non muscle invasive bladder cancer, but the operation the recurrence rate can be as high as 67%. so Chinese, Department of Urology guide to Europe and the United States suggest that all non muscle invasive bladder cancer patients should be adjuvant intravesical therapy. BCG (Bacillus Calmette-Guerin BCG) is a biological agent used for bladder perfusion, a live attenuated bacteria, has a certain the antigenicity and allergenicity and toxicity. After years of practice, BCG has been recognized as immune agents in the prevention of recurrence of bladder tumor is the most effective, suitable for superficial, non muscle invasive bladder cancer after reperfusion therapy.
Intravesical instillation of BCG although the exact curative effect, but the adverse reaction rate is high, limiting its clinical application. And studies have reported that 30 to 50% of patients with bladder cancer BCG perfusion failure due to intolerable side effects, poor response to BCG treatment, tumor progression or recurrence, eventually stopping BCG perfusion. Increase perfusion dose BCG is the most direct method to enhance the response to BCG treatment, but the contradiction is more BCG perfusion dose, although the anti-tumor effect is obvious, but the side effect increases, how to improve the efficacy of BCG and reduce the adverse reaction, has become a hot research topic.
Due to periodic urination, drug perfusion quickly excreted in the urine, the bladder mucosa and the relatively short time, usually only 1~2 hours. Bladder instillation of drug, in a relatively short period of time, differences in response to individual patient on intravesical drug large part of patients did not even bladder reaction. Instillation effect is related with the drug concentration and duration of bladder perfusion, rather than the dose of the drug. Therefore, critical duration in the bladder of the curative effect of BCG after reperfusion.
Based on chitosan (CS) and p- sodium glycerophosphate (beta -glycerophosphate, GP) the temperature sensitive gel is a kind of biological material, under the condition of room temperature, mixed solution of CS and GP is a liquid is heated to 37 DEG C temperature conditions, which is transformed into CS/GP gel.CS gel in the drug carrier, is widely used in fine cell encapsulation and tissue engineering and so on, is an excellent and promising medical delivery system. So we assume that CS/GP thermosensitive gel can be loaded BCG bladder perfusion, in order to prolong the residence time of BCG in the bladder, improve the curative effect of BCG.
CS/GP temperature sensitive gel carrier into the bladder will still be excreted in the urine. We envision the use of magnetic particles and magnetic field to prevent the gel carrier urine.Fe304 magnetic nanoparticles (Fe3O4magnetic nanoparticle Fe3O4-MNP) is a kind of nano ferrite, with good magnetic response, can be uniformly dispersed in the liquid, and in vivo can be degraded, no toxic side effect and other characteristics, as the magnetic material has been widely used in biological field. So we will Fe3O4-MNP packed into CS/GP thermosensitive gel, in the presence of an external magnetic field, using the Fe3O4-MNP provided by the magnetic pull, the gel carrier in the bladder wall adhesion.
research objective
We used CS/GP gel matrix in this paper, the design of a carrier complex equipped with BCG, Fe3O4-MNP (Fe3O4-BCG-CS/GP), is a liquid at room temperature, after entering the body can be transformed into gel, bladder perfusion of BCG to BCG in the bladder to prolong the time, and ultimately enhance the antitumor effect of BCG.
Method
Construction of 1. Fe3O4-BCG-CS/GP gel carrier system, characterization detection and feasibility evaluation of application in vivo
Preparation of 1.1Fe3O4-BCG-CS/GP carrier complex
According to the previous literature and our previous experiments, the preparation methods are as follows:
1) the CS deacetylation of 95% was dissolved in the dilute hydrochloric acid of 0.1mol/L.
2) continuous stirring for 2 hours at room temperature;
3) filtration of insoluble particles in CS solution;
4) p-GP powder was dissolved in distilled water.
5) the CS solution and the beta -GP solution were refrigerated in the 4 C C refrigerator for 10 minutes.
6) in the condition of continuous stirring, the p-GP solution is added to the CS solution by drop by drop, until the homogeneous solution is formed.
7) then a certain amount of BCG and Fe3O4-MNP are added into the mixed solution, and the method of mechanical agitation and ultrasonic dispersion are mixed uniformly.
In vitro gel time determination of 1.2CS/GP solution and Fe3O4-BCG-CS/GP complex solution
The gelation time of CS/GP and Fe3O4-BCG-CS/GP carrier complexes with different concentration ratio was determined according to the following classical methods:
Add the solution to a centrifuge tube; heating in a water bath at a temperature of 37 DEG C; according to certain time intervals, remove the centrifuge tube, inverted from the observation of water bath; inverted 30s sample flow, can complete the gelation that record CS/GP solution; the gelation time of different CS and GP ratio, Fe3O4-BCG-CS/GP carrier complex solution gelation time similarly determined.
Gelation time determination and frozen section examination in 1.3 rats
In vivo determination of gelation time using rats. Before perfusion, intraperitoneal injection anesthesia, epidural catheter 3Fr catheter with self-made rat. Taking the empty needle bladder of rats. The 0.1ml Fe3O4-BCG-CS/GP carrier complex solution was injected into the rat bladder. According to step 2 Determination of gelation time, rats were killed remove the bladder, observe the gelation completion. Then the rat bladder for frozen section examination, observation of compound gel form.
1.4 digital microscope and scanning electron microscope
The surface morphology of.Fe3O4-BCG-CS/GP complex solution, we use the digital microscope and scanning electron microscope to observe Fe3O4-BCG-CS/GP gel gelation completed in 37 DEG C water bath. A small amount of sample freeze drying. Placed on the microscope digital radiography. The precious metal of silver plating, enhanced conductivity, surface morphology after the treatment was observed in complexes in gel under scanning electron microscope.
Determination of the retention time in the bladder of 1.5Fe3O4-BCG-CS/GP complex gel rat
The retention time of female rats' Wistar 36 is used to determination of Fe3O4-BCG-CS/GP compound gel in the bladder. Intravesical instillation of 2 hours before the rats were forbidden to drink. After anesthesia for bladder perfusion, all rats in the 4kG magnetic field in feeding every 4 hours after reperfusion. 3 rats from the bladder; frozen section check immediately, HE staining was observed in bladder residual gel form, acid fast staining and double staining HE staining was used to observe the remaining BCG gel form.
Study on the effect of 2. Fe3O4-BCG-CS/GP gel carrier system on bladder tumor
2.1 experimental design
This part of the experiment used 24 female Wistar rats. According to previous mature experimental design, using the BBN induced rat bladder cancer model after anti tumor experiment.BBN added daily drinking water, the concentration of 0.05%, a total of 8.10 weeks after feeding, 24 rats according to the different perfusion drugs were randomly divided into 4 group:
The first group (Group1): for the control group, the PBS perfusion of 0.1ml was given.
The second group (Group2): the BCG of 1mg/0.1ml was given to the bladder.
Third groups (Group3): CS/GP solution perfusion group, each infusion of 0.1ml;
The fourth group (Group4): Fe3O4-BCG-CS/GP complex solution 0.1ml and 1mg BCG. four groups were perfused once a week for 6 times. The 1-8 week of the experiment was induced by tumor and 11~16 weeks as the treatment period. Four groups of rats were fed in the magnetic field with the intensity of 4kG.
2.2 antitumor effect verification
After 20 weeks, all rats were killed under anesthesia, collection of bladder tissue were observed. The number of records with volume of each rat tumor and each tumor, calculate each rat tumor incidence and the average tumor volume of each rat. The diameter of more than 0.5mm was prepared for the tumor, included in the statistics. The tumor volume calculated by the following formula:
V (mm3) =1/2XaXb2 (a is the largest diameter of the tumor, and B is the shortest diameter of the tumor).
2.3 urine cytokine analysis
After intravesical instillation of 4 groups, will be transferred to metabolism cages of rats to collect urine. First after intravesical instillation of 24 hours urine was collected daily collected 1 times, a total of 6 times. According to the experimental plan, collect urine in 1 rats were killed before. Each collection time of about 2 hours. The centrifugal tube containing urine protease inhibitors, placed on the collection of ice. Collected after centrifugation, stored in the refrigerator -80 C for inspection. This experiment using double antibody sandwich ELISA method to detect the concentration of IL-2 and IFN- y in the urine of rats, describe the change curve. By calculating the area under the curve (area under the curve, AUC) to estimate the amount of cytokine secretion in rats received single bladder perfusion in urine, assessing the immune response in a rat bladder in strength.
2.4 immunohistochemical examination
Intravesical instillation of BCG rats caused by intravesical CD4+ lymphocyte infiltration, play an important role in the antitumor mechanisms of BCG. All rats were sacrificed after collection of bladder tissue of rats by immunohistochemical examination. We used the CD4 antibody of CD4+ lymphocyte infiltration was detected in bladder mucosa.
2.5 statistical analysis
All data read detection steps repeated three times, indexes were statistically analyzed, using Prism5 statistical software, the measurement values were expressed as the X + s, according to the analysis were used for statistical analysis paired t test or ANOVA, using two-sided test.P0.05 the difference was statistically significant.
Result
Construction of 1. Fe3O4-BCG-CS/GP gel carrier system, characterization detection and feasibility evaluation of application in vivo
The experiment was colorless and translucent, CS/GP solution prepared by homogeneous properties, in vitro is heated to 37 DEG C under the condition of C, CS/GP successfully completed the solution gelation solution loses fluidity after gelation. The dumping of the centrifuge tube, fixed flow.Fe3O4-BCG-CS/GP compound gel gel samples were successfully completed, black, uniformity experiments. Found that the concentration of CS and GP can affect the gelation time. Because of the Fe3O4-MNP complex in the gel, the gel retains a certain magnetic response. The frozen section, HE staining, Fe3O4-BCG-CS/GP carrier complex also completed the gelation in the rat bladder. Because CS is organic and frozen section method preserve the complex gel in the bladder shape, showing micro structure, Fe3O4-BCG-CS/GP composite gel carrier was grid size, diameter. A digital microscope and electron microscope can be observed by complex coagulation The glue surface showing the same grid structure in different sizes, similar to the structure shown in frozen sections. Under the action of magnetic field, composite gel in the bladder attached to the bladder wall surface. With the prolongation of time gel in the bladder, the increase in the degradation of the mucosal surface, loss of urine. The urine and dissolution, the concentration and density also decreased, gel swelling grid expansion, grid wall thinning. After 48 hours, we are still in the bladder to detect a small amount of gel retention, so we can think that the magnetic gel as a BCG carrier, in the external magnetic field, the bladder retention time up to 48 hours.
Study on the effect of 2. Fe3O4-BCG-CS/GP gel carrier system on bladder tumor
In the study of rat bladder tumor in rats after perfusion of Fe3O4-BCG-CS/GP complex, all the 24 rats were scheduled to complete the experiment for 20 weeks. At the end of the twentieth weeks, the rats were killed, the bladder tissue of rats were examined with ordinary.Fe3O4-BCG-CS/GP compound BCG showed significant antitumor effects induced by the average tumor.Fe3O4-BCG-CS/GP complex volume group rats compared to BCG rats decreased significantly, but the difference in the number of tumors was not statistically significant.Fe3O4-BCG-CS/GP complex after reperfusion in rat urine IL-2 and IFN- gamma concentration was compared with ordinary BCG group increased, and the secretion of large amount. The CD4 staining of rat bladder mucosa Fe3O4-BCG-CS/GP complexes the perfusion with more CD4+ lymphocytes.
conclusion
In this experiment

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.14

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