胆管放射性粒子支架与普通塑料支架堵塞物成分分析的对照研究
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摘要
恶性梗阻性黄疸是指由原发性胆管癌、壶腹癌、胰腺癌、肝癌或其它部位的癌肿淋巴结转移等压迫胆道而引起的梗阻性黄疸。其早期诊断困难,手术切除率低,重度黄疸是患者死亡的重要原因之一,而化疗联合放疗是目前公认的最佳保守治疗方案。上个世纪70年代发明的内镜下胆管支架引流术,可有效缓解胆管梗阻的症状。经过30余年的发展,目前内镜下胆管支架置入被认为是对良性梗阻患者创伤程度最小、对不能外科手术治疗的恶性梗阻性黄疸患者的首选治疗方案。
     然而,支架置入后的首要问题是支架的阻塞,同时并发反复发作的黄疸和(或)胆管炎,使得支架需要频繁更换。这不仅影响了患者的生活质量,同时还增加了患者的经济负担。因此,探讨支架置入后发生堵塞的原因和机制,是研究的热点所在。
     我科发明的胆管放射性粒子支架,即将放射性125I粒子置于特殊设计的胆道塑料支架上,使介入技术同管腔内近距离放射治疗相结合,发挥两者的优势,在保持胆道引流的同时,通过放射性125I粒子的内照射起到治疗肿瘤的作用。这就为恶性梗阻性黄疸患者的治疗开辟了新思路,提供了新手段。前期的动物实验及临床应用均证明其安全性及有效性。
     然而,放射性粒子支架同样存在着置入后出现堵塞的问题,并且由于放射线对周围组织的影响,使支架的堵塞因素更为复杂化。对于其可能的堵塞机制,目前还没有相应的研究报道。本研究以比较胆管放射性粒子支架和普通塑料支架的堵塞物成分为切入点,进一步探讨放射性粒子支架的堵塞机制。
     研究目的
     1、通过检测胆管放射性粒子支架与普通塑料支架的堵塞物成分,分析其物质组成,探讨二者堵塞机制的不同;
     2、通过胆管放射性粒子支架与普通塑料支架堵塞物成分的分析,进一步对放射性粒子支架的安全性及有效性进行评价;
     3、为延长放射性粒子支架的开放时间,进一步优化放射性粒子支架的设计打下基础。
     研究方法
     回收长海医院内镜中心自2008年6月至2009年12月,经ERCP术置入的已经发生堵塞的恶性梗阻性黄疸患者的胆管塑料支架共20根。其中A组设为内径10Fr的放射性125I粒子支架,共10根;B组设为COOK公司生产的10Fr、9-15cm,无侧孔的胆道塑料支架,共10根。
     1、微生物学的对照研究
     将所有的样本使用无菌刀片按三等份切开,分为上段、中段、下段三部分。并做以下标记:(1)上段:近肝段;(2)中段;(3)下段:近十二指肠段。以上三段均单独分析,即对每一段支架分别行堵塞物微生物培养及鉴定,对照两组的微生物种类及种类数目。
     2、理化性质的对照研究
     (1)扫描电镜技术
     使用扫描电镜在20kV的工作条件下,将支架内外表面放大不同的倍数进行观察,探讨两种支架内外表面情况的不同。
     (2)傅里叶变换红外光谱(FTIR)技术
     将支架内堵塞物经真空冷冻干燥12小时后回收。使用傅立叶变换红外光谱仪测定产物的红外吸收光谱(IR),对所含物质逐一鉴定。
     (3)热裂解-气相色谱-质谱(PY/GC-MS)检测技术
     将堵塞物样品置于石英裂解管中,在750℃的裂解温度下对其进行裂解,采用Nist谱库进行搜索。标准化(纯度>95%)的胆红素、胆汁酸盐、棕榈酸、胆固醇、角鲨烯(胆固醇的前体物质)五种物质的PY/GC-MS谱图作为参照标准。
     (4)组织学研究
     在患者行ERCP更换胆道支架的过程中,收集从胆道中清理出的组织及坏死物,行组织学检查,分析其成分并判断其来源。
     研究结果
     1、微生物研究结果提示,引起支架堵塞的微生物主要以革兰阴性杆菌、革兰阳性球菌及真菌为主,主要有大肠杆菌、肺炎克雷伯杆菌、阴沟杆菌、变形杆菌、鲍曼不动杆菌以及粪肠球菌、屎肠球菌、铜绿假单胞菌、摩根摩根菌和白色念珠菌等。放射性粒子支架与普通支架内的微生物种类及种类数量无明显统计学差异(P>0.05),支架的不同部位之间的微生物种类数量有统计学意义,即近十二指肠段较其他两段含微生物种类多(P<0.05)。
     2、经肉眼及扫描电镜观察,放射性粒子支架的外表面可见一层较厚的蛋白样物质沉积,而在普通塑料支架的外表面几乎没有看到这层物质。与微生物培养结果基本一致,在两种支架的内表面都可见微生物生物膜形成,支架内可见大量的杆状和球状细菌。同时,在两种支架的内表面均可见大量的纤维样物质及胆固醇结晶样物质沉积。
     3、FTIR结果分析:FTIR检测发现所有的回收样本均表现了相似的谱图,谱图的特征峰主要归属于蛋白质、游离胆红素、棕榈酸、角鲨烯及胆固醇,且两组样品的特征峰在视图上没有显著的差异。
     4、PY/GC-MS结果分析:堵塞物的裂解结果表现出类似的谱图,裂解产物由多种小分子物质组成,分子量介于120-400之间。参照标准品裂解谱图后发现,其裂解产物主要由胆红素类、棕榈酸类及胆固醇类三类物质组成。通过计算每组样本中的各类物质的含量百分比,其中每种物质的含量在不同的样本中均有差异,但其差异在两组之间不显著,无明显的统计学意义(P>0.05)。
     5、组织学分析:放射性粒子支架组中一例胆囊癌患者在更换支架的过程中,行气囊胆道清理术,术中清理出大量的胆道结石和组织坏死物。收集清理出的胆道坏死组织,行组织病理学检查。病理检查结果提示胆囊腺癌。
     结论
     125I放射性粒子释放出的低剂量的γ射线对胆道内细菌的影响较弱,即不能产生抑制及杀灭细菌的作用或这种作用很弱;其次,肉眼及扫描电镜结果可知,在大部分A组样品中,支架外表面有较多的蛋白样物质附着,据文献报道,这层物质的蛋白含量较支架内高。因此我们认为,在堵塞的胆管支架外表面,放射性粒子支架的蛋白含量较普通塑料支架多,这是由于放射性粒子的内照射造成肿瘤细胞坏死脱落所致。因此,脱落物沉积后造成支架管腔堵塞可能是粒子支架堵塞的机制之一。但同时认为,由于蛋白样物质的脱落和沉积,造成放射性粒子支架较普通塑料支架可能更易于发生堵塞,因此,这为今后改进放射性粒子支架的设计提供了有利的依据。使用FTIR和PY/GC-MS两种化合物的检测方法分析可知,两组支架内部的堵塞物成分主要包括蛋白质、游离胆红素、棕榈酸、角鲨烯及胆固醇,且组成成分在含量上没有显著差异(除蛋白质未定量测量外),这进一步证实了放射性粒子支架的安全性;组织学的结果显示,放射性粒子杀灭肿瘤细胞从而造成其坏死脱落,进一步证实了放射性粒子支架的有效性。本研究目前的结果来自于较小样本,所得统计学结论的可靠性和实用性有待于大样本实验的验证,目前本项研究仍在进行之中,更为准确和理想的结果将在后续工作中展示。
Malignant obstructive jaundice is a symptom which is caused by primary carcinoma of bile duct, ampullary, pancreas, hepar and metastatic lymph nodes of other position compressing bile duct. Because of the difficult of the early diagnosis and surgical removal, chemotherapy combine radiotherapy are recognized as the best palliative treatment. But severe jaundice is a major cause of death. ERBD which was invented in 70s of last century can effectively alleviate the symptoms of bile duct obstruction. ERBD is considered to be the minimum degree of trauma in patients with benign obstruction and the best choose for the malignant obstructive jaundice after more than 20 years of development. However, the most important issue after the stent placement is stent obstruction, simultaneous recurrent jaundice, and (or) cholangitis, making stents require frequent replacement. It not only affect the patient's life quality, but also increase economic burden. Therefore to investigate the reason and mechanism of the stent obstruction after ERBD, is becoming hotspot research in recent years. Radioactive Stents which is invented by our department is a special plastic bile stent combined radioactive seeds with stent drainage, which associate intervention and intraluminal brachytherapy (ILBT). It has advantage both in bile drainage and treatment of carcinoma. It improve the life quality of the patients notablely and establish new treatment method.Our previous animal study and clinical application demonstrated that radioactive stent appeared to be safe and potentially effective for the treatment of malignant obstructive jaundice which was unresectable. But the radioactive stent also has the problem about stent obstruction, and until now there isn't any corresponding research report investigating the possible mechanism of it. This study focus on the different obstructive components in radioactive stent and common plastic stent, to investigate the obstructive mechanism of radioactive stent.
     Objective
     1、To test the obstructive component in bile radioactive stent and common plastic stent, then analysis material compositions and discuss the different obstructive mechanism.
     2、According to the obstructive compositions of the two kinds of stents, to appraise the security and the efficiency of the radioactive stent.
     3、To do the groundwork for optimize the design of the radioactive stent, intend to prolong the patent time of the stent.
     Methods
     From June 2008 to December 2009,20 occluded bile duct stents which were inserted by ERCP were retrieved from 20 malignant obstructive jaundice patients in the centre of endoscope of Changhai hospital. Radioactive stents which are 10Fr in group A(n=10), and the common plastic stents which are 10Fr and length between 9 to 15 cm producted by COOK in group B (n=10).
     1、A comparative study of microbiology All the samples were cut in three parts, and the segments were labeled as follows:(1) Upper segment:part closest to the liver, (2) Medium segment, (3) Lower segment:part closest to the duodenum. The three parts were analysed separately for microorganisms identification.
     2、A comparative study of physicochemical analysis
     (1) Scanning Electron Microscope
     The scanning electron microscope was operated at 20kV. To investigate the different between the two kinds of stents according to the surface of internal and external stent amplifying in different multiples.
     (2) FTIR Spectroscopy
     Dried the clogging material under vacuum for 12 hours and used the FTIR spectroscopy to analyses of the clogging material.
     (3) Pyrolysis derivatisation/gas chromatography/mass spectrometry (Py/GC-MS)
     The samples were deposited on a quartz capillary, The pyrolysis wire was set at 750℃. Pure commercially available bilirubin, cholesterol, palmitate were used as standards.
     (4) Histological study
     Though the histological examination to determine their sources and composition of duct cleaning from ERCP.
     Results
     1、The result of bacterial culture and identification showed that, there were many kinds of bacteria and Fungi can cause the obstruction of the stents, such as escherichia coli, klebsiella pneumoniae, bacillus cloa, caebacillus proteus, acinetobacter baumannii, enterococcus faecalis, enterococcus faecium, pseudomonas aeruginosa, morganella morganii and chaplet. The bacteria's types and the quantity of types are not significant difference between the radioactive stent and the common stent (P>0.05).But the quantity of bacteria's types are significant in different part of the stent, the lower segments have more kinds of bacteria than other segments (P<0.05).
     2、According to the images of SEM, there are a layer like protein on the the most surfaces of external stents, but there are not on the common ones. Are basically consistent with the results of bacterial culture, there are bacterial biofilms on the surface of internal stents and there are a lot of bacilli and coccus in the stents. Meanwhile, cholesterol crystals and dietary fibers were seen by scanning electron microscope in the stents.
     3、The results of FTIR showed all the retrieved samples showed similar spectra, and the analysis of the clogging material showed that it mainly consists of calcium palmitate, bilirubinate, bilirubinate and protein. And the two characteristic peaks of the samples in view there is no significant difference.
     4、The results of PY/GC-MS showed split product formed by a variety of small molecules, molecular weight ranging between 120-400.The results from the Py/GC/MS analysis of the clogging material showed split product are bilirubinoid, palmitic acidsand, cholesterol. The sludge from different patients showed that the relative amount of each substance varied randomly between the patients.
     Conclusion
     The low dose ofγ-rays released by 1251 radioactive seeds can not kill and inhibit the bacteria or this role is very weak; followed by the naked eye and scanning electron microscopy results suggest that in most samples of group A, the external surface of stents have more protein-like substances attached, those were reported in the literature, this layer of material with high protein content than in the stents. Therefore, we believe that protein content of the deposition in the external surface of radioactive stents are more than the common stents. This is due to internal exposure of radioactive particles cause necrosis of tumor cells, therefore, shedding sediment resulting from the stent lumen may one mechanism of the radioactive stents obstruction. But that, due to protein-like substance sediment, the radioactive stents may be easily obstructed than the common plastic stents. This result provide a favorable basis for improvement of the design of radioactive stent in the future.
     The results of FTIR and PY/GC-MS analysis showed that the clogging material were mainly consisted of calcium palmitate, bilirubinate, bilirubinate and protein.and composition had no significant differences between two groups(except not quantitative measurement of protein), other than quantitative is not outside), which further confirmed the safety of radioactive seeds stent. Histological results showed radioactive seeds to kill tumor cells resulting in the necrotic, confirming the effectiveness of radioactive stent. the results of this study comes from small samples, statistical findings from the reliability and availability is subject to verification of large sample tests, this study is ongoing at present, more accurate and satisfactory results will follow work display.
引文
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