食管癌患者样品的光谱检测与分析

【作者】 吴俊富；

【导师】 郭茂田；

【作者基本信息】 郑州大学 ， 光学， 2007， 硕士

【摘要】 癌症是以细胞异常增殖及转移为特点的一类疾病，已成为困扰人类身体健康的首要问题。食管癌是全球最常见的十大恶性肿瘤之一，发病率呈快速上升趋势。食管癌的诊断、早期诊断对于患者的有效治疗，延长生命具有十分重要意义。目前，临床上食管癌虽有多种诊断方法，但最终确定肿瘤性质主要还是依据病理学诊断，此举不但过程繁琐，常受人为因素影响，诊断过程也会给患者带来痛苦，而且确诊时，肿瘤往往已发展到中晚期。若能通过非破坏性、非侵入性的检测手段早期发现疾病，则对食管癌的诊断治疗具有重大意义。光谱技术的出现，为食管癌患者早期诊断、无损或微损诊断提供了可能。光谱技术就是利用分析物质的光谱来了解物质结构信息的光学技术。食管癌患者组织的癌变是一个十分复杂的过程，患者正常食管组织、肿瘤边界组织和肿瘤中心组织的形态、成分构成在细胞和分子水平上都存在差异。组织细胞在癌变过程中，可能会影响到核酸、蛋白质等生物大分子含量、结构的变化，同时，食管癌患者的血清及胃液在组织癌变的不同时期也会发生相应的变化。但是，常规的方法和仪器无法对此进行精确检测和分析。光谱技术由于精确度和灵敏度很高，为癌变信息检测提供了方便、快捷的途径。本研究采集了食管癌患者三处不同部位的食管组织样品、三个不同时期的血清样品和胃液样品并进行相应的处理；对影响食管癌患者组织样品拉曼光谱的因素进行了深入的实验研究；对患者血清样品的拉曼光谱和吸收光谱及胃液等样品的光谱进行了分析，检测到样品物质分子水平上的变化，为食管癌的早期诊断提供了有力的实验依据。本文的主要工作内容及结论如下：1．设计出一套食管癌患者样品的采集与处理方案。样品种类全面丰富，不仅有食管癌患者的组织样品，还包括血清与胃液样品，且均来自同一患者；2．建立了食管癌患者组织样品模型。通过采集食管癌患者正常食管组织、肿瘤边界组织和肿瘤中心组织三处不同部位的样品，分别合理模拟肿瘤的产生（萌芽）、发展、成熟的过程；3．建立了食管癌患者血清和胃液样品模型。通过采集食管癌患者肿瘤手术切除后8天、手术切除出院后3个月随访时和确诊入院后三个不同时期的血清和胃液，分别合理模拟肿瘤的产生（萌芽）、发展、成熟的过程；4．对影响食管癌患者组织样品拉曼光谱的因素进行了较为深入的实验分析。指出影响组织样品拉曼光谱的因素有：组织形态、组织保存的条件、组织切片的厚度、组织的离体时间以及激光波长；5．得出获取食管癌患者组织样品较佳拉曼光谱（拉曼光谱携带的信息较多）的条件：（1）激光激发波长为532nm的绿光；（2）组织样品离体时间应在1小时内；（3）组织样品应处理成冰冻切片；（4）组织样品冰冻切片厚度应在10μm～60μm之间；6．食管癌患者组织样品的拉曼光谱可见，肿瘤组织的谱线强度比正常组织的谱线强度弱的多；癌变组织的拉曼光谱和正常组织的拉曼光谱在1649cm^-1波峰附近处均出现了拉曼峰，癌变组织在2932cm^-1处有一明显拉曼峰，而正常组织在2903cm^-1处有一微弱的拉曼峰；食管正常组织和食管肿瘤组织的拉曼峰在1650cm^-1处有明显差异；强度之比I₁₆₅₀／I₂₉₃₂同样存在显著不同；从血清吸收光谱在412nm处峰强的大小可以初步作为早期诊断食管癌的依据，并且符合率较高。更多还原

【Abstract】 Cancer is the disease with characteristics of abnormal cell breeding and metastasizing, and become primary threat to the human health. Esophagus cancer is one of the top 10 common malignant tumors in the world with the even rapidly morbidity. The diagnosis of esophagus cancer, especially, early diagnosis is very important to patients’ effective treatment and extending their lives. Although at present there are many kinds of clinical diagnosis methods for esophagus cancer, ultimately the type of tumor is determined mainly by pathological diagnosis. They are minute and complicated, influenced by individual factor, bring suffering to patients in the process. And when diagnosed, the tumor often has been advanced. If we can diagnose the disease through non-destructive, non-invasively means in early days, it has great meanings for diagnosis of esophagus cancer and treatment. The appearance of the spectroscopy, it gives the possibility of early and non-destructive diagnoses of esophagus cancer.Spectroscopy is the optics technology by analyzing samples’ spectra to find out material structure information. The concretization of esophageal tissue is a very complicated process, and there exist different shapes and Constitutes between normal esophageal tissue, tumors border tissue and center tissue in cellular and molecular level. The changes of the content and structure of biological macromolecules, such as nucleic acid, protein, etc. might be influenced by histiocytes in the course of canceration, meanwhile, the corresponding changes will take place in different periods in the serum and gastric juice of the patient of cancer of the esophagus. However, these could not be accurately measured or analyzed by routine methods and instruments. Convenient, swift way was provided to measure the canceration information by the spectroscopy for it’s accuracy and sensitivity.In this research, gathered three tissue samples of different position, three serum or gastric juice samples of different periods of the patient of esophagus cancer and carry on corresponding treatment. Measured and analyzed deep the influencing factor of Raman spectrum of samples. Analyzing the spectrum of samples, such as serum and gastric juice etc, and the samples’ changes on the molecule level, try hard to offer the effective experiment basis for early diagnosis of esophagus cancer.The groundwork content and conclusion of this paper are as follows:1. Design the scientific collection and treatment scheme of the samples of esophagus cancer firstly. The kind of samples is overall and abundant, there are not only tissue samples of the patient of esophagus cancer, but also serum and gastric juice samples, and both come from the same patient.2. Have set up the tissue samples’ model of the patient of esophagus cancer . Gathering the patient’s normal tissue, tumor border tissue and tumor centre tissue, which simulate the course of tumor’s sprouts, tumor’s development, and tumor’s ripe.3. Have set up the serum and gastric juice samples’ model of the patient of esophagus cancer . Gathering the serum and gastric juice samples at 8 days after surgical resection, at the follow-up time of 3 months after leaving hospital and at the time admitted to hospital, which simulate the course of tumor’s sprouts, tumor’s development, and tumor’s ripe.4. The influencing factors to the tissues’ Raman spectra were measured and analyzed deep. Point out the factors which include tissue modality, preservative condition, section thickness, and laser wavelength.5. The conditions of better Raman spectrum, in which there is more information: （1） the exciting laser is green which wavelength is 532nm. （2） Exsomatizing time is within 1h. （3） The samples is Frost sections. （4） the thickness is between 10μm～60μm.6. From Raman spectroscopic study on tissues, we could arrive that the intensity of fluorescence of esophageal tumor tissues is less than normal tissues. The Raman peak at the 2932cm^-1 and 1650cm^-1 of esophageal tumor tissues is prominent. The Raman peak at 1650cm^-1 was found in normal tissues, not found of the Raman peak at 2932cm^-1, and found a new Raman peak at 2903 cm^-1. There is obvious difference about the peak at the 1650cm^-1 between normal tissues and tumor tissues, and the ratio of the intensity (I₁₆₅₀/I₂₉₃₂) is the same. We could diagnose the disease on the grounds of the intensity of apex in 412nm of spectrum of serum, and the coincidence rate is relatively high.更多还原