| Background:Gastric cancer is one of the most common malignant tumors in the world,accounting for the seeond rank in the carcinomatous decease.The annual mean death rate of gastric cancer is16per one hundred thousand in our country, which accounts for the first rank among the gastrointestinal tumors.The total five-year survival rate of gastric cancer is about10%. Gastric cancer is one of the worst five-year survival rate of malignancy in the world.The incidence of gastric cancer of male is1.5-2.5times that of women, and can occur at any age, With increasing age, the incidence of gastric cancer was significantly increased,its incidence peak age was between50and80years old. Its prognosis is closely related to the classification stage,Patients with early gastric cancer without lymph node metastasis have good prognosis, and the5-year survival rate can reach95%If only involving the mucous membrane and at the same time with local lymph node metastasis,5years survival rate is about82%; If the tumor has serosal involvement and transfer, the prognosis is poorer,Therefore early detection and early diagnosis and treatment can effectively improve the survival rate in patients with gastric cancer.In malignant tumor in the stomach, stomach cancer accounts for about95%or more. Although gastric cancer incidence and mortality rates have fallen in some countries and regions, but so far, gastric cancer still is the common malignant tumors in the world.At present, although gastric cancer the diagnostic techniques (the electronic endoscopic techniques, imaging techniques and tumor markers for detecting an object, etc.) for gastric cancer is developing rapidly, but eventually depends on the the pathomorphism diagnosis to determine the nature of the tumor, the degree of differentiation and the prognosis,etc.The method is single and has certain subjectivity, Therefore to establish a real-time, nondestructive, accurate and objective methods and techniques for early diagnosis of gastric cancer has extremely important significance. The emergence of spectrum technology provides a new way for the early diagnosis of gastric cancer. Raman spectroscopy can provide a variety of information about the branch structure directly, it is the application were reported in the study of protein secondary structure, DNA and carcinogen molecules. Malignant transformation of a cell or tissue, is due to the structure or composition of the biological molecules occurred changes, such as DNA, lipid, protein, etc. Cells and tissues are composed of protein, nucleic acid, carbohydrate, lipid, coenzyme, vitamins, and other ingredients, a biological molecule of the complex structure of the basic unit of life has its corresponding characteristic Raman peaks. The development of Raman spectroscopy provide the possibility for the study of gastric cancer occurrence and development of different lesions stage., and these biochemical changes reflected in the corresponding changes in Raman spectra can as a reliable indicator for early diagnosis, prevention and early treatment to patients with gastric cancer.Laser Raman Spectroscopy (Raman Spectroscopy, RS) as a non-invasive nondestructive testing technology, can provide rich information about the molecular structure characteristics and material composition,which is usually referred to as molecular fingerprint of the materials (molecular fingerprints).With material specific molecular vibration spectrum,it can identify and distinguish between different material structure, and becomes an effective means to study the molecular structure of the substance.To some groups of atoms in biological molecules, the structure and the configuration of the chemical bonds of is sensitive to vibration frequency, when the intramolecular structure change and internal interactions change,it will affect the frequency and intensity of the Raman spectra. This indicates that the Raman spectrum of the biological molecules contains information about the structure and dynamics of these molecules.Near-infrared Raman spectroscopy is particularly attractive because it can effectively reduce autofluorescence, deeply penetrate into the tissue and cells. To various tissues and cells of people, morphology and biological functions differ from one another, but they’re composed of biological molecules,such as proteins, nucleic acids, lipids and carbohydrates,ect. Each material has its characteristic Raman spectrum.Tissues and cells undergo malignant transformation always starting form their molecules, the configuration and conformation and the composition of each component all changed in all kinds of biological molecules in the process of malignant transformation.These early changes do not cause changes in clinical symptoms and medical imaging, but Raman spectra is very sensitive to molecular structure, conformation and the environment, and can reflect the fine structure of the molecule, the structure of the vibration transfer,so it can detect these changes from the molecular level.Compared with fluorescence and scattering spectroscopy, Raman spectroscopy have higher molecular specificity and has a unique advantage in early diagnosis of precancerous lesions and cancer.At the same time, because the Raman spectra of water is very weak and simple, so the Raman spectra can get close to nature, be active state to study biological macromolecular structure and its change.In the process of tumor growth and development,the structure, conformation and quantity of proteins, lipids, carbohydrates, and nucleic acid in tissues and cells will change significantly.Raman spectroscopy can achieve high sensitivity, high resolution to detect this information changes, which reveals the differences of the structure and composition between cancerous tissues and normal tissues at the molecular level, by comparing raman spectroscopy of the cancerous tissues, precancerous tissues and normal tissues,we can find the characteristic spectrum information of organization pathological changes from the differences between them,thus providing a basis for diagnosis of cancer.These features of Raman spectra make researchers launched a wide application in tumor diagnosis. At present scholars have done research in the Raman spectra of lung cancer, liver cancer, esophageal cancer, breast cancer and so on. The results show that Raman spectroscopy technology in detecting molecular changes associated with histopathological aspects have special potential and advantages, and make the nondestructive, real-time, accurate, efficient and objective optical diagnosis possible,Such already can reduce biopsy to the harm to patients, and can realize real-time and effective diagnosis, increase the chances of patients survival.Especially with the development of quantitative and semi-quantitative Raman spectroscopy and applications in the medical field has laid a solid foundation for quantitative diagnosis of early tumor in clinical.Raman spectroscopy is expected to become an important means for the early diagnosis of malignant tumors and malignant cancerous mechanism, and is a larger challenge to the traditional biomedical analysis technology.Purpose:The purpose of this research topic based on the Raman spectroscopy is a non-invasive detection technology, sensitive to detect changes in material composition and has the advantages of fingerprint features, using near infrared laser Raman spectra for detecting the differences of Raman spectra between gastric cancer and precancerous tissue and normal gastric tissue, and collecting their Raman spectra,The comparative analysis of the specificity of Raman spectroscopy in between them as well as the Raman peak wavelength and peak changes,in search of the spectral characteristics which reflects material changes.And further joint histopathology test results of the control group and validate the use of near-infrared Raman spectroscopy on the feasibility of early diagnosis of gastric cancer.lt provides strong scientific basis for clinical application,and to verify a new optical non-destructive method for early diagnosis of gastric cancer,Fundamentally,to get rid of traditional doctors experience sampling observation, diagnosis and biopsy histopathology diagnosis method.According to the peculiar optical characteristics of different tissues, different cells,we can identify and diagnose the physiological status of tissues and cells which were tested. So as to realize the early diagnosis of tissues and cells, and further demonstrate that near infrared laser Raman spectroscopy could be a new method for the screening of gastric cancer,the monitoring of prognosis and the judgment of therapeutic efftiveness.Establish a nondestructive, real-time, accurate, efficient and objective of the gastric cancer optical diagnostic method.Materials and methods:1. experimental apparatus and consumables:(1).The In Via type+Plus the confocal micro Raman spectrometer (Renishaw British company)(2). The binocular inverted microscope (Japan OLYMPUS)(3). He-Ne laser Renishaw (British company)(4). Raman-Wire3.2for raman spectra processing, Matlab for data analysis,and origin8.5for drawing software (Microcal companies in the United States)2.experiment instrument parameter Settings:(1)Light source:semiconductor laser (2)excitation wavelength:785nm(3)Standard output power:300mW,10%laser power, laser power on the surface about10mW(4)Stray light resistance is greater than1014(5)Incident and exit slit width:0.15mm(6)Scanning step length:0.2nm(7)Negative pressure:8(8)Integration time:150ms(9)Spectral resolution of lcm"1(10)20X objective lens focusing laser and collecting Raman signal(11)Take the spectral range:700-1800cm-1(1300cm-1as the center)The experiments were tested by the same person in the darkroom.3.The research object:Collected105cases of gastric normal tissue, precancerous lesions and gastric cancer tissues through gastroscopy and histopathologic examination confirmed in our hospital from June2011to August2012.105subjects with complete clinical information, all subjects were in strict compliance with the above inclusion and exclusion criteria, this research conforms to human trials ethics standard, approved by the hospital ethics committee.Gastric cancer group:33cases, male:23cases, female:10cases, mean age:(54.52±10.52)yeaes old. precancerous lesions group:27cases, male:19cases, female:8cases, mean age:(53.07±11.40)yeaes old.Gastric normal group:45cases, male:23cases, female:22cases, mean age:(52.59±9.83). No statistical difference was found between age groups.33cases of gastric cancer tissues, including14cases of poorly differentiated adenocarcinoma,11cases of moderately differentiated adenocarcinoma,8cases of well-differentiated adenocarcinoma;27cases of precancerous lesions, including6cases of atrophic gastritis with intestinal metaplasia,9cases of adenomatous plyps with low level intraepithelial neoplasia,12cases of adenomatous plyps of high-grade intraepithelial neoplasia.4. sample collection and preservationAfter specimens removed from the stomach, wash2-3times immediately with physiological saline to remove necrotic tissue and blood.Each tissue sample are divided into two portions, each size of about0.5cm3, a placed inside filled with the same type of plastic pipe in10%formalin solution, then labeled,Each tissue samples were divided into two portions, each size of0.5cm3,One tissue sample fixed in the same type of plastic pipe which was filled with10%formaldehyde solution, then label,Then routine paraffin embedding, prepared20um thick serial sections, parallel Another into the freezing tube, and then labeled, placed in a liquid nitrogen tank, saved. The label should be marked with a number and sample collection time. Such as:2011082516(August25,2011,16).5. sample processing(1) equipped with tissue samples frozen pipe is removed from the liquid nitrogen tank, natural thawing at room temperature30minutes, then use sterile tissue forceps tissue samples taken out of the freezing tube placed in the same flat after surface treatment the Raman interference signal of the models on a slide (slide glass substrate can be ignored);(2) slides, together with the tissue sample on the sample stage;(3) Adjust the two switches so that the natural light to the sample;(4) adjust the controller to move the sample stage;(5) with30times the eyepiece tissue sample, so that the focus to the tissue sample;(6) again to adjust the two switch allows the laser through at the same time shielding the white;(7) by a set of parameters to obtain the Raman spectra under the different wave number, repeat the acquisition, for the realization of spectral reproducibility, the measurement process is performed within2hours;(8) spectra entered into the computer, and then use the Raman-Wire3.2, Matlab and Origin8.5software for data and graph processing.6.statistical analysisUse One-Way ANOVE and independent samples t-test, with P<0.05for the difference was statistically significant. The above statistical analyzes were performed using SPSS19.0software.Results:Experimental results show that the Raman spectrum of the vast majority of similar tissue sample has a good reproducibility, the Raman spectral characteristics is basically similar. The Raman spectral distribution of different types of tissue samples have bigger difference. The study results show that the prominent Raman peaks located at around853cm-1,936cm-1,1003cm-1,1032cm-1,1174cm-1,1208cm-1,1323cm-1,1335cm-1,450cm-1,1655cm-1raman spectrum bands (p<0.05,one-way ANOVA with confeidences interval at95%).Compared with normal group,precancerous group show higher intensities at around853cm-1,936cm-1,1003cm-1,1032cm-1,1174cm-1,1208cm-1,raman spectrum bands,while lower at1323cm-1,1335cm-1,1450cm-1,1655cm-1.Compared with gastric cancer group,precancerous group show lower intensities at around1323cm"1,1335cm-1,1450cm-1,1655cm-1raman spectrum bands,while higher at936cm-1,1032cm-1,1174cm-1,1208cm-1.Two inversion spectrum are found at around1230cm-1and1250cm-1wavelengths.The intensity of the Raman signal is higher in normal tissues than in pathological tissues (both precancerous lesions and gastric cancer tissues) below1230cm-1, and vice versa above1230cm-1.Comparison of the Raman signal intensity between precancerous lesions and gastric cancer tissues indicates that the former is notably higher below1250cm-1, and vice versa above1250cm-1, except for the ranges around700to790,1000to1008, and1480to1640cm-1that yield an opposite difference. The discrimination results based on NIR RS using PCA-LDA and PCA-NBC algorithms associated with leave-one-out, cross-validation method are summarized in Table3. Diagnostic sensitivities of81.5%,85.3%, and100%,and specificities of86.4%,100%, and97.4%, respectively,are achieved by using PCA-LDA algorithms to discriminate the normal, precancerous lesions and gastric cancer,while PCA-NBC algorithms provide the diagnostic sensitivities of96.3%,96.9%, and96.9%, and specificities of93%,100%, and95.2%, respectively, for identifying those three kinds of tissues.Conclusion:There’re significant differences of Raman Spectrum between Gastric carcinoma and precancerous lesions and normal gastric tissue.It is found that NIR RS combined with PCA-LDA,PCA-NBC techniques and the Raman peak intensity ratio results can obtain a rich source of diagnostic information about specific biochemical alterations within tissues, it can, therefore, help us further understand the biological processes involved in the transitional period from normal to cancer. Hence, NIR RS can not only be utilized to differentiate cancer tissues from normal gastric tissues but also provides a potential way for the diagnosis of premalignant lesions. |
PDF Full Text Request