| According to the WHO statistical data report, new cases of lung cancer around the world every year are more than1.2million, among which the incidence of non-small cell lung cancer is relatively higher, which is more than1.01million persons per year. In China, new cases of cancer amount to2.8million, of which lung cancer cases top the list. The death rate of lung cancer during the past30years has risen from5.46per100000persons to30.83per100000persons, which is the NO.1common cancer mortality. At present, the survival rate of lung cancer every year is merely12%on average, and its high metastasis and recurrence rate is the biggest confine to clinic treatment. As a result, the mechanism and integrated control measures of non-small cell lung cancer invasion and metastasis has been the focal point of research. Invasion and metastasis is the most fundamental bio-characteristic of non-small cell lung cancer. Increasing evidences has shown that the invasion and metastasis of non-small cell lung cancer is a complex dynamic procedures with multiple factors, multiple stages, multiple links, and gradual development. Not only does it involve the abnormal expression of various genes of cancer cells as well as the activation of several signal channels, but also the interaction between cancer cells and stromal microenvironment, namely, the tumor stroma microenvironment. But researches purely at the level of molecule or cell with various categories, distinct functions and effects or the overall level of clinical can hardly precisely reflect the interaction between the cancer cells and the stromal microenvironment. In the histological level, most of the cells and molecules as well as structure changes of tissues can be observed, which reflects the panorama of the coevaluation of cancer cells and the stromal microenvironment. However, there is no satisfying research technique to reveal its evolution characteristics.Quantum dots has excellent photoelectric characteristics, such as adjustability in terms of the size and compenent of the transmitted wave, excellent light resistance and bleaching property, which are the important material foundations of molecular imaging. It has already shown the excellent application prospect in terms of bio-imaging and molecular marker. The fusion of positioning characteristics of multispectral imaging analysis technique and optical imaging techniques can provide distribution diagram of different kinds of chemical or biochemical components in analytical sample and obtain the qualitative, quantitative, and positioning analytical information. Thus, the multispectral imaging analysis technique based on quantum dots molecular probes will hopefully break the technical barrier. In the premise of ensuring high sensitivity and high specificity, it is able to display many components in tumor stroma microenvironment.This project starting from clinical oncology, focused on a crucial scientific issue which is the characteristic of the key factors in the tumor stromamicroenvironment as well as its effect on the process of invasion and metastasis of non-small cell lung cancer makes full use of the unique advantage of quantum dots molecular probes in application field and uses quantum dots molecular probes combining with multi-spectral analysis technology to establish multi-spectral imaging of quantum dots molecular probe analysis technology that can be applied to the microenvironment of tumor stroma in order to realize the extract, excavation, and analysis of the information of the microenvironment and multi-molecular home-position, real-time, and joint imaging technique. And on this basis it in-depth studies the characteristics of the tumor stroma microenvironment in the process of non-small cell lung cancer development as well as analyzes the coevaluation of cancer cells and other key tumor molecules and factors in the tumor stromamicroenvironment. It enriches the tumor microenvironment theory, and at the same time puts forward the policy of using the characteristic of quantum dots molecular probe techniques along with detection of tumor associated protein and key factors in the tumor stromamicroenvironment as the development direction and related comprehensive analytical policy. It lays foundation for expanding the research of invasion and metastasis of non-small cell lung cancer and promotes the conversion of fundamental achievement.This research mainly includes3parts:Part One:Research based on the primary application of quantum dot probe technology on the marker protein molecules in NSCLCObjective:To probe the simplicity, feasibility and practicability of quantum dots immunofluorescence technique through taking advantage of QDs together with tissue microarray technology to detect the presentation of CEA, CK19, VEGF, and P53protein in non-small lung cancer and to contrast the result with that of immunohistochemistry.Method:QDs-IHC is used to detect the protein presentations of CEA, CK19fragment, VEGF, and P53respectively, and the joint presentations of CEA and CK19and the results are contrasted with that of traditional immunohistochemistry.Results:After comparing the presentations of CEA, CK19protein fragment between the experimental group of cancer tissnes and the control group of non-cancer tissues, the differences emerge (P<0.01), but they are irrelevant to the age, sex, different histological types and clinical stages of lung cancer and whether there is lymphatic metastasis (P>0.05). There are differences on the presention of VEGF between the experimental group and control group (P<0.05), but they are irrelevant to the age, sex, different histological type, and clinical stages of lung cancer while they are relevant to lung cancers with lymphatic metastasis (P<0.05). However, there are no differences on P53between the experimental group and control group (P>0.05), which are irrelevant to the age, sex, different histological types and clinical stages of lung cancer, P53protein and lung cancer with lymphatic metastasis are positively correlated (P<0.05). There are no differences on the positive rate results between QDs-IHC and4types of protein expressions detected by IHC (P>0.05).Conclusion:The background of QDs-IHC detection is clearer, and the false positive rate is low. There is no difference between results concluded by2types of detection (P>0.05). In addition, QDs-IHC can detect2or3types of protein molecules and is also practical as well as economical in saving samples. Part Two:Research on simultaneous imaging and correlation between tumor associated protein molecules and key factors in microenvironment of stroma based on quantum dots molecular probe techniquesObjective:To study the simultaneous imaging and correlation of the tumor-associated protein molecules and key factors in the stromal microenvironment by establishing a type of stable home-positioning, real-time, co-localizing, and multi-molecule imaging technique system as well as quantitative analytical techniques system.Method:The quantum dots molecular probes and multispectral imaging analysis technique are used to establish a multicolor imaging method for microenvironment of tumor stroma to realize the molecule imaging of the microenvironment of stroma of non-small cell lung cancer, to analyze the correlation of molecule expression of those4types of CEA, CK19fragments, VEGF, and P53tumor protein in a multi-parameter, multi-dimensional, and quantitative way, and to detect the joint expression of CEA and CK19Results:Among97cases of non-small cell lung cancer tissues, there are81cases of positive co-expression of CK19fragment and CEA protein and4cases of negative co-expression and they are positively correlated (P<0.01, r=0.358), which reveals that they may jointly promote the invasion and metastasis of lung cancer. Other3types of tumor related protein are not correlated. Meanwhile, it also proves that QDs-IHC can detect2types or even more than3types of protein molecules at a time. This method is practical and more economical in terms of saving samples. It also provides a basis of method for later research.Conclusion:This research establishes the multi-spectral imaging of quantum dots molecular probe analysis techniques system applied to the research on microenvironment of tumor stroma, which has the advantages of home-position, real-time, co-localization, multi-molecule imaging and multispectral quantitative analysis. It can reveal the change of tumor associated protein molecules and many components of the microenvironment of stroma in the premise of high sensitivity and high specificity. Part Three:Research on co-evolution of non-small cell lung cancer and microenvironment of stroma based on quantum dots molecular probe techniquesObjective:To study the correlation of co-evolution of non-small cell lung cancer cell and microenvironment of stroma, and to deepen the understanding towards the fundamental changing rules of microenvironment in the process of invasion and metastasis of NSCLC.Method:A tissue chip containing97cases of NSCLC tissues is built, and the established multi-spectral imaging quantum dots molecular probe analysis system is used to study the relation between the characteristics of microenvironment of stroma and NSCLC prognosis through analyzing the co-evolution of cancer cell, microenvironment, and different types of related protein molecule in tumor in the process of tumor invasion and metastasis from the angle of morphology and statistics.Results:The main components of basilar membrane of cancer nest-type IV collagen are regarded as the research object. And through the research on spatial features of cancer cell and microenvironment of tumor stroma in the process of tumor development, it not only shows that this technique is stable, reliable, and widely-spread, but gives a better presentation of the change of structure and molecule in the process of invasion. It can present the continuity, smoothness, integrity, and thickness of type IV collagen in a quantitative and qualitative way. On this basis, we have discovered4new types of pattern of tumor invasion, including flush type, amoeboid typt, polar type, and line type. These4types of invasion patterns represent the co-evolution process of cancer cell and microenvironment of stroma in the point of spatial structure change which enriches tumor invasion theory and broadens the development direction of quantum dots molecular probes in the field of tumor research. And we use double color imaging quantum dots molecular probes technique as the basis, and mark the type IV collagen and VEGF in ECM in order to study the dynamic change of the degradation of ECM when VEGF is positive during the period of invasion of non-small cell lung cancer.Conclusion:Multi-spectral imaging of quantum dots molecular probe analysis technology is an effective method for the research on microenvironment of tumor stroma, and it provides new experimental evidence for the effect that microenvironment of tumor stroma has on invasion and metastasis, namely, the unique ECM degradation characteristics in every stage of cancer development. |
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