Application Of Quantum Dots Fluorescent Probes To Detect Specific Protein In Tissue Of Transplanted Tumor Of Nude Mice And Human Tongue Squamous Cell Carcinoma

In recent years the incidence of oral cancer in China, US, Japan and Germany increased, particularly in the young women. It is estimated that there are 300,000 new cases every year. Although the treatment for cancer has made great important progress, the oral cancer mortality remains unchanged and the 5-year survival rate remains at around 50% since the 1960s.Currently more and more attention were paid to the etiology, diagnosis, treatment and prognosis of oral cancer. With the development of tumor molecular biology, it revealed that malignant tumors were relevant to activation of various oncogene and inactivation of tumor-suppressor gene and apoptosis of regulating disorders. Oncogene activation and tumor-suppressor gene inactivation got involved in the development process of cancers. Studies have shown that it has close relationship between the expression level of oncogene and treatment effect and prognosis of cancers. The research of oncogene is very important to the prevention, diagnosis and treatment of the cancers. But the oncogene may not be identical in different parts, phenotype and stages of cancers. Therefore it has practical value to study oncogene in different types and stages of cancers.More than 90% of oral cancer are squamous cell carcinoma and the tongue squamous cell carcinoma is of the majority. The occurrence and development of the tongue squamous cell carcinoma is a complicated process involving multiple factors. In the process of malignant transformation, tumor suppressor gene p53 and apoptosis-suppressing gene Bcl-2 play an important role. The current study showed that apoptosis occurred in the development of oral cancer plays an important role and Bcl-2 is one of the most important apoptosis regulatory genes. Many scholars confirmed that Bcl-2 and other apoptosis-related protein have abnormal expression in oral squamous cell carcinoma. P53 is one of the most widely and deeply studied oncogene, a tumor cells suppressor gene that exists in almost all human tumors. Therefore, the research on Bcl-2 and p53 will help to understand the development and progression of malignant tumors. The organic dyes have been used to label the biomacromolecule for a long time, which was to study the interaction between various proteins and cells function. However, because of the shortcomings of organic dyes such as narrow excitation bands, low photostability, short fluorescence lifetimes,the applications as markers in many areas were limited. Recently, nanoparticles fluorescent probe based on the semiconductor quantum dots (Quantum dot, QD) is widely observed because of its unique optical properties. QD technology has a very promising prospect in the imagery of the biomedical field of molecular, cellular and endosomatic imaging, particularly in the study of biological imaging of oncology. We used optical advantages of semiconductor quantum dots as a fluorescent probe for detecting specific protein expression on human tongue squamous cell carcinoma and its tumor in nude mice at different stages. Using quantum-dot fluorescent probe to detect Bcl-2, p53 protein in human tongue squamous cell carcinoma and to detect Bcl-2 protein in tumor-bearing nude mice have not been reported.Chapter oneObjectsTo study if the quantum dot fluorescent probe can be applied to detect specific protein in tumor-bearing nude mice Tca8113 tissue sections and its value.Methods1. The establishment of tumor-bearing animal model and specimen preparation: Human tongue Tca8113 cells were cultured and inoculated subcutaneously in nude mice to establish tumor-bearing animal model then observe tumor growth situation. The nude mice were sacrificed when the tumor grew to about 1.0cm3 Then the tumor was divided into two parts. One part of it was fixed with 4% paraformaldehyde and paraffin-embedded, the other part was made into frozen section.2. Paraffin-embedded tissue sections were tumor tissues confirmed by HE staining.3. Bcl-2 protein was confirmed in paraffin-embedded tissue sections by immunohistochemical staining.4. Quantum dots(QD605) were applied to tag Bcl-2 in paraffin-embedded tissue sections by immunofluorescence staining and the expression was observed under fluorescence microscope.5. Quantum dots(QD605) were applied to tag Bcl-2 in frozen tissue sections by immunofluorescence staining and the expression was observed under fluorescence microscope.Results1. HE staining showed that tissue was composed of a large number of tumor cells. Tumor cells vary in size tightly arranged and there are little intercellular substance, high nucleocytoplasmic ratio, heteromorphism. The nuclear shape and size varied. Tumor-bearing nude mice animal models were established successfully.2. HRP-DAB immunohistochemical staining showed that Bcl-2 protein was expressed in tumor cells, showed brown-colored mainly located in cytoplasm.3. In the paraffin-embedded tissue sections using quantum dot (QD605) labeled Bcl-2 protein by immunofluorescence staining showed that quantum dots combined with the Bcl-2 protein and the complex located in the cytoplasm showed specific red fluorescence under UV excitation. And Bcl-2 protein located where the same as what HRP-DAB immunohistochemical staining showed. Blank control showed no obvious specific red fluorescence.4. In the frozen sections using quantum dot (QD605) labeled Bcl-2 protein by immunofluorescence staining showed that quantum dots combined with the Bcl-2 protein and the complex located in the cytoplasm, showed specific red fluorescence under UV excitation. And Bcl-2 protein located where the same as what paraffin-embedded tissue sections showed, but its fluorescence in frozen sections showed higher brightness.Chapter twoObjectsTo explore if quantum dot fluorescence probe can be applied to do quantitative detection of specific protein in the tissue of transplanted tumor in nude mice and study its value.Methods1. The establishment of tumor-bearing animal model and specimen preparation: Human tongue Tca8113 cells were cultured and inoculated subcutaneously in 20 nude mice to establish tumor-bearing animal model then observe tumor growth situation and two died on the half-way.18 nude mice were transplanted tumor successfully and they were grouped according to their inoculated time.18 nude mice were divided into 6 groups(5th day,10th day,15th day,20th day,25th day,40th day). The nude mice were sacrificed and the tumors size were measured then were made of paraffin-embedded tissue sections.2.18 paraffin-embedded tissue were made HE staining respectively to confirm the tumor-bearing nude mice tumor tissue.3. Quantum dots (QD605) were applied to tag Bcl-2 protein in every group tissue sections of human tongue squamous cell carcinoma with indirect immunofluorescence under the same condition and shot 5 non-duplicate view map of fluorescence imaging each slice under confocal laser microscope. Then analyze the fluorescence intensity using IPP professional image analysis software.4. HRP-DAB immunohistochemistry staining was applied to tag Bcl-2 protein in every group tissue sections of human tongue squamous cell carcinoma under the same condition and shot 5 non-duplicate view map each slice under microscope. Then analyze the optical density of positive products using IPP professional image analysis software.5. Statistical analysis:statistical analysis was performed by using spssl3.0 statistical software, The significance level was set at p<0.05. The results of statistical analysis were measured, and the trend curve of Bcl-2 protein was made.Results1. HE staining showed tissue was composed of a large number of tumor cells.18 tumor-bearing nude mice animal model were established successfully.2. The analysis results of quantum dot fluorescence intensity showed that difference among groups was significant. The difference of quantum dots fluorescence intensity was not statistical significant (P>0.05)between 5th day group and 10th day group, between 15th day group and 20th day group, between 20th day group and 25th day group,between 25th day group and 40th day group,but statistical significant (p<0.05)between 10th day group and 15th day group,between 15th day group and 25th day group,between 15th day group and 40th day group, between 20th day group and 40th day group. It proved that Bcl-2 protein in nude mice reached a peak at about 15th days and then decline slowly.3. The analysis results of optical density of positive products with HRP-DAB immunohistochemistry staining showed the similar trend.4. The result showed that the trend of Bcl-2 in nude mice got by quantum dots immunofluorescence staining and by HRP-DAB immunohistochemistry staining is similar. Bcl-2 protein in nude mice reached a peak at about 15th days and then decreased gradually.Chapter threeObjectsTo study if quantum dots fluorescent probes can be applied to detect p53 protein and Bcl-2 protein in tissue of human tongue squamous cell carcinoma and evaluate their clinical value.Methods 1. Source:10 cases pathology confirmed squamous cell carcinoma of the tongue tissue were selected.2. Paraffin sections:10 tongue squamous cell carcinoma tissue samples were routinely paraffin-embedded.3. Quantum dots with the same diameter(QD605) were applied to tag p53 and Bcl-2 protein respectively in tissue sections of human tongue squamous cell carcinoma with indirect immunofluorescence and the expression was observed under UV fluorescence microscope4. QD605 was used to tag p53 protein while QD545 was used to tag Bcl-2 protein in the same tissue section of human tongue squamous cell carcinoma with indirect immunofluorescence and the expression was observed under UV fluorescence microscope.Results1. Under UV fluorescence microscope QD605 quantum dots markered p53 and Bcl-2 expression were clearly seen in human tongue squamous cell carcinoma. P53 was clearly seen in nuclear and Bcl-2 in cytoplasm, Showed red fluorescence. And there is no specific red fluorescence in the blank control.2. Under UV fluorescence microscope QD605 quantum dots markered p53 and QD545 quantum dots markered Bcl-2 expression were clearly seen in human tongue squamous cell carcinoma. P53 was clearly seen in nuclear, showed red fluorescence, while Bcl-2 in cytoplasm, showed green fluorescence.Conclusion1. Quantum-dot fluorescent probe can be applied to detect a special protein in tissue of tumor-bearing nude mice.2. Quantum-dot fluorescent probe can be applied to analyze the trend of Bcl-2 protein in tumor-bearing nude mice.3. Quantum-dot fluorescent probe can be applied to detect a special protein in human tongue squamous cell carcinoma tissue. 4. Quantum-dot fluorescent probe can be applied to detect a variety of proteins in human tongue squamous cell carcinoma tissue.