| BackgroundCancer is one of the leading causes of death in human, the study on the carcinogenesis, development, invasion, metastasis and therapeutics of cancer has been a hot spot in the field of medical research. Thyroid cancer is the most common thyroid malignant tumor, about 1% of systemic malignant tumors, according to the foreign literature report, the incidence of about 0.5-10/10 thousand. And in recent years, with the development trend of rising. According to thyroid tumor differentiation degree, Thyroid carcinoma thyroid carcinoma can be classified as differentiated thyroid cancer and anaplastic thyroid cancer. According to the histogenesis, differentiated thyroid cancer can be classified as papillary thyroid carcinoma and follicular thyroid carcinoma. The former accounts for 75% of all thyroid cancer, the other accounted for 16%. Otherwise, medullary thyroid carcinoma accounted for 5%, anaplastic thyroid cancer accounting for 3%. On different pathological types of thyroid cancer, their biological characteristics, clinical manifestation, diagnosis, treatment and prognosis are different. The prognosis of the differentiation carcinoma in thyroid carcinoma is good.However, the anaplastic thyroid cancer prone to distant metastases. Patients often have entered the late level, at the time of diagnosis. Therefore, on current clinical treatment, including surgery, radiation iodine and levothyroxine replacement therapy, prognosis is poorer. So study on the pathogenesis of thyroid cancer has very important clinical significance.According to the traditional theory, cancer came from mature cell mutation. It is composed of uniform tumor cell proliferation, mutation and development. However, The study shows that in recent years that The tumor cells have heterogeneity. In another words, Not all tumor cells have the ability to form new tumors. Recent researches had discovered that there were some stem-like subpopulations in tumor tissues which had the potential to proliferate infinitely and played much crucial role in initiating carcinogenesis and development, while the other cells would die after transitory differentiation. It was thought that the tumor growth resulted from the proliferation of some tumor stem cells with special surface markers. For this reason, the tumor treatment must aim directly at the tumor stem cells but not at the bulk of tumor cells with little ability to proliferate and differentiate. More and more evidences suggest that, malignant tumor growth and metastasis need those stem-like subpopulations. They have the characteristics of stem cells, so call as cancer stem cells.The characteristics of malignant tumor progressive growth, metastasis and recurrence with the development of stem cells have many similarities. Firstly, they both have the ability of Self-renewal and proliferation. Secondly, they both have the Phenotypic and heterogeneity. Thirdly they both have the similar growth regulating mechanism and signal transduction pathways. Fourthly they both have the Multiple differentiation potential. Fifth, they both have the same surface markers, like as CD34 and CD90.Cancer stem cell theory elaborated of the malignant tumor origin of the disease from a brand-new angle. The line of sight of research start from tumor cells into cancer stems cells. And to understand the development of the malignant tumor biological behavior from this new perspective. The occurrence of cancer stem cells not only dominated the malignant tumor, but also with the further development of malignant tumor, including the recurrence, metastasis, and the tolerance of tumor in chemotherapy are closely related. According to the cancer stem cell theory suggests that the occurrence of thyroid cancer may come from thyroid cancer stem cells. Separation and identification thyroid cancer stem cells from thyroid cancer tissue play a key role in the biological mechanisms and clinical prevention, diagnosis and treatment on thyroid cancer.Cancer stem cell screening the most commonly used method is to use the specific cancer stem cell surface markers that can be divided into fluorescence activated cell sorting and immune magnetic bead separation technique. Side population cells separation method, which does not depend on the cell surface markers, is to use stem cells to the characteristics of the fluorescent Hoechst33342 discharge. a group of cells refuse to dye or light dye on Hoechst33342, sorting through the flow cytometry instrument on this part of the cell or detection, using this feature sorting this part of the tumor cells known as side population cells. Side population cells a special group of subpopulation, its differentiation state between embryonic stem cells and adult stem cells. It has a multi-directional differentiation, unlimited proliferation and self-renewal potential. Now, it confirms that a variety of tumors such as gallbladder, lung cancer, breast cancer, and gastrointestinal tract tumor can sort out side population cells, and at the same time confirmed these cells with stem cell self-renewal and multi-directional differentiation properties.Compared with using specific cancer stem cell surface markers screening method, side population cells sorting method more is more economical, convenient and at the same time can also be used in phenotypic markers unknown sorting cancer stem cells.AS side population cells sorting method has mature sorting method. This study intends to use thyroid cancer organizations of human being as the research object, SP and non-SP cells were sorted from human thyroid carcinoma cells by fluorescence-activated cell sorting (FACS). The expression of Oct-4 and ABCG2 in SP cells and non-SP cells was detected by Western blot. To explore whether there is side population cells with stem cell properties in thyroid cancer organizations of human being, which provides new thinking for the pathogenesis of thyroid cancer.Objectives1. Using flow cytometry sorting technology, explore whether there is a side group of cells with stem cell properties in human thyroid carcinoma tissues.2. To isolate, culture and identify the stem-like subpopulations cells from human thyroid carcinoma tissues in vitro, and to investigate the basic characteristics of stem-like thyroid cells.Methods1. To isolate and culture thyroid carcinoma cells from human thyroid carcinoma tissues in vitro.Tumor tissue samples were obtained from a patient with thyroid carcinoma after surgery, and then thyroid carcinoma cells were cultured in vitro by primary tissue culture technique. Thyroid carcinoma cells were grown in RPMI 1640 supplemented with 5% fetal bovine serum and penicillin/streptomycin at 37 ℃ in a humidified atmosphere with 5% CO2.2. Using flow cytometry sorting side population cells and non side population cellsThe cells were detached by trypsinization and washed with ice-cold PBS/2% FBS. Cells were labeled in the growth medium with5.0 g/ml Hoechst33342 dye either alone or in combination with 50g/ml verapamil at 37℃ for 90 min. After washing with PBS/2% FBS,the cells were then incubated with 2 g/ml propidium iodide to exclude dead cells. The Hoechst dye was excited with UV laser and its fluorescence was measured with both 675 filter (Hoechst Red) and 450 filter (Hoechst Blue).3. Using Western blot method to detect the expression of ABCG2, Oct-4 protein in side population cells and non side population cellsTo extract the total protein of side population cells and non side population cells, use Western blot method to detect the expression of ABCG2, Oct-4 protein.4. Statistical analysisDifferences between groups were examined for statistical significance using X ±S and T test,P value not exceeding 0.05 was considered statistically significant.Results1. SP cells with stem cell potency were isolated from human thyroid carcinoma cells, experimental group sorting out the content of SP cells is 1.40%,and the control group sorting SP cells content is 0.155% (p<0.05)2. The expression of Oct-4 and ABCG2 is significantly higher in SP cells than in non-SP cells.ConclusionThe origin of the human thyroid cancer is closely related to cancer stem cells. SP cells, which have the potency of stem cells, exist in human thyroid carcinoma cells.The expression of Oct-4 and ABCG2 is significantly higher in SP cells. Therefore, side group of thyroid carcinoma cells can be thyroid cancer drug resistance, the root cause of recurrence and metastasis, suggests future research direction is more targeted therapy against the side group of cells. |
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