Identification Of Stem Cells Of Osteosarcoma In Chinese People And Its Biological Characteristics

Object:With the improvement of living standards, extension of lifetime and aggravation of environmental pollution, the incidence of tumor has been gradually increasing.The tumor has been the most important threat to human life together with cardiovascular disease.According to statistics, about 265 million people lost their lives because of tumor every year in China. Therefore, people paied great attention and put huge manpower and material to cancer research in domestic and foreign. Although cancer diagnosis technology have made great progress, but the prognosis of most malignant tumors is still not ideal.The main reason lies in shortage understanding of the pathogenesis and biological characteristics of tumor,lack of effective clinical treatments.Osteosarcoma is primary malignant bone tumors originating in mesenchymal cells.In primary bone tumors, the incidence of osteosarcoma is only second to plasma-cell myeloma,with a higher predilection for young in 10-25 years, a male to female ratio of 1.5:1. With high malignance, fast growing,early transfer, strong invasion in osteosarcoms, about 80% of patients had been found the transfer of lung or other parts at diagnosis in clinical work.The prognosis of patients with osteosarcoma is poor in the past, the 5-year survival rate is only about 20%.Although the surgery and chemotherapy has made significant progress in recent years, but the long-term survival rate of patients with osteosarcoma still lingers at 65%, most patients eventually died because of local recurrence or distant metastasis of osteosarcoma. At present, the treatment of osteosarcoma is still mainly aimed at tumor whole, most osteosarcoma cells can be removed or killed through the adjuvant chemotherapy or surgical excision of tumor, but it is very difficult to prevent recurrence and metastasis of osteosarcoma. The theory of tumour stem cells provides a new interpretation and treatment direction for happen, transfer and attack of osteosarcoma. According to the theory, threr is only a handful of tumor cells (tumor stem cells) rather than all tumor cells with tumorigenicity.Tumour stem cells are not easily killed by conventional radiotherapy or chemotherapy because it is regular in dormant period similar to normal stem cells,even 99.99% tumor cells were killed, but as long as there is the the rest 0.01% cells will become the root of tumor recurrence.This theory is very well explain the clinical confused of high recurrence rate and poor long-term curative effect of osteosarcoma by bone tumor doctor. Aiming at high relapse and metastasis of osteosarcoma in this study, collecting Chinese osteosarcoma specimen, isolating and culturing tumour stem cells of osteosarcoma and obtaining tumor stem cell samples of Chinese osteosarcoma.understanding the osteosarcoma stem cell tumorigenicity and metastatic, finding specifically expressed genes and proteins of osteosarcoma stem cells, exploring the stem cell tumor-specific markers, providing the basis for further research to pathogenesis, early diagnosis and targeted therapy of osteosarcoma.Methods:Collecting countrymen fresh specimens of osteosarcoma;culturing primary osteosarcoma cells;inducing osteosarcoma sphere cells by adding variety of different growth factors in serum-free medium;using immunofluorescence technique to detect the expression of Oct4,Nanog and Sox2 on osteosarcoma sphere cells;exploring and obtaining the optimum condition of cultivation; detecting the expression of CD73,CD90, CD105 by flow cytometry; sorting CD105~+ cells by immunomagnetic beads and studing its biological characterstics.Results:1.Collecting countrymen fresh specimens of osteosarcoma and culturing primary cells Chinese osteosarcoma cells,sucultivating the cells.2.culturing primary Chinese osteosarcoma cells in serum-free medium,adding respectively four groups of growth factors: EGF,IGF-1,EGF,bFGF,EGF,IGF-1,TGF-β1,EGF,TGF-β1,bFGF and each group successfully induced osteosarcoma sphere; using immunofluorescence technique to detect the expression of Oct4,Nanog and Sox2 on four groups of osteosarcoma sphere cells;the positive expression of Oct4,Nanog and Sox2 was found on the fourth group(EGF,TGF-β1,bFGF)of most osteosarcoma sphere cells;the positive expression of Oct4,Nanog and Sox2 was found on the other three groups of a few osteosarcoma sphere cells.3. Detecting the expression of CD73,CD90,CD105 on osteosarcoma sphere cells by flow cytometry The positive expression of CD73,CD90,CD105 were 95.5%,93.9%,1.4%, respectively.4.Sorting osteosarcoma sphere cells with CD105 marker by immunomagnetic beads;obtaining CD105~+ and CD105- osteosarcoma cells and inoculating respectively. CD105~+ cells can be induced patent osteosarcoma cell sphere. Using immunofluorescence technique to detect the expression of RunX2,Osx,Oct4,Sox2, Nanog on CD105~+ and CD105- osteosarcoma sphere cells,the positive expression of RunX2,Oct4,Sox2,Nanog was found on most CD105~+ cells,the negative expression of Osx was found on a few CD105~+ cells. the negative expression of RunX2,Osx,Oct4,Sox2,Nanog was found on nearly all CD105- cells.5. The results of cell invasion Assay, cell proliferation test and tumorigenicity experiments showed that CD105~+ cells has stronger invasion and proliferation, the more ability of tumor formation in mice.Conclusions:Fresh specimens of osteosarcoma wrer collected to culture and obtain primary Chinese osteosarcoma cells.The cells successfully induced osteosarcoma sphere in serum-free medium with growth factors of EGF,TGF-β1,bFGF.Immunofluorescence showed the positive expression of Oct4,Nanog and Sox2 was found on the majority of the osteosarcoma sphere cells.CD105 is the important surface marker of tumor stem cells of osteosarcoma by flow cytometry.Sorting osteosarcoma sphere cells with CD105 marker by immunomagnetic beads.The positive expression of RunX2,Oct4,Sox2,Nanog and negative expression of Osx was found on most CD105~+ cells.The results of cell invasion assay, cell proliferation test and tumorigenicity experiments showed that CD105~+cells has stronger invasion and proliferation, the more ability of tumor formation in mice. The results has revealed that the osteosarcoma cells,isolated and cultured in this study, have stem cells characteristics.