| Background: cancer stem cells(tumor stem cells,TSC)theory suggests few cancer stem cells with main characteristics of the potential abilities of self-renewal,unlimited proliferation and tumorigenicity existing in the tumor tissue.Cancer stem cells are closely related to the tumor malignant biological behaviors,such as development,drug resistance and migration.Lung cancer is the highest incidence of malignancies in China,with lung adenocarcinoma accounting for 30 to 40 percent of lung cancer,one of the most common types of tissue for lung cancer.Researchers found that lung adenocarcinoma originated in the airway stem cells,and airway stem cells belong to the end of the airway bronchioles alveolar ducts joint bronchioles alveolar stem cells.Lung adenocarcinoma is considered to be a stem cell disease,and the researchers think targeted killing lung cancer stem cells may be an effective and radical treatment to lung cancer.Especially in recent 10 years,these studies found that cancer stem cells can play the role of “seed” cells of metastases.Researchers think that “metastatic tumour cells is equal to the cancer stem cells”.Currently,tumor cells was detected from the circulatory blood have been recognized as a clinical technique that was approved,and tumor stem cells can be isolated and cultivated from the circulatory blood.Although the technology can detect and isolate circulating tumor cells(circulating tumor cells,CTCs),but expensive,which limits its application and spread in scientific research and clinic,and currently there is a lack of a simple and cheap method which is used to isolation and cultivation,and there is also a lack of a technique that can be used to subsequent amplification and cultivation of tumor stem cells.Because the tumor stem cells of lung adenocarcinoma originates from normal tissue stem cells,there are many similar biological characteristics in both.Therefore,this study attempts to adopt methyl cellulose medium that is semisolid and used to cultivating human hematopoietic stem cells to cultivate the colonies of the single nuclear cells of peripheral blood from patients with lung adenocarcinoma,and attempts to cultivate colony cells in vitro.The purpose of this study is providing preliminary experimental basis for definitude the characteristics of cancer stem cell.Objective: this study adopt methyl cellulose medium that is semisolid and used to cultivating human hematopoietic stem cells to create an growing environment for stem cell colony,and cultivate the colonies of the single nuclear cells of peripheral blood from patients with lung adenocarcinoma and use DMEM medium to cultivate colony cells in vitro,in order to achieve the following objectives:(1)Exploring the possibility of the existence of non hematopoietic stem cells of peripheral blood from patients with lung adenocarcinoma.(2)Exploring the possibility which colonies of non hematopoietic stem cells were cultivated by methyl cellulose medium that is semisolid and used to cultivating human hematopoietic stem cells and the caseof colony cultivation.(3)Exploring the extracorporeal cultivation case of colny cells originated in non hematopoietic stem cells of peripheral blood from patients with lung adenocarcinoma with DMEM fluid medium.Methods: 10 patients with lung adenocarcinoma were selected,and the clinical TNM stage was T2N1M1a-T4N3M1 c.Draw the peripheral blood of 10 ml from every patient and use Ficoll-PaqueTM PLUS to isolate peripheral blood mononuclear cells,and cultivate these cells by methyl cellulose semi solid medium(including of factors such as recombinant human stem cell factor etc.suitable for stem cells' growth).Then observe the growth situation of the cells and colonies dynamically,and describe the colonies' growth morphology and growth curve,and differentiate the non hematopoietic stem cells' cloned types,and select the non hematopoietic stem cells' colonies for detection with flow cytometry,and the detection indexes are CD34 and CD44.Pick the non hematopoietic stem cell colonies and cultivate these cells with complete medium in vitro after mechanical separation and observe the extracorporeal cultivation case.Results: this study conducted the research which is colonies cultivation of the single nuclear cells of peripheral blood from 10 patients with lung adenocarcinoma.Join cell quantity that is(2.2±0.149)x 105 cells into every culture,and there are(120.70±27.893)colonies in every culture on average after two weeks cultivation.The growth curve of cell colonies shows: cells begin to gather like balls in 5-7 days cultivation,and small? preliminary cell colonies formed in 8-10 days,and cell colonies formed in 14-16 days.Colony types are mainly the sample like BFU-E,the sample like CFU-G and the sample like CFU-GEMM,among them,there are some cell colonies look like “paving stones” appeared surrounding the sample like BFU-E and the sample likeCFU-GEMM colonies,and cell colonies look like “paving stones” present intensive flake and boundaries among cells are not clear.Pick these cell colonies to detect CD34 and CD44 double standard with flow cytometry,and found that the group of cells has the CD34 low CD44high characteristic.Further pick up these different types of colonies and cultivate them in vitro using complete medium,and observe them lasting 8 weeks,and the formation and proliferation of the adherent cells are not found.Conclusion:(1)Among the single nuclear cells of peripheral blood from patients with lung adenocarcinoma,the possibility of non-hematopoietic stem cells' existence is there.(2)The possibility of non-hematopoietic stem cells' colonies are cultivated with methyl cellulose medium that is used to cultivating human hematopoietic stem cells is very large,maybe beause of "circulating tumor cells".(3)The extracorporeal cultivation of non-hematopoietic stem cells probably existing should be carried out further on the condition of the adjustment of the medium composition and the improvement of experimental technology. |
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