| BackgroundsTumor is a serious disease which seriously impairs people's health, which accounts for 20% of the total number of all the people who have lost their lives. As the scientists know more about growth, invasion, recurrence, metastasis and the organization of micro-environmental factors of primary tumors, the diagnosis and treatment of cancer has made great progress. Nowadays, there are several existing treatments such as operation, radiotherapy and chemotherapy, yet there are still some problems, such as low cure rate, high drug side-effect, high recurrence rate and so on. In the recent years, more and more research suggest that there remain a small number of tumor cells which are the real reason of origin, occurrence, metastasis and recurrence of the tumor, and this kind of cells have the characteristics the same with the stem cells, so they are called Tumor Stem Cells (TSCs).Tumor stem cells have the following characteristics:First, unlimited self-renewal ability:this kinds of cells can proliferate and produce daughter cells of the same property with the previous generation after inoculated into the SFM; Second, differentiation potential:this kinds of cells can differentiate into different phenotypes of tumor cells after inoculated into the SSM, but this potential has directive property in certain circumstances; Third, high tumorigenicity:even very small number of the cells inoculated into immunodeficient mices can produce tumor in them; Fourth, they have the similar signaling pathway with the normal stem cells:Wnt, Shh, Notch, Bmi and HH-GLI, and so on; Fifth, high migration and metastasis ability:the metastasis of tumors is largely due to the existence and metastasis of the tumor stem cells through a variety of ways; Sixth, not sensitive to radiation and drugs:the majority of tumor stem cells are in a quiescent stage, while the rays and drugs which aim at the tumor cells in a proliferation stage; Seventh, multi-drug resistance:researches have show that there is a kind of protein on the surface of the tumor stem cells, which is called ABCG5 and can efflux drugs, so they can resistant many kinds of drugs by identification and combination with the common antigens or antibodies.As a surface marker of tumor stem cells, CD 133 was firstly discovered in brain tumors. Singh successfully isolated CD133+ tumor stem cells from the ganglioglioma and glioblastoma. CD133+ tumor cells cultured in vitro can form neurospheres-like cloning, and have self-renewal and differentiation ability, they can produce tumors in immunodeficiency mices with the same subtypes of the tumors in situ, and can passage succesionally. In addition, studies have shown that CD133+ cells play an important role in the study of liver cancer, pancreatic cancer, colon cancer, prostate cancer, breast cancer, laryngeal cancer, gastric cancer, renal cell carcinoma and other solid tumors, making CD133 to be a specific molecular marker in the isolation cultivation and identification of tumor stem cells. In recent years, researches pay more attention on the molecular and genetic mechanisms of tumor stem cells. The results show that as a cell surface adhesion molecule, CD133 and other cytokines as well as the formation of the tumor microenvironment, participate in the growth, recurrence, metastasis, cell signaling pathway regulation and resistance to the chemoradiotherapy. Therefore, we can block the cell cycle of the tumor stem cells, block the activation of signal transduction pathways, change the targets in radiotherapy and chemotherapy, disrupt the microenvironment of tumor stem cells and so on, then to develop new methods of tumor targeting therapy aming at the tumor stem cell surface marker CD133 and other tumor-related pathway.Objective1. Discuss the existence and growth of the CD133+ tumor stem cells in the digestive system tumors;2. Discuss the self-renewal capability of the CD133+ tumor stem cells;3. Discuss the differentiation capability of the CD133+ tumor stem cells;4. Discuss the tumorigenicity of the CD133+ tumor stem cells;5. Discuss the clinical meanings and study prospects of CD133 and tumor molecular targeting therapy.Materials and Methods1. Source of specimen:A total of 15 tumor samples from Department of Hepatobiliary Surgery and Department of General Surgery of Henan Provincial People's Hospital and The First Affiliated Hospital of Zhengzhou University,2010.5-2010.11, including 5 cases of gastric cancer,5 cases of colon cancer,5 cases of liver cancer.2. Methods2.1 Take fresh tumor tissue, and collect cells through mechanical separation, collagenase digestion and filteration; After removing red blood cells by erythrocyte lysing solution, resuspend it using RPMI1640 medium (SFM) as a single cell suspension.2.2 Prepare for the 2×107/ml cell suspension, and add FcR blocking agent and PE-antihuman CD133 antibody; Then detect the proportion of CD133+ cells and sort CD133+ and CD133- cells using the flow cytometry.2.3 The sorted CD133+ cells, CD133- cells and unsorted cells are incubated in RPMI1640 SFM, and placed in 37℃,5% CO2 incubator for cultivation. Observe the cell morphology and the condition of CD133+ cells forming into tumor spheres and conducting the subculture.2.4 The sorted CD133+ cells, CD133- cells and unsorted cells are incubated in 96 well plates respectively, detect the cellular proliferation capacity with MTT assay in the first 1,3,5,7,9,15 days, and draw cell growth curve, then compare the proliferation rate of the three kinds of cells.2.5 The sorted CD133+ cells are inoculated in RPMI1640 SSM containing 10% fetal bovine serum, and placed in 37℃,5% CO2 incubator respectively for cultivation. Detect the percentage of CD133+ cells at the first 1,4,8,12 days in cultivation through the flow cytometry dynamically.2.6 Take the CD133+ and CD133- cell of the logarithmic growth phase into cell suspension, and inject 500 of CD133+,2×104 of CD133- cells into armpit and dosum of NOD/SCID mices subcutaneously to observe tumor formation.Results1. Flow cytometry detects the expression of CD 133+ cells:The positive rate of CD133+ cells in gastric cancer is 0.6%-1.0%, colon cancer is 1.9%-2.6%, and liver cancer is 0.8%-1.3%.2. CD133+ tumor cells in vitro cultivation:CD133+ cells in SFM can grow in suspension, proliferate, form into suspended tumor cell spheres, and propagate successionally.3. Test for the proliferation ability of the CD133+ cells in vitro:CD133+ cells in SFM have a strong proliferation ability in vitro, the curve is steep, and it is even more significant in 1-3 d and 5-7 d; whereas the unsorted cells take the second place, whose growth curve is gentle; and the CD133- cells is lowest, whose growth curve is relatively flat, suggesting a weak proliferation ability in vitro.4. Test for the differentiation ability of the CD133+ cells in vitro:CD133+ cells in SSM can differentiate into the adherent cells which are similar to the original tumor tissues, and after cultivated for 12 days, the proportion of the CD133+ cells is from 92.01% down to 4.33%.5. Observation of the tumorigenicity of CD133+ cells:CD133+ tumor cells even if a small number of cells have high tumorigenicity, 500 CD133+ tumor cells incubated into NOD/SCID mices can copy into tumors, and the CD133- cells are not tumorigenic.Conclusions1. CD133+ cells exist in tumors of the digestive system, but only of a small number;2. CD133+ cells in serum-free medium can grow up to tumor spheres, and can propagate successionally;3. CD133+ cells in serum-supplemented medium gradually differentiate into cells which are similar to the original tumors;4. Only a small amount of CD133+ cells can be high tumorigenicity;5. CD133 may be one of the surface markers of tumor stem cells in the digestive system tumors, Using CD133 to be a molecular target for tumor therapy symbol can provide ways for tumor molecular targeting therapy. |
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