| ObjectiveCervical cancer is one of the main malignant tumors harm to women health, Although human has never stopped about cervical cancer research, and made a great breakthrough, the cervical cancer is also one of the serious threat risk factors to women’s physical and mental health.With the development of theory of cancer stem cells, cervical cancer stem cell research also started gradually, researchers trying to looking for exploring new paths of diagnosis and treatment of cervical cancer stem cells for cervical cancer。Markers of the study is the precondition of cervical cancer stem cell research, already found some potential markers for cancer stem cells, such as CD44and CD24, CD133, ABCG2, etc. but whether these markers can be a cervical cancer stem cell markers, still need more research to confirm. Combine the study of tumor stem cells, this research is to isolation and culture of primitive cells of cervical cancer, and to detect the cancer stem cell markers CD44and CD24expression of primitive cells in cervical cancer, while detecting the expression of the markers in the blood, in addition, this experiment adopts the immunofluorescence double staining and immunohistochemical localization expression in cervical cancer detection of this two kinds of markers to enrich the research of entity tumor stem cells.MethodsCollect on October1,2011to October1,2012in first affiliated hospital of zhengzhou university gynecological surgery in patients with cervical cancer specimens(including cancer tissue and peripheral blood). Clinical satge (I A～ⅡB) in16cases, According to the international union of gynecology and obstetrics staging criteria:ⅠA period (4cases),IB period (5cases),ⅡA period (4cases) and ⅡB period (3cases). Cancer histopathological types of invasive squamous cell carcinoma, Patients with a median age56years (32～71years old), at the same time10cases of normal ccrvical tissues as control,patients with median age43(26-63). First, application of collagen enzyme protein separation method of cervical cancer and using cancer stem cell culture medium, culturing the primary cells, After a period of time, due to the primary cell magazine is more, with lymphocyte separation medium separation can be obtained relatively pure cells. Lymphocyte liquid separation in the peripheral blood mononuclear cells.Second,application of flow cytometry to detect the expression of CD44and CD24in cervical cancer primary cell and peripheral blood.At last, immunofluorescence double staining and immunohistochemical staining are applied to detect CD44and CD24expression on cervical cancer tissue surface。Results1. Application of collagen enzyme protein separation method of cervical cancer and using cancer stem cell culture medium, culturing the primary cells,we can get cells suspended ball. but not in normal cervical tissue.2. Flow cytometry detection of tumor markers CD44and CD24, results showed that primitive cell surface expression of CD44and CD24of cervical cancer, but in normal cervix CD44and CD24negative; Patients with cervical cancer in the peripheral blood expression of CD44and CD24, healthy controls in the peripheral blood of CD44and CD24negative;3. Immunohistochemistry showed that CD44and CD24can express in cervical cancer tissue at the same time, but no expression in normal tissue, difference have statistical significance.Immunofluorescence double dye showed that CD44and CD24can express in cervical cancer cell surface at the same time, the two marks may be in the process of the occurrence of cervical cancer in a common mechanism of action.Conclusion1. we can obtain cervical cancer cells suspended ball through the protease digestion method and the cancer stem cell cultures.2. Cancer stem cell markers CD44and CD24expression in cervical cancer tissue and peripheral blood, but no expression in the normal control group. |
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