Development Of The Mechanism Of CD44 Occur In Ovarian Cancer

Background:In gynecological disease,ovarian epithelial cancer is in the highest mortality rate of malignant tumor. Seventy-five percents of patients in the hospital for diagnosis have entered the late stage of cancer (?-?). The mainstream in the clinical treatment method is based on to platinum combining chemotherapy.There are still about 60-80%of patients will relapse.If patients in the late stages of ovarian cancer, roughly only 16-22 months for survival time. The 5 year survival probabilities are approximately only 30%. The treatment is not ideal, mainly in the course of chemotherapy.Tumor cells produced drug resistance. Recently there has been a clinical trial of small molecule compounds and targeted drugs, but the prognosis of patients with ovarian has not been improved. In revealing the tumor genome mapping, for the study of ovarian, many researchers have studied the content is mainly a table view of genetics and genomic changes in clinical prognosis, study of mechanism for tumor recurrence and treatment of cancer of the ovaries to produce drug resistance is not clear, in this way, the treatment of ovarian blocked.In recent years, researchers to find new therapeutic avenues in ovarian cancer, the attention to cancer stem cells, according to the study of other solid tumors, based on summing up the experience of open up a new approach to treat ovarian cancer. Now for the ovarian cancer research, stem cell selection is based on the experience of other cancers related research in the field of to, choose marker protein separation technology,mainly by the use of CD44, CD133, CD90 CD117, CD24, ALDH1 and EpCAM these markers were marked, there is now some results, but because the markers are mainly derived from other solid tumor stem cell therapy research, for ovarian cancer stem cells research role is not obvious, so controversial. It is very important to study the specific marker proteins, and it is very important to study the stem cells of ovarian cancer by using these marker proteins.In the preparation of the study, the use of stable isotope labeling and proteome analysis, the proteome analysis is based on mass spectrometry, using proteomics technology of SP and non SP cells in the membrane protein analysis, the ovarian cancer stem cells labeled candidate protein transmembrane protein CD44 screened out, and in vitro experiments to show the positive stem cell characteristics were identified. This study used is flow cytometry sorting and serum suspension culture method, through two experiments in vitro and in vivo and in the organization and ascites of the patients and the cell line inspection of CD44 showed marked positive EOC cell sphere forming ability and its ability to update and differentiation and oncogenic ability to verify its cisplatin for resistance, and to analyze the relationship between its expression and EOC patients with clinical outcome. And preliminary study on the role of estrogen in the microenvironment of cancer stem cells.Objective:It will be screened out CD44 + cells in ovarian cancer marker, and in vitro experiments showed positive cells were identified in the initial stem cell characteristics.This study using flow cytometry and serum-free suspension culture method, two vitro and in vivo experiments, testing CD44-positive ovarian cancer cells in the tissues of patients with ascites in cell lines and in the sphere forming ability itself renewal,differentiation and carcinogenesis as well as the ability to verify that it has the resistance to cisplatin, but to analyze the relationship between clinical prognosis and its expression in ovarian cancer patients.Method:1. The use of flow cytometry analysis of ovarian cancer cell lines was applied in SKOV3?A2780?ES2?IGROV1?OC3.CD44+ in the proportion of cells. Using the method of cell instrument of CD44 and CD44 cell sorting prove it in the culture medium after training again to differentiate subpopulations HG-DMEM/ 10%FBS.Without the use of serum suspension culture method to test the CD44 and CD44 cell sphere forming ability and its passage capability, and the use of immunofluorescence and the method to detect the accumulation of SKOV3 sphere for CD44.2. Flow cytometry was used to CD44 and CD44 to sort out, after inoculation with two kinds of cells with gradient in NOD / SCID the two mice subcutaneously to observe the in vivo tumor formation rate,observed for both a rate of tumor or tumor size appears statistically significant differences. After the mice into the tumor, and then sorted out the two group of cells inoculated into the mice, once again to observe. Flow cytometry was used to analyze the proportion of CD44+ cells in the transplanted tumor, and to verify the ability of CD44+ and CD44- in the differentiation of the two cells in vivo.3. Using various degrees of ES2 cell lines SKOV3 and CD44 + and CD44 + cells related to cisplatin resistance MTT method to verify, by flow method to test different concentrations of cisplatin on cells SKOV3/CD44+ produce differences in enrichment.Verification flow method of cisplatin chemotherapy for EOC patients in organization of cancer cells in the CD44 + cells produce enrichment; NOD / SCID mice ovarian cancer xenograft model is built up to verify cisplatin chemotherapy in the transplanted tumor in the CD44 + cells produced enrichment.Results:1. In the use of flow cytometry found that there are five kinds of cancer cell lines in the amount of 0.4%-2% cell population, it is relatively stable in the proportion of CD44+.Using flow cytometry method to select the CD44 + cell subsets, in the condition of cell differentiation were two weeks of in vitro culture, then again after flow cytometry analysis found that CD44 + cells from differentiated cells CD44, its positive rate is still the original level,but CD44 cells but can differentiate the CD44 + cells. This shows that the CD44 + cells have the capacity to differentiate. Using serum-free suspension technology to cultivate SKOV3sphere. Flow cytometry was used to both CD44 and CD44 of the cells to sorting out, sorting out by suspension technique to culture, this time found number of CD44 + cells than CD44 cells formed sphere, and CD44 + cells into the sphere to in vitro at least four generation, but CD44 cells basically is cannot be passaged. Immunofluorescence and flow cytometry analysis can prove that CD44 +cells in the expression of SKOV3sphere significantly paste than Wall SKOV3 cells to high; by in vivo experiments once again proved to the CD44 + cells than CD44 cells tumorigenic ability and loz the former cells can let NOD / SCID mice tumor, CD44 cells to achieve this effect requires at least 104 cells. CD44+ cells can be transplanted in vivo in mice, and the latter can not be passed. HE staining showed the same histological types as the two generation of transplanted tumors in CD44+ cells. Flow cytometry analysis can see the positive rate of CD44 in CDCs OA+ cell xenografts in nude mice is unchanged, but also in CD44 cell transplanted tumor in basically no CD44 + cells,suggesting that CD44 + cells in vivo with differentiation ability.2. In the primary cancer cells and ascites will be separated from the cancer cells, flow analysis can be seen in the above tissues and ascites cells which have CD44+ cell group.And establish training system of primary ovarian cancer cell sphere, from found in primary tissue CD44 + cells can form sphere, and can be passaged, but CD44 cells basically unable to form a sphere, by immunofluorescence and flow cytometry verification can see expression in primary cancer cells sphere has significantly improved. Through in vivo experiments again show that CD44 + cells to CD44 cells tumorigenic ability stronger than and 103 of the former cells can still let the NSG mice tumor, the latter to achieve this effect must have at least 105 cells to. The transplanted tumor of the former can carry on the continuous transmission in the mouse, and the latter can not be passed. HE staining showed the same histological types as the two generation of transplanted tumors in CD44+ cells. Flow cytometry analysis can see the positive rate of CD44 in CDCs OA+ cell xenografts in nude mice is unchanged, but also in CD44 cell transplanted tumor in basically no CD44 + cells, suggesting that the CD44+ cells in vivo and differentiation ability.3. Using MMT method to detect the drug resistance of the cells, in the SKOV3 and ES2 cell line CD44+ cells in the drug resistance to cisplatin is much higher than the CD44-cells. See, SKOV3 cells after different concentrations of cisplatin for total cell decreased. The positive rate of CD44 anti but increased, and is almost constant the absolute number of CDCs OA+ cells by flow cytometry assay. The positive rate of CD44 in cancer cells of the same patients before and after chemotherapy could be seen that the proportion of CD44+ cells was significantly increased after cisplatin chemotherapy. In vivo animal experiments show that after cisplatin chemotherapy mice transplanted tumor growth rate obviously to than not the treatment of slow, but the transplanted tumor in the positive rate of CD44 have to be higher than the group, which proves that cisplatin for CD44 has enrichment effect.Conclusion:1. The expression of CD44+ cells in 5 cancer cell lines was basically stable at 0.4%-2%,which was consistent with the proportion of stem cells. In ovarian cancer cell line CD44+ cell population is the ability of differentiation, the formation of sphere, the ability to update their own CSCs features. The cells have a stronger ability to form tumors, and can be continuously cultured in mice.2. In EOC patients with primary cancer cells can be separated from the CD44+ cell group, the cell population is the ability to differentiate in vivo and sphere formation ability and self renewal capacity and other characteristics of CSCs. CD44+ cells in primary cancer cells have a stronger ability to form tumors,and can be continuously cultured in vivo.3. Ovarian cancer cell lines, primary ovarian cancer cells, as well as animal experiments can verify the CD44+ cells have a role in the enrichment of cisplatin.