Establishment Of Ovarian Clear Cell Cancer Model

BackgroundOvarian clear cell carcinoma(OCCC)is a special subtype of epithelial ovarian cancer(EOS)and its incidence in epithelial ovarian cancer is 4%to 25%of EOC.Related to race,up to 25%in Japanese population,with high degree of malignancy,easy to relapse early,natural resistance to first-line platinum chemotherapeutic drugs,is the worst prognosis in EOC In the subtype,despite the efforts of the OCCC treatment for several decades and with encouraging progress,the 5-year survival rate of the disease is still low,about 20%.Ovarian clear cell carcinoma has unique characteristics different from other epithelial ovarian cancers,but currently there is a lack of reliable and specific methods for the diagnosis and treatment of OCCC.Basic researches are mostly based on in vitro monolayer cell culture conditions,and Lack of relevant research in the body.This requires an appropriate animal model to fill in the status quo of research vacancies in vivo,but also requires a more convenient and stable OCCC model,accelerate the basic research on OCCC,and provide broader ideas for clinical diagnosis and treatment.A good animal model can provide a good theoretical basis for early clinical diagnosis and interventions.The cancer model study mainly includes animal models and in vitro models.The main experimental objects of animal models are nude mice,mice,and rats.Nude mice and mice have immunodeficiencies,and cancer models are easy to establish,but they cannot simulate cancer cells in vivo.Growth-related immune responses and other related microenvironments are relatively costly.Rats use a model of abdominal cavity transplantation to study cancer chemotherapy treatment.They do not respond well to the development of tumors.There are embedding methods for orthotopic transplantation in rats.Transplantation with tissue blocks,embedding method using xylene thorium as inducer,but the tumor formation time is long,about 5-8 months,and the ovarian cancer type and survival rate are uncertain,the tissue block can ensure the model type,but it needs to be transplanted.The complex process of tumor formation,implantation,and embedding.The in vitro model studies are two-dimensional(2D)culture and three-dimensional(3D)culture,while less research is based on the molecular level in the 3D model.Studies have shown that in cultured cells in 3D mode,the resulting structure can more closely mimic the complex disease microenvironment in vivo,and the cell growth pattern is closer to that in vivo.At present,the use of normal immune rats to establish ovarian cancer model is less,the use of cell suspension in situ injection method is also less,and there is a blank in the in vivo study of OCCC.ARID1A is a tumor suppressor gene,localized in the nucleus,and expressed in many tissues throughout the body,such as the pancreas,spleen,small intestine,prostate,rectum,and ovary.The ARID1A mutation is the most common among gynecologic tumors,with the highest proportion in OCCC.In OCCC,the expression of this gene is missing,and in different stages,the frequency of ARID1A mutation is also different,and it is also related to the chemical resistance of OCCC.ObjectiveTo explore the feasibility of establishing Wistar rat orthotopic xenograft model of human ovarian clear cell carcinoma,and to explore the feasibility of establishing animal models of OCCC by using the method of orthotopic transplantation of cell suspension in normal immunized rats to make the establishment of OCCC animal models feasible.The method is simple and convenient,which provides the basis for the in vivo study of OCCC and fills in the vacancy in vivo research.It provides a broader platform for exploring early detection and detection methods for human ovarian clear cell carcinoma,effective targeted therapy,and predicting prognosis.MethodsAccording to the purpose of this experimental study,the main research contents and methods are divided into three major parts,which are summarized as follows:1.Two-dimensional and three-dimensional culture of ES2 cellsThe ES2 cells were cultured in two dimensions(normal monolayer cell culture).ES2 cells were subcultured and counted,and the growth curve of ES2 cells was plotted to provide a basis for the establishment of OCCC animal models.During the experiment,using rat tail collagen as a scaffolding material,we tried to establish three-dimensional culture of ES2 cells.Even though ES2 cells showed a three-dimensional structure growth pattern,the possibility of ES2 cells being used as OCCC in vitro models was explored.2.Establishment of OCCC model using Wistar female ratsTwenty-four experimental female Wistar rats that had been purchased were adapted for 7 days and were randomly divided into three major groups of 8 rats each.The ES2 cells in the logarithmic growth phase were prepared into different concentrations of cell suspensions according to the doses of 1×108(group E1),5×107(group E2),and 1×107(group E3)cells.Microsyringe The cell suspension was injected in situ and inoculated into the left ovarian capsule of each group of female Wistar rats.From the 14th day after the surgery was established on the model,one rat was randomly selected from each group to obtain an ovarian sample for each 7 days.The samples were subjected to pathological examination to identify the model.3.Identification of ovarian clear cell carcinoma modelThe ovaries of the removed Wistar rats were fixed,embedded,sectioned,stained with HE and stained with ARID1A immunohistochemistry.Results1.ES2 cells grow in an adherent manner in the two-dimensional model,exhibit an irregular shuttle type,and grow into small cell clusters of unequal size in the three-dimensional mode,eventually forming larger cell clumps.This clump can be maintained.2 weeks or so.2.The same concentration of ES2 cells were inoculated into the ovarian capsule of Wistar rats.E1 group was modeled at about 7 weeks,E2 group was about 7 weeks,and E3 group was about 8 weeks.3.The ovarian samples were stained with HE and compared with the histopathological morphology of human OCCC,with higher similarity.4.ARID1A immunohistochemical staining in the ovary showed a lack of ARID1A expression and was highly consistent with human OCCC tissue.Conclusion1.The pathological and molecular biological characteristics of ovarian tissue after identification as a model are similar to those of human OCCC.The OCCC model establishment method for this experiment is feasible,it is simple,inexpensive,stable,and has a high survival rate.With the advantages of considerable modulus,etc.,if the number of inoculated cells is increased and fluorescence-infected or imaging techniques are used to assist in the dynamic observation of ovarian tissue changes,the survival rate and the rate of molding may be further accurate.It is preliminarily considered that the OCCC animal model established by this experimental method can be used in the OCCC basic molecular biological analysis and biological treatment research model to fill the OCCC in vivo research gap,thereby providing more preclinical studies for the diagnosis and treatment of OCCC.2.Two-dimensional and three-dimensional cell cultures were performed using the same strain of ES2 cells,but the growth patterns showed significant differences,mainly due to different cell culture patterns.Moreover,ES2 cells grow well in 3D mode and may become an important means for OCCC in vitro studies.