| Objectives:At present,the treatment of Epithelial ovarian cancer(EOC)is still a major challenge in the clinical works.For the first time that the radical treatment of cytoreductive surgery and postoperative chemotherapy based on platinum are sensitive to the vast majority of patients with ovarian cancer and the first remission rate was about 80%.However,the residual tumor cells are easy to be hidden because of recurrence.The probability of second debulking surgery is still lower for the recurrences often invased the ambient normal organs or distant metastasis,or it may confers a high risk of perioperative morbidity in some cases.The majority of patients will choose to cycle chemotherapy to control the disease progression.However,repeated administration of chemotherapy causes numerous intolerable cumulative toxicities,which worsen until continuation of chemotherapy finally becomes impossible,and also the therapy eventually relevant to chemoresistance and it is unclear whether all patients benefit from a comprehensive surgical intervention in the same way.Repeated chemotherapy,multidrug combination therapy often lead to multidrug resistance.The two main factors of relapse and drug resistance make ovarian cancer at the first place in the incidence of gynecologic cancer in recent years.How to predict and reverse the occurrence of drug resistance,and how to develop treatment strategies have became difficulty in the field of treatment of ovarian cancer.The ascites of ovarian cancer predicts poorer prognosis and creates a special tumor microenvironment.So far there is no effective means of control.The main treatment methods were repeated puncture drainage and intraperitoneal perfusion chemotherapy.Malignant ascites forms a special tumor microenvironment of ovarian tumor,which is closely related to tumor heterogeneity and tumor biological behavior.The purpose of studys are to analyze the relationship between the factors from ascites and the prognosis of ovarian cancer.Many scholars separate and identify the components of ascites.The results showed that the cytokines in malignant ascites were closely related to the prognosis of ovarian cancer,and could be used as a specific marker of tumor,the soluble components in malignant ascites are involved in the activation of multiple signaling pathways,promoting cell proliferation,invasion,immune escape,metastasis and drug resistance,the components of cells in ascites can express the phenotype of tumor stem cells,and have high degree of malignancy.In spite of ascites has been of great concern in recent years,there are few studies on ascites as a whole fect on ovarian cancer.Purpose :In this study,we obtained the non cellular components of ascites from ovarian cancer patients,and use it as cell culture conditions.To investigate the effection and mechanisms of ascites on proliferation,apoptosis and drug resistance.To provide experimental basis for the effect of ascites on the progression of ovarian cancer.Methods :1.38 cases of ovarian cancer ascites were obtained by ultrasound guided puncture,and the ascites non-cell component were obtained by high speed centrifuged and filtrated.Use it as culture conditions for ovarian cancer cell SKOV3.They were divided into two study groups,experimental group is the cell cultured by ascites non-cell components and control group that cultured by fetal bovine serum.2.MTT assay was used to detect the proliferation of SKOV3 cells cultured by ascites non-cell component,and the cell growth curve should be drawn.3.The experimental groups and the control groups were treated with different concentrations of chemotherapeutic drugs(DDP and LY294002).MTT assay was used to detect the sensitivity of SKOV3 cells to DDP in different groups.4.Treatment of SKOV3 cells with different culture conditions by chemotherapy drugs(DDP,LY294002 and DDP combined with LY294002)about 24 h,and flow cytometry was used to detect the changes of apoptosis in different groups.5.Western Bloting was used to detect the expression level of drug resistance protein,cell cycle protein and apoptosis related protein in the groups for serum cultured and ascites non-cell component cultured.6.Western Bloting was used to detect the expression level of PI3K/Akt pathway related protein in the groups for serum cultured and ascites non-cell component cultured.In order to understand the role of this pathway in this study.Results:1.MTT experiment results show that the non ascites non-cell components can promote tumor cell proliferation.There is a close relationship between ascites microenvironment and tumor proliferation.According to the cell proliferation curve,the SKOV3 cells proliferated rapidly in the ascites non-cell components group.The proliferation stage was also earlier and the cell doubling time was shortened.2.The cells in the experimental group(ascites cell culture group)and the control group(serum culture group)were treated with different concentrations of chemotherapeutic drugs.MTT assay was used to detect the sensitivity of SKOV3 cells to cisplatin.The results showed that the inhibition of SKOV3 cells in the ascites non-cell components group was significantly lower than that in serum culture group.The IC50 value of DDP in the experimental group was 16.23±2.45 ug/ml,and the IC50 value of DDP in the control group was 7.91±0.19 ug/ml(P<0.001).The sensitivity of cells to cisplatin was decreased by 1.05 times.Under the same concentration of cisplatin,and the inhibition rate of cells in the experimental group was lower than that of control group(P <0.05).3.Detection of cell inhibition rate on the experimental group and control group cell inhibition rate in different concentrations of LY294002.The inhibition rate of SKOV3 in each group increased with the increase of concentration.The inhibition rate of the experimental group was lower than that of the control group under different concentrations of LY294002(P <0.05).4.Stimulate SKOV3 cells in experimental group and control group by LY294002 combined with cisplatin showed that LY294002 can reverse cisplatin resistance caused by non cellular components of ascites.5.Flow cytometry showed that there was no significant difference between the experimental group and the control group(P>0.05).With the cisplatin treatment for24 hours,the apoptosis of non cellular components of ascites was significantly lower than that of control group.After cisplatin combined with LY294002 treated,the anti apoptotic effect induced by the non cellular components of ascites can be reversed.6.The non cellular components of ovarian cancer ascites can increase the drug resistance related protein P-gp and MRP1,cyclin Aurora-A and 53BP1,and the expression of anti apoptotic protein Bcl-2 and PI3K/Akt related proteins expression.However,there was no significant increase in the expression of pro-apoptotic protein Bax,and apoptotic pathway protein was not cleaved.7.After LY294002 stimulation,the expression of Bcl-2 protein was decreased in the SKOV3 cell line cultured by ascites non-cell components group,but there was no significant effect on P-gp and MRP1.Conclusions:1.The non cellular components of ascites from ovarian cancer can significantly improve the proliferation ability of SKOV3 cell lines and shorten the proliferation cycle.The process can be reversed by the PI3 K inhibitor LY294002.2.The non cellular components of ascites in ovarian cancer can reduce the inhibition rate of SKOV3 cell line to cisplatin and reduce the apoptosis of caused by cisplatin.The drug resistance and anti apoptotic process may be related to PI3K/Akt pathway.3.The non cellular components of ovarian cancer ascites can promote the expression of P-gp and MRP1,and also promote the expression of cyclin Aurora-A and 53BP1 and the same to expression of anti apoptotic protein Bcl-2.However,it has nothing to do with pro-apoptotic protein Bax. |
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