The Study Of Progesterone Therapy On Orthotopic Nude Rat Model For Human Endometrial Carcinoma Infused Via Internal Iliac Artery

The human endometrial carcinoma inclined to occur in younger ages in last two decades.The percentage of patients younger than 40 years increased from 1%-8%to 13.3%.Most of these patients combine with nuUiparity,infertility,amenorrhea, obesity and polycystic ovarian syndrome,which belongs to the typeⅠor estrogen-dependent endometrial carcinoma.Recently,the conservative therapy with progesterone failed in 30%of all cases.The dose-response relationship existed in progesterone therapy on endometrial carcinoma in a concentration range.The concentration at the site of tumor may be compromised by the side-effect of oral dose.The deficiency of oral dose,such as gastrointestinal discomfort,resulted in the drug malabsorption and inadequate effective concentration in tumor.Change the route of drug administration may acquire better effect than the oral dose by direct infusion via the vessels supplying the tumors with high dose of progesterone.Thus,according to the nude mouse model,we initiated to establish the orthotopic nude rat model for human endometrial carcinoma which facilitates the basic research of progesterone intervention therapy.The soluble progesterone is infused via internal iliac artery in order to verify the feasibility of progesterone intervention therapy for endometrial carcinoma,moreover,to explore a new route of the therapy preserving the reproductive function.This study is categorized in three parts as below:PartⅠThe establishment and biological features observation of the orthotopic nude rat model for human endometrial carcinomaObjective:To establish the orthotopic nude rat model for the human endometrial carcinoma,and observe its biological features.Methods:Ishikawa cell strain was cultured in-vitro and inoculated subcutaneously into 8 female nude rats.Two weeks later,2 nude rats were sacrificed from which the subcutaneous tumors were excised.The subcutaneous tumors were cut into carcinoma mass of 2mm~3 volume and then implanted into the left uterine cavity of 10 nude rats. All 10 nude rats were sacrificed 4 weeks after the implantion.The left uterus were excised and the carcinoma mass were studied in histopathology, immunohistochemistry and flow cytometer. Results:The subcutaneous tumors were generated in all 8 nude rats.The implanted carcinoma was generated in the left uterine cavity in 7 rats.The immunohistochemistry confirmed the estrogen receptor and progesterone receptor were both expressed in the ubcutaneous tumors.The flow cytometer indicated the cells in S-phase accounted for 30.85±6.05%of the nude rat orthotopic tumor.Conclusion:The orthotopic nude rat model were successfully established and can serve as an optimal animal model for in vivo study and contribute to explore the new therapy of human endometrial carcinoma.Furthermore,this model is adapt for the study of the intervention therapy for human endometrial carcinoma via internal iliac artery.PartⅡThe Selection and Security Observation of Progesterone SolventsObjective:To screen the progesterone solvents administrated in artery infusion and observe the toxicity initially.Methods:Calculate and compare the maximal solubility of Megestrol acetate in 95% ethanol,75%ethanol,80%dimethylsulfoxide and N-Methyl pyrrolidone,respectively. Sixteen female nude mouse were infused via tail vein with Megestrol acetate and multiple solvents,respectively.The survival rate and toxicity were compared after 1 week observation,then the toxic progesterone solutions were excluded.Eight nude rat underwent the security experiment by the infusion via artery and oral intake of progesterone solution.Results:Sudden death of the nude mouse occurred during the infusion of Megestrol acetate-95%ethanol and Megestrol acetate-75%ethanol via the tail vein.No toxicity effect was observed during infusing low dose Megestrol acetate-dimethylsulfoxide via the tail vein.Hematuria and subsequent death occurred during infusing middle and high dose Megestrol acetate-dimethylsulfoxide via the artery.No death of nude mouse and rat occurred in the group of Megestrol acetate- N-Methyl pyrrolidone whose ingestion,excretion and activity are normal.Conclusion:The security of Megestrol acetate-N-Methyl pyrrolidone is warranted in the acute toxicity test.Thus,the safe solvent of big dose progesterone is discovered for the intervention therapy on endometrial carcinoma. PartⅢThe Effect of Progesterone infused via Internal lliac Artery on Human Endometrial Carcinoma in Orthotopic Nude Rat ModelObjective:To discuss the therapy effect of progesterone infusion via internal iliac artery on the human endometrial carcinoma.Methods:Thirty human endometrial carcinoma models made of female four-weeks old nude rat were categorized into five groups randomly according to the method described in PartⅠ.Group A is to infuse the Megestrol acetate-N-Methyl pyrrolidone via internal iliac artery(n=6).Group B is to infuse the N-Methyl pyrrolidone via internal iliac artery(n=6).Group C is to infuse the saline via internal iliac artery(n=6). Group D is to feed the Megestrol acetate-N-Methyl pyrrolidone(n=6).Group E is to feed the N-Methyl pyrrolidone(n=6).0.5ml blood was obtained via the tail vein to test the progesterone level 8 hours after the therapy.The progesterone levels were compared among the groups.All 30 nude rats were sacrificed and dissected to observe the pelvic cavity and left side uterus 14 days after the therapy.The cyclic stage of carcinoma cells were analyzed by the microscope observation of HE stain and flow cytometer.The apoptosis of carcinoma cells was analyzed by the Annexin V.Results:All the thirty nude rats survived.The carcinoma were successfully implanted in 22 rats(73.3%).The progesterone level of group A(39.64±0.72 ng/ml) is higher than group D(31.23±4.00 ng/ml)(P＜0.05).The percentage of S-stage cells in group A(6.18±0.40%) is less than group D(7.99±0.66%)(P＜0.05).The apoptosis rate in group A(26.52±2.79%) is higher than group D(21.00±2.66%)(P＜0.05).The progesterone level,the percentage of S-stage cells and the apoptosis rate in group B, group C and group D were of no statistically significant difference.Conclusion:The single high dose progesterone via intemal iliac artery increase the blood concentration,promote the apoptosis and inhibit the splinter of tumor cells. Accordingly,the progesterone intervention therapy may improve the efficacy on human endometrial carcinoma.N-Methyl pyrrolidone is of stability and no toxicity in controllable dose as the solvent,and does not intervene the test results.