| Endometriosis is an common benign gynecological disease but the clinical character of deep infiltration, metastasis and repeat recurrence of some cases always puzzled our gynecological clinician. Methotrexate, a commonly used chemotherapeutics, not only being used for gynecological gestational trophoblastic disease (GTD) but also used for benign diseases such as ectopic pregnancy, placenta increta etc al, and the previous epidemiological investigation about risk factors of pelvic endometriosis of our group also showed that previous history of chemotherapy maybe a protective factor for the morbidity of endometriosis. Accordingly, we applied the in vivo fluorescence imaging / analysis technology, immunohistochemistry and flow cytometry and observed the effect of methotrexate on ectopic endometrium and expression of adherency related cytokines by ectopic endometrium during the process of development of endometriosis to offer the primary groundwork of animal experiment for the clinical application of methotrexate for some refractory endometriosis. The studies were proceded in 3 steps:Partâ… : Reproduction of the mouce model of human endometriosisObjective: To reproduce the mice model of human endometriosis. Methods: 1．Enhanced green fluorescent protein transgenic and wild-type C57 BL/6J mouse were used to reproduce the homologous and variant endometrium intraperitoneal injection mouse model of endometriosis. 2．Two weeks later model mice were sacrificed and under the Olympus SZX16 fluorescent microscope general and 3D fluorescence image of ectopic endometrium were made and optical density of fluorescence of ectopic endometrium was calculated. 3．The ectopic endometrium, uterus and ovaries were examined histologically.Results: 1．Under fluorescent microscope all ectopic endometrium was alive and the green cake-like or punctiform ectopic endometrium was found between the intestines, around uterus, under abdominal wall or under hepatic lobules or spleen in the model mice and adhering zone was also seen between the intestines, between the ectopic endometrium and abdominal wall and between the ectopic endometrium and spleen. The achievement ratio of this operation was 100%. 2．Typical endometrial gland and mesenchymal was seen under the microscope, this tell us that the mouse model was successful.Conclusion: 1．We succeeded in reproducing the homologous and variant endometrium intraperitoneal injection mouse model of endometriosis. 2．This mouse model was more sensitive in detection of ectopic endometrium and made the quantification of intensity of fluorescence enabling. This quantification made our results more accurate and objective.Partâ…¡. The effect on ectopic endometrium of model mice induced by methotrexate injected intraperitoneally after operationObjective: To observe the effect on ectopic endometrium of model mice induced by methotrexate injected intraperitoneally after operation.Methods: 1．Transgenic and wild-type C57 BL/6J mouse were used to reproduce the mouse model of endometriosis. 2．Two weeks after the operation the control group (D group) was intraperitoneally treated with sodium chloride while the experimental mice were intraperitoneally treated with methotrexate of 25mg/kg (group A), 50mg/kg (group B), 75mg/kg (group C), respectively. 3．One week later all model mice were sacrificed, under Olympus SZX16 fluorescent microscope general and 3D fluorescence image of ectopic endometrium were made and optical density of fluorescence of ectopic endometrium was calculated. 4．The ectopic endometrium, uterus and ovaries were examined histologically, FCM analysis was performed to access cell apoptosis and the ectopic endometrium was processed for immunohistochemistry to evaluate the changes on expression of correlated cytokines VEGF,ICAM-1,E-cad by ectopic endometrium.Results: 1．Under fluorescent microscope all ectopic endometrium was alive. 2．The volume and optical density of fluorescence of ectopic endometrium of the control group were significantly higher than that of experimental group (P < 0．01) and the difference was dose-dependent (P < 0．01) . 3．The cell apoptosis of experimental group was significantly higher than that of control group (P < 0．01) and also the difference was dose-dependent (P < 0．01) . 4．The expression of correlated cytokines VEGF and ICAM-1 in control group was significantly higher than that of experimental groups (P < 0．05) , but the difference of E-cad expression between these two groups was not significant (P > 0．05) . VEGF and ICAM-1 expression were weakly positive in experimental group A and B, and the the difference of VEGF expression between group A and group B was not significant (P > 0．05) but the difference of ICAM-1 expression between the two groups was significant (P < 0．05) . VEGF expression was extremely weakly positive in group C, while ICAM-1 expression in group C and E-cad expression in group B and C was also negative.Conclusion: Methotrexate can exert growth-inhibitory and apoptosis-inducing effect on ectopic endometrium of model mice. Accordingly, this effect made ectopic endometrium grow downwards and lessen the fluorescence expression, this effect was dose-dependent. Methotrexate can exert inhibitory effect on expression of cytokines VEGF,ICAM-1,E-cad by ectopic endometrium and this effect was related to drug dose.Partâ…¢: The effect on ectopic endometrium of model mice induced bymethotrexate injected intraperitoneally before operationObjective: To observe the effect on ectopic endometrium of model mice induced bymethotrexate injected intraperitoneally before operation.Methods: 1．Mice were divided randomly into four groups and the control group (Dgroup) was intraperitoneally treated with sodium chloride while the experimentalmice were intraperitoneally treated with methotrexate of 25mg/kg (group A), 50mg/kg(group B), 75mg/kg (group C), respectively. 2．Three days and one week after thedosage some untouched mice of C group were sacrificed respectively and the drugconcentration in serum and tissues was determined by HPLC. At the same time the mouse model was also reproduced. 3．Two weeks later all model mice were sacrificed, under Olympus SZX16 fluorescent microscope general and 3D fluorescence image of ectopic endometrium was made and optical density of fluorescence of ectopic endometrium was also calculated. 4．The ectopic endometrium, uterus and ovaries were examined histologically, FCM analysis was performed to access cell apoptosis and the ectopic endometrium was processed for immunohistochemistry to evaluate expression of correlated cytokines VEGF,ICAM-1,E-cad by ectopic endometrium.Results: 1．Under fluorescent microscope all ectopic endometrium was alive. 2．Three days and one week after the dosage the drug concentration in serum and tissues was undetectable. 3．The volume and optical density of fluorescence of ectopic endometrium of the control group were significantly higher than that of experimental group (P < 0．01) and the difference was dose-dependent (P < 0．01) . 4．The cell apoptosis of three experimental groups was also significantly higher than that of control group (P < 0．01) and the difference between group A and C and between group B and C was significant (P < 0．05) , while the difference between group A and B was not significant (P > 0．05) . 5．VEGF expression in control group was significantly higher than that of group B and C (P < 0．05) but was equal to group A (P > 0．05) , and ICAM-1 expression in control group was significantly higher than that of experimental groups (P < 0．05) . E-cad expression was weakly positive in control group but was negative in experimental groups. ICAM-1 expression in group A was weakly positive and ICAM-1 and E-cad expression in group B was negative while these three cytokines were negative in group C.Conclusion: Methotrexate injected intraperitoneally before operation can exert growth-inhibitory and apoptosis-inducing effect on ectopic endometrium of model mice. Methotrexate also can exert inhibitory effect on expression of cytokines VEGF,ICAM-1,E-cad by ectopic endometrium.In summary, our data showed that methotrexate intraperitoneal injection before or after ectopic endometrium implantation can exert growth-inhibitory and apoptosis inducing effect on ectopic endometrium of model mice and also can exert inhibitory effect on expression of adherency related cytokines VEGF,ICAM-1,E-cad by ectopic endometrium. This tells us that with further understanding and prevention strengthening of side effect of chemotherapy drug methotrexate may be used as remedy and recurrence prophylaxis drug for some refractory endometriosis... |
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