| Chapter One The establishment of the mice model of autoimmune premature ovarian failure and expression of Foxp3 in the thymus and ovariesObjective To establish and evaluate the mice model of premature ovarian failure, and understand the expression of Foxp3 in the thymus and ovaries.Method Thirty female C57BL/6 mice were randomly divided into three groups, the POF model group (the premature ovarian failure group), the adjuvant group and the blank group, ten mice in each group. The POF model was set up based on methods reported by Fu Li et al. The mice in the model group were subcutaneously injected with CFA-Pzp3 mixture 0.1ml in bilateral centers of the heel (0.05 ml on each side), accordingly/in the same way,0.1 ml CFA-triple-distilled water mixture was injected in the adjuvant group and the blank group respectively. We observed the sexual cycle by means of vaginal smears. All mice were sacrificed on the 14th day after the injection; the peripheral blood, ovaries and thymus tissues were collected for further examination. The AzpAb concentrations were determined by ELISA method; the proportion of growing follicles in each group was calculated on HE stain slices; CD45 expression in the ovaries reflecting the severity of autoimmune oophoritis and Foxp3 expression in the thymus and ovaries of each group were both determined by immunohistochemical method.Results:?The proportion of the mice that suffered from sexual cycle disorder, the average serum AzpAb concentration and the incidence of autoimmune oophoritis in the model group were significantly higher than those in the adjuvant group and the blank group (P<0.05). The proportion of growing follicles in the ovaries in the model group was significantly lower than that in the adjuvant group and the blank group (P <0.05). While there is no statistical difference between the two control groups in the aforesaid indications (P> 0.05).?The immunohisto-chemistry scores and positive expression rate of Foxp3 in the thymus and ovaries in the model group were much lower than those in the adjuvant group and the blank group (P<0.05). While there is no statistical difference between the two control groups in the aforesaid indications (P >0.05).Conclusion:?Pzp3 was a nice antigen to induce animal model of premature ovarian failure;?The expression of Foxp3 in the thymus and ovaries of mice with autoimmune ovarian failure was significantly declined. Chapter Two Therapeutic effect of different doses of testosterone propionate on mice premature ovarian failureObjective:To discus therapeutic effect of different doses of testosterone propionate on autoimmune ovarian failure of mice.Method:Fifty female C57BL/6 mice were randomly divided into five groups, the low-dose testosterone propionate therapy group (the LDTP group), the moderate-dose testosterone propionate therapy group (MDTP group), the high-dose testosterone propionate therapy group (the HDTP group), the glucocorticoids therapy group (the GC group) and the model group, ten mice in each group. The mice model of autoimmune ovarian failure was established according to our previous study. We observed the sexual cycle by vaginal smears. Each mouse was respectively intraperitoneally injected with 0.02 mg TP,0.1 mg TP,0.5 mg TP,0.2 mg GC, and 0.1ml physiological saline daily according to the group it belonged to. The treatment lasted for two weeks in all. All mice were sacrificed on the 28th day after subcutaneous injection; the blood and ovaries tissues were collected. The serum AzpAb concentrations were determined by ELISA method; the proportions of growing follicles were calculated on HE stain slices; CD45 expression in the ovaries were determined by immunohistochemical method to understand the severity of autoimmune oophoritis.Results:The proportion of the mice that suffered from sexual cycle disorder, the serum AzpAb concentrations and the incidence of autoimmune oophoritis in the MDTP group were significantly lower than those in the model group (P<0.05), but the proportion of growing follicles in the ovaries in the MDTP group was significantly higher than that in the model group (P<0.05). The proportion of the mice that suffered from sexual cycle disorder, the serum AzpAb concentrations in the LDTP and the HDTP groups were significantly higher than those in the MDTP group (P<0.05), but the proportion of growing follicles in the ovaries in the LDTP and the HDTP groups was significantly lower than that in the MDTP group (P<0.05). While there is no statistical difference between the MDTP and the GC groups in the aforesaid indications (P> 0.05).Conclusion:Moderate-dose testosterone propionate could ease premature ovarian failure.Chapter Three The mechanism of therapeutic effect of testosterone propionate on mice premature ovarian failureObjective:To discuss the mechanism of therapeutic effect of testosterone propionate on mice premature ovarian failure.Method:Forty female C57BL/6 mice were randomly divided into four groups, the testosterone propionate therapy group (the TP group), the flutamide therapy group (the Flu group), the flutamide plus testosterone propionate therapy group (the TP+Flu group) and the model group, ten mice in one group. The mice model of autoimmune ovarian failure was established according to our previous study. We observed the sexual cycle by vaginal smears. Each mouse was respectively intraperitoneally injected with 0.1 mg TP,0.5mg Flu,0.1 mg TP+0.5mg FLu, and 0.1ml physiological saline daily according to the group it belonged to. The treatment lasted for two weeks in all. All mice were sacrificed on the 28th day after subcutaneous injection; the blood, ovaries and thymus tissues were collected. The serum AzpAb concentrations were determined by ELISA method; the proportion of growing follicles was calculated on HE stain slices; CD45 expression in the ovaries reflecting the severity of autoimmune oophoritis, AR and Foxp3 expressions in the thymus and Foxp3 in the ovaries were determined by immunohistochemical method. Relative protein expressions of AR and Foxp3 were determined by Western-Blot method; while relative mRNA expression was determined by Real-time PCR method.Results:?The proportion of the mice that suffered from sexual cycle disorder, the serum AzpAb concentrations and the incidence of autoimmune oophoritis in the TP group were significantly lower than those in the model group (P<0.05), while the proportion of growing follicles in the ovaries in the TP group was significantly higher than that in the model group (P<0.05). The proportion of the mice that suffered from sexual cycle disorder, the serum AzpAb concentrations and the incidence of autoimmune oophoritis in the Flu and the TP+Flu group were significantly higher than those in the TP group (P<0.05), while the proportion of growing follicles in the ovaries in the aforesaid two groups was significantly lower than that in the TP group (P<0.05). There is no statistical difference between the Flu and the TP+Flu groups and the model group in the aforesaid indications (>0.05).?The relative protein and mRNA expression of AR in thymus? in the TP group were significantly higher than those in the model group (P< 0.05). The mRNA expression of AR in the TP+Flu group and the Flu group were significantly lower than that in the TP group (P<0.05). While there is no statistical difference among the Flu group and the TP+Flu group and the model group in the aforesaid indications (P>0.05); the difference in the positive expression rate of AR and the immunohistochemistry scores among the four groups was not statistically significant (P>0.05).?The immunohistochemistry scores, the positive expression rate, the relative protein and mRNA expression of Foxp3 in the thymus in the TP group were respectively higher than those in the model group (P<0.05). While all the aforesaid indications in the TP+Flu group and the Flu group were significantly lower than those in the TP groups (P<0.05). While the difference in the aforesaid indications among the Flu group, the TP+Flu group and the model group was not statistically significant (P>0.05).?The immunohistochemistry scores and the positive expression rate of Foxp3 in ovaries in the TP group were significantly higher than those in the model group (P<0.05).While the aforesaid indications in the TP+ Flu group and the Flu group were significantly lower than those in the TP group (P<0.05). While the difference in the aforesaid indications among the Flu group, the TP+Flu group and the model group was not statistically significant (P>0.05).Conclusion:Testosterone propionate could improve mice premature ovarian failure though up-regulating the expression of AR and Foxp3. |
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