| Objective:In order to prevent and treat the graft rejective reaction,and to restore the function of transplanted ovary,gene therapy with indoleamine2,3-dioxygenase(IDO)is used in animal model of allogeneic ovarian transplantation.Methods:1.Ovarian autotransplantation was carried out in 9-11 week-old Lewis female rats(A group).Allogeneic ovarian transplant was performed in 9-11 week-old SD female rats with donors' ovaries from Lewis female rats of the same age,and was randomly classed into B(blank control),C(treated with cyclosporin A),D(treated with recombinant lentivirus containing IDO gene)and E(treated with lentivirus vector containing no target gene)groups.There were 20 cases of Lewis female rats received ovarian transplant in each group and the survival rate was observed among the rats after operation.2.The success rate of ovarian transplantation were compared among the group.3.From the fifth postoperative day,the vaginal Smears(Hematoxylin and Eosin Staining)were obtained from the rat for evaluating the changes of the vaginal keratinocytes and counting the days of estrus.The female rats which received ovarian transplant mated with syngeneic male during the days of estrus,and the pregnancy was observed.4.Postoperative 40 and 70 days,the serum E2 and FSH levels of the female rats was determined respectively.5.Five female rats which received ovarian transplant were randomly selected and were executed postoperative 40 day,and the other were executed postoperative 70 day.The transplanted ovaries from executed rats were made histopathological examination.In order to observe effect of infection,the rapid frozen tissue sections from fresh transplanted ovaries of executed rats in group D were checked under fluorescence microscope.6.The IDO expression in transplanted ovaries was detected by immunohistochemical method.Results:1.The survival rates of rats which received ovarian transplant were 95%(19/20),55%(11/20),80%(16/20),95%(19/20)and(12/20)in group A,group B,group C,group D and group E respectively.The survival rate in group D were higher than that in group B and group E(p<0.05,respectively),however,no significant difference was observed between group D and group A(p>0.05).2.The success rates of ovarian transplantation were 94.74%(18/19),27.27%(3/11),37.5%(6/16),73.68%(14/19)and 25.00%(3/12)in group A,group B,group C,group D and group E respectively.The success rates in group D were higher than that in group B,group C and group E(p<0.05,respectively),but no significant difference was observed between group D and group A(p>0.05).3.The days to reach estrus in group D were earlied than that in group B,group C and group E(p<0.05,respectively).There were two rats which received ovarian transplant naturally conceived and delivered,one in group A,the other in group D.No conception was found in group B,group C,and group E.4.In the fortieth days after the opreation,we had measured the levels of FSH and E2 in serum of every groups,the FSH level of D group was significantly lower than that of B group,C group and E group(p<0.05),and the E2 level of D group was significantly higher than that of B group and E group(p<0.05).In the seventieth days after the opreation,there was no significant difference between FSH and E2 in the D group compared with the other four groups.5.The transplanted ovaries from executed rats were made histopathological examination and the results showed that a small number of follicles and corpora lutea were found in group B and group E,quite a number of follicles and corpora lutea were detected in group C,and a large number of follicles and corpora lutea were found in group A and group D.A large amount of green fluorescence in frozen tissue sections was observed under fluorescence microscope.6.By immunohistochemistry,the IDO was not detected in normal ovaries,but detected in follicles(cytoplasm)and corpora lutea of transplanted ovaries,and the expression of IDO in group D was higher than other groups.Conclusion:Transplanted ovaries can express corresponding protein after treated with recombinant lentivirus containing IDO gene,the survival rate of the transplanted ovaries has improved,and the reproductive and endocrine function of transplanted ovaries have been restored after the graft express IDO protein,in animal model of allogeneic ovarian transplantation. |
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