| Objective:Establish allograft ovary subcutaneous free transplantation model with SD rats and Wistar rats, and set autologous skin free transplantation model with Wistar rats as the control group. Using the recipe of "Shoutai Wan" which has the function of invigorating the kidney and preventing miscarriage to these rats, observe the histological and ultrastructural changes of the rats'ovaries, the apoptotic index, the expression of the apoptotosis related genes—Bcl-2,Bax,Fas,FasL and the estradiol level of the ovaries so as to explore the possible mechanisms of anti acute rejection of ovary transplantation of the Shoutai Wan.Method:210 female Wistar rat were randomly divided into the following seven groups (30 in each group):①Autologous transplantation group②autologous transplantation+Shoutai Wan group,③allograft transplantation group,④allograft+Shoutai Wan group,⑤allograft+Ciclosporin A (CsA) group,⑥allograft+CsA and Shoutai Wan group,⑦normal controls. Establishing autologous subcutaneous free ovarian transplantation model in Group①and group②, and allograft ovary transplantation model in group④,⑤and⑥, with Wistar rats as the receptor and SD rats the donor. As for the normal controls, group⑦just received incision and suture with sham operating presudure.After the operation, the autologous transplantation+Shoutai Wan group(group②) and the allograft+Shoutai Wan group(group④) received Shoutai Wan decoction, which was intragastric administrated with the dosage of 26.8g/kg/d for 28 days. The allograft+CsA group(group⑤) received CsA, intragastric administrated with the dosage of 10mg/kg/d for 14 days and then reduced to the dosage of 6mg/kg/d for another 14 days. The allograft+CsA and Shoutai Wan group (group⑥) received Shoutai Wan decoction and CsA at the same time, and the dosage was the same as group④and group⑥. The normal controls(group⑦) received sodium chloride with the dosage of 5ml/kg/d for 28 days.On the d7,d14,d21,d28,d35 after the transplantation operation, sacrificed 6 rats in each group and collected the bilateral ovaries to observe the ultrastructural changes of the transplanted ovaries and the morphological features of the neo-vascular with electron microscope. Using light microscope to observe the ovarian follicles, number of corepus luteum and the changes of the stroma, detecting the apoptotic index of transplanted ovaries with TUNEL kit, and the expression of apoptosis related genes Bcl-2/Bax, as well as Fas and FasL were detected with immunohistology method. Blood samples were collected by retro-orbital puncture one day before the operation to detect the serum estradiol (E2)level, and also collected blood sample for 3 times between day 6-15, day 16-25 and day 26-35 after the operation, which was chosen according to the vaginal smear which indicated the summit secretion of the estradiol. The general state sch as the weight and food intake condition of each group were also observed and recorded.Results:1. Changes of the serum E2 levelThe preoperative serum E2 level of the other 6 ovary transplanted groups were slightly lower than that of the normal group, while the differences has no significance (P>0.05).6-15 days after the operation, the serum E2 level of the group①,②,⑤and⑥were greatly increased, and the differences were insignificant (P>0.05), while that of the group③and④were reduced instead of elevated, respectively compared with group⑤and⑥, the difference were both meaningful (P＜0.05). The serum E2 level were as follows: group⑥> group⑤> group④> group③>group①,16-25 days after the operation, the differences of the serum E2 level between group①,②,⑤,⑥and group③and④were significant (P＜0.05), while that of the group①and group②has no significance (P>0.05),26-35 days after the operation, the serum E2 level of the group①,②,⑤and⑥were still higher than that of the group③and④(P＜0.05), The serum E2 level of group④was a bit higher than group③, while the difference was insignificant (P>0.05), and the difference between group①and group②was insignificant (P>0.05)2. The morphological features of the transplanted ovary tissuesWe used hematoxylin and eosin (HE) staining to observe the different stages of ovarian follicles and corepus luteum number. The different stages of ovarian follicles and corepus luteum number in group①,②,④,⑤and⑥were significantly more than that of the group③and less than that of group⑦. The primary follicles of group②was obviously more than group①, The ovarian follicles and corepus luteum number in group⑥were significantly larger than that of group⑤and group④. We just noticed corepus luteum 7 days after the operation in group③, which has no follicles and corepus luteum in any other checking time.According to the ultrastructural analysis, the ultrastructure of the survived autologous transplanted ovarian cells was generally normal, which just showed the slight dilation of the mitochondrion and endoplasmic reticulum(ER), and the stagnant condition of neovascular. There were severe cell necrosis and apoptosis in the allograft ovaries. The nuclei of the necrotic cells deformed, with margination of chromatin, un-integrated cell membrane less organelle, vacuolization of the mitochondrion and plentiful lipid droplets. The apoptotic cells showed karyopyknosis with enlarged perinuclear space, the chromatin agglutinated into massive with different shapes and size, and gradually split into fragments. Pykno-cytoplasm with condensed organelle, the cell membrane sagged to pack the nuclear debris and organelle to form apoptotic body. There were plenty of macrophage, lymphocyte and neutrophilic granulocyte with stagnant condition of neovascular in the transplanted ovaries. The ovarian tissues indicated fibrosis in the later period of the transplantation. While with positive anti rejection treatment, the ultrastructure of the allograft ovaries were generally normal, with much intracytoplasm organelle and swelling of the mitochondrion and ER.3. Changes of the apoptotic cells, Bcl-2, Bax, Fas and FasLWe Applied TUNEL kit to detect the apoptotic cell number. The result indicated that group③had the most apoptotic cells and group⑦had the least apoptotic cells, group③had much more apoptotic cells than that of group①(P＜0.05), and number of apoptotic cells in group④,⑤and⑥were less than that of group③(P＜0.05), number of apoptotic cells in group②was less than that of group①(P＜0.05)The examination of Bcl-2 expression of transplanted ovaries indicated that Bcl-2 level of group①and③were much lower than group⑦(P＜0.05).7 days after the transplantation, the Bcl-2 level of group⑤was higher than that of group③and④(R＜0.05).35 days after the transplantation, the Bcl-2 level of group⑤and⑥were much higher than that of group③(P＜0.05) The examination of Bax expression of transplanted ovaries indicated that Bax level of group①was much lower than group③and⑦(P＜0.05) 14 days after the transplantation, and there were no obvious differences among the three groups in other time (P>0.05). The Bax level of group②was lower than that of group①(P＜0.05).14 days after the transplantation, the Bax level of group④⑤and⑥were much lower than that of group③(P＜0.05). CsA and Shoutai Wan had a negative interaction on the expression of Bax.The examination of Fas expression of transplanted ovaries indicated that Fas level of group①was much lower than group⑦(P＜0.05) 14 days after the transplantation, and there were no obvious differences among the three groups in other time (P>0.05).The Fas level of group①and②didn't have significant difference at any time after the transplantation (P>0.05).28 days after the transplantation, the Fas level of group⑤was much lower than that of group③and④(P＜0.05), and Fas level of group⑥was much lower than that of group④(P＜0.05). CsA and Shoutai Wan had a positive interaction on the expression of Fas.The examination of FasL expression indicated that The FasL level of group①and③didn't have significant difference with that of group⑦at any time after the transplantation (P>0.05), and there were no significant difference between group①and②either. FasL level of③,④,⑤and⑥also had no obvious differences (P＞0.05).Conclusion:1. It is possible to perform autologous ovary transplantation which was being frozen. The transplanted ovaries also had endocrine secretion function, and they can preserve plenty of ovarian follicles. allograft ovaries also had endocrine secretion function, but some follicles were injured.2. ShouTai Wan have the function of anti-acute rejection of the allograft ovaries, ShouTai Wan and CsA have synergism action in improving the E2 level and preserving ovarian follicles.3. Apoptosis is greatly related to rejection reaction. Both ShouTai Wan and CsA can inhibit the apoptosis of ovarian cells during the acute rejection action, and CsA's efficacy was stronger than ShouTai Wan. As for the function of inhibiting the apoptosis of ovarian cells, they don't have synergism action 4. The mechanisms of ShouTai Wan's action on anti acute rejection of ovary transplantation may be relate to its mediation of cell apoptosis. Both ShouTai Wan and CsA can elevate the expression of Bcl-2 and lower the expression of Bax, so the Bcl-2/Bax rate also elevated, which induce the function of inhibiting the apoptosis of ovarian cells. However, ShouTai Wan doesn't have manifested function of mediating the expression of Fas,FasL of the ovaries.5. ShouTai Wan can reduce the injury of primordial follicle of the autografting ovaries, which may be relate to its function of inhibiting the apoptosis of ovarian cells. However, ShouTai Wan can't improve the survival rate of autografting ovaries and the E2 level in a short period of time.
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