摘要
目的
建立SD大鼠和Wi star大鼠同种异体卵巢皮下游离移植模型,并以Wi star大鼠自体卵巢皮下游离移植模型作为对照组,以补肾安胎中药复方“寿胎丸”进行干预,观察卵巢移植模型组织形态学与超微结构的变化、凋亡指数及凋亡相关基因Bcl-2、Bax. Fas. FasL的表达、卵巢激素E2的水平,探讨“寿胎丸”抗卵巢移植急性排斥反应的机理。
方法
210只Wistar雌性大鼠随机分为7组,每组30只。①自体移植模型组；②自体移植+寿胎丸组；③异体移植模型组；④异体移植+寿胎丸组；⑤异体移植+CsA组；⑥异体移植+CsA及寿胎丸组；⑦正常对照组。①、②建立自体卵巢皮下游离移植模型,③、④、⑤、⑥建立同种异体卵巢移植模型,受体Wistar大鼠,供体SD大鼠,⑦行开腹缝合假手术处理,为正常对照组。
术后给药,②自体移植+寿胎丸组和④异体移植+寿胎丸组予寿胎丸水煎剂,按26.8g/kg/d剂量灌胃,连用28天；⑤异体移植+CsA组予CsA,按10mg/kg/d剂量灌胃,连用14天,然后减量,按6mg/kg/d剂量连用14天；⑥异体移植+CsA及寿胎丸组同时予CsA和寿胎丸水煎剂,剂量同前；①自体移植模型组、③异体移植模型组、⑦正常对照组予生理盐水,按5ml/kg/d剂量灌胃,连用28天。
各组于移植术后d7、d14、d21、d28、d35分批处死Wi star大鼠各6只,取双侧移植卵巢：在电镜下观察移植卵巢超微结构变化,新生血管的形态学特征；光镜下计算各个发育阶段卵泡、黄体的数量,观察间质的变化；TUNEL试剂盒检测移植卵巢凋亡指数；免疫组化检测凋亡相关基因Bcl一2／Bax.凋亡因子Fas及其天然配体FasL的表达；移植术前1天眼眶后静脉丛取血测血清E2,移植术后取三次血,第6-15天、第16-25天以及第26-35天,根据阴道涂片,选雌激素分泌最高峰当日,取血测血清E2。观察记录各组动物的体重及进食等一般情况。
结果
1、血清内分泌激素E2的变化
各组卵巢移植大鼠移植术前的血清E2水平稍低于正常对照组静止期的E2水平,差异无统计学意义(P＞0.05)。移植术后6-15天,①自体移植模型组、②自体移植+寿胎丸组、⑤异体移植+CsA组、⑥异体移植+CsA及寿胎丸组大鼠的E2水平有大幅度升高,①自体移植模型组、⑥异体移植+CsA及寿胎丸组大鼠的血清E2与正常对照组血清E2比较,差异无统计学意义(P＞0.05)；③异体移植模型组、④异体移植+寿胎丸组大鼠的E2水平不升反降,分别与⑤异体移植+CsA组、⑥异体移植+CsA及寿胎丸组大鼠的血清E2比较,均有统计学意义(P＜0.05)；血清E2水平,⑥异体移植+CsA及寿胎丸组>⑤异体移植+CsA组>④异体移植+寿胎丸组、③异体移植模型组；③异体移植模型组与①自体移植模型组E2水平相比较,差异有统计学意义(P<0.05)。
移植术后16-25天,①自体移植模型组、②自体移植+寿胎丸组、⑤异体移植+CsA组、⑥异体移植+CsA及寿胎丸组E2水平分别与③异体移植模型组、④异体移植+寿胎丸组比较,差异均有统计学意义(P<0.05)；①自体移植模型组和②自体移植+寿胎丸组E2水平比较,差异无统计学意义(P＞0.05)。
移植术后26-35天,①自体移植模型组、②自体移植+寿胎丸组、⑤异体移植+CsA组、⑥异体移植+CsA及寿胎丸组E2水平仍高于③异体移植模型组和④异体移植+寿胎丸组,有统计学意义(P<0.05)；④异体移植+寿胎丸组E2水平稍高于③异体移植模型组,但差异无统计学意义(P>0.05)；①自体移植模型组和②自体移植+寿胎丸组E2水平比较,差异无统计学意义(P＞0.05)。
2、移植卵巢组织形态学的变化
石蜡切片HE染色观察移植卵巢内各级卵泡和黄体数目,①自体移植模型组、②自体移植+寿胎丸组、④异体移植+寿胎丸组、⑤异体移植+CsA组、⑥异体移植+CsA及寿胎丸组卵巢内各级卵泡和黄体的数目,均多于③异体移植模型组,少于⑦正常对照组；②自体移植+寿胎丸组的原始卵泡明显多于①自体移植模型组；⑥异体移植+CsA及寿胎丸组的卵泡和黄体多于⑤异体移植+CsA组、④异体移植+寿胎丸组；③异体移植模型组大鼠卵巢仅在移植后7天出现黄体,其余各时间点,均未发现各级卵泡和黄体。
电镜观察发现,存活的自体移植卵巢细胞,超微结构基本正常,仅见线粒体和内质网轻度扩张,新生血管有扩张瘀血现象。异体移植卵巢的细胞坏死和凋亡严重,坏死细胞细胞核变形,染色质边集,细胞膜不完整,细胞器极少,线粒体空泡化,脂滴丰富。凋亡细胞的细胞核固缩,核周间隙增宽,染色质凝集,形成不同性状和大小的块状,并逐步分裂为碎片；胞质致密,细胞器浓缩,失去水分；细胞膜下陷,包裹核碎片和细胞器,形成凋亡小体。移植卵巢内聚集大量巨噬细胞、淋巴细胞和中性粒细胞,新生血管有扩张瘀血现象,移植后期卵巢组织纤维化。经积极抗排斥治疗的大鼠,卵巢细胞超微结构基本正常,胞浆内细胞器较多,线粒体与内质网有不同程度肿胀。
3、凋亡细胞数、Bcl-2、Bax、Fas、FasL的变化
TUNEL法检测移植卵巢内的细胞凋亡数,③异体移植模型组凋亡细胞最多,⑦正常对照组凋亡细胞最少；③异体移植模型组多于①自体移植模型组(P<0.05)；④异体移植+寿胎丸组、⑤异体移植+CsA组、⑥异体移植+CsA及寿胎丸组的凋亡细胞均少于③异体移植模型组(P<0.05)；②自体移植+寿胎丸组的凋亡细胞少于①自体移植模型组(P<0.05)。
检测移植卵巢内的Bcl-2表达,发现移植术后①自体移植模型组与③异体移植模型组大鼠卵巢内的Bcl-2水平与正常组比较明显降低(P<0.05)；②自体移植+寿胎丸组的Bcl-2水平高于①自体移植模型组(P<0.05)；移植后7天,⑤异体移植+CsA组的Bcl-2高于③异体移植模型组和④异体移植+寿胎丸组(P<0.05)；移植后35天,④异体移植+寿胎丸组、⑤异体移植+CsA组、⑥异体移植+CsA及寿胎丸组的Bcl-2水平均高于③异体移植模型组(P<0.05),寿胎丸与CsA的的交互作用是负向的。
检测移植卵巢内的Bax表达,移植14天,①自体移植模型组的Bax水平低于③异体移植模型组和⑦正常对照组(P＜0.05),其余各时点,三组比较,均无明显差异(P>0.05)；②自体移植+寿胎丸组的Bax水平低于①自体移植模型组(P<0.05)；移植后14天,④异体移植+寿胎丸组、⑤异体移植+CsA组、⑥异体移植+CsA及寿胎丸组的Bax水平低于异体移植模型组(P＜0.05),CsA和寿胎丸的交互作用是负向的。
检测移植卵巢内的Fas表达,经统计分析,移植后14天,①自体移植模型组的Fas水平低于⑦正常对照组(P<0.05),其余各时间点,①自体移植模型组、③异体移植模型组与⑦正常对照组比较,均无明显差异(P>0.05);①自体移植模型组和②自体移植+寿胎丸组的Fas水平,在移植后各个时间点比较,差异均无统计学意义(P>O.05)：移植术后28天,⑤异体移植+CsA组的Fas水平低于③异体移植模型组和④异体移植+寿胎丸组,⑥异体移植+CsA及寿胎丸组Fas水平低于④异体移植+寿胎丸组(P<0.05)。寿胎丸、CsA的交互作用为正向效应。
检测移植卵巢内的FasL表达,①自体移植模型组、③异体移植模型组大鼠卵巢内的FasL水平,与正常对照组在各时间点比较,均无明显差异(P＞0.05)；①自体移植模型组和②自体移植+寿胎丸组大鼠的FasL水平,在移植后各个时间点比较,差异均无统计学意义(P>0.05)；异体卵巢移植各组大鼠卵巢内的FasL水平比较,在移植术后各时间点,差异均无统计学意义(P>0.05)。
结论
1白体卵巢冻融后移植是可行的,移植后的卵巢具有内分泌功能,并能保存较多的卵泡；异体卵巢移植经积极抗排斥治疗后也有内分泌功能,但卵泡丢失较多。
2寿胎丸有抗异体卵巢移植排斥反应的作用。在提高内分泌激素E2水平和保存卵泡方面,寿胎丸与CsA有协同作用。
3细胞凋亡与排斥反应相关,寿胎丸与CsA均能抑制急性排斥反应中卵巢细胞的凋亡,但CsA作用更明显,在抑制卵巢细胞凋亡方面,未发现寿胎丸和CsA有协同作用。
4寿胎丸抗大鼠同种异体卵巢移植急性排斥反应的机理,可能与其调控细胞凋亡有关,CsA和寿胎丸均能升调Bcl-2表达,降调Bax表达,升调Bcl一2／Bax的比值,从而发挥抑制卵巢细胞凋亡的作用；寿胎丸调控卵巢细胞内Fas.FasL因子的作用不明显。
5寿胎丸可减少自体移植卵巢内原始卵泡的丢失,可能与其抑制卵巢细胞凋亡的作用相关,而但其在短期内提高自体移植卵巢成活率、提高内分泌激素E2水平的作用尚不明显。
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 level
The 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 tissues
We 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 FasL
We 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.
引文
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