胚胎—胎仔发育毒性试验（Ⅱ段）中动物易感阳性对照品优化匹配性及其作用机制的研究
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摘要
1.目的
比较敌枯双和环磷酰胺两种阳性对照药物致畸敏感性,为大鼠胚胎-胎仔发育毒性试验(Ⅱ段生殖毒性)中设置适当的优化匹配的阳性对照品作依据,并对其发生的可能机制进行研究。
2.方法
挑选成年健康雌雄SD大鼠以1:1的比例在当日下午17:00时后合笼交配,于次日晨检查阴栓和涂片,查到阴栓或涂片查到精子者的确定为交配成功,交配成功日为孕D_0。雌性SD大鼠60只,分为6组,其中设阴性对照两组,分别给予0.5%的羧甲基纤维素钠(CMC)灌胃和生理盐水尾静脉注射;敌枯双两组于孕D_(10),分别灌胃给予敌枯双11.0 mg/kg和13.8mg/kg;环磷酰胺两组于孕D_(10),分别尾静脉给予环磷酰胺7.2mg/kg和9.0mg/kg;从孕D_0开始称重,以后每3d称体重1次。在孕D_(20)用3%戊巴比妥纳注射液麻醉处死孕鼠,解剖,取出胎仔,检查黄体数、子宫连胎仔重、胎盘重量、活胎数、死胎数、吸收胎数、活胎重量、项臀长及外观和骨骼畸形,制备孕鼠血清用于血SOD(超氧化物歧化酶)、MDA(丙二醛)、NO(一氧化氮)的测定。每组1/3的胎仔经茜素红染色后进行骨骼畸形检查,2/3的胎仔去除内脏后放入4%甲醛溶液固定两周后做脊髓病理切片观察脊髓病理组织学改变;采用免疫组化SABC法检测敌枯双组胎仔脊髓PCNA(增殖细胞核抗原)蛋白表达和环磷酰胺组胎仔脊髓凋亡相关基因Bcl-2、Bax蛋白的表达,以及神经细胞凋亡状态。神经细胞凋亡用凋亡指数(AI)表示,AI=凋亡细胞数/总细胞数×100%。基因Bcl-2、Bax蛋白的表达强度用平均灰度值G表示,蛋白表达越强G值越小。
3.结果
敌枯双低剂量组和高剂量组胎仔发生的骨骼畸形率分别是92.59%和100%,分别与环磷酰胺低剂量和高剂量组胎仔骨骼畸形率78.5%和100%相比无统计学差异(P＞0.05),敌枯双低剂量组和高剂量组胎仔发生的外观畸形率分别是84.62%和86.84%,外观畸形率比环磷酰胺低剂量和高剂量组2.6%和0.0%增高(P＜0.01),而且敌枯双组胎仔畸形种类较多,如:四肢短小、心脏外露、脐疝、腹裂、显性脊柱裂、尾畸形(无尾、卷尾、短尾)、肛门闭锁等,骨骼畸形有颅骨发育不全、肋骨畸形(分叉肋、融合肋、缺肋)、脊柱骨缺失(尾椎缺失、腰椎缺失)、胸骨发育迟缓等;环磷酰胺组外观畸形发生的较少,唇裂;骨骼畸形较明显,以颅骨发育不全、胸骨发育迟缓为主。
从光镜下观察,敌枯双低剂量组胎仔的胸髓和腰髓病理切片中分别发现有82.35%(14/17)和75%(12/16)出现中央管消失或偏移,基板、翼板分界不清、神经细胞明显减少,脊髓严重萎缩等病理改变(P＜0.01)。敌枯双高剂量组的胸髓和腰髓病理切片中分别发现有66.67%(4/6)和100%(7/7)出现上述病理改变(P＜0.01)。环磷酰胺两个剂量组胎仔的脊髓病理切片均未发现畸形病变。
脊髓凋亡的神经细胞核体缩小散在于脊髓中,凋亡神经细胞核内清晰可见深蓝色颗粒,凋亡细胞与周围细胞分离,阴性对照CMC组胎仔胸髓和腰髓前角凋亡指数为0.8%和0.6%,与敌枯双低剂量组胎仔的胸髓和腰髓前角凋亡指数0.7%和0.5%和敌枯双高剂量组胎仔的胸髓和腰髓前角凋亡指数0.5%和0.9%相比,均无统计学差异(P＞0.05)。环磷酰胺低剂量组胎仔的胸髓和腰髓细胞凋亡指数分别为3.1%和3.9%,与生理盐水组胎仔的胸髓和腰髓细胞凋亡指数0.5%和0.7%相比明显增加(P＜0.01)。
通过计算阳性细胞百分率,结果显示PCNA主要在脊髓细胞核上表达呈黄褐色,并在灰质和白质中均有表达,CMC组胎仔胸髓和腰髓细胞与敌枯双低剂量组和高剂量组胎仔胸髓和腰髓细胞的PCNA表达无明显差别(P＞0.05)。
Bcl-2和Bax主要在胎仔脊髓的前角细胞的胞浆中表达,生理盐水组胎仔胸髓和腰髓前角细胞的Bcl-2和Bax表达灰度值G值分别是Bcl-2(120.46±10.08和124.84±11.23),Bax(141.76±10.13和139.86±13.89);环磷酰胺低剂量组胎仔胸髓和腰髓前角细胞的Bax表达明显增强(P＜0.01),灰度值G值分别为106.68±8.76和107.30±11.27。环磷酰胺低剂量组胎仔胸髓前角细胞的Bcl-2表达明显减弱(P＜0.01),灰度值G值为141.93±13.73;环磷酰胺低剂量组胎仔腰髓前角细胞的Bcl-2表达较弱(P＜0.05),灰度值G值为134.82±9.67。
环磷酰胺低剂量组孕鼠血清中MDA含量与生理盐水组MDA无差别(P＞0.05),环磷酰胺高剂量组孕鼠血清中MDA含量均显著高于生理盐水组和环磷酰胺低剂量(P＜0.01)。环磷酰胺低剂量组和高剂量组孕鼠血清NO浓度显著高于生理盐水组(P＜0.05),环磷酰胺低剂量组孕鼠血清中的SOD活性明显高于生理盐水组(P＜0.01),而环磷酰胺高剂量组孕鼠血清中的SOD活性明显低于生理盐水组(P＜0.01)。
4.结论
(1)与环磷酰胺比较,敌枯双为SD大鼠较为敏感的致畸阳性药物,在孕D_(10)给予11mg/kg敌枯双一次性灌胃为最理想的阳性对照设计方案。
(2)敌枯双和环磷酰胺均有母体毒性及胚胎毒性,敌枯双和环磷酰胺用药途径、剂量差异较大,在用药时间相同情况下导致不同类型畸形,提示两种药物在体内代谢途径不同,可能通过不同途径导致畸形的发生。
(3)同等条件下,敌枯双诱导胎仔产生的多个靶组织(尾、肋骨和胸骨)的缺失或畸形和脊髓畸形,推测两者可能有内在联系。在本研究条件下,我们发现敌枯双导致神经管未闭和脊髓畸形与细胞凋亡机制没有明显关系。
(4)环磷酰胺的神经管致畸剂量在7.2mg/kg至9.0mg/kg之间,若能选择一个合适的剂量很有可能诱导出脊髓畸形的模型。在环磷酰胺作用下脊髓神经细胞过度凋亡,提示神经细胞的过度凋亡与脊椎裂畸形的发生密切相关。
(5)环磷酰胺组脊髓神经细胞Bax蛋白表达强,Bcl-2蛋白表达弱,有促进细胞凋亡的作用;而正常胎仔脊髓神经细胞Bcl-2蛋白表达强,Bax蛋白表达弱,抑制神经细胞过度凋亡;可见,在脊椎裂畸形发生过程中,Bcl-2和Bax基因对脊髓神经细胞凋亡进程的发生起重要作用。
(6)实验发现环磷酰胺组孕鼠体内氧化反应水平较高,提示环磷酰胺的致畸作用可能与氧化损伤有密切关系,并发现脊髓神经细胞凋亡,推测氧化应激-神经细胞凋亡-神经管畸形的发生可能具有因果关系。
1.Objective
To search a better positive control drug by comparing the malformation sensitivity of N,N'-methylene-bis-(α-amino-1,3,4-thiadiazole)(Bis-A-TDA) and cyclophosphamide(CP) on SD rats for Embryo-fetal development toxicity test.(Ⅱsection reproduction toxicity),of which we investigate the mechanism.
2.Methods
Healthy adult female rats were choosed by 1:1 mated with male rat at 17:00 every afternoon,and the vagina embolism and vagina smear checked in next morning.Once the vagina embolism or sperm in vagina smear were detected,we determined they were mated successfully.60 female SD rats were divided into 6 groups at random which included two negative control group administrated with 0.5 %CMC intragastric or physiological saline by injected in the caudal vein respectively;two Bis-A-TDA experimental groups were administered with Bis-A-TDA 11.0 mg/kg or 13.8mg/kg;two CP experimental groups were injected with CP 7.2 mg/kg or 9.0mg/kg in the morning at 10~(th) of gestation.Pregnant rats were weighed per triduum from the first day of gestation,checked up corpus luteum, foetus weight(including uterus),placental weight,living foetus,dead foetus, absorbed foetus,foetus weight,crown rump length,appearance and skeletal malformation of foetus,and incidence of malformation on the day 20 of gestation. Collected pregnant rats' blood serum were used for mensuration of SOD,MDA,NO. 1/3 foetus were dealed with alizarin red stain for skeletal malformation examination, and 2/3 foetus were put into 4%Formaldehyde Solution after removing internal organs to make internal organs pathological section for myeleterosis examination. We detected the protein expression of PCNA of spinal cord of Bis-A-TDA experimental groups compared with control group by immunohistochemistry SABC methods.We detected the apoptotic cells and the protein expression of Bcl-2 and Bax in spinal cord of CP group compared with control group with immunohistochemistry SABC methods.
3.Results
There was no statistics difference in skeletal deformity incidence between low-dose Bis-A-TDA group,high-dose Bis-A-TDA group(92.59%and 100%) and low-dose CP group,high-dose CP group(78.5%and 100%)(P＞0.05).The appearance deformation incidence of two Bis-A-TDA groups(84.62%and 86.84%) were much higher than which of two CP groups(2.6%and 0.0%)(P＜0.01).A variety of malformation was induced by Bis-A-TDA,like limbs microsoma,heart revealed,exomphalocele,abdominal cleft,spina bifida,tail malformation,anal atresia,cranial anostosis,rib deformity,vertebra absence,breast hypoevolutism,et al,but less malformation was appeared in CP experimental groups,which were mainly skeletal hypoevolutism.
Under the electron microscope,82.35%(14/17) thoracic cord and 75%(12/16) isthmus cord pathological section of low-dose Bis-A-TDA group appeared changed, including central canal disappearance,offset,basal plate and alarpalate fuzziness, nerve cells obviously reduction,spinal cord seriously emarcid(P＜0.01),the same to the high-dose Bis-A-TDA group with observing 66.67%(4/6)thoracic cord and 100%(7/7)isthmus cord pathological section.There were no pathological changes in spinal cord of CP group.
Apoptotic cells appeared typical morphological changes,spinal cord nerve cell caryosome minished,chromatin condensation,nuclear fragmentation,apoptotic body and apoptotic cells dissociated from peripheral cell.There was no difference among negative control CMC group,low-dose Bis-A-TDA group and high-dose Bis-A-TDA group in apoptotic index of thoracic cord and isthmus cord anterior horn cell.low-dose CP group was higher than negative control physiologic saline group in apoptotic index of thoracic cord and isthmus cord angulus anterior (P＜0.01).
Calculating masculine cell showed that there was no difference between negative control CMC group and Bis-A-TDA group in PCNA expression(P＞0.05).
Bcl-2 and Bax mainly expressed in anterior horn cell endochylema.Compared with negative control physiologic saline group,Bax expression in thoracic cord and isthmus cord anterior horn cell of low-dose CP group increased significantly(P＜0.01),and Bcl-2 expression in thoracic cord and isthmus cord anterior horn cell of low-dose CP group are lower(P＜0.05).
MDA density in pregnant rats' blood serum had no difference between negative control physiologic saline group and low-dose CP group,but was higher in high-dose CP group(P＜0.01).Density of NO in pregnant rats' blood serum of low-dose CP group and high-dose CP group are both higher than negative control physiologic saline group(P＜0.05),activity of blood serum SOD in low-dose CP group is higher than which of negative control physiologic saline group,while is lower in high-dose CP group(P＜0.01).
4.Conclusion
1.Compared with cyclophosphamide,(Bis-A-TDA) was an excellent positive control drug on SD rats which posses high malformation sensitivity.It is the best positive control design proposal that rats were intragastric administered with Bis-A-TDA 11.0 mg/kg in the morning of 10~(th) of gestation
2.We could state that Bis-A-TDA and CP may possibly induce malformation via different mechanism in the rats from the below viewpoints:the different methods and the different doses required at the same time of drug applieation, to produce malformation and the difference on anatomy and spina cord pathology.
3.There maybe an internal relation between multi-target tissue defection and deformity of spinal cord.Under the certain condition,we found that Bis-A-TDA induced NTDs(neural tube defect) and Deformity of spinal cord was not by apoptosis.
4.NTDs teratogenic dose of CP maybe between 7.2mg/kg and 9.0mg/kg.Excessively apoptotic nerve cells appearing in the fetal spina cord were closely related to the occurrence of the spina bifida malformation in fetal rats.
5.The stronger expression of Bax in spina cord facilitates nerve cell apoptosis of the CP group,and the stronger expression of Bcl-2 in the normal spinal cord inhibiits the programmed cell death.The correlative genes Bcl-2 and Bax participate in the apoptosis process.
6.That the density of MDA and NO in blood serum of CP groups were higher than control group accounted that oxidative damage played significant roles in teratogenic effect of CP.There were cause-effect relation among oxidation, neurons apoptosis and spina cord deformity.
比较敌枯双和环磷酰胺两种阳性对照药物致畸敏感性,为大鼠胚胎-胎仔发育毒性试验(Ⅱ段生殖毒性)中设置适当的优化匹配的阳性对照品作依据,并对其发生的可能机制进行研究。
2.方法
挑选成年健康雌雄SD大鼠以1:1的比例在当日下午17:00时后合笼交配,于次日晨检查阴栓和涂片,查到阴栓或涂片查到精子者的确定为交配成功,交配成功日为孕D_0。雌性SD大鼠60只,分为6组,其中设阴性对照两组,分别给予0.5%的羧甲基纤维素钠(CMC)灌胃和生理盐水尾静脉注射;敌枯双两组于孕D_(10),分别灌胃给予敌枯双11.0 mg/kg和13.8mg/kg;环磷酰胺两组于孕D_(10),分别尾静脉给予环磷酰胺7.2mg/kg和9.0mg/kg;从孕D_0开始称重,以后每3d称体重1次。在孕D_(20)用3%戊巴比妥纳注射液麻醉处死孕鼠,解剖,取出胎仔,检查黄体数、子宫连胎仔重、胎盘重量、活胎数、死胎数、吸收胎数、活胎重量、项臀长及外观和骨骼畸形,制备孕鼠血清用于血SOD(超氧化物歧化酶)、MDA(丙二醛)、NO(一氧化氮)的测定。每组1/3的胎仔经茜素红染色后进行骨骼畸形检查,2/3的胎仔去除内脏后放入4%甲醛溶液固定两周后做脊髓病理切片观察脊髓病理组织学改变;采用免疫组化SABC法检测敌枯双组胎仔脊髓PCNA(增殖细胞核抗原)蛋白表达和环磷酰胺组胎仔脊髓凋亡相关基因Bcl-2、Bax蛋白的表达,以及神经细胞凋亡状态。神经细胞凋亡用凋亡指数(AI)表示,AI=凋亡细胞数/总细胞数×100%。基因Bcl-2、Bax蛋白的表达强度用平均灰度值G表示,蛋白表达越强G值越小。
3.结果
敌枯双低剂量组和高剂量组胎仔发生的骨骼畸形率分别是92.59%和100%,分别与环磷酰胺低剂量和高剂量组胎仔骨骼畸形率78.5%和100%相比无统计学差异(P＞0.05),敌枯双低剂量组和高剂量组胎仔发生的外观畸形率分别是84.62%和86.84%,外观畸形率比环磷酰胺低剂量和高剂量组2.6%和0.0%增高(P＜0.01),而且敌枯双组胎仔畸形种类较多,如:四肢短小、心脏外露、脐疝、腹裂、显性脊柱裂、尾畸形(无尾、卷尾、短尾)、肛门闭锁等,骨骼畸形有颅骨发育不全、肋骨畸形(分叉肋、融合肋、缺肋)、脊柱骨缺失(尾椎缺失、腰椎缺失)、胸骨发育迟缓等;环磷酰胺组外观畸形发生的较少,唇裂;骨骼畸形较明显,以颅骨发育不全、胸骨发育迟缓为主。
从光镜下观察,敌枯双低剂量组胎仔的胸髓和腰髓病理切片中分别发现有82.35%(14/17)和75%(12/16)出现中央管消失或偏移,基板、翼板分界不清、神经细胞明显减少,脊髓严重萎缩等病理改变(P＜0.01)。敌枯双高剂量组的胸髓和腰髓病理切片中分别发现有66.67%(4/6)和100%(7/7)出现上述病理改变(P＜0.01)。环磷酰胺两个剂量组胎仔的脊髓病理切片均未发现畸形病变。
脊髓凋亡的神经细胞核体缩小散在于脊髓中,凋亡神经细胞核内清晰可见深蓝色颗粒,凋亡细胞与周围细胞分离,阴性对照CMC组胎仔胸髓和腰髓前角凋亡指数为0.8%和0.6%,与敌枯双低剂量组胎仔的胸髓和腰髓前角凋亡指数0.7%和0.5%和敌枯双高剂量组胎仔的胸髓和腰髓前角凋亡指数0.5%和0.9%相比,均无统计学差异(P＞0.05)。环磷酰胺低剂量组胎仔的胸髓和腰髓细胞凋亡指数分别为3.1%和3.9%,与生理盐水组胎仔的胸髓和腰髓细胞凋亡指数0.5%和0.7%相比明显增加(P＜0.01)。
通过计算阳性细胞百分率,结果显示PCNA主要在脊髓细胞核上表达呈黄褐色,并在灰质和白质中均有表达,CMC组胎仔胸髓和腰髓细胞与敌枯双低剂量组和高剂量组胎仔胸髓和腰髓细胞的PCNA表达无明显差别(P＞0.05)。
Bcl-2和Bax主要在胎仔脊髓的前角细胞的胞浆中表达,生理盐水组胎仔胸髓和腰髓前角细胞的Bcl-2和Bax表达灰度值G值分别是Bcl-2(120.46±10.08和124.84±11.23),Bax(141.76±10.13和139.86±13.89);环磷酰胺低剂量组胎仔胸髓和腰髓前角细胞的Bax表达明显增强(P＜0.01),灰度值G值分别为106.68±8.76和107.30±11.27。环磷酰胺低剂量组胎仔胸髓前角细胞的Bcl-2表达明显减弱(P＜0.01),灰度值G值为141.93±13.73;环磷酰胺低剂量组胎仔腰髓前角细胞的Bcl-2表达较弱(P＜0.05),灰度值G值为134.82±9.67。
环磷酰胺低剂量组孕鼠血清中MDA含量与生理盐水组MDA无差别(P＞0.05),环磷酰胺高剂量组孕鼠血清中MDA含量均显著高于生理盐水组和环磷酰胺低剂量(P＜0.01)。环磷酰胺低剂量组和高剂量组孕鼠血清NO浓度显著高于生理盐水组(P＜0.05),环磷酰胺低剂量组孕鼠血清中的SOD活性明显高于生理盐水组(P＜0.01),而环磷酰胺高剂量组孕鼠血清中的SOD活性明显低于生理盐水组(P＜0.01)。
4.结论
(1)与环磷酰胺比较,敌枯双为SD大鼠较为敏感的致畸阳性药物,在孕D_(10)给予11mg/kg敌枯双一次性灌胃为最理想的阳性对照设计方案。
(2)敌枯双和环磷酰胺均有母体毒性及胚胎毒性,敌枯双和环磷酰胺用药途径、剂量差异较大,在用药时间相同情况下导致不同类型畸形,提示两种药物在体内代谢途径不同,可能通过不同途径导致畸形的发生。
(3)同等条件下,敌枯双诱导胎仔产生的多个靶组织(尾、肋骨和胸骨)的缺失或畸形和脊髓畸形,推测两者可能有内在联系。在本研究条件下,我们发现敌枯双导致神经管未闭和脊髓畸形与细胞凋亡机制没有明显关系。
(4)环磷酰胺的神经管致畸剂量在7.2mg/kg至9.0mg/kg之间,若能选择一个合适的剂量很有可能诱导出脊髓畸形的模型。在环磷酰胺作用下脊髓神经细胞过度凋亡,提示神经细胞的过度凋亡与脊椎裂畸形的发生密切相关。
(5)环磷酰胺组脊髓神经细胞Bax蛋白表达强,Bcl-2蛋白表达弱,有促进细胞凋亡的作用;而正常胎仔脊髓神经细胞Bcl-2蛋白表达强,Bax蛋白表达弱,抑制神经细胞过度凋亡;可见,在脊椎裂畸形发生过程中,Bcl-2和Bax基因对脊髓神经细胞凋亡进程的发生起重要作用。
(6)实验发现环磷酰胺组孕鼠体内氧化反应水平较高,提示环磷酰胺的致畸作用可能与氧化损伤有密切关系,并发现脊髓神经细胞凋亡,推测氧化应激-神经细胞凋亡-神经管畸形的发生可能具有因果关系。
1.Objective
To search a better positive control drug by comparing the malformation sensitivity of N,N'-methylene-bis-(α-amino-1,3,4-thiadiazole)(Bis-A-TDA) and cyclophosphamide(CP) on SD rats for Embryo-fetal development toxicity test.(Ⅱsection reproduction toxicity),of which we investigate the mechanism.
2.Methods
Healthy adult female rats were choosed by 1:1 mated with male rat at 17:00 every afternoon,and the vagina embolism and vagina smear checked in next morning.Once the vagina embolism or sperm in vagina smear were detected,we determined they were mated successfully.60 female SD rats were divided into 6 groups at random which included two negative control group administrated with 0.5 %CMC intragastric or physiological saline by injected in the caudal vein respectively;two Bis-A-TDA experimental groups were administered with Bis-A-TDA 11.0 mg/kg or 13.8mg/kg;two CP experimental groups were injected with CP 7.2 mg/kg or 9.0mg/kg in the morning at 10~(th) of gestation.Pregnant rats were weighed per triduum from the first day of gestation,checked up corpus luteum, foetus weight(including uterus),placental weight,living foetus,dead foetus, absorbed foetus,foetus weight,crown rump length,appearance and skeletal malformation of foetus,and incidence of malformation on the day 20 of gestation. Collected pregnant rats' blood serum were used for mensuration of SOD,MDA,NO. 1/3 foetus were dealed with alizarin red stain for skeletal malformation examination, and 2/3 foetus were put into 4%Formaldehyde Solution after removing internal organs to make internal organs pathological section for myeleterosis examination. We detected the protein expression of PCNA of spinal cord of Bis-A-TDA experimental groups compared with control group by immunohistochemistry SABC methods.We detected the apoptotic cells and the protein expression of Bcl-2 and Bax in spinal cord of CP group compared with control group with immunohistochemistry SABC methods.
3.Results
There was no statistics difference in skeletal deformity incidence between low-dose Bis-A-TDA group,high-dose Bis-A-TDA group(92.59%and 100%) and low-dose CP group,high-dose CP group(78.5%and 100%)(P＞0.05).The appearance deformation incidence of two Bis-A-TDA groups(84.62%and 86.84%) were much higher than which of two CP groups(2.6%and 0.0%)(P＜0.01).A variety of malformation was induced by Bis-A-TDA,like limbs microsoma,heart revealed,exomphalocele,abdominal cleft,spina bifida,tail malformation,anal atresia,cranial anostosis,rib deformity,vertebra absence,breast hypoevolutism,et al,but less malformation was appeared in CP experimental groups,which were mainly skeletal hypoevolutism.
Under the electron microscope,82.35%(14/17) thoracic cord and 75%(12/16) isthmus cord pathological section of low-dose Bis-A-TDA group appeared changed, including central canal disappearance,offset,basal plate and alarpalate fuzziness, nerve cells obviously reduction,spinal cord seriously emarcid(P＜0.01),the same to the high-dose Bis-A-TDA group with observing 66.67%(4/6)thoracic cord and 100%(7/7)isthmus cord pathological section.There were no pathological changes in spinal cord of CP group.
Apoptotic cells appeared typical morphological changes,spinal cord nerve cell caryosome minished,chromatin condensation,nuclear fragmentation,apoptotic body and apoptotic cells dissociated from peripheral cell.There was no difference among negative control CMC group,low-dose Bis-A-TDA group and high-dose Bis-A-TDA group in apoptotic index of thoracic cord and isthmus cord anterior horn cell.low-dose CP group was higher than negative control physiologic saline group in apoptotic index of thoracic cord and isthmus cord angulus anterior (P＜0.01).
Calculating masculine cell showed that there was no difference between negative control CMC group and Bis-A-TDA group in PCNA expression(P＞0.05).
Bcl-2 and Bax mainly expressed in anterior horn cell endochylema.Compared with negative control physiologic saline group,Bax expression in thoracic cord and isthmus cord anterior horn cell of low-dose CP group increased significantly(P＜0.01),and Bcl-2 expression in thoracic cord and isthmus cord anterior horn cell of low-dose CP group are lower(P＜0.05).
MDA density in pregnant rats' blood serum had no difference between negative control physiologic saline group and low-dose CP group,but was higher in high-dose CP group(P＜0.01).Density of NO in pregnant rats' blood serum of low-dose CP group and high-dose CP group are both higher than negative control physiologic saline group(P＜0.05),activity of blood serum SOD in low-dose CP group is higher than which of negative control physiologic saline group,while is lower in high-dose CP group(P＜0.01).
4.Conclusion
1.Compared with cyclophosphamide,(Bis-A-TDA) was an excellent positive control drug on SD rats which posses high malformation sensitivity.It is the best positive control design proposal that rats were intragastric administered with Bis-A-TDA 11.0 mg/kg in the morning of 10~(th) of gestation
2.We could state that Bis-A-TDA and CP may possibly induce malformation via different mechanism in the rats from the below viewpoints:the different methods and the different doses required at the same time of drug applieation, to produce malformation and the difference on anatomy and spina cord pathology.
3.There maybe an internal relation between multi-target tissue defection and deformity of spinal cord.Under the certain condition,we found that Bis-A-TDA induced NTDs(neural tube defect) and Deformity of spinal cord was not by apoptosis.
4.NTDs teratogenic dose of CP maybe between 7.2mg/kg and 9.0mg/kg.Excessively apoptotic nerve cells appearing in the fetal spina cord were closely related to the occurrence of the spina bifida malformation in fetal rats.
5.The stronger expression of Bax in spina cord facilitates nerve cell apoptosis of the CP group,and the stronger expression of Bcl-2 in the normal spinal cord inhibiits the programmed cell death.The correlative genes Bcl-2 and Bax participate in the apoptosis process.
6.That the density of MDA and NO in blood serum of CP groups were higher than control group accounted that oxidative damage played significant roles in teratogenic effect of CP.There were cause-effect relation among oxidation, neurons apoptosis and spina cord deformity.
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