吗替麦考酚酯对大鼠胚胎耳郭发育的影响

Effects of mycophenolate mofetil on the development of rat embryonic auricle

背景:先天性小耳畸形发病机制尚不明确,目前研究认为小耳畸形与环境及遗传因素共同作用有关,药物致畸是环境因素中的一个重要方面。目的:观察大鼠孕鼠暴露在不同剂量吗替麦考酚酯环境中的胚胎耳郭发育指标,以期建立一种药物诱导的小耳畸形模型,为进一步探讨小耳畸形的发病机制提供基础。方法:将成年SD大鼠按雌雄比2∶1合笼,次日检查雌鼠,发现阴栓定为孕0 d。将32只SD大鼠孕鼠随机分为4组,分别记为对照组、吗替麦考酚酯50,100,200 mg/kg组,每组8只。孕9,10 d每天早上8点分别用50,100,200 mg/kg吗替麦考酚酯玉米油灌胃1次,对照组仅灌胃玉米油,于孕20.5 d麻醉后脱颈处死各组孕鼠,大体观察各组胚胎发育情况及耳郭发育指标,micro-CT影像学观察中耳及内耳发育情况,组织学检测耳郭软骨及软组织改变。结果与结论:(1)与对照组相比,各吗替麦考酚酯组死胎数、吸收胎数增加,活胎数及活胎率降低;耳横长、耳纵长、耳纵长/耳横长、鼻枕距/双顶径、眼距、体长、体质量等指标存在明显统计学差异(P<0.05)。吗替麦考酚酯50 mg/kg组与吗替麦考酚酯100,200 mg/kg组相比,各指标存在明显统计学差异(P<0.05)。吗替麦考酚酯100 mg/kg组与吗替麦考酚酯200 mg/kg组相比,耳纵长、体长、体质量、鼻枕距存在明显统计学差异(P<0.05)。(2)与对照组相比,吗替麦考酚酯组micro-CT影像学主要改变为颅骨发育不良、听泡发育不完整。(3)苏木精-伊红染色、甲胺蓝染色、Ⅱ型胶原免疫组化结果示吗替麦考酚酯影响软骨细胞增生、分化以及Ⅱ型胶原表达。(4)结果表明,吗替麦考酚酯可导致大鼠胚胎耳郭组织发育不良且呈剂量依赖性,可能是因为其阻止了软骨细胞增生、分化及Ⅱ型胶原表达,吗替麦考酚酯可诱导建立小耳畸形动物模型。

BACKGROUND:The pathogenesis of congenital microtia is not clear.Current studies believe that microtia is related to the joint action of environmental and genetic factors,and drug te ratogenesis is an important aspect of environmental factors.OBJECTIVE:To observe the embryonic auricle development indexes of pregnant rats exposed to different concentrations of mycophenolate mofetil,in order to establish a drug-induced microtia model and provide a basis for further exploring the pathogenesis of microtia.METHODS:Adult Sprague-Dawley rats were caged acco rding to the ratio of male to female at 2:1,and the female rats were checked the next day.The presence of a vaginal plug was designated as gestational day 0.Thirty-two pregnant rats were randomized into four groups(n=8 per group):control group,50,100,and 200 mg/kg mycophenolate mofetil groups.Rats in the four groups were intragastrically given corn oil,and 50,100,and 200 mg/kg mycophenolate mofetil corn oil respectively at 8 a.m.on gestational days 9 and 10.The pregnant rats in each group were decapitated under anesthesia on gestational day 20.5.The embryonic development and auricle development indexes were generally observed.The development of middle and inner ears was observed by micro-CT imaging.Auricle ca rtilage and soft tissue changes were histologically observed.RESULTS AND CONCLUSION:(1)Compared with the control group,the numbers of dead fetuses and absorbed fetuses were increased and the number and rate of live fetuses decreased in different mycophenolate mofetil groups.There were significant differences in ear transverse length,ear longitudinal length,ear longitudinal length/ear transverse length,nasal occipital distance/biparietal diameter,eye distance,body length and body mass between different mycophenolate mofetil groups and control group(P<0.05).The above-mentioned indexes were significantly different in the 50 mg/kg mycophenolate mofetil group from 100 and 200 mg/kg mycophenolate mofetil groups(P<0.05).There were also significant differences in ear length,body length,body mass,and nasal occipital distance between 100 and 200 mg/kg mycophenolate mofetil groups(P<0.05).(2)Compared with the control group,the main micro-CT changes in each mycophenolate mofetil group were dysplasia of the skull and incomplete development of auditory vesicle.(3)The results of hematoxylineosin staining,toluidine blue staining and typeⅡcollagen immunohistochemistry staining showed that mycophenolate mofetil affected the proliferation and differentiation of chondrocytes and the expression of typeⅡcollagen.(4)To conclude,mycophenolate mofetil can cause the dysplasia of rat embryonic auricle in a dose-dependent manner,because mycophenolate mofetil may inhibit the prolife ration and differentiation of chondrocytes and the expression of typeⅡcollagen.Mycophenolate mofetil can be used to establish an animal model of microtia.