维甲酸诱导大鼠脊髓脊膜膨出动物模型的建立及相关蛋白表达的研究

Retinoic acid induced MMC in fetal rats：Characterization by histopathological and immunohistochemical analysis

目的探索利用全反式维甲酸（ATRA）诱导大鼠胎鼠脊髓脊膜膨出（MMC）建立神经源性膀胱（NBD）的大鼠动物模型，并观察相关病理表现及蛋白表达。方法探索在不同给药时间及不同ATRA剂量下，大鼠胎鼠MMC的发生情况，得到最佳诱导致畸时间及ATRA剂量，建立动物模型；所得胎鼠分为MMC组（胎鼠出现MMC，不合并其他严重畸形）、RA组（胎鼠外观正常，无明显畸形）、OIL组（母鼠同期灌胃2ml橄榄油，得到正常胎鼠）。观察该模型胎鼠相关组织的病理表现，并通过免疫组化方法测定脊髓组织GFAP、膀胱组织a-SMA、膀胱tubulin-β-Ⅲ和nNOS表达情况。结果在母鼠孕10d（E10）以120mg／kg的剂量灌胃ATRA能获得最佳建模效果；MMC组胎鼠脊髓组织病理表现存在异常，膀胱组织病理表现基本正常；MMC组胎鼠脊髓组织GFAP蛋白表达（5．5±1．6）×10^4，较RA组的（2．5±2．0）×10。增加，膀胱组织mSMA蛋白无明显差异，MMC组膀胱组织tubulin-β-Ⅲ和nNOS蛋白表达分别为（22．4±2．7）×10^4、（16．1±4．1）×10^4，较RA组的（28．4±8．3）×10^4、（25．6±7．1）×10^4减少。结论ATRA能诱导大鼠胎鼠产生MMC，得到MMC相关NBD动物模型；其MMC胎鼠具有典型的脊髓损伤表现，尽管膀胱肌层发育正常但存在明显的神经分布异常。

Objective The aims of this study were to create a reliable method of inducing an iso- lated MMC-like lesion in fetal rats by maternal administration of all trans-retinoic acid （ATRA） and to analyse its histopathological and immunohistochemical characterizations. Methods Time-dated primi- gravida Sprague-Dawley rats were fed different doses of ATRA on the different days in order to find the best way to create MMC-associated NBD in fetal rats. Fetuses harvested from pregnant rats and were examined histologically. The expression patterns of GFAP, a-SMA, tubulin-[3-11I, nNOS were analyzed by immunohistochemistry. Results A single administration of 120 mg/kg of ATRA at em- bryonic dayl0 resulted in the highest incidence of MMC-like defects. Similar to the findings in human fetuses with MMC, the severity of the lesion spanned the spectrum of severity observed in MMC fetu- ses. H-E staining of bladders from MMC fetuses demonstrated normal arrangement of the outer and inner layer of smooth muscle. The staining intensity of GFAP of MMC fetuses of spinal cord were in- creased. Immunoreactivity for α-SMA , tubulin-β-Ⅲ and nNOS was observed within the bladder wall. Staining intensity for α-SMA did not vary between MMC and the other bladders. Meanwhile, the den- sity of tubulin-β-Ⅲpositive nerve fibers and nNOS-positive nerve fibers within the detrusor muscle of MMC bladders were both reduced. Conclusions ATRA has been shown to be highly effective and reliable in inducing MMC-associated NBD in fetal rats. MMC fetuses presented with typical spinal cord damage. The smooth muscles of bladders from MMC fetuses were normal while the distribution of nerves were abnormal.