产前汉防己甲素干预对膈疝肺血管和结缔组织生长因子的作用

The effects of prenatal tetrandrine treatment on pulmonary vascular development and CTGF expression in rats with congenital diaphragmatic hernia

目的产前对孕晚期膈疝模型雌鼠给予汉防己甲素（Tet）干预后，观察Tet对胎鼠膈疝（CDH）模型的肺血管发育和结缔组织生长因子（CTGF）的作用和影响。方法9只健康怀孕的SD大鼠雌鼠孕9．5d被随机平均分为3组：即对照组（C组）、除草醚组（N组）和汉防己甲素组（NT组）。N和NT组灌胃给予除草醚。NT组于孕第18．5～20．5天，每天给予30mg／kg Tet灌胃，C组和N组仅给予生理盐水。孕第21．5天于麻醉下剖宫取胎鼠双肺，通过对胎肺组织行HE染色观察肺组织及血管发育，行免疫组织化学染色和RT-PCR方法观察CTGF在肺组织中的表达高低。结果本实验中N组和NT组总膈疝发生率为50％，两组间差异无统计学意义（P〉0．05）。N组肺血管数目（2．5±1．1）个较C组（4．5±1．1）个有明显减少，差异有统计学意义（P＜0．01）；两组与NT组（3．7±1．4）个比较，差异没有统计学意义（P〉0．05）。血管外径（ED）在各组间差异没有统计学意义（P〉0．05）。血管内径（ID）表现为N组（24．8±4．7）μm＜NT组（42．7±4．0）μm＜C组（51．9±8．5）μm，各组间差异有统计学意义（P〈0．01）；血管中层（平滑肌细胞）增殖、变厚，血管中膜厚度百分比N组（58．1％±6．0％）〉NT组（25．4％±8．3％）≈C组（16．6%±9．7％），N组与另两组比较，差异有统计学意义（P〈0．01），NT组与C组间差异没有统计学意义（P＞0．05）；血管外膜厚度百分比N组（134．0%±11．9%）〉NT组（86．6G±3．8％）〉C组（66．9％±16．7％），各组组间差异有统计学意义（P〈0．01）。CTGF免疫组织化学染色阳性表达强度（IOD值）结果为N组〈NT组〈C组，差异有统计学意义（P〈0.01），根据△Ct值分析基因的表达，RT-PCR结果示CTGF mRNA肺组织表达由低到高为：N组％NT组〈C组，差异有统计学意义（P〈0.01）。结论Tet对实验大鼠动物模型的胚胎期肺血管有抑制血管重构、促血管生成和发育作用，其作用机制可能与Tet上调CTGF的表达有一定关系。

Objective To explore the effects of prenatal tetrandrine treatment on vascular development and CTGF expression in the lungs of rats with congenital diaphragmatic hernia （CDH） induced by Nitrofen. Methods Nine pregnant Sprague-Dawley rats were randomly and evenly divided into 3 groups according the treatment： C group （control group）, NT group （tetrandrine group） and N group （nitrofen group）. The rats of the N and NT group were administrated with 125 mg of nitrofen dissolved in 2 ml of olive oil by gastric gavage on day 9. 5 of gestation, whereas those in C group only received same volume of olive oil. The rats of the NT group were administered with Tet （30 mg/kg） once a day from gestational day 18.5 to 20. 5, while their counterparts of the C and N group were only treated with saline. On day 21 of gestation, all fetuses were delivered by cesarean section. The rat fetuses were sacrificed and examined for diaphragmatic hernia. Lung vascular dysplasia or remodeling was evaluated on H^E staining slides. Immunohistochemical staining and real time polymerase chain reaction （RT-PCR） were performed to detect the expression of CTGF in lung tissues. Results Twenty-seven （50%） fetuses with CDH were harvested from the N and NT rats. No significant difference of CDH incidence was seen between the two groups （P〉0. 05）. More pulmonary vessels were ohserved in the fetuses of C group, and these vessels had thinner medial smooth muscle and adventitia, and larger lumina compared with the counterparts of N and NT group. The pulmonary vascular development of the fetuses of NT group was better than that of the N group. There were significant differences between the C and the other two groups in internal diameter （ID） （P〈O. 01 ） and percentage of adventitial thickness （AT%） （P〈0. 01）. The ID of N group was smaller than that in the other 2 groups （P〈0. 01）, but the medial thickness （MT%） and ATG of N group were larger than those in the other 2 groups （P〈0. 01 ）. The highest protein and mRNA expression of CTGF were observed in N group, followed by NT group and C group （P〈0. 01）. Conclusions Prenatal treatment with tetrandrine can promote lung development and inhibit pulmonary arterial structurai remodeling in fetal rats with nitrofen-induced CDH via the unregulated expression of CTGF in the lungs.