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目的 探讨有机磷农药 (敌百虫 ,LD50 )在先天性巨结肠症发病中的作用。方法 4 0只妊娠Wistar大鼠随机分为 4组。在大鼠妊娠期 8～ 14d ,Ⅰ组每日以生理盐水灌胃作为阴性对照 ;Ⅱ组每日以 5 0mg/kg敌百虫 (LD50 的 1/ 10 )灌胃 ;Ⅲ组给予 10mg/kg敌百虫 (LD50 的 1/ 5 0 ) ;Ⅳ组给予 2mg/kg敌百虫 (LD50 的 1/ 2 5 0 )。分娩前处死母鼠 ,取出胎鼠 ,每只母鼠取 4只体重相似的胎鼠 ,取其远段 1cm结肠组织 ,提取DNA和RNA。采用RT PCR方法检测RET基因表达 ,采用PCR SSCP对RET基因第 15外显子 (E15 )进行基因突变检测 ,阳性PCR产物进行直接测序。结果 RET基因在所有的标本中均稳定表达。在Ⅲ组 (10mg/kg敌百虫 )的 2只胎鼠 (由不同母鼠所产 )和Ⅱ组 (5 0mg/kg敌百虫 )的 1只胎鼠的DNA标本在PCR SSCP分析时发现泳动变位 ,经测序证实均为V90 6密码子存在GTG→GTT的同义突变。结论 有机磷农药敌百虫可能导致RET基因突变
Objective To investigate the role of organophosphorus pesticides (trichlorfon, LD50) in the pathogenesis of Hirschsprung’s disease. Methods 40 pregnant Wistar rats were randomly divided into 4 groups. Rats in group I were treated with normal saline intragastrically for 8-14 days during gestation. Group II was intragastrically administered with 50 mg / kg of trichlorfon (1/10 of LD50) every day in group II and 10 mg / kg Trichlorfon (LD50 1/50); Group IV given 2mg / kg trichlorfon (LD50 1/250). Before sacrifice, the mother rats were sacrificed and fetuses were taken out. Four fetuses with similar body weight were taken from each fetus, and 1 cm colonic tissues were taken from the distal part to extract DNA and RNA. The expression of RET gene was detected by RT-PCR. The mutation of exon 15 of RET gene was detected by PCR SSCP. The positive PCR products were directly sequenced. Results The RET gene was stably expressed in all specimens. DNA samples from one fetus of two fetuses of group III (10 mg / kg trichlorfon) (produced by different mother mice) and one group II (50 mg / kg trichlorfon) were found by PCR SSCP analysis The results of sequencing showed that both of them were synonymous with GTG → GTT in V90 6 codon. Conclusion Organophosphorus pesticide trichlorfon may cause RET gene mutation