TCDD暴露对小鼠在位子宫内膜hMLH1表达的影响及其甲基化程度的研究

Influence of TCDD exposure on hMLH1 expression in eutopic endometrium and hypermethylation study in mice

目的探讨自胚胎期至成年期氯代二恶英(TCDD)暴露后小鼠子宫内膜组织、hMLH1的表达及错配修复基因1(hMLH-1)基因启动子区CpG岛甲基化程度。方法选60只C57BL/6雌性小鼠以2:1与30只C3H雄性小鼠随机合笼交配,见到阴道栓为受孕当日,60只已孕母鼠于受孕第8天(胎儿期)以鼻饲法暴露TCDD,且被随机分为对照组(TCDD:0μg/kg)及实验组(TCDD:3μg/kg),实验组所产雌性子鼠于出生后21天(青春期)再次分别暴露TCDD,每组均选12只雌性子鼠(体重6.40±0.20g),依据暴露剂量分为A组:0μg/kg,B组:3μg/kg,C组:10μg/kg。实验各组于出生后49天(成年期)再次暴露TCDD(3μg/kg),出生后70天再次给予TCDD 3μg/kg同时行子宫内膜移植术构建小鼠子宫内膜异位症模型,术后3、6、9周各组再次追加暴露TCDD(3μg/kg),术后12周脱臼处死雌性子鼠。免疫组化SP法检测在位内膜组织中hMLH1的表达。甲基化特异性PCR(M SP)方法检测各组hMLH1基因启动子区CpG岛的甲基化程度。结果 hMLH1在对照组中的表达与A组相比无显著性差异(t=0.561,P>0.05),但其他各组间两两比较有显著性差异,其中A组表达高于B组(t=3.056,P<0.05),B组表达高于C组(t=2.228,P<0.05)。各组hMLH1基因启动子区CpG岛甲基化程度比较:对照组与A组比较无显著性差异(χ~2=1.339,P>0.05),其他各组间两两比较有显著性差异,其中A组低于B组(χ~2=4.444,P<0.05),而B组低于C组(χ~2=6.316,P<0.05)。结论自胚胎期至成年期持续暴露TCDD有可能使hMLH1基因启动子区CpG岛发生超甲基化改变,导致hMLH1在组织中表达下降,从而影响成熟期子宫内膜的错配修复基因的功能,这些可能促进了成年期子宫内膜异位症的发生与发展。

Objective To explore the expression of hMLH1 in eutopic endometrium of mice exposed to TCDD from embryonic stage to adulthood and to study hypermethylation of CpG island in promoter region of hMLH-1 gene. Methods Sixty C57BL/6 female mice were mated with 30 C3H male mice at ratio 2： 1 randomly. The day on which pessulum was seen was taken as gestational day. On the 8th gestational day（ fetal stage）,60 pregnant mice were exposed to TCDD with nasal feeding method,and divided into control group（ exposed in TCDD with 0μg/kg） and experimental group（ exposed in TCDD with 3μg/kg）. Female offsprings of mice in the experimental group were exposed to TCDD again on the 21 st day after birth（ puberty stage）,and according to different exposure dose divided into group A with exposure dose of 0μg/kg,group B with dose of 3μg/kg and group C with dose of 10μg/kg with 12 female mices（ 6. 40 ± 0. 20 g of weight）in each group. Mices in the experimental group were exposed to TCDD at 3μg/kg on 49 th day and on 70 th day after birth,mouse endometriosis model was established through endometrium transplantation. Mices were exposed to TCDD again at 3,6 and 9 weeks after transplantation and killed by cervical dislocation at 12 weeks after surgery. Expression of hMLH1 in endometrium was detected with SP immunohistochemical method and methylation of CpG island in promoter region of hMLH1 gene was detected using methylation-specific PCR（ MS-PCR） in each group. Results Difference in expression of hMLH1 between the control group and group A was not significant（ t =0. 561,P 0. 05）. Pairwise comparison carried out in other groups showed significant difference. Expression level in group A was higher than that in group B（ t = 3. 056,P 0. 05）,and that in group B was higher than that in group C（ t = 2. 228,P 0. 05）. Comparison of methylation of CpG island in promoter region of hMLH1 gene among groups showed that there was no significant difference between the control group and group A（ χ~2 = 1. 339,P 0. 05）,but pairwise comparison of methylation in other groups found significant difference and methylation in group A was lower than that in group B（ χ~2 = 4. 444,P 0. 05） and methylation in group B was lower than that in group C（ χ~2 = 6. 316,P 0. 05）. Conclusion Persistent exposure to TCDD from embryonic period to adulthood perhaps triggers methylation of CpG islands in promoter region of hMLH1 gene,resulting in decrease of hMLH1 expression and influencing function of mismatch repair gene in mature endometrium,which may promote occurrence and development of endometriosis in adulthood.