甲状腺素对甲状腺功能减低大鼠子代脑组织中同源盒基因Nkx6．1 mRNA表达的影响

Effects of thyroid hormone on the expression of homeobox gene Nkx6.1 in the cerebrum tissue of offspring of hypothyroidism rat

目的通过对妊娠期甲状腺功能减低（简称甲低）大鼠补充甲状腺素，探讨妊娠不同时间补充不同剂量甲状腺素对子代大鼠脑组织中同源盒基因Nkx6．1 mRNA表达的影响。方法1月龄Wistar大鼠240只，雌雄各半，体质量100—120g。按体质量将雌性大鼠随机分为8组：对照组，甲低组，甲低孕鼠妊娠早期（1～17d）补充甲状腺素（妊早甲低）高、中、低剂量组，甲低孕鼠妊娠晚期（18～20d）补充甲状腺素（妊晚甲低）高、中、低剂量组，每组15只。高、中、低剂量组每天补充的甲状腺素分别为3．5、2．0、0．5μg／100g体质量。8组大鼠均饲以重度缺碘地区粮食配制的饲料，对照组饮用含碘200μg／L的碘酸钾溶液，7个甲低组饮用去离子水。雄性大鼠用正常食料饲养，3个月后按1：1交配。8组大鼠分别取孕17d、新生、生后20d子代大鼠脑组织，采用实时荧光定量PCR法检测脑组织中Nkx6．1 mRNA表达。结果①成功建立了甲低动物模型，8组大鼠血清TT3、TT4、FT3、FT4组间比较差异有统计学意义（F值分别为4．08、31．99、5．79、26．34，P均〈0．01）。②在孕17d、新生、生后20d，Nkx6．1 mRNA表达组间比较差异有统计学意义（F值分别为758．720、1121．589、144．716，P均〈0．01）；组内不同时间比较差异有统计学意义（F值分别为2898．863、325．605、716．285、56．329、236．727、196．678、7115．752、9152．306，P均〈0．01）。③时间因素和剂量因素对Nkx6．1 mRNA表达有影响（F值分别为1176．655、246．530，P均〈0．01）；时间与剂量因素间存在交互作用（F=1249．934，P〈0．01）。④在孕17d、新生、生后20d，甲低组Nkx6．1 mRNA表达与对照组比较，差异有统计学意义（P均〈0．01）。⑤在新生、生后20d，6个甲低补充甲状腺素组Nkx6．1 mRNA表达与甲低组比较，差异有统计学意义（P均〈0．01）。⑥在孕17d、新生、生后20d，除妊早甲低中剂量组外，其余5个甲低补充甲状腺素组Nkx6．1 mRNA表达与对照组比较，差异有统计学意义（P均〈0．01）。⑦在孕17d、新生、生后20d，妊早甲低高、低剂量组Nkx6．1 mRNA表达与中剂量组比较，差异有统计学意义（P均〈0．01）；在孕17d、生后20d，高剂量组与低剂量组比较，差异有统计学意义（P〈0．01）。⑧在新生和生后20d，妊晚甲低组组间Nkx6．1 mRNA表达比较，差异有统计学意义（P均〈0．01）。结论甲低孕鼠子代大鼠脑组织同源盒基因Nkx6．1的表达受补充甲状腺素剂量及时间影响。

Objective To explore the effects of thyroid hormone on the expression of homeobox gene Nkx6.1 in offspring of hypothyroidism rats and the relationship between gene expression and hormone level by supplying their hypothyroidism pregnant mother with thyroid hormone. Method A total of 240 Wistar rats were half male and half female. Female rats were randomly divided into eight groups： control, hypothyroidism group, hypothyroidism groups which were supplied with thyroid hormone in high, medium and low dosage in early stage（1 - 17 d） and in late stage（18 - 20 d）. According to 100 grams of body weight, the concentration of thyroid hormone were 3.5,2.0,0.5 μg/d in high, medium and low dosage group. All the rats were fed with low-iodine food. The normal control group was given KIO3 solution and the other groups were given deionized water. After three months female rats were mated with male rats. The content of Nkx6.1 mRNA in brain tissue of 17-day fetal rats, new-born and 20- day old offspring by real-time fluorescence quantitative PCR techniques. Results ①A rat model of hypothyroidism was successfully established, there were statistical significance between 8 groups in TT3,TT4, FF3, FT4（F = 4.08, 31.99,5.79,26.34, all P 〈 0.01 ）. ②The expression of Nkx6.1 mRNA had significant difference （F = 758.720, 1121.589,144.716, all P 〈 0.01 ） between groups in 17-day fetal rats, new-born and 20-day old offsprings and intragroups in different time （F = 2898.863,325.605,716.285,56.329,236.727,196.678,7115.752,9152.306, all P 〈 0.01）. ③The time factor and dosage factor had influence on Nkx6.1 mRNA expression （F = 1176.655,246.530, all P 〈 0.01 ）. There were interaction between time and dosage factor（F = 1249.934, P 〈 0.01 ）. ④Comparison of Nkx6.1 mRNA expression between hypothyroidism group and normal control group had significant difference in the above three time points（all P 〈 0.01 ）. ⑤Comparisons of Nkx6.1 mRNA expression between 6 hypothyroidism groups which were supplied with thyroid hormone and hypothyroidism group had significant difference （all P 〈 0.01 ） in new-born and 20-day old offspring; comparisons of Nkx6.1 mRNA expression between hypothyroidism groups which were supplied with high and medium thyroid hormone and hypothyroidism group had significant difference in 17-day fetal rats（all P 〈 0.01 ）. ⑥Comparison of Nkx6.1 mRNA expression between hypothyroidism groups which were supplied with medium thyroid hormone in early stage and normal control group had no statistical significance （all P 〉 0.05）, while between the other 5 groups which were supplied with thyroid hormone and normal control group had significant difference（all P 〈 0.01 ） in the above three time points.⑦Muhiple comparison of early stage groups which were supplied with thyroid hormone showed that the expression of Nkx6.1 mRNA had significant difference（all P 〈 0.01 ） between high, low dosage groups and medium group in 17-day fetal rats, new-born and 20-day old offspring and between high dosage groups and low dosage groups .in 17-day fetal rats and 20-day offspring（all P 〈 0.01）. ⑧Muhiple comparison of late stage groups supplied with thyroid hormone showed that the expression of Nkx6.1 mRNA had significant difference （all P 〈 0.01 ） between three groups in new-born and 20- day old offspring. Conclusion The expression of Nkx6.1 in rats offspring is highly related to the supply dosage and supply time of thyroid hormone in hypothyroidism pregnant rats.