甲减胎鼠脑发育中Shh信号通路与Nkxs表达的相互影响

发布时间：2018-01-12 23:32

  本文关键词：甲减胎鼠脑发育中Shh信号通路与Nkxs表达的相互影响　出处：《天津医科大学》2016年硕士论文　论文类型：学位论文

  更多相关文章： 甲减 胎鼠 脑发育 Shh Nkx2.2 Nkx6.2 大脑皮层 星形胶质细胞

【摘要】：目的:Shh与Nkxs基因家族与脑发育密切相关,分析甲减母鼠在孕20天时胎鼠脑发育过程中Shh信号通路对Nkx2.2和Nkx6.2在基因和蛋白水平的表达影响,进一步明确甲减状态下Nkxs影响脑发育的作用机制,并检测大脑皮质皮层在早期脑发育中对甲状腺激素的依赖性。此外,筛选在甲减模型中可以不受处理因素影响稳定表达的管家基因作为内参。方法:建立甲减大鼠模型,用低碘饲料和KClO3水溶液进行喂养,正常组用KIO3水溶液补偿碘摄入;三个月后,设置正常组、正常抑制组、甲减组、甲减治疗组、甲减治疗抑制组、甲减激活组,按2μg/100g/d L-T4治疗,通路抑制组按1mg/100g/d腹腔注射Cyclopamine,通路激活组按0.3mg/100g/d腹腔注射Purmorpamine。利用荧光定量PCR法和Western blot法检测各组胎鼠脑组织中Gli-1、Nkx2.2、Nkx6.2的mRNA相对表达量和蛋白表达水平,用HE染色法观察脑皮层分层生长情况和神经元细胞迁移情况。体外培养大鼠星形胶质细胞,按12h、24h、48h分为高激素水平、正常激素水平和低激素水平,观察体外条件下甲状腺激素对Shh、Nkx2.2和Nkx6.2的表达影响。结果:1.内参基因UBC可以在甲减模型中稳定表达;2.本次体内实验大鼠妊娠期对DMSO的最大耐受量为1ml/100g/d;1mg/100g/d Cyclopamine注射量可抑制Shh信号通路;0.3mg/100g/d Pumorpamine注射量可激活Shh信号通路;3.正常激素水平下,抑制Shh信号通路后,Nkx2.2和Nkx6.2的表达水平会下降;4.TH水平低水正常水平时,Gli-1、Nkx2.2和Nkx6.2的表达会下降;5.Shh在早期脑发育中对TH很敏感,当TH水平低时,外源性补充通路调控剂来特异性激活Shh通路后,会提高Gli-1表达水平,但对Nkx2.2和Nkx6.2的表达没有影响;通过注射左旋甲状腺素恢复TH水平后,Gli-1、Nkx2.2和Nkx6.2表达与正常组无差异;当恢复后抑制Shh信号通路后,Gli-1表达水平明显低于正常组,但Nkx2.2和Nkx6.2的表达与甲减时无差异;6.1ml/100g/d DMSO对早期脑发育胎鼠的三种基因的表达水平均没有影响;7.体外培养星形胶质细胞在12h、24h和48h时,低激素组Shh表达水平均明显低于正常组,高激素组与正常组相比,12h时表达无差异,24h和48h时Shh表达量均高于正常组,且在48h表达最多;8.Nkx2.2在正常组中12h时表达量最高,48h时表达量最低;在低激素组中,12h时表达量最高,之后表达水平未见差异;激素水平正常时,Nkx6.2表达水平未见波动;在低激素组中,12h和24h明显低于正常组;在高激素组中,12h和24h表达量高于正常组,48h低于正常组;说明TH可以在细胞水平明显影响Shh和Nkx2.2、Nkx6.2的表达;9.Shh通路和TH水平正相关影响皮层分化。结论:甲减大鼠的Shh信号通路与Nkx2.2和Nkx6.2的表达具有相关性。
[Abstract]:Objective to study the relationship between the Nkxs gene family and brain development. To investigate the effects of Shh signaling pathway on the expression of Nkx2.2 and Nkx6.2 at gene and protein levels during the development of fetal brain in hypothyroidism rats at 20 days of gestation. The mechanism of Nkxs affecting brain development in hypothyroidism state was further clarified, and the dependence of cortical cortex on thyroid hormone in early brain development was detected. Methods: the rat model of hypothyroidism was established and fed with iodine deficiency feed and KClO3 aqueous solution. In normal group, iodine intake was compensated with KIO3 aqueous solution. Three months later, normal group, normal inhibition group, hypothyroidism group, hypothyroidism treatment group, hypothyroidism inhibition group, hypothyroidism activation group were treated with 2 渭 g / 100 g / d L-T4. In the pathway inhibition group, Cyclopamine was injected intraperitoneally at 1 mg / 100 g / d. In the pathway activation group, Purmorpamine was injected intraperitoneally at 0.3 mg / 100 g / d. Fluorescence quantitative PCR and Western were used. Blot method was used to detect Gli-1 in fetal brain tissue. The relative expression of mRNA and the protein expression of Nkx6.2 were observed in Nkx2.2 and Nkx6.2 respectively. The stratified growth of cerebral cortex and neuronal cell migration were observed by HE staining. Astrocytes were cultured in vitro and divided into high hormone levels according to 12h ~ 24h ~ 48h. The effect of thyroid hormone on Shh in vitro was observed. The expression of Nkx2.2 and Nkx6.2. Results: 1. The UBC gene could be expressed stably in hypothyroidism model. 2.The maximum tolerance to DMSO during pregnancy was 1 ml / 100 g / d; 1 mg / 100 g / d Cyclopamine injection could inhibit the Shh signaling pathway. 0.3 mg / 100 g / d Pumorpamine injection could activate the Shh signaling pathway. 3. Under normal hormone level, the expression of Nkx2.2 and Nkx6.2 decreased after inhibition of Shh signaling pathway. 4. The expression of Gli-1 nkx2.2 and Nkx6.2 decreased when th level was low and water was normal. 5. Shh is sensitive to th during early brain development. When the level of th is low, exogenous supplemental pathway regulators can specifically activate the Shh pathway, which will increase the level of Gli-1 expression. But there was no effect on the expression of Nkx2.2 and Nkx6.2. There was no difference in the expression of Nkx2.2 and Nkx6.2 between the control group and the control group after the restoration of th level by injection of levothyroxine. The expression of Gli-1 was significantly lower than that of normal group after inhibition of Shh signal pathway after recovery, but the expression of Nkx2.2 and Nkx6.2 had no difference with hypothyroidism. 6.1 ml / 100g / d DMSO had no effect on the expression of three genes in early brain development fetal mice. 7. The expression of Shh in the low hormone group was significantly lower than that in the normal group at 24 h and 48 h after cultured astrocytes in vitro, but there was no difference between the high hormone group and the normal group at 12 h. At 24h and 48h, the expression of Shh was higher than that of normal group, and the highest expression was at 48h. 8. The expression of Nkx2.2 in normal group was the highest at 12h and the lowest at 48h. In the low hormone group, the expression level was the highest at 12 h, but there was no difference in the expression level after 12 hours. When the hormone level was normal, the expression of Nkx6.2 did not fluctuate. In the low hormone group, 12h and 24h were significantly lower than those in the normal group. The expression levels of 12 h and 24 h in the high hormone group were higher than those in the normal group at 48 h and lower than that in the normal group at 48 h. The results showed that th could significantly affect the expression of Shh and Nkx6.2 at cell level. 9. Shh pathway and th level positively correlated with cortical differentiation. Conclusion: the Shh signaling pathway in hypothyroidism rats is correlated with the expression of Nkx2.2 and Nkx6.2.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R714.256
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本文编号：1416509