本文關(guān)鍵詞：甲減胎鼠腦發(fā)育中Shh信號(hào)通路與Nkxs表達(dá)的相互影響　出處：《天津醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 甲減 胎鼠 腦發(fā)育 Shh Nkx2.2 Nkx6.2 大腦皮層 星形膠質(zhì)細(xì)胞

【摘要】：目的:Shh與Nkxs基因家族與腦發(fā)育密切相關(guān),分析甲減母鼠在孕20天時(shí)胎鼠腦發(fā)育過(guò)程中Shh信號(hào)通路對(duì)Nkx2.2和Nkx6.2在基因和蛋白水平的表達(dá)影響,進(jìn)一步明確甲減狀態(tài)下Nkxs影響腦發(fā)育的作用機(jī)制,并檢測(cè)大腦皮質(zhì)皮層在早期腦發(fā)育中對(duì)甲狀腺激素的依賴性。此外,篩選在甲減模型中可以不受處理因素影響穩(wěn)定表達(dá)的管家基因作為內(nèi)參。方法:建立甲減大鼠模型,用低碘飼料和KClO3水溶液進(jìn)行喂養(yǎng),正常組用KIO3水溶液補(bǔ)償?shù)鈹z入;三個(gè)月后,設(shè)置正常組、正常抑制組、甲減組、甲減治療組、甲減治療抑制組、甲減激活組,按2μg/100g/d L-T4治療,通路抑制組按1mg/100g/d腹腔注射Cyclopamine,通路激活組按0.3mg/100g/d腹腔注射Purmorpamine。利用熒光定量PCR法和Western blot法檢測(cè)各組胎鼠腦組織中Gli-1、Nkx2.2、Nkx6.2的mRNA相對(duì)表達(dá)量和蛋白表達(dá)水平,用HE染色法觀察腦皮層分層生長(zhǎng)情況和神經(jīng)元細(xì)胞遷移情況。體外培養(yǎng)大鼠星形膠質(zhì)細(xì)胞,按12h、24h、48h分為高激素水平、正常激素水平和低激素水平,觀察體外條件下甲狀腺激素對(duì)Shh、Nkx2.2和Nkx6.2的表達(dá)影響。結(jié)果:1.內(nèi)參基因UBC可以在甲減模型中穩(wěn)定表達(dá);2.本次體內(nèi)實(shí)驗(yàn)大鼠妊娠期對(duì)DMSO的最大耐受量為1ml/100g/d;1mg/100g/d Cyclopamine注射量可抑制Shh信號(hào)通路;0.3mg/100g/d Pumorpamine注射量可激活Shh信號(hào)通路;3.正常激素水平下,抑制Shh信號(hào)通路后,Nkx2.2和Nkx6.2的表達(dá)水平會(huì)下降;4.TH水平低水正常水平時(shí),Gli-1、Nkx2.2和Nkx6.2的表達(dá)會(huì)下降;5.Shh在早期腦發(fā)育中對(duì)TH很敏感,當(dāng)TH水平低時(shí),外源性補(bǔ)充通路調(diào)控劑來(lái)特異性激活Shh通路后,會(huì)提高Gli-1表達(dá)水平,但對(duì)Nkx2.2和Nkx6.2的表達(dá)沒(méi)有影響;通過(guò)注射左旋甲狀腺素恢復(fù)TH水平后,Gli-1、Nkx2.2和Nkx6.2表達(dá)與正常組無(wú)差異;當(dāng)恢復(fù)后抑制Shh信號(hào)通路后,Gli-1表達(dá)水平明顯低于正常組,但Nkx2.2和Nkx6.2的表達(dá)與甲減時(shí)無(wú)差異;6.1ml/100g/d DMSO對(duì)早期腦發(fā)育胎鼠的三種基因的表達(dá)水平均沒(méi)有影響;7.體外培養(yǎng)星形膠質(zhì)細(xì)胞在12h、24h和48h時(shí),低激素組Shh表達(dá)水平均明顯低于正常組,高激素組與正常組相比,12h時(shí)表達(dá)無(wú)差異,24h和48h時(shí)Shh表達(dá)量均高于正常組,且在48h表達(dá)最多;8.Nkx2.2在正常組中12h時(shí)表達(dá)量最高,48h時(shí)表達(dá)量最低;在低激素組中,12h時(shí)表達(dá)量最高,之后表達(dá)水平未見差異;激素水平正常時(shí),Nkx6.2表達(dá)水平未見波動(dòng);在低激素組中,12h和24h明顯低于正常組;在高激素組中,12h和24h表達(dá)量高于正常組,48h低于正常組;說(shuō)明TH可以在細(xì)胞水平明顯影響Shh和Nkx2.2、Nkx6.2的表達(dá);9.Shh通路和TH水平正相關(guān)影響皮層分化。結(jié)論:甲減大鼠的Shh信號(hào)通路與Nkx2.2和Nkx6.2的表達(dá)具有相關(guān)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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R714.256
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本文編號(hào)：1416509