【摘要】：目的研究以全反式維甲酸誘導(dǎo)構(gòu)建的神經(jīng)管畸形SD胎鼠生長發(fā)育、脊髓神經(jīng)干細(xì)胞生物學(xué)特性及H3K27me3的變化與葉酸對其干預(yù)作用之間的關(guān)系。探討神經(jīng)管畸形發(fā)生及葉酸干預(yù)作用的機制，為神經(jīng)管畸形的防治提供更為可靠的理論依據(jù)。 方法1.動物模型的建立：將性成熟健康雌鼠隨機分為①正常組，②致畸組，，③致畸葉酸干預(yù)組。其中③致畸葉酸干預(yù)組，于交配前兩周開始飼喂含葉酸大鼠顆粒飼料至剖宮取胎；②致畸組、③致畸葉酸干預(yù)組于E10d天時，按100mg/kg一次性給予濃度為40mg/ml的的ATRA，①正常組予同樣體積的橄欖油。于E15d時剖宮取胎，觀察胎鼠大體形態(tài)，并記錄數(shù)量、身長、體重及畸形類型。 2.病理學(xué)觀察：取明顯神經(jīng)管畸形的胎鼠及正常胎鼠相同部位組織，包埋蠟塊進行HE染色，顯微鏡下觀察其病理學(xué)變化。 3.脊髓神經(jīng)干細(xì)胞分離培養(yǎng)：取三組E15d胚胎的脊髓，分離培養(yǎng)其中的神經(jīng)干細(xì)胞，觀察各組中神經(jīng)干細(xì)胞的比例、生長活性以及分化特性的變化。 4.各組神經(jīng)干細(xì)胞中H3K27me3表達(dá)檢測：通過免疫細(xì)胞學(xué)技術(shù)定性判斷三組神經(jīng)干細(xì)胞中H3K27me3的表達(dá)，并通過Western blot技術(shù)定量分析H3K27me3在三組細(xì)胞核中的表達(dá)量。 結(jié)果1.①正常組共獲得胎鼠117只，其中死胎一只，其余均活胎，外觀無明顯畸形；②致畸組其中死胎或吸收胎12只，活胎93只，顯性脊柱裂45.2%(42/93)，無尾、環(huán)尾或短尾畸形66.7%(62/93)，足畸形22.6%(21/93)，無腦及腦膨出等畸形8.6%(8/93)；③致畸葉酸干預(yù)組其中死胎或吸收胎7只，活胎106只，顯性脊柱裂26.4%(28/106)，無尾、環(huán)尾或短尾畸形42.5%(45/106)，足畸形11.3%(12/106)，無腦及腦膨出等畸形5.7%(6/106)。對照組共獲得胎鼠125只，②組較①組發(fā)育差，身長、體重經(jīng)t檢驗分析，差異有統(tǒng)計學(xué)意義（P0.05），③組較②組發(fā)育好，差異有統(tǒng)計學(xué)意義（P0.05）。 2.病理觀察發(fā)現(xiàn)正常胎鼠可觀察到脊髓形態(tài)正常，椎體錐弓發(fā)育良好，被覆皮膚連續(xù)，E15d胎鼠神經(jīng)管基本閉合，中央管形態(tài)規(guī)則。顯性脊柱裂胎鼠脊髓改變復(fù)雜，脊柱背側(cè)（位置較致畸葉酸干預(yù)組高）被覆皮膚不連續(xù)，組織缺損，沿脊柱縱軸呈現(xiàn)一個橢圓形缺損區(qū),長約0.3~0.4cm（范圍較致畸葉酸干預(yù)組大），深部脊髓組織缺失、形態(tài)改變，脊髓脊膜向外膨出，多有炎性細(xì)胞浸潤，自尾側(cè)向頭側(cè)，尾側(cè)端見到完全紊亂多極性排列的神經(jīng)管上皮形成多個管狀結(jié)構(gòu)混亂排列，形成神經(jīng)組織錯構(gòu)腫瘤。 3.免疫細(xì)胞化學(xué)顯示所獲得細(xì)胞Nestin陽性，并可誘導(dǎo)分化出GFAP、NSE陽性細(xì)胞，具有NSCs特性。①正常組，②致畸組，③致畸葉酸干預(yù)組鏡下神經(jīng)球計數(shù)分別為：29.4±2.07、17.8±2.17、23.4±1.52（P0.05），直徑：140.2±4.82、106.6±3.85、121.8±4.09（P0.05）；誘導(dǎo)分化后Nestin表達(dá)量分別為：（6±0.9）%、（21±4）%、（32±3）%（P0.05）；免疫細(xì)胞化學(xué)顯示三組神經(jīng)干細(xì)胞中均有H3K27me3表達(dá)，Western blot結(jié)果顯示畸形組中H3K27me3明顯較正常組高表達(dá)，葉酸干預(yù)組較其表達(dá)下調(diào)（P0.05）。 結(jié)論1.全反式維甲酸可以誘導(dǎo)多種神經(jīng)管畸形的發(fā)生； 2.神經(jīng)管畸形鼠生長發(fā)育遲緩，葉酸能夠有效改善這一現(xiàn)象； 3.無血清培養(yǎng)條件下脊髓源性神經(jīng)干細(xì)胞生長良好，保持了干細(xì)胞特性； 4.神經(jīng)管畸形鼠中神經(jīng)干細(xì)胞比例較低，且增殖分化能力弱，葉酸的干預(yù)可以起保護作用； 5.葉酸與H3K27的三甲基化狀態(tài)及神經(jīng)干細(xì)胞的生物學(xué)特性具有內(nèi)在聯(lián)系。
[Abstract]:Objective To study the growth and development of neural tube malformations in SD fetal rats induced by all-trans retinoic acid, the biological characteristics of spinal neural stem cells and the relationship between the changes of H3K27me3 and folic acid intervention. According to it.
Methods 1. Establishment of animal model: Sexually mature and healthy female rats were randomly divided into normal group, teratogenic group and teratogenic folic acid intervention group. ATRA of 40 mg/ml was administered to the normal group. The same volume of olive oil was administered to the normal group.
2. Pathological observation: The tissues of fetal rats with obvious neural tube malformation and normal fetal rats were embedded with wax and stained with HE. The pathological changes were observed under microscope.
3. Isolation and culture of neural stem cells from spinal cord: Three groups of E15d embryonic spinal cord were isolated and cultured, and the proportion of neural stem cells, growth activity and differentiation characteristics were observed.
4. Detection of H3K27me3 expression in neural stem cells of each group: Qualitative determination of H3K27me3 expression in three groups of neural stem cells by immunocytology, and quantitative analysis of H3K27me3 expression in three groups of nuclei by Western blot.
Results (1) 117 fetuses were obtained in the normal group, including one dead fetus and the other live fetuses without obvious deformities; (2) In the teratogenic group, 12 dead or absorbed fetuses, 93 live fetuses, 45.2% (42/93) of dominant spina bifida, 66.7% (62/93) of tail circumference or short tail deformity, 22.6% (21/93) of foot deformity, 8.6% (8/93) of encephalocele and other deformities; In the acid intervention group, there were 7 stillbirths or absorbed fetuses, 106 live fetuses, 26.4% (28/106) dominant spina bifida, 42.5% (45/106) tailless, circumcaudal or short tail deformities, 11.3% (12/106) foot deformities, 5.7% (6/106) anencephaly and encephalocele deformities. 0.05), group III developed better than group II, the difference was statistically significant (P0.05).
2. Pathological observation showed that normal fetal rats had normal spinal cord morphology, well developed vertebral pyramidal arch, continuous covered skin, closed neural canal and regular central canal morphology on E15 days. Spinal cord changes in dominant spina bifida fetuses were complicated, and the covered skin was discontinuous on the dorsal side of spine (higher than that in teratogenic folic acid intervention group). There was an oval defect, about 0.3-0.4 cm long (larger than that of the teratogenic folic acid intervention group). The deep spinal cord tissue was absent and the morphological changes were observed. The spinal cord meninges swelled outward. Inflammatory cells infiltrated into the spinal cord. From the tail side to the head side, the completely disordered multipolar arrangement of neural tube epithelium was seen at the tail side, forming a number of tubular structures disordered arrangement, forming nerves. Tissue hamartoma.
3. Immunocytochemistry showed that Nestin-positive cells could induce differentiation of GFAP and NSE-positive cells with NSCs characteristics. The expression of Nestin was (6 65
Conclusion 1. all trans retinoic acid can induce a variety of neural tube defects.
2. the growth retardation of mice with neural tube defects can be effectively improved by folic acid.
3. in the absence of serum culture, spinal cord derived neural stem cells grew well and maintained the characteristics of stem cells.
4. The proportion of neural stem cells in neural tube malformation rats is low, and the ability of proliferation and differentiation is weak, folic acid intervention can play a protective role.
5. folic acid is closely related to the trimethylation state of H3K27 and the biological characteristics of neural stem cells.
【學(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R741

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 李紅麗,蔡文琴,楊忠,李澤桂,李巍,付曉嵐;維甲酸誘導(dǎo)神經(jīng)管畸形發(fā)生過程中神經(jīng)干細(xì)胞的變化研究[J];第三軍醫(yī)大學(xué)學(xué)報;2003年01期

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