神经管畸形鼠神经干细胞特性及叶酸对其干预作用的研究

发布时间：2018-09-13 12:10

【摘要】：目的研究以全反式维甲酸诱导构建的神经管畸形SD胎鼠生长发育、脊髓神经干细胞生物学特性及H3K27me3的变化与叶酸对其干预作用之间的关系。探讨神经管畸形发生及叶酸干预作用的机制，为神经管畸形的防治提供更为可靠的理论依据。方法1.动物模型的建立：将性成熟健康雌鼠随机分为①正常组，②致畸组，，③致畸叶酸干预组。其中③致畸叶酸干预组，于交配前两周开始饲喂含叶酸大鼠颗粒饲料至剖宫取胎；②致畸组、③致畸叶酸干预组于E10d天时，按100mg/kg一次性给予浓度为40mg/ml的的ATRA，①正常组予同样体积的橄榄油。于E15d时剖宫取胎，观察胎鼠大体形态，并记录数量、身长、体重及畸形类型。 2.病理学观察：取明显神经管畸形的胎鼠及正常胎鼠相同部位组织，包埋蜡块进行HE染色，显微镜下观察其病理学变化。 3.脊髓神经干细胞分离培养：取三组E15d胚胎的脊髓，分离培养其中的神经干细胞，观察各组中神经干细胞的比例、生长活性以及分化特性的变化。 4.各组神经干细胞中H3K27me3表达检测：通过免疫细胞学技术定性判断三组神经干细胞中H3K27me3的表达，并通过Western blot技术定量分析H3K27me3在三组细胞核中的表达量。结果1.①正常组共获得胎鼠117只，其中死胎一只，其余均活胎，外观无明显畸形；②致畸组其中死胎或吸收胎12只，活胎93只，显性脊柱裂45.2%(42/93)，无尾、环尾或短尾畸形66.7%(62/93)，足畸形22.6%(21/93)，无脑及脑膨出等畸形8.6%(8/93)；③致畸叶酸干预组其中死胎或吸收胎7只，活胎106只，显性脊柱裂26.4%(28/106)，无尾、环尾或短尾畸形42.5%(45/106)，足畸形11.3%(12/106)，无脑及脑膨出等畸形5.7%(6/106)。对照组共获得胎鼠125只，②组较①组发育差，身长、体重经t检验分析，差异有统计学意义（P0.05），③组较②组发育好，差异有统计学意义（P0.05）。 2.病理观察发现正常胎鼠可观察到脊髓形态正常，椎体锥弓发育良好，被覆皮肤连续，E15d胎鼠神经管基本闭合，中央管形态规则。显性脊柱裂胎鼠脊髓改变复杂，脊柱背侧（位置较致畸叶酸干预组高）被覆皮肤不连续，组织缺损，沿脊柱纵轴呈现一个椭圆形缺损区,长约0.3~0.4cm（范围较致畸叶酸干预组大），深部脊髓组织缺失、形态改变，脊髓脊膜向外膨出，多有炎性细胞浸润，自尾侧向头侧，尾侧端见到完全紊乱多极性排列的神经管上皮形成多个管状结构混乱排列，形成神经组织错构肿瘤。 3.免疫细胞化学显示所获得细胞Nestin阳性，并可诱导分化出GFAP、NSE阳性细胞，具有NSCs特性。①正常组，②致畸组，③致畸叶酸干预组镜下神经球计数分别为：29.4±2.07、17.8±2.17、23.4±1.52（P0.05），直径：140.2±4.82、106.6±3.85、121.8±4.09（P0.05）；诱导分化后Nestin表达量分别为：（6±0.9）%、（21±4）%、（32±3）%（P0.05）；免疫细胞化学显示三组神经干细胞中均有H3K27me3表达，Western blot结果显示畸形组中H3K27me3明显较正常组高表达，叶酸干预组较其表达下调（P0.05）。结论1.全反式维甲酸可以诱导多种神经管畸形的发生； 2.神经管畸形鼠生长发育迟缓，叶酸能够有效改善这一现象； 3.无血清培养条件下脊髓源性神经干细胞生长良好，保持了干细胞特性； 4.神经管畸形鼠中神经干细胞比例较低，且增殖分化能力弱，叶酸的干预可以起保护作用； 5.叶酸与H3K27的三甲基化状态及神经干细胞的生物学特性具有内在联系。
[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.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741

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