三碘甲状腺原氨酸对人神经干细胞分化过程中甲状腺激素受体表达的干预效应

背景:三碘甲状腺原氨酸是脑发育临界期的重要调控因素,在中枢神经系统发育过程中可能决定着神经干细胞的世系分化命运。目的:观察三碘甲状腺原氨酸诱导人神经干细胞分化情况以及分化过程中甲状腺激素受体mRNA的表达变化。设计:开放性实验。单位:天津市武警医学院病理教研室,天津医科大学内分泌研究所。材料:实验于2003-01/2005-03在天津医科大学完成。由天津医科大学总医院提供孕10～12周流产胎儿,产妇及家属均签署知情同意书,实验经医学伦理委员会批准。方法:①无菌条件下取人胚胎两侧大脑半球,剥除脑膜,剪碎脑组织,过滤制备细胞悬液。以20μg/L碱性成纤维细胞生长因子 30nmol/L三碘甲状腺原氨酸联合诱导神经干细胞增殖,按1×109L-1密度分别接种于涂有多聚左旋赖氨酸的24孔板和25mL培养瓶中常规培养,培养液为含N2添加剂的DMEM/F12无血清培养基。48h后半量换液,7d后撤除碱性成纤维细胞生长因子,单独用三碘甲状腺原氨酸诱导分化。②培养1,2,3周时取细胞,RT-PCR半定量检测神经干细胞诱导分化不同阶段甲状腺激素受体mRNA表达的变化,免疫细胞化学鉴定分化后的细胞类型。主要观察指标:①三碘甲状腺原氨酸诱导神经干细胞分化前后细胞形态观察及类型鉴定。②神经干细胞分化过程中甲状腺激素受体mRNA表达的变化。结果:①诱导前细胞呈圆形,表面光滑,并逐渐聚集成神经细胞球,Nestin单染及Nestin BrdU双染均呈免疫细胞化学反应阳性。三碘甲状腺原氨酸诱导分化1周时,细胞大多呈单极或双极,有细长突起,神经丝蛋白、胶质纤维酸蛋白、半乳糖脑苷免疫细胞化学染色呈阳性;诱导3周后可见细胞核呈圆形,突起多且粗,细胞整体呈蜘蛛状,髓鞘碱性蛋白阳性率达80%。②甲状腺激素受体α1mRNA在诱导前神经干细胞状态时表达量最高,三碘甲状腺原氨酸诱导后表达量逐渐下降,至2周时达最低,此后表达量有所回升,但仍低于神经干细胞状态(F=32.49,P=0.008)。甲状腺激素受体α2mRNA表达变化趋势与甲状腺激素受体α1相同。甲状腺激素受体β1mRNA在神经干细胞状态时表达量最低,三碘甲状腺原氨酸诱导后表达量逐渐升高,2周时达最高,且超过同时间点甲状腺激素受体α1的表达(t=15.64,P=0.001),至诱导3周时表达水平降至最低。甲状腺激素受体α3mRNA在三碘甲状腺原氨酸诱导后呈下降趋势,2周时接近干细胞状态(F=51.94,P=0.378),此后又降至较低水平。结论:三碘甲状腺原氨酸能诱导神经干细胞分化为神经元、少突胶质细胞和星形胶质细胞,且分化过程中甲状腺激素受体mRNA存在不同时间顺序的表达。

Interventional effect of triiodothyronineon thyroid hormone receptor mRNA expression during the differentiation of human embryonic brain-derived neural stem cells

BACKGROUND: Triiodothyronine (T3) is an important regulation factor at the critical period of brain development. It maybe control the successive differentiation during the development of central nervous system (CNS).OBJECTIVE: To monitor the differentiation of neural stem cells (NSCs) induced by T3 and the thyroid hormone receptor (TR) mRNA expression changes.DESIGN: Open experiment.SETTING: Department of Pathology, Tianjin Medical College of Chinese People's Armed Police Force; Institute of Endocrinology of Tianjin Medical University.MATERIALS: This study was carried out in the Tianjin Medical University between January 2003 and March 2005.Ten-to-twelve-week-old aborted fetuses were obtained from the General Hospital of Tianjin Medical University with the approval of the local ethical committee. Informed consents were obtained from the mothers and their relatives.METHODS: ①Under the aseptic condition, the bilateral cortex of human fetal brain was removed and dissociated by brief mechanical trituration in D-Hanks. Then, 20 μg/L bFGF and 30 nmol/L T3 were used to induce the proliferation of NSCs and inoculated to poly-L-lysine-coated 24-well plate and 25 mL culture flask for routine culture at 1 ×109 L-1. The culture medium was DMEM/F12 serum-free complemented with N2. Half of the culture medium was changed every 48 hours.Seven days later, bFGF was discarded, only T3 was used for induction and differentiation. ② At 1, 2 and 3 weeks of culture, cells were collected, and RT-PCR was semiquantitatively used to detect TR mRNA expression changes at different stages of differentiation of NSCs. Isoforms were identified by immuocytochemistry.MAIN OUTCOME MEASURES: ①Cellular morphology observation and isoforms identification before and after differentiation of NSCs induced by T3. ② TR mRNA expression changes during the differentiation of NSCs.RESULTS: ①The hNSCs were round and had a smooth surface and gradually gathered to neurospheres. The proliferative hNSCs were nestin-positive and incorporated BrdU. When NSCs were induced by T3 for one week, most of the cells took on monopole or double poles, and had long and thin processes. The differentiated cells were neurofilament protein (NFP)-positive, galactocerebroside (GC)-positive or glial fibrillary acidic protein (GFAP)-positive. When NSCs were induced by T3 for three weeks, most of the cells were big, with unclear cell membrane, round nucleus, many thick processes which had many branches. The spider-like cells were scattered, and 80% of the cells were myelin basic protein-positive. ② TRα1 mRNA expression level was the highest before inducing NSCs. With the induction of T3, the expression level was decreased gradually, and was the lowest at 2 weeks, and then was rebounded gradually, but the final level was still lower than that of NSC (F =32.49, P =0.008). The tendency of TRα2 mRNA expression alteration was identical with that of TRα1 mRNA. TRβ1 mRNA expression level was the lowest in NSC, was increased gradually with the induction of T3 and attained the highest level at 2 weeks of induction of T3. Furthermore, the expression level of TRβ1 mRNA was also higher than that of TRα1 at the same time (t =15.64,P =0.001), and it reached the lowest level at 3 weeks of the induction. TRα3 expression level was firstly decreased after the differentiation induced by T3, and was close to the expression level of NSC at 2 weeks of induction (F =51.94, P =0.378), then was decreased to lower lever.CONCLUSION: T3 can induce NSC to differentiate into neurons, oligodendrocyte and astrocytes. TR mRNAs are expressed in different time intervals during the differentiation of NSCs.