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

Chunrong LIU; Lanying LI; Ben LIU; Xiaoyi ZANG; Zupei CHEN

ABSTRACT

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.