| Objective Platelet-Derived Growth Factor (PDGF) and Thyroidhormone(T3) have important effects on regulating the proliferation and destined differation of the neural stem cell (NSC). There is an interaction between them. The Thyroid hormone receptors(THR) is the hinge of the interaction. PDGF and Tj guarantee the CNS normal development through co-regulating the THRs . So we do experiments to understand ulteriorly the relationship amomg NSC\ PDGF, T3 andTHR..M ethods We successfully expanded human embryonic brain-derivedNSC into spheres with mitogens. The NSC were identified by immunocytochemistry method, BrdU labeling and cell cloning were used to observe the proliferation ability of NSC. PDGF x T3 were separately used to induce the differetiation of NSC. After the NSC differentiated. We detected the transcription and translation change of different THRs genes by semiquantitative reverse transcription-polymerse chain reaction(RT-PCR) analysisJELISA respectively. At same time the differentiated cell were identified by immunocytochemistry method.Results (l)NSC induced by mitogens were nestin-positive andincorporated by BrdU. When NSC differentiated, the cell were NF-positive > GC-positive or GFAP-positive.(2) It was the best culture condition to promote the growth of NSC clone that seeding the cell at a density of 2X 105cells/mL in the conditioned medium containing FGF(10ng/mL) and PDGF(20 ng/mL) (3) In the first and second trimester human fatal brain, different THRs were expressed incerebrunu cerebellunu brain stenu hippocampuSx spinal cord x thalamus. Inthe first trimester human fatal brain, the THR a isoforms were higher expressed in cerebrum, cerebellum, hippocampus than other brain region, however, THR P i was lower expressed than THR a i and THR a 3- In the second trimester human fatal brain, The expression of THR a , has no difference among the different brain region. THR 3 i was relatively higher expressed in cerebrum, cerebellum, hippocampus, but THR a 2 was relatively higher expressed in brain stem, spinal cord. Furthermore, THR u , was lower expressed than THR P i and THR a 2. (4) When we amplified the THR a 2 by PCR, the other sequence was amplified at same time, it is about lOObp less than the THR a 2, so we cloned it into pGEM-T vector to definitude its sequence. The results show the sequence is identical to THR a 2 with the exception that it is missing amino acids 371-412 in the a 2 sequence, we assure that it is the human cerebrum THR a 3. (5) In neuron, THRs expression is much higher,THRa 2> a ,> p ,,In astrocyte,THRs expression is lower, THR a 2> 3 , > a ,; (5) After the NSC was induced by TV MBP positive cell is about 80%. The spatial and temporal expressions of different THRs mRNAs exist in the oligodendrocyte development. (6) After the NSC was induced by PDGF,NF positive cell is about 80%, THR a ,, u 2, Pi transcription was up-regulated about 30-50%, 20-40%, 10-30%,respectively compared with the NSC induced by 10%serum.THRs protein expression is NSOPDGF Induced>T3 Induced..Conclusion (1) NSC cultured in our experiment have the potential toself-renew and proliferation , as well as differentiation (2) The spatial and temporal expressions of different THRs mRNAs exist in CNS development. (3) We firstly prove the nucleotide sequence of human cerebrum THR a 3. (4) Different THRs were highly expressed in NSC and down-regulated once theNSC differentiate. THRs expression is different in the neuron and astrocyte. (5) Ta can induced NSC developing to oligodendrocyte. TI exerts distinct effects through the spatial and temporal expressions of different THRs mRNAs. (6) PDGF may initiate neuronal differentiation in NSC. NSC induced by PDGF, transcription and translation level of different THRs genes were up-regulated compared with the NSC induced by serum.The results made a great foundation for the further research of the important role of THRs during the CNS development, and have much clinical significance on therapy of cretinism^... |
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