Gene Expression Changes In Oligodendrocytes After Ionizing Radiation In Vitro

Ionizing radiation remains a major treatment modality for primary and metastatic tumors of the central nervous system (CNS) and the head-neck. In addition, application of radiation in some other nonneoplastic disorders of the brain is increasing. In the treatment of tumors within or adjacent to the brain and spinal cord, this complications is associated with a high risk of morbidity, which can be especially devastating to patients. For this reason, this effect of radiation on the CNS has been the basis for extensive research. The pathogenesis and the early stage diagnosis of the radiation-induced CNS injury were insufficient at the present time. Studying extensively will be significantly to the development of radiation oncology.Pathological analysis of radiation-induced CNS injury confirmed that vascular abnormalities appeared atypical in the early stage. Late changes of the brain can be distinguished: Demyelination, proliferative and degenerative glial reactions, capillary occlusion and white matter necrosis. Because vascular abnormalities and demyelination dominate the histological presentation of radiation injury in the CNS, the vasculature and the oligodendrocytes lineage have traditionally been considered the primary radiation targets in the CNS. With the development of these research works, at least four factors contribute to the development of CNS toxicity: Damage to vessel structures; deletion of oligodendrocyte-2 astrocyte progenitors (O-2A) and mature oligodendrocytes; deletion of neural stem cell populations in the hippocampus, cerebellum and cortex; generalized alterations of cytokine expression.Both recent laboratory and clinical data have been used to establish the mechanisms of oligodendrocytes apoptosis, radiation effects of O-2A and mature oligodendrocytes, the response to dose per fraction and number of fractions. In contrast, the relationships between the radiation response to oligodendrocytes and the mechanisms of demyelination remain unclear. The cellular and molecular processes responsible for the histological presentation of radiation induced CNS injury remain poorly defined. There are a lot of reports in neurological and neurobiological research that oligodendrocytes were also targets of injury in several pathological conditions, such as trauma, ischemia, hypoxia, abnormal immune reaction. Recent studies indicated that the oligodendrocytes lineage express the cell-specific antigens in the specific stage. The methods have been established to obtaining more pure O-2A progenitor cells. After pathological stimulation these O-2A progenitor cells were proliferated, migrated and differentiated, which provided new avenues to explore the new therapy strategy for the CNS injury.PartⅠThe method for culture of rats'pure oligodendrocytesObjective To establish the method of obtaining more pure O-2A progenitor cells, so as to build oligodendrocytes culture for subsequentant microarray research.Methods Based on the differential properties of cellular adhesions and developmental time–course, O-2A progenitor cells were isolated at the onset of the layer of T1A with a standard shaking method and then purified with the differential adhesion method combining with using the growth factors, and further O-2A progenitor cells differentiated into oligodendrocytes depending on the culture medium. Immunocytochemistry labeling for cell-specific antigens had been done to characterize oligodendrocytes.Results The mature oligodendrocytes expressed MBP or GalC, whereas did not express the A2B5 antigens. The purity of oligodendrocytes was 98% and the quantities were 5×106. PartⅡEarly gene expression profile in oligodendrocytes after exposure to ionizing radiation in vitroObjective To characterize the cellular functions associated with the altered transcript profiles of oligodendrocytes exposed to in vitro X-ray irradiation.Methods The total RNA was extracted from oligodendrocytes in unirradiated control (Group 1) and at 1 hour (Group 2), 4 hour (Group 3) after 6MV X-ray irradiation at 10Gy, hybridized to Affymetrix RAT230 2.0 Array, and evaluated the difference and screening the genes expressed differently.Results The expression rate of gene chips in three groups was 42.81%, 41.64% and 45.31% respectively. The genes with 2-fold expressed differentially include 27 genes up-regulated and 164 genes down-regulated in Group 2 vs. Group 1, 295 genes up-regulated and 302 genes down-regulated in Group 3 vs. Group 1, 510 genes up-regulated and 320 genes down-regulated in Group 3 vs. Group 2. According to functional categories results, there were 1079 genes expressed. The numbers of functional classification were 79. The annotated genes functional classification was associated with oligodendrocytes injury, repair and protection, such as cellular physiological process, apoptosis or tumor, cell cycle, metabolism, cell communication, receptor binding, et al.PartⅢReal-time RT-PCR analysis of functional gene expression in early radiation-induced CNS injuryObjective To validate the microarray results and discuss these significantly modulated genes in oligodendrocytes.Methods Quantitative real-time RT-PCR was performed to analysis 9 genes mRNA for selected radiation-modulated genes.Results Comparing with the results between the mircoarray and the real-time RT-PCR, the coincidence rate was 81.5%.ΔCt value of 9 genes included:①glial fibrillary acidic protein: 1.3777, 2.2266, 5.3992;②myelin basic protein: -3.4061, -2.6133, -2.0232;③apolipoprotein E: 12.1203, 14.7452, 17.2264;④thyrotropin releasing hormone: 5.8994, 8.2740, 2.4709;⑤thyroid hormone receptorα: 6.3269, 5.8081, 6.4793;⑥transforming growth factor, beta 2: 4.6782, 5.2108, 5.1181;⑦early growth response 2: 10.0840, 7.4766, 8.5754;⑧neural cell adhesion molecule 1: 3.7024, 3.9211, 5.6716;⑨S100 protein, beta polypeptide: 2.5607, 5.5696, 4.2581.Conclusions1. Pure oligodendrocytess expressed MBP or GalC positive and A2B5 negative epitope. The purity and the quantities matched the criterion of microarray.2. Radiation-induced changes in oligodendrocytes gene expression took place early after irradiation. The findings suggest that irradiation of the oligodendrocytes induced the expression of genes involved in damaging, protective and reparative functions, while the dynamic balance of these modulated gene decided the ultimate radiation-induced pathophysiological effects.3. The results of real-time RT-PCR coincided with the results of microarray. Both of them were approximate in the testing particularity, while real-time RT-PCR was superior to microarray in the testing sensitivity. These real-time RT-PCR results revealed that gene expression changed during the early radiation-induced CNS injury, and that, down-modulating GFAP, MBP, apoE, TGFβ2, NCAM-1 mRNA coincided with the mechanisms of the oligodendrocytes damaging, while up-modulating TRH, THRα, EGR2 mRNA and down-modulating S100βmRNA coincided with the mechanisms of the oligodendrocytes protective and reparative functions.