The Expression Of TRAIL And Its Receptors On Developing Oligodendrocyte And Their Effects On The Apoptosis Of OPC

Premature white matter injury is a major contributor to neonatal mortality and morbidity, often leading to mental retardation and sensory-motor impairment, which seriously endanger the life quality of preterm children. Currently, there is no effective way to prevent and manage premature white matter injury. Oligodendrocyte precursor cells (OPC) is the major target cells during white matter damage in preterm children, while apoptosis is the main form of OPC death. Death receptor pathway is the main way to OPC apoptosis, which is mediated by tumor necrosis factor superfamily. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of tumor necrosis factor superfamily. Recent evidences showed that TRAIL and its receptors were widely expressed in normal cells and were involved in cell survival and immune surveillance. Despite lots of researches have focused on the role of TRAIL receptors in the demyelinating diseases, little is known about the expression of TRAIL receptor during oligodendrocyte development and the protective effect on OPC by regulating TRAIL receptors. Therefore, further investigation of the role of TRAIL and its receptors in oligodendrocyte development can surely get a better understanding to the pathogenesis and molecule mechanisms of PWMI, thus lead us to some bright new ideas and more effective ways to prevent and manage this life-threatening diseases.Part Ⅰ. Cultivation and identification of human oligodendrocyte lineage cells in vitroHuman oligodendrocyte precursor cell-oligospheres were cultured in vitro by different medium. Oligodendrocyte lineage cells were identified by immmunofluorecence technique. Oligoendrocyte progenitor cells were PDGFRa-positive; pre-oligodendrocytes were O4-positive; mmature oligodendrocytes were O1-positive and mature oligodendrocytes were MBP-positive. The purity of cultured cells were assessed by using flow cytometry. In our cell culture systems, the human OPC spheres which cultured in OsM contained 97.06% PDGFRa+/O4-cells. When human OPCs had differentiated for 1 week in the differential CDM, the cells were 96.01% of O4+/O1-pre-OLs. OPCs in differential CDM supplemented with the growth factor cocktail survived and differentiated into immature OLs by 3 weeks, with 95.30% of the cells were both O4+ and O1+ positive. OPC spheres cultured in OPCDM and had differentiated into mature OLs by 4 weeks. Under this culture condition,91.60% of the cells were MBP+mature OLs. Human oligodendrocyte precursor cell-oligospheres have the biological characteristics of oligodendrocyte progenitor cells, and can different into mature oligodendrocytes in particular medium. High ratios of purified oligodendrocyte lineage cells can be obtained by cultured in particular medium.Part II. The expression and the role of TRAIL/-Rs on developing human oligodendrocyteNow, we got highly purified oligodendrocyte lineage cells, the expression of TRAIL and its receptors were detected by Real-Time Reverse Transcription-PCR and westernblot. Four TRAIL receptor isoforms could be identified in the OL lineage, conversely, no TRAIL expression could be identified in all OL developmental stages. TRAIL-R1 and -R2 were expressed strongly on OPCs and pre-OLs, while TRAIL-R3 and -R4 were expressed relatively high on immature OLs and mature OLs. Annexin and PI staining analyzed by flow cytometry was performed to detect the sensitivity of OL lineage to TRAIL induced apoptosis. TRAIL-induced human OL lineage was apoptotic in both a time-and a concentration-dependent manners. OPCs and pre-OLs were very sensitive to a higher concentration of TRAIL, whereas immature and mature OLs showed resistance to TRAIL treatment.Part Ⅲ. The protective effect on OPC by down regulating DR4/DR5 or up regulating DcR1/DcR2To test whether the discrepancy of sensitivity of human OL lineage cells to TRAIL-induced apoptosis was due to the different expression of TRAIL receptors, OPCs were transfected with TRAIL-DR4/DR5 siRNA or TRAIL-DcR1/DcR2 pcDNA3.1. Annexin V-FITC and PI staining were used to determine the percentage of cells undergoing apoptosis after exposure to TRAIL. The cells were incubated with TRAIL at a concentration range of 20-20000 ng/ml before Annexin V-FITC and PI staining being carried out. Human OPCs transfected with TRAIL-DR4 siRNA or TRAIL-DcR2 pcDNA3.1 showed significant reduction of cell death compared to empty plasmid-treated cells. TRAIL-induced human OPCs death is mediated by TRAIL-DR4, and TRAIL-DcR2 is the main protective receptor of OPC.