| Recently, there are great improvements on the researches of axon regeneration after spinal cord injury (SCI). Cell transplantation was thought to be one of the hopeful treatments. Olfactory ensheathing cells (OECs) have attracted great attention in recent years because of its special biological characteristics. Previous studies have demonstrated that after transplanted into the injured spinal cord, OECs can arrange into cell-chain, which promotes axon regeneration and lead it passing the injury part. OECs can also promote neurons growth and axonal extension. It has showed that OECs can promote descent conducting pathway regeneration. OECs transplantation is supposed to be the most promising treatment of SCI.There are five parts in this study. The first part is to study the effects of OECs on growth of the cultured DRGn. DRGn from newly-born rat were digested with trypsin to produce cell suspension, and then co-cultured with OECs of different densities (8×105/ml, 4×105/ml, 2×105/ml, 105/ml, 104/ml). The growth conditions of cells were observed by phase-contract microscope, NSE and GAP43 immunocytochemical staining. The activities of neurons were then detected. After 3d co-cultured, we found that the cell body of neurons were significantly larger with longer prominences and elevated activities than negative control. Conclusion: When co-cultured, OECs could promote the growth of DRGn.The second is to study the effects of OECs on apoptosis of cultured DRGn induced by H2O2. Cultured DRGn were induced apoptosis by treated with 1mmol/L H2O2, and then co-cultured with OECs of different density (104/ml, 105/ml, 2×105/ml, 8×105/ml), or with OECs at different time after adding H2O2 (0h, 4h, 8h, 12h, 24h). The neurons apoptosis were observed with Tunel staining and the rates of apoptosis were measured by flow cytometry (FCM). The activities of neurons were measured using MTT assays. We found that OECs significantly inhibited apoptosis of neurons. The rates of apoptosis of DRGn which was co-cultured with OECs were obviously lower than positive control. The apoptosis rates of DRGn which was co-cultured with OECs at 0h, 4h, 8h and 12h after adding H2O2 were obviously lower than the 24h group and positive control. The activities of neurons have the similar results. Conclusion: OECs can prevent DRGn from H2O2 induced apoptosis with density-dependent effect and time-dependent effect.The third is to study the effects of OECs on apoptosis-related genes expression in DRGn with H2O2 induced apoptosis. Cultured DRGn were induced apoptosis by treated with 1mmol/L H2O2, and then co-cultured with olfactory ensheathing cells of 2×105/ml. The apoptosis rates were measured by FCM. The expression of apoptosis-related genes (FADD, caspase-3, Bcl-2, BAX, and Bim) were measured using RT-PCR and Western-blot. We found that the apoptosis rates of DRGn which was co-cultured with OECs were obviously lower than the positive control. The expression of caspase-3, BAX and Bim in DRGn which was co-cultured with OECs were obviously lower than positive control, and the expression of Bcl-2 were obviously higher than positive control. Conclusion: OECs could prevent DRGn from apoptosis by down-regulating expression of caspase-3, BAX, Bim and up-regulating expression of Bcl-2.The fourth is to study the effects of OECs after transplanted into the injuried spinal cord of rats. The SCI model of rat was established, then 5ul cell suspension of OECs stained by Hoechst 33342 were injected into the injured spinal cord. The growth conditions of OECs were observed two weeks after transplantation. The axon regeneration was observed by NSE and GAP43 immunocytochemical staining. The motor function of animal was estimated by BBB values. We found that the OECs were alive in spinal cord at 2w after transplantation. The apoptosis of neurons in spinal cord of transplantation group were obviously less than the positive control. The number of regeneration of axon of transplantation group was significant higher than positive control. But the BBB values of transplantation group were similar with positive control. Conclusion: After transplantation, OECs can promote the growth of axon, prevent cavity formation and glial scar formation, and prevent neurons from apoptosis.The fifth is the isolation, culture and purification of OECs from human olfactory mucosa. OECs were obtained by dissection of olfactory mucosa and purified based on the different attachment rates among various types of cells. Immunocytochemistry were used to study the morphological characteristics of OCEs. The purity of OECs was about 75% after 14 days culture. Immunocytochemistry results revealed that the purified OCEs were immunoreactive to NGFR p75. Most cells were bipolar or tripolar cells with thin and long prominences, and only a few were flat cells. Conclusion: We successfully obtained OECs from human olfactory mucosa. The OECs from olfactory mucosa is an accessible source of tissue for autologous grafting in treatment of human SCI.
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