| Spinal cord injury (SCI) is a common disease in clinic, which is caused by all kinds of accidents and degeneration of spine, followed by hemorrhage, edema, necrosis, capsule formation and gumnosis of spinal cord. All these result in different disabilities. For a long time, attempting to find satisfied approaches to treat SCI is a challenge. Current treatments include operation and drugs, the drugs we use should be able to protect neurons and promote function recovery. Neurotrophic factor (NTF) is the target drug in recent researches.The NTFs that always used include nerve growth factor (NGF), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3(NT-3), neurotrophin-4/5(NT-4/5) et al. NTFs can protect neurons, stimulate neurite outgrowth, promote regeneration and functional recovery. NGF, BDNF and bFGF have served as neurotrophic factors in clinic and researches for many years, their functional mechanisms and signal transduction pathways have been studied by various researchers. Additionally, production of these NTFs is much easier than before, and the prices are much lower. So NGF, BDNF and bFGF are chosen as target NTFs.In this study, neuron culturing technology was used, different NTFs and their couples were added in each group. We evaluated the biological characteristics of neurons, observed the outgrowth of neurites. The purpose is to determine which combined NTF couple produce the best results, in order to guide the use of NTF in clinic and research.In the first part of this study, cells were obtained from new born SD rats by enzymatic digestion. During the process of culture, cells were purified by cytarabine. After that different NTFs and their couples were added in each group with the concentration of 50ng/ml, while DMEM/F12 as control. Phase contrast observation was done. Cells were evaluated byβ-tubulin3 and Hoechst staining, percentage of neurons was determined, the length of neuritis was measured,and the numbers of neuron cells and nuclears were determined.The results showed that:1. After purified by cytarabine, the percentage of neurons was above 90%. The neurons presented high adherence rate and uniform appearance. So we can obtain purified rat spinal cord neurons by this method.2. Different NTFs and their couples were added in each group, while DMEM/F12 as control. The results showed that NGF, bFGF and BDNF can enhance rat neuronal neurite outgrowth and survival. Combined use of any two can produce better results, especially for BDNF and bFGF. In the second part, 9 groups of BDNF+bFGF with different concentrations were added in cultured neurons. The same observation and evaluation were done.We concluded that:1. Combined use of BDNF and bFGF could effectively enhance rat spinal cord neuron survival. To certain extent of concentration (10-100 ng/ml), the effect was dose-dependent. High concentration of NTF produced better results.2. The length of neurites distinguished between any two groups. The results of following groups: BDNF50ng/ml+bFGF100ng/ml, BDNF100ng/ml+bFGF50ng/ml, BDNF100ng/ml+bFGF100ng/ml were superior to others. According to the two parts, we can conclude that: BDNF100ng/ml+bFGF100ng/ml produced the best results.In conclusion, we successfully obtained purified spinal cord neurons from new born SD rats. We evaluated the biological characteristics of neurons, observed the outgrowth of neuritis, determined which group of NTFs produced the best results. The results showed that: BDNF+bFGF presented better characteristics than other combined effects of NTF, additionally, to certain extent of concentration, the effect of neuron protection was dose-dependent, BDNF100ng/ml+bFGF100ng/ml produced the best results.
|
PDF Full Text Request