| 1. SummarizeNeural stem cells (NSCs) are such a kind of cells which have the abilities of replicating themselves and multiple differential potential. NSCs can be considered as a substitute method to cure some diseases in central nerve system(CNS), including degeneration disease, brain (or spinal cord) wound and brain tumours. Therefore, it is great important either to built a stable and credible NSCs cultivation model in vitro or to explore the proliferation regulation and differentiation mechanism. For the embryonic NSCs constrained by it's source, immunological repulsion and ethical moral problems, so the bone marrow stromal cells-derived NSCs (BMSCs-D-NSCs) play an important role in the utilization of "self-derived neural stem cells" and would be a ideal cells resources for cells-replacing therapy of the nerve obstacle diseases.This experiment aimed at exploring the conditions of BMSCs differentiating into NSCs and mature neural cells, observing the effects of different cell growth factors on NSCs proliferation and differertiation. BMSCs were obtained from the marrow of rhesus monkey thighbone and cultured under conditions of NSCs medium. Nestin, CD 133, Neuron-specific endase (NSE) , p-tublin and Glial fibrillary acidicprotein(GFAP) were served as makers to identify the antigen character of cells. Patch-clamp was used to detect the electrophysiological properties of differentiated neural cells. These results can provide evidence for adult NSCs utilization in neural science field.The objective of NSCs' studying is to restore the wound or recession function of CNS with their plasticity in structure and function. So the function maturity of neuron-like cells differentiated from the BMSCs-D-NSCs is the most important. We had separated and cultivated NSCs from BMSCs, and induced them into neuron-like cells in our previous experiment. But we didn't know whether these neuron-like cells possess the intrinsic electrophysiological functions as neurons besides their neuron-like morphological characteristic. The patch-clamp is a technique developed from voltage-clamp by Nehetr and Sakmann in 1976, which can record the ion-channels currents and electric activity of cell-membrane, and shows some distinct virtues such as recording the cell-membrane ion-currents directly at the same time of maintaining the membrane voltage invariability during the course of cell activity, reflecting the activity of single (or multiple) channel(s) consequently. It is a great change to connect cell science with molecule physiology, which makes various fields of neural science into a whole. The patch-clamp can provide credible evidence and effective test way for future utilization and study of NSCs by detecting the electrophysiological properties of NSCs and the differentiated cells in various developmental phases, and then evaluate whether these neuron-like cells possess the similar function to normal neurons. This experiment detected the electrophysiological properties of BMSCs-D-NSCs and their differentiated neuron-like cells, for the first time in domestic. The results showed that the neuron-like cells derived from BMSCs-D-NSCs have active electrophysiological properties. It displayed not only tetrodotoxin(TTX) sensitive and quickly activatedVoltage-dependent inward Na+ channels, but also outward K+ currents. The K+ showed two types: instantaneous K+ currents and delay rectified one, which were similar to normal neuron's K+ currents. In conclusion, BMSCs could differentiate into mature neuron-like cells in vitro in appropriate conditions and these neuron-like cells posesssed the electrophysiological properties similar to normal neuron. 2. Main methods and results(1) Separation, purification and identification of BMSCs-D-NSCs:Object: Getting the BMSCs from rhesus bone marrow.Method: The BMSCs were separated and purified by gradient density-centrifugation methods, cultivated in NSCs medium, and then induced into NSCs in vitro. The antigen properties of BMSCs-D-NSCs and the differentiated cells at various periods were identified with immunocytochemistry method.Result: BMSCs could proliferate and differentiate into NSCs in vitro, which showed middling or big round with possitive express of Nestin and CD133 antigens. The BMSCs-D-NSCs also could further differentiate into neural cells including neuron- and glia-like cells, expressing Nestin, CD 133, NSE and GFAP respectively. This showed that BMSCs could be induced into NSCs in appropriate conditions in vitro, and they could further differentiate into neural cells.(2) The stimulative effect of Basic Fibroblast Growth Factor (bFGF) and Epidermal Growth Factor (EGF) on BMSCs-D-NSCs in vitro:Object: To explore the conditions of BMSCs differentiated into NSCs and to compare the effects of some growth factors such as bFGF and EGF on NSCs growth curve.Methods: BMSCs were obtained from the bone marrow of monkey thighbone and inoculated in 24-aperture cultivable utensils. The concentration of cultivated cells was 0.5xl06/mL, the serum concentration was 15%. Cells were cultivated at37°C, 50mL/L C02 and saturated humidity, then changed the culture medium completely at the 72nd hour after cultivated and added bFGF(20ng/mL) and/or EGF(20ng/mL) to induce BMSCs proliferation and differentiation. The cells with growth factors-free were as the control. Nestin and CD 133 antigen were served as makers to identify NSCs. Take count of cells with cell-counter everyday for 18d consecutively and protract cells growth curve.Results: Most cells began to express Nestin and CD 133 antigen at the 6th day after culture. Henceforth, these cells began to proliferate rapidly. The experimental cells numbers increased much more rapidly than the control. The cells numbers of bFGF+EGF group increased from (96.30±8.78) xlO4/mL to (1407.90±26.62) xlO4/mL. The beginning proliferation time of the cells in the bFGF+EGF group is earlier 2 days than in the control. In conclusions, bFGF combining with EGF could effectively improve the proliferation effects of NSCs, and make the proliferation beginning time earlier than the control. bFGF and EGF also had the effects on making BMSCs differentiate into NSCs and then neural cells in vitro. (3) Studies on conditions which induce BMSCs-D-NSCs to differentiate into neural cells in vitro:Object: To explore the conditions of inducing BMSCs differentiate into NSCs and further mature neural cells, and to observe the stimulative effects of Retinoic acid (RA), glial cell-line derived neurotrophic factor (GDNF), brain derived neurotrophic factor (BDNF) and 2-mercaptoethanol (2-ME) at different concentration of serum on BMSCs-D-NSCs differentiation.Method: BMSCs were obtained from the monkey thighbone and cultured in NSCs culture medium at 37°C, 50mL/L C02 and saturated humidity. The concentration of cultivated cells was 0.5xl06/mL. The serum was at 10% of final concentration. At the 72nd hour after cultivation, bFGF and EGF were added intoculture medium to induce BMSCs proliferation and differentiation. When the cells began to proliferate largely, they were subcultured at a rate of 1 = 2. And then, different concentration of serum(0, 2.5, 5, 10%), RA, GDNF, BDNF and 2-ME were added into culture medium according to various groups. The cells in most of groups were induced for 96h except for 2-ME group, which was preinduced with lmmol/L of 2-ME for 24h and then induced by 2mmol/L 2-ME with serum-free medium for 72h. Nestin, CD 133, NSE, P-tublin and GFAP were served as makers to identify the antigen character of all these cells.Results: Most cells expressed the antigens of Nestin and CD133 at the 8th day after cultured. Neuron-like cells were found at the 3rd days after induced by RA, GDNF, BDNF and 2-ME, which expressed NSE, p-tublin and GFAP respectively. RA+GDNF with low concentration serum (2.5%) was the most effective conditions for cells' differentiation compared to other induced conditions in vitro in this experiment. This suggested that BMSCs-D-NSCs could differentiate into mature neural cells efficiently in vitro by combining RA+GDNF with low concentration of serum (2.5%).(4) The study on electrophysiological properties of neuron-like cells differentiated from monkey BMSCs-D-NSCs:Object: To compare the electrophysiological properties between NSCs and neuron-like cells differentiated from BMSCs.Method: BMSCs were obtained from rhesus bone marrow, cultured and induced under conditions of NSCs medium with some nerve growth factors, including bFGF, EGF, RA, BDNF and GDNF, etc. Nestin, NSE, P-tublin and GFAP were respectively served as makers to identify the antigen characters of various phases cells. Patch-clamp was used to detect the electrophysiological properties of BMSCs-D-NSCs and differentiated neuron-like cells.Result: The member properties changed a lot between undifferentiated cells and differentiated neuron-like cells. Some neuron-like cells showed TTX-sensitive and quickly activated Voltage-dependent Na+ channels, but all the undifferentiated cells did not express inward currents. In conclusion, BMSCs-D-NSCs Could differentiate into mature neuron-like cells in vitro in appropriate conditions and these cells have the electrophysiological properties similar to normal neuron. 3. ConclusionBMSCs could be induced to NSCs in appropriate conditions in vitro. bFGF combining with EGF could effectively improve BMSCs-D-NSCs proliferation, the beginning time of which was earlier than the one in control. bFGF and EGF also possessed the effects on making BMSCs-D-NSCs differentiate into neural cells in vitro in this experiment. In addition, BMSCs could differentiate into mature neuron-like cells efficiently in vitro under conditions of combining RA+GDNF with low concentration of serum (2.5%), and these neural-like cells showed the electrophysiological properties similar to normal neuron. This experiment implies clearly that these neuron-like cells not only have the neuron-shape, expressed the neuron's antigen character, but also have the electrophysiological properties similar to normal neuron.
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