| ObjectiveTo induce adult adipose-derived stromal cells(ADSCs) differentiate into neurons in vitro, to detect the physiological function of neurons, and the apoptosis which occurred in the process of adult ADSCs differentiating into neurons.Methods1. With reference to methods of Zuk's, Ye CQ's and others', adult adipose- derived stromal cells were isolated and cultured, then subcultured once the cells were confluent on the wall. Taking the 3rd-6th passaged cells into induction and differentiation, the cell morphology was observed under inverted phase contrast microscope.β-mercaptoethanol was used to induce adult ADSCs to differentiate into neurons. And according to various induction time, the cells were divided into several groups, including un-induction group, pre-induction group, 1 hour induction group, 3 hours induction group, 5 hours induction group and 8 hours induction group. The morphology of the cells in each group was found with inverted phase contrast microscope and then the percentage of differentiation of the cells was calculated .2. By MTT method to detect the growth curve of cells for induced adult ADSCs.3. By immunocytochemistry staining method to detect the expression of positive cells of neural precursor cells marker neuroepithelial stem cell protein, neuronal marker microtuble-associated protein-2, neuron specific endolase and glial cell marker glial fibrillary acidic protein of each time point.4. The ultrastructure of neural precursor cells, neurons, and apoptosis cells were observed with transmission electron microscope. 5. Membrane potential changes of adult ADSCs and neurons induced for 5 hours were observed under the inverted fluorescence microscope.6. By TUNEL technique to detect the apoptosis index after induction for 1, 3, 5, 8 hours.7. Image acquisition and statistical analysis: To calculate the rate of immunocytochemistry and TUNEL positive cells per high-power light microscope, the numbers of total cells and positive cells were counted, each slice was counted five times with a total number of three slices (that is to count 15 times), and then the average rate of neural cell markers were calculated positive expression. MicroplateReader was used to detect the optical density of induced adult ADSCs for each group, each one has six samples, every group was detect 3 times. The cell ultrastructure was observed through a H7500 transmission electron microscope. Membrane potential of cells were observed by Olympus confocal laser scanning microscopy. All the experimental data were built a database with EXCEL 2003, and then analyzed statistically with the statistical software package SPSS 13.0. The mean of different time points in the same group was analyzed through single factor analysis of variance. The acquired data was characterized byχ±s, and deviation for P<0.05 had statistical significance.Results1. Adult ADSCs primary cultures began to adhere within 24 hours, the morphology of cells was irregular, round and short spindle, cells appeared to be fusiform at 48 hours, and some cells had processes, exhibiting a fibroblast-like morphology. At 7-10 days, a large number of cells showed a long spindle shape, revealing a whirlpool arrangement. Cells were adherent at 12 hours after being passaged. The cells showed a fibroblast-like morphology at 24 hours, exhibiting uniform morphology after 3-6 passages, at the same time cells proliferation was active. After induction for 24 hours by pre-induction medium, some adult ADSCs began to change, with short processes extending. Then the primary medium was replaced with formal induction medium. After 1 hour, the nuclear was rounded, and theˉ cytoplasm retracted towards the nuclear, some cells extended long axon-like processes. At 3 hours, the cytoplasmic refractive index increased, extracellular halation was clearly observed. At 5 hours, the cells exhibited a typical morphology of neuron cells, that was a large round nuclear, a small conical cell body, which had retracted to the nuclear, heighening refraction of the cytoplasmic, more longer processes, and at the end of processes some of which appeared to be branched, and connected as network. At 8 hours, the morphology of differentiated cells was similar to that at 5 hours. After 8 hours, the cells began to die gradually, the intracytoplasmic emerged vacuoles, and some cells separated from the culture bottle and floated. To about 12 hours most cells died.2. MTT method was used to detect the living cells of each time point. With induction time extending, survival cells reduced. However, the cell growth in induction for 1 to 5 hours was stable(P>0.05). But the number of cells inducted for 8 hours was significantly lower than that before(P<0.05).3. The result of immunocytochemistry showed, that the positive cells of neuroepithelial stem cell protein expression in 3 hours induction group, the ratio of positive expression reached the peak, was (86.25±4.82)%, then the expression of positive cells decreased(P<0.05). The expression of microtuble-associated protein-2 and neuron specific endolase peaked at 5 hours induction, were (77.69±1.53)% and (85.92±3.07)% (P<0.05). The expression of glial fibrillary acidic protein in un-induction, pre-induction, induction for 1, 3, 5, 8 hours groups were negative.4. There were many protuberance on the cell membrane under transmission electron microscopy. There were plenty of organelles in the neural precursor cells. The neural precursor cells had a large size nucleus, large nucleoplasmic index, much extended chromatin, and less condensed chromatin. The nucleus had double-layer nuclear envelope, more nuclear pore on the nuclear envelope. At the time point of 5 hours of differentiation,the specific organelle Nissl body of neurons was seen under the transmission electron microscopy, and the apoptosis cells with typical ultrastructural features were also seen.5. Results of membrane potential showed that at the time point of 5 hours of differentiation, neurons have higher resting membrane potential, it could be produce rapid polarization after stimulation of high potassium solution(P<0.05).6. The result indicated that as the induction time extending, the survival cells gradually reduced. In the process of induction, the percentage of apoptosis cells was significantly increased(P<0.05).Conclusions1. At the time point of 5 hours of differentiation, neurons expressed mature K+ channels, when they were stimulated by high potassium solution, the membrane depolarized.2. Neurons differentiated from adult ADSCs could live short time, as the induction time extending the survival cells gradually reduced. |
