Induce Rat Bone Marrow Mesenchymal Stem Cells Differentiate Into GABAergic Neurons In Vitro

Bone marrow mesenchymal stem cells(BMSCs), as the somatic stem cells derived from the mesoderm which have multi-directional differentiation potential , easily purified from the culture and can avoid the immune barriers to transplantation, and break through the ethical constraints of NSC and ESC research, recently have become a hot trend in stem cells study. Under appropriate conditions BMSCs can differentiating into bone cells, cartilage cells, fat cells, neural cells and so on, especially the direction of the trend of differentiating into neural cells which has brought new gospel for patients who are central nervous system injured in clinical work. Moreover recent studies have shown that BMSCs have some effects on rat epilepsy model, gama aminobutyric acid(GABA)ergic neurons as a key pathogenic factors to epilepsy happened, whether BMSCs own the ability of differentiating into GABAergic neurons have not a definite coverage, while the function of the induced GABAergic neurons in vitro remains to be further studied. In this paper, resort to the previous scholars about studying the differentiation of NSC and BMSCs and characteristics of self-induced differentiation of GABAergic neurons, successfully induced the BMSCs to GABAergic in vitro. Although studies have shown that the efficiency and corresponding function of the differentiated GABAergic neurons is limited, the initial results of this research has laid a theoretical foundation for the treatment of epilepsy and opened a new direction of the cell transplantation for brain lesions. In this paper, the two aspects of isolation and culture of BMSCs, induced differentiation of BMSCs were researched as following:1. Isolation and culture of BMSCs.Objective To obtain purified BMSCs. Methods took 10ml sterile syringes to wash rat bone marrow cavity with PBS towards the SD rats age of 4-6 week, weighing about 100g, collected about 5ml of bone marrow fluid in the tube, along the tube wall slowly added an equal volume of Percoll (1.073g/ml) separation of liquid, then to take the speed of 2500rpm to high-speedly centrifuge 20min, carefully swabbed the thin interface of the cells, transferred the cells to tube containing PBS, blew the cells with pipette and centrifuged with 1000rpm about 5min, drained the PBS and added DNEM/F12 containing 10% FBS, transferred to the 40ml plastic bottle after wind and percussion uniform and got the primary cultured cells. Static cultured for 3 days to change the first half of the amount of fluid, the normal exchange of medium later, about 10-12 days closely 70% of bottom of the bottle was covered with cells and then passaged to obtain the first generation of the cells, so did the later passage. Obtained cells with morphological observation and immunohistochemical analysis. Results Separated cells began to attach the wall around 48h, most morphology was spindle, primary cells were nested growth. The cells proliferated rapidly after passage and found a few amount of suspended cells in the medium, the second generation of the cells became purified generally. Meanwhile immunohistochemical analysis showed the situation of surface marker antibodies for CD29 (91.3±5.0)%, CD105 (72.1± 6.3)%, and CD34 (2.4±0.8)%. Conclusion Isolation and culture of rat bone marrow can be obtained the relatively purified BMSCs at the second generation. This conclusion provided a favorable prerequisite for the induction of differentiation of BMSCs.2. Induce rat bone marrow mesenchymal stem cells differentiate into GABAergic neurons in vitro.Objective To explore the trend of BMSCs differentiate into GABAergic neurons. Methods Added neural stem cells(NSC) medium to the favorable conditions of BMSCs for 1 week, then induced with 10ng/ml brain-derived neurotrophic factor(BDNF) and 10ng/ml bone morphogenetic protein-2(BMP-2) for 3-4 days, next cultured with 1μmol/L all trans retinoic acid(ATRA) for 3-4 days to complete the differentiation. Observed the morphological change of the induced cells, analyzed with Immunofluorescence and Western blot to the differentiated cells, statistical disposal to the last results. Results The cells'morphology began to change in the NSC culture medium after three days, existed a clear spherical body for about 1 week and increasing transparent. The cells began to appear bulge after adding BDNF and BMP-2, clear body and processes following the differentiation completed, showing a typical neuron-like cell morphology. Immunofluorescence manifested the positive neural cells: Nestin( 27.3±5.6 ) %, NSE (37.6±4.6) % and GAD67 (18.7±5.7 ) %. Western blot analysis showed that the relative gray value of protein bands of GAD67 and NSE was significantly higher than non-induced differentiated cells. Conclusion BMSCs had the trend of differentiating into GABAergic neurons in vitro, even more pronounced trend adding in BMP-2. The results of this research laid a certain theoretical basis for BMSCs transplantation for the treatment of epilepsy.