Marrow stromal cells to neural cells for culture and in vivo transplantation experiments
|School||Jiangxi Medical College|
|Keywords||Bone marrow stromal cells Nerve cells Directed differentiation Focal cerebral ischemia In vivo transplantation|
Objective: Bone marrow stromal cells (Marrow stromal cells, MSCs) is a presence in the non-hematopoietic stem cells in the bone marrow, which has the capacity of self-renewal and differentiation potential, in the repair of the central nervous system injury shows a good prospect and times by concern neuroscience workers. This experiment vitro cultured MSCs understanding of the biological characteristics of growth and exploration into nerve cells induced conditions; observed MSCs transplanted into ischemic brain injury in rats, cell survival, migration; and MSCs to differentiate into possible mechanism of nerve cells is discussed. In order to provide a theoretical basis for MSCs transplantation repair of central nervous system injury. Method: 1. Separated by density gradient centrifugation MSCs, repeated adherent purified amplification of MSCs microscope to observe the morphological characteristics and draw the growth curve. Sodium ferulate (Sodium Ferulate, SF) concentration on the growth of MSCs by MTT assay, and to determine the concentration of the of sodium ferulate best induced culture. A certain concentration of sodium ferulate MSCs induced culture, β-mercaptoethanol induced culture group also set up as a positive control. Changes in cell morphology observed two groups of MSCs. Immunocytochemical staining techniques detect induced the cultured cells neurofilament protein (NF), Nestin (expression of Nestin) neurons enolase enzyme (NSE), glial cells acidic protein (GFAP) nerve cell marker expression . 4 different doses in the nerve cells in the induction medium mitogen-activated protein kinase (MAPK) pathway specificity blocker PD98059, observed the MSC morphological changes and nerve cells express specific markers of change. lt; WP = 13 gt; 5. making the rat model of focal cerebral ischemia in vitro has first marker BrdU MSCs by ipsilateral internal carotid artery transplanted into a mouse model, were sacrificed one week after the mice by BrdU immune staining observed survival distribution of MSCs. Results: 1.MSCs after inoculation 24h, part-adherent cells were fibroblast-like cell morphology 3d small colony of cells seen after the formation of the rapid increase of about 10 to 14 days, confluent cells to shuttle the formation of fibrous like cells. Subcultured cells grow faster than primary cells. The MSCs growth of the S-shaped curve, the doubling time of 72h. 2. Containing sodium ferulate 0.5mg/ml and 1mg/ml medium of no significant impact on the growth of MSCs in culture 7d, the survival rate of both MSCs were 95%, 94%; while containing 2mg/ml 3mg medium / ml sodium ferulate significant growth inhibition of MSCs cultured 7d, both survival rates were 54% and 20%, respectively. 3.MSCs by medium containing 1mg/ml sodium ferulate induced 6h, the cell body shrinkage rounded, three-dimensional enhancement, protrusions grow, the subsequent processes gradually increased, after 24 hours, the cell protrusions interconnected, staggered into network, showed nerve cell-like morphology. This phenomenon until seven days after the gradual aging of cells; induced culture 6h of MSCs Nestin expression after 24 hours failed to detect its expression of mature nerve cell markers NF, NSE and GFAP expression increased induced cultured for 3 days The highest positive rate of NSE and GFAP, were 67 ± 3.5%, 39 ± 1.8%. induced culture 3h β-mercaptoethanol control group, MSCs are the expression of Nestin, 6h, the majority of MSCs into a typical neuron-like cells, NSE-positive cells was 68.8 ± 3.3% for 24h induced cells detached from the culture plate. Inducing medium containing PD98059, MSCs still on the nerve cell-like morphological changes can be detected nerve cell marker positive rate of less blocker group did not change significantly. Would be induced by sodium ferulate 24h and has been in the BrdU-labeled MSCs by the ipsilateral internal carotid artery implanted in a rat model of focal cerebral ischemia in the brain, BrdU-positive cells was observed after one week is mainly distributed in the ischemic side of the cortex, a small distribution center in the infarct area (caudate putamen). Conclusions: 1. Vitro culture conditions, MSCs growth proliferated rapidly, with the capacity of self-renewal and differentiation potential of nerve cells. lt; WP = 14 gt; Chinese medicine monomer sodium ferulate induced to differentiate into neural progenitor cells, and thus differentiation into neurons and glial cells, and induction of differentiation of the cells survived for up to 7 days. 3 mitogen activated protein kinase (MAPK) pathway was not involved in the process of MSCs to differentiate into neural cells. 4. Sodium ferulate in vitro directed differentiation of MSCs in vivo transplantation, in focal cerebral ischemia in the rat brain can survive.