ABSTRACT
BACKGROUND:PA6 cells are bone marrow stromal stem cells from the mouse skul , and scientists have found that PA6 cells co-cultured with some kinds of stem cells have shown neural differentiation, based on which, PA6 cells can be used for repair of nerve injury. Therefore, researchers give more and more attentions to PA6, but few studies have addressed isolation and culture methods of bone marrow stromal stem cells from the skul . OBJECTIVE:To isolate and culture bone marrow stromal stem cells from the skul of Sprague-Dawley rats and to observe cellular morpholopy in vitro and perform immunofluorescence identification. METHODS:Under sterile conditions, newborn Sprague-Dawley rat’s skul was cut into pieces. Smal skul pieces were washed using Dulbecco’s modified Eagle’s medium containing 10%fetal bovine serum. Single cellsuspension was made, and placed in culture flask. The culture medium was changed many times for cellpurification. The second passage of cells was obtained for morphology observation under an inverted microscope. cellsurface markers were detected by using immunofluorescence staining. RESULTS AND CONCLUSION:After the primary culture for 24 hours, cells exhibited adherent growth;after 3 days, cells were increased in number, and presented with irregular shapes, such as polygon, triangle, spindle and flat shape. After passage, cells were uniform in morphology, arranged in radial and bunch patterns, and exhibited strong adherent ability. Some cells grew in cluster, and proliferated faster than primary cells. Immunofluorescence staining showed that cells were positive for CD105, CD73, CD44, CD90, but negative for CD45, D34,CD14, HLA-DR. Results indicate that this method can obtain bone marrow stromal stem cells.
ABSTRACT
BACKGROUND:Studies have shown that Schwann cells form a Bunger band in the basement tube and guide the extension of regenerating axons after peripheral nerve injury, but the exact mechanism remains to be explored. OBJECTIVE:To explore the effect of Wal erian degeneration on biological characteristics and secretory function of Schwann cells in rats with sciatic nerve injury. METHODS:A rat model of sciatic nerve injury was established and divided into two groups:sciatic nerve transection group and surgical control group. Schwann cells were isolated and cultured from sciatic nerve segments by one enzyme digestion. The cellmorphology was observed under light microscope and S-100 protein expression was determined by immunofluorescence staining. After subculture, the first generation of Schwann cells were chosen to draw the growth curve by the counting method within 14 days. The cellactivity was detected by MTT assay. The adhesion of Schwann cells was examined by acid phosphatase analysis and the concentration of nerve growth factor was detected by ELISA method. RESULTS AND CONCLUSION:At 14 days after primary culture, a great number of Schwann cells were observed near the edges of nerve segments in the sciatic nerve transection group, but only smal number of Schwann cells scattered around nerve segments in the control group. Schwann cells in both groups showed S-100 positive expression. At 3 days after subculture, Schwann cells reached the logarithm proliferative phase, the cellnumber and proliferation absorbance values in both groups were increased along with time extension. Furthermore, the number of Schwann cells and absorbance value in the sciatic nerve transection group were significantly higher than those of control group (P<0.05). The adhesion ability in the sciatic nerve transection group was also significantly higher than those in the control group (P<0.05). ELISA results showed that, the concentrations of nerve growth factor in the sciatic nerve transection group were significantly higher than those in the control group at 4, 6, 8, 10, 12 and 14 days (P<0.05). After sciatic nerve injury, Wal erian degeneration can induce Schwann cells dedifferentiate into the precursors, significantly influence the biological function of Schwann cells, promote the proliferation of Schwann cells within the short term, secrete large amounts of neurotrophic factors, enhance celladhesion, and provide a suitable microenvironment for regenerated axons. In addition, it creates the necessary microenvironment for peripheral nerve regeneration.
ABSTRACT
BACKGROUND:Schwann cells transplantation can change the local micro-environment and help to repair the injured neural tissue, so getting a large number of highly purified and active Schwann cells is the key of the study. OBJECTIVE: To search for a simple and rapid method to extract and purify the Schwann cells.METHODS: Rats were divided randomly into two groups, namely, in vivo pre-degeneration of sciatic nerve resection group and untreated control group, with 20 rats in each group. Under sterile conditions, the rat sciatic nerves were cut off at post-operative 7 days, Schwann cells were extracted by using mixed enzyme digestion and tissue mass transplantation; through low enzyme digestion and twice inoculation to differential adhesion, Schwann cells were purified. Cell morphology was observed under phase contrast microscope and identified by mmunofluorescence staining; cell purity was calculated; MTT method assay was used to determine the capacity of cell proliferation.RESULTS AND CONCLUSION: At 7 days of the culture, the experimental group showed the typical bipolar or bipolar Schwann cells, with connections between cells; in control group, cell processes were shorter and less associated with the surrounding cells. Following S-100 immunofluorescence staining, cells were positive for green expression.Cells proliferated rapidly in the experimental group and formed a swirling shape at 15 days, there were a relatively small number of fibroblasts, at the purity of 96.1%; in the control group, the cells proliferated slowly, with many fibroblasts at a low purity. MTT assay showed that primary cultured Schwann cell proliferated weakly in both groups; compared with the control group, the proliferation of subcultured Schwann cells in the experimental group was markedly increased (P < 0.05 or 0.01), and reached a peak 3 4 days later. The results confirmed that in vivo denaturing, in vitro hybrid enzyme digestion, tissue mass transplantation combined with low enzyme digestion, separation of double-differential adhesion of Schwann cells is a simple and rapid method to extract and purify Schwann cells.