ABSTRACT
Objective To investigate the effects of the fibrin scaffold on the differentiation and the proliferation of neural stem cells and astrocytes. Methods Neural stem cells and the gliocytes derived from spinal cord were cultured in vitro respectively. The purified neural stem cells or gliocytes were seeded separately onto the fibrin scaffolds as experimental group and the glass slides modified with poly-L-lysine(PLL)as control group. At different time in culture the neural stem cells were immunofluorescence stained with antibodies against the marker of neurons I.e. Neurofilament(NF).The length of NF-positive neuritis was masured and the average value was calculated in the culture well (n=4). The gliocytes were immunofluorescence stained with antibodies against the marker of astrocytes I.e. Glial fibrillary acidic protein (GFAP ). The total number of the cells and the GFAP-positive cells were counted from 5 different fields of vision in the culture well (n=4), then the average ratio of GFAP-positive cells was calculated. The differentiation of neural stem cells, the extension of neurites and the proliferation of astrocytes on the fibrin scaffolds were compared with those on the slides. The protein of GFAP was detected by Western blotting to analyse the mature degree of astrocytes. All above experiments were repeated 3 times respectively. Results Immunofluorescence staining showed that the NF-positive neurites in the fibrin scaffold group were longer than those in the control group, whereas GFAP-positive cells were fewer than those in the control group. The expression of GFAP in the cells on the scaffold was lower than that in the control group.Conclusion The fibrin scaffold could promote differentiation of the neural stem cells to neurons and extension of the neurites. Meanwhile, the scaffold could inhibit proliferation and mature of the astrocytes.
ABSTRACT
Objective To isolate and identify smooth muscle progenitor cells(SPCs) from rat bone marrow and to observe specific expression of smooth muscle progenitor cells during proliferation and differentiation in vitro.Methods MNCs were isolated by density gradient centrifugation from rat marrow and cultured in conditioned nutrient medium,identification was performed by immunofluorescent staining(?-SMA,CD14).Smooth muscle cells specific markers(?-SMA) were checked with Western blotting and Real-time PCR at different time.Results During culturing,cells adhered and became spindle shaped with outgrowth at 4 d and 7 d,and showed typical "peak" "valley" at 14 d.Both ?-SMA and CD14 were positive after 4 d.Expression of ?-SMA was not found at 1 d with Western blotting,but it gradually enhanced at 4 d and reached the peak from 10 d to 14 d,then maintaned high-level at 21 d.The results with Real-time PCR indicated that no expression of ?-SMA mRNA within non-induced cells was found,but after being induced it gradually enhanced at 4 d and got to peak at 14 d,then kept the high-level at 21 d,low-expression at 1 d was significantly different from the other ones(P