Olfactory Ensheathing Cells In Culture Medium Of Nerve-like Cells Rhoa Activity And Axonal Growth

ObjectiveTo observe the expression of olfactory ensheathing cells culture medium(OECCM)on RhoA activity in nerve-like cells PC12 cells, and investigate the relationship between Olfactory ensheathing cells promote axon growth and RhoA / ROCK signaling pathway. Methods(1) Primary olfactory ensheathing cells were cultured, purified and identified, and the purified cell conditioned medium was collected into the OECCM.(2) PC12 cells were cultured and treated with OECCM and/or LPA, and then the amount of GTP-RhoA was detected by Western Blotting.(3) PC12 cells were cultured and treated with OECCM, and then the morphological changes were observed .(4) PC12 cells were cultured and treated with OECCM and/or LPA, and then the absorbance value changes of RhoA were observed by immunofluorescence.(5) PC12 cells were cultured and treated with OECCM and/or LPA, and then the cells were stained with phalloidin and the formation of stress fiber was visualized under fluorescent microscope.(6) PC12 cells were cultured and treated with OECCM, and then the expression and distribution of neurofilament protein was detected by immunofluorescence.(7) PC12 cells were cultured and treated with OECCM, and then the relative content of neurofilament proteins, synapsin, synaptophysin were detected by Western Blotting.Results(1) Cultured olfactory ensheathing cells were bipolar, tripolar and multipolar , and the cell bodies were smaller,stereoscopic, and good refraction. The cell processes are intertwined into a network structure, and the olfactory ensheathing cells in more than 85% purity.(2) Olfactory ensheathing cell culture medium could inhibit the activation of RhoA, and could antagonize RhoA activity caused by LPA.(3) In the PC12 cell line, by the role of olfactory ensheathing cell culture medium, the cells have long processes of varying lengths, and with time woven into nets.(4) In the PC12 cell line , by the role of olfactory ensheathing cell culture medium and/or LPA, the immunofluorescence staining showed the absorbance value of RhoA in olfactory ensheathing cell culture medium group was significantly lower than the control group, while the LPA group was significantly higher.(5) Olfactory ensheathing cell culture medium was able to antagonize the LPA-induced stress fiber formation.(6) In the PC12 cell line, by the role of olfactory ensheathing cell culture medium, immunofluorescence staining showed the distribution of neurofilament protein gradually increased and the distribution of size and size distribution ofβ-tubulin ratio was significantly higher than the control group.(7) In the PC12 cell line, by the role of olfactory ensheathing cell culture medium, the Western blotting showed the expression level of NF, synapsin and synaptophysin in olfactory ensheathing cell culture medium group was significantly higher than the control group.ConclusionOlfactory ensheathing cell culture medium could promote the axonal growth of PC12 cells, the structural and functional differentiation into neurons, and in the process inhibit RhoA activity, which makes olfactory ensheathing cells promote neurite growth mechanism through the RhoA / ROCK signaling pathway may exist.