Experimental Study On Cyclic Tensile Strain Modulating Neurotrophic And Myelinating Gene Expression Of Schwann Cells

【Objective】This study aimed to apply the ElectroForce 3200 tension measuring machine equipped with BioDynamic system to load cyclic tensile strain(CTS)of different frequency parameters on Schwann cells(SCs)that were seeded in Sylgard184 silicone rubber in vitro.Through observing morphology,distribution and proliferation of SCs,detecting neurotrophic,myelinating gene expression and cytoskeletal protein changes,we aimed to investigate the response of SCs about nerve regeneration related biological behavior under CTS.To optimize stimulation frequency of CTS and further offer experimental foundation and the direction for the effective treatment of spinal cord injury(SCI)by the application of CTS combined with SCs.【Method】SCs were cultured on Sylgard184 silicone rubber which has better biocompatibility and flexibility in vitro.With use of ElectroForce 3200 mechanics test instrument,strain input included 0Hz,0.25 Hz and 0.5Hz was loaded circularly,all group has 5% strain for 6 hours with one voice.Cell morphology and distribution were observed under phase contrast microscope;proliferation of SCs was investigated by using CCK-8 method after loading.By conducted quantitative real-time PCR and Western blot,we detected the change of neurotrophic and myelinating gene expression after loading.Moreover,immunofluorescence staining was performed to visualize the changes of cytoskeleton proteins and their relationship with myelinating gene expression after loading.To comprehensive data and do statistical analysis.【Result】This study succeeded to construct SCs culture model in vitro under CTS stimulation.After appling different frequency parameters of CTS on SCs,results showed that cells in the control group were in normal growth and the distribution has no clear change.SCs loaded by 0.25 Hz were plumpness,moreover,cells arranged in polar distribution(consistent with the pull direction)and the proliferation was higher than the other two groups on the 7 day(P < 0.05);A small amount of SCs loaded by 0.5Hz were detached and adherent cells remained no polar.The results performed by qPCR and Western blot showed that cells treated with frequency of 0.25 Hz CTS expressed and secreted significantly more neurotrophic factors(BDNF,NGF,NT-3),down-regulated expression of Krox-20 and MAG.(P < 0.05).Meanwhile,enhancement of fluorescence intensity of Krox-20 gene was observed after stimulation of CTS.Fluorescence staining of F-actin was strong and concentrated in peri-nuclear regions in the tensile group.【Conclusion】Biological function of nerve regeneration of SCs is affected by CTS.The results showed that the morphology,distribution and proliferation of SCs were affected by CTS: appropriate frequency of CTS stimulation can make SCs form more plump,cell arrangement also showed a certain polarity.The high frequency of CTS can induce the local cells to fall off,while the cells that are not shed are scattered and have no obvious polarity.The effect of cyclic tensile stress on SCs can promote cell proliferation.The appropriate frequency CTS promote Schwann cells neurotrophic factors(BDNF,NGF,NT-3)synthesis and secretion;up-regulated gene Krox-20 and MAG expression of myelin.SCs after CTS stimulation lead into myelin Krox-20 gene changes may be mediated by F-actin cytoskeleton.But there still need to further explore and find specific signal pathway.