Effects Of Laminaria Japonica Polysaccharide On Radiation Induced Injury Of Nerve Cells And Endothelial Cells In Vitro

Radiation brain injury?RBI?is a serious complication after radiotherapy for malignant tumors of head and neck.Laminaria japonica polysaccharide?LJP?is a bioactive polysaccharide isolated from Laminaria japonica.Our previous study found that LJP can improve the memory function of RBI,but its mechanism is not clear.In view of this,the project develops nerves in vitro The effects of LJP on radiation-induced neural cell and cerebral vascular endothelial cells were observed from cell and molecular level,and the mechanism of LJP in radiation-induced brain injury was revealed.1.The first part:isolation,primary culture and identification of neural cells and cerebrovascular endothelial cells from neonatal SD ratsNeonatal SD rats born within 12 hours of clean grade were harvested from the cortex,neurons,glial cells and microvascular endothelial cells in the cortex were isolated and cultured.The morphological and quantitative changes of the cells were observed under inverted microscope.After cell maturation in vitro,neuronal cells,glial cells and cerebrovascular endothelial cells were labeled with NSE?GFAP?vWF antibody,and identified by immunofluorescence staining.The results showed that:?1?Neuron cells:most of the cells adhered to the wall for 4-5 hours,the cell bodies were round,the number of the small processes increased in 24hours,the processes became longer,and the cell bodies of the processes between the few cells increased in 2-3 days,which were oval.The neurites became longer and thicker,and the connections between the processes increased.After 4-6 days,the cell bodies gathered together to form a typical neural network between the processes of 7 to 8 days.Fluorescence immunohistochemical staining showed that the nucleus was large,round and bright,and NSE was expressed in the cytoplasm and processes.The protuberances interweave into a network.?2?Glial cells:when cultured for 3 to 5 hours,the cells adhered to the wall,and the halos were obvious.After 1-2 days,the cells began to spread out,and the slender processes grew for 4-5 days.The volume of the cells was obviously increased,and the number of the cells growing out of the process was obviously increased for 7 days,and the bottle-bottom of the culture bottle was filled with the cells.After purification and passage,the glial cells were stellate or irregular,with abundant processes and many branches,and the primary processes were thicker,often with secondary branches.Immunofluorescence staining showed that GFAP was expressed in cytoplasm and processes.?3?Cerebrovascular endothelial cells:cultured for 24 hours,the vascular segments and cells were adhered to the wall,and the cells were spindle-shaped.From 3 to 5 days,a large number of cells grew along the microvascular segment and formed island like hyperplasia at the center of inoculation.There was no overlap between the different communities.The cells in the colony were arranged in a whirlpool shape and gradually converged with each other for 6 to 8 days,and the cells began to converge into pieces.The size and shape of the cells are more uniform,showing a"paving stone"appearance.24 hours after passage,the fusiform cells scattered in the adherent cells were fused into pieces at 5 days,and the cells after further passage still retained the characteristics of endothelial cells,which were typical"paving stones".Form.Immunofluorescence staining showed that vWF was expressed in cytoplasm.2.Part two:effects of LJP on radiation induced injury of neurons and cerebrovascular endothelial cells in VitroThe cells with good growth and no pollution were randomly divided into control group,LJP group,radiation group,radiation+LJP group.The radiation group and the LJP group were exposed to ⁶⁰Co?irradiation at a dose of 10 Gy,while the control group was exposed to the same environment without irradiation.After 24 hours of irradiation,the cell culture medium was taken and centrifuged.The expression of cytokines was detected by ELISA.The number of positive cells and the absorbance value were analyzed by cell immunofluorescence staining and image analysis.?1?The protective effect of LJP on radiation-induced injury neurons:.?1?the number of neuron cellsComparison with control group,absorbance decreased significantly after radiation?P<0.05?,but there was no significant difference?P>0.05?between the LJP group and the control group?P>0.05?.There was no significant difference between the LJP group and the control group?P>0.05?.?2?the expression of Bax/Bcl-2in the cell culture medium of the radiation group was significantly lower than that of the control group?P>0.05?.However,the expression of Bax was increased,but no change was found in the radiation LJP group?P>0.05?.Bcl of LJP group compared with control group There was no significant difference in the expression of Bcl-2/Bax?P>0.05?.?2?the effects of LJP on glial cells induced by radiation:?1?glial cell numbers there was no difference in the number of glial cells between groups before and after irradiation.?2?the expression of Bax?Bcl-2 and TNF-?in the culture medium of each group was not significantly different after radiation?P>0.05?.?3?the effects of LJP on radiation induced endothelial cells:?1?number of cerebrovascular endothelial cellThe number and absorbance of vWF positive cells in LJP group were significantly lower than those in control group?P<0.05?.After LJP intervention,the number and absorbance of vWF positive cells in LJP group were not significantly different from those in control group?P>0.05?.?2?the expression of vWF PAI-1 and t-PA factor Compared with the control group,the contents of vWF and PAI-1 in the endothelial cell culture medium after radiation were significantly increased?P<0.05?,while the level of t-PA factor was significantly lower than that in the control group?P<0.05?.There was no significant difference between the control group,the radiation+LJP group and the LJP group.To sum up,the conclusion of this study1.In this experiment,primary neurons,glial cells and cerebrovascular endothelial cells were cultured successfully in neonatal SD rat cortex within12 hours.2.⁶⁰Co?-rays could induce Bax expression to promote the injury of cultured neurons in vitro,while LJP could induce the expression of Bcl-2and inhibit Bax production to protect the neurons from radiation-induced injury.3.⁶⁰Co?-rays can promote the release of vWF,PAI-1 factor and inhibit the release of t-PA by endothelial cells,while LJP can promote the release of t-PA factor by inhibiting the release of vWF?PAI-1 factor,and protect endothelial cells from radiation injury.4.There was no effect on glial cells cultured in vitro by 60 Co?-ray at 10 Gy dose.