| Objective: The cell state of microglia in different concentrations of polyethylene glycol was observed,and the suitable concentration for microglia transplantation was determined.Then the combined transplantation of polyethylene glycol and microglia was used to treat spinal cord injury in rats,and the effect of this treatment on spinal cord injury was observed.Methods: In the first part,primary microglia were isolated and purified from the brain tissue of 1-2-day-old rats.Microglia were cultured in PEG2000 medium containing 5%,10% and 30% polyethylene glycol,respectively.The cell viability and migration ability of microglia to scratch injury were detected by CCK-8 method,scratch test,flow cytometry,Western Blot and immunocytochemical staining,respectively.Phagocytic ability and activation phenotype in response to lipopolysaccharide(LPS)stimulation.In the second part,the optimal concentration of polyethylene glycol selected by the first part of the in vitro culture experiment was used to explore the transplantation conditions of microglia in vivo,and the survival status of transplanted cells in vivo was observed.The third part,polyethylene glycol was used as the medium to transplant microglia into the lesions of rats with spinal cord injury.BBB score was used to evaluate the motor function of hindlimbs of rats with spinal cord injury.Samples were taken 21 days after operation,and immunohistochemical staining was used to evaluate the lesion area,scar formation and the number of apoptotic cells in each group.Results: In the first part,the cell viability was detected by CCK-8 method.Compared with the control group,the cell culture medium with 5% and 10%concentration of polyethylene glycol had no effect on the activity of microglia,while the activity of microglia in 30% polyethylene glycol cell medium decreased significantly.The results of immunoblotting and immunocytochemical staining showed that different concentrations of PEG2000 did not affect the level of NF-κ Bp65 in resting primary microglia,but the expression of i NOS decreased with the increase of PEG2000 concentration in the medium.Therefore,from the point of view of western blotting,PEG2000 can inhibit the inflammatory response of microglia.In addition,the expression of Arg-1 in microglia in 30% PEG2000 medium was significantly higher than that in the control group,and there was a tendency of anti-inflammatory transformation.More importantly,when LPS was stimulated for 6 h,the expression of i NOS and NF-kappa B-p65 in microglia in different concentrations of PEG2000 medium decreased compared with the control group,and the levels of i NOS and NF-kappa B-p65 decreased gradually with the increase of PEG2000 concentration.After 12 hours of LPS stimulation,the results were similar to those of 6 hours of stimulation.Immunocytochemical staining showed that the result of immunocytochemical staining was consistent with that of westernblot.After 6 hours of lipopolysaccharide stimulation,the expression of i NOS in microglia in the medium containing polyethylene glycol cells decreased,on the contrary,the expression of Arg-1 in microglia increased.After 12 hours of lipopolysaccharide stimulation,with the increase of polyethylene glycol concentration in the medium,the expression of i NOS in microglia decreased gradually,on the contrary,the expression of Arg-1 in microglia increased.In the inflammatory stimulation environment,lower concentrations of PEG2000(e.g.5%,10%)tend to inhibit the inflammatory response of microglia and promote the transition of microglia to anti-inflammatory phenotypes.In the second part,the polyethylene glycol concentration for the survival of microglia selected by the first part of the in vitro culture experiment was used in the in vivo transplantation experiment.It was found that the depth of the tip into the spinal cord was 1.2mm,and the sample injection rate was50nl/min.The survival status of microglia in vivo was observed within 21 days after transplantation.The third part,from the experimental results of the first part,it is concluded that the concentration of polyethylene glycol less than 10% has no effect on the survival of microglia.When PEG2000 and microglia were mixed into the lesions of rats with spinal cord injury,it was found that the BBB score of rats with spinal cord injury was significantly recovered,and the area of focus and the number of apoptotic cells in each group were better than those in other groups.Conclusion: Polyethylene glycol has good biological safety,microglia can survive well in 5% and 10% concentration of PEG2000,and 30% concentration of polyethylene glycol can induce the transition of microglia to M2 antiinflammatory phenotype,and this phenomenon is further enhanced in the inflammatory environment stimulated by lipopolysaccharide.Stereotactic injection is feasible for cell transplantation.The mixed transplantation of PEG2000 and microglia can promote the recovery of motor function in rats with spinal cord injury.The transformation of transplanted microglia to M2anti-inflammatory phenotype induced by polyethylene glycol may play an important role in the recovery of motor function in rats with spinal cord injury. |
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