Human umbilical cord mesenchymal stem cell-derived exosomes reduce the permeability of blood-spinal cord barrier after spinal cord injury / 中国组织工程研究

ABSTRACT

BACKGROUND:Endothelin has been found to be involved in the breakdown of the blood-spinal cord barrier after spinal cord injury,and stem cell-derived exosomes can reduce the permeability of the blood-spinal cord barrier and repair spinal cord injury. OBJECTIVE:To investigate whether exosomes produced by human umbilical cord mesenchymal stem cells can reduce the permeability of the blood-spinal cord barrier by inhibiting endothelin-1 expression,thus repairing spinal cord injury. METHODS:Exosomes were extracted from the cultured supernatant by the hyperspeed centrifugation method.The morphology of exosomes was observed by transmission electron microscope.The expression levels of tsg101 and CD63 were detected by western blot assay.Eighty SD rats were randomly divided into sham operation group,model group,exosome group,and endothelin-1 group(n=20).The modified Allen's method was used to create the rat model of spinal cord injury.In the endothelin-1 group,10 μL(1 μg/mL)endothelin-1 was injected directly into the injured area with a microsyringe.Immediately,1 day,2 days after operation,sham operation group and model group were injected with 200 μL PBS solution through the tail vein;the exosome group and endothelin-1 group were injected with 200 μL exosome(200 μg/mL)solution through the tail vein,respectively.Hind limb motor function scores were performed on days 1,3,7,14 and 21 after spinal cord injury.The blood-spinal cord barrier permeability was observed by Evans blue staining on day 7 after injury.The expression levels of tight junction proteins β-Catenin,ZO-1,Occludin and endothelin-1 in the spinal cord were detected by western blot assay. RESULTS AND CONCLUSION:(1)Basso-Beattie-Bresnahan score in the exosome group was significantly higher than that in the model group at 3-21 days after injury(P<0.05).Hematoxylin-eosin staining showed that spinal cord injury was greatly reduced in the exosome group compared with the model group.Basso-Beattie-Bresnahan score in the endothelin-1 group was significantly decreased compared with the exosome group(P<0.05).Spinal cord injury was more severe in the endothelin-1 group than that in the exosome group.(2)The expression of endothelin-1 in the model group was significantly increased compared with the sham operation group(P<0.05),and the expression of endothelin-1 in the exosome group was significantly decreased compared with the model group(P<0.05).(3)The blood-spinal cord barrier Evans blue exudate in the exosome group was significantly decreased compared with the model group(P<0.05).The expression levels of the tight junction proteins β-Catenin,Occludin and ZO-1 in the exosome group were increased(P<0.05);the Evans blue exudate in the endothelin-1 group was significantly increased compared with the exosome group(P<0.05).The expression level of tight junction protein was significantly decreased compared with the exosome group(P<0.05).(4)The results show that human umbilical cord mesenchymal cell-derived exosomes protect the permeability of the blood-spinal cord barrier by down-regulating the expression of endothelin-1 and play a role in the repair of spinal cord injury.