| Objective:After bone marrow mesenchymal stem cell(BMSC)and nucleus pulposus cell(NPC)are co-cultured,the exosomes secreted by BMSC can affect the autophagy level of NPC and improve the degeneration of the intervertebral disc.The purpose of the study is to explore the therapeutic effect of BMSC and NPC on degenerative nucleus pulposus cells after co-culture without contact,and to study its specific mechanism of action.Methods:Rat nucleus pulposus cells and bone marrow mesenchymal stem cells were isolated and cultured in vitro,and oxygen deprivation(Oxygen and glucose deprivation,OGD)model was established to simulate the pathological state of intervertebral disc degeneration in vivo,and non-contact co-culture was carried out.The nucleus pulposus cells were randomly divided into 7 groups: 1.Blank control group,2.Degeneration group(OGD),3.Co-culture group(OGD+BSMC),4.Autophagy inhibition group(OGD+BSMC+3-MA)),5.RAPA-induced co-culture group(OGD+RAPA-induced BMSC),6.Knock out mi R-155 BMSC+NPC glyco-oxygen deprivation group(OGD+BSMC+knock out mir-155),7.Knock out mi R-21 BMSC+NPC glucose and oxygen deprivation group(OGD+BSMC+knockout mir-21).RT-PCR was used to detect the m RNA and mi R-155 expression changes of Beclin1,LC3,BACH1 in each group of NPC cells,and the content of Beclin1,LC3,BACH1,fusion protein in each group of NPC cells was detected by immunofluorescence staining(GFP);The Westren-blot method quantitatively detects the expression of Beclin1,LC3,BACH1,protein and apoptosis-related proteins Bcl-2 and Bax,and evaluates autophagy by the change of LC3II/LC3 I ratio.Use SPSS22.0 statistical software for data analysis.The results are expressed as mean values±SD,and statistical differences between groups are compared by t test,and p<0.05 is regarded as statistically different.By detecting the expression of Beclin1,LC3,BACH1 and other proteins in the nucleus pulposus cells of each group,and detecting the expression of BACH1 and autophagy in the co-cultured nucleus pulposus cells after mi R-21 and mi R-155 are knocked out,the co-culture of BMSC and NPC can be studied.The role of intervertebral disc degeneration.Results:1.Compared with the blank control,after OGD modeling,the expression of Beclin1,LC3,BACH1 and other proteins in NPC in the degeneration group decreased,while the expression of m TOR increased;compared with the blank control group,Beclin1 in NPC in the co-culture group The expression of proteins such as,LC3 and BACH1 increased,while the expression of m TOR decreased.Compared with the degeneration group,the expression of Beclin1,LC3,BACH1 and other proteins in the co-culture group increased significantly,while the expression of m TOR decreased significantly.2.Compared with the blank control,after using 3-MA to inhibit autophagy,the expression of Beclin1,LC3,BACH1 and other proteins in NPC decreased,and the expression of m TOR increased;while the level of BMSC autophagy induced by RAPA,Beclin1,LC3,BACH1 in NPC The expression of other proteins increased,while the expression of m TOR decreased,and the degree was greater than that of the co-culture group.3.Compared with the blank control group,after knocking out the mi R-155 gene,the expression of Beclin1,LC3,BACH1 and other proteins in NPC decreased,while the expression of m TOR increased.This trend was similar to that of the degenerative group;compared with the blank control group,There was no significant difference in the expression of Beclin1,LC3,BACH1 and other proteins in NPC after mi R-21 was knocked out.4.Compared with the degeneration group,the expression of Beclin1,LC3,BACH1 and other proteins in NPC in the co-culture group increased significantly,while the expression of m TOR was significantly reduced;compared with the degeneration group,the use of 3-MA to inhibit Beclin1 in NPC after autophagy The expression of proteins such as,LC3,BACH1 decreased,and the expression of m TOR increased,and there was a significant difference;compared with the degeneration group,the expression of Beclin1,LC3,BACH1 and other proteins in NPC was significantly increased after the autophagy level of BMSC was induced by RAPA.The expression of m TOR was significantly reduced,and the difference was obvious.5.Compared with the degeneration group,after the mi R-155 gene was knocked out,there was no significant difference in the expression of Beclin1,LC3,BACH1 and other proteins in NPC;compared with the degeneration group,after the mi R-21 gene was knocked out,Beclin1,The expression of proteins such as LC3 and BACH1 were significantly increased,while the expression of m TOR was significantly reduced.6.Compared with the co-culture group,the expressions of Beclin1,LC3,BACH1 and other proteins in NPC were significantly decreased after autophagy was inhibited by3-MA,and the expression of m TOR was increased,and there were significant differences;compared with the co-culture group,RAPA induced BMSC autophagy After the level of phagocytosis,the expression of Beclin1,LC3,BACH1 and other proteins in NPC increased significantly,while the expression of m TOR decreased significantly.7.Compared with the co-culture group,after knocking out the mi R-155 gene,the expression of Beclin1,LC3,BACH1 and other proteins in NPC decreased significantly,and the expression of m TOR increased,and there were significant differences;compared with the co-culture group,knocking out mi R-21 After gene,the expression of Beclin1,LC3,BACH1 and other proteins in NPC increased,while the expression of m TOR decreased,and the degree was lower than that of the mi R-155 knockout group.Conclusion:1.This experiment confirmed that after OGD modelling,the level of autophagy-related proteins in NPC decreased,the number of nucleus pulposus cells decreased,the degree of apoptosis increased,and the nucleus pulposus cells showed a certain degree of degeneration.2.After BMSC and NPC modeled by OGD are co-cultured,the level of autophagy-related proteins in nucleus pulposus cells increases,and the proliferation ability of nucleus pulposus cells is enhanced.The co-culture can improve the degeneration of NPC to a certain extent,showing a certain degree Therapeutic effect.3.The co-culture system increases the autophagy level of NPC through mi R-155 in BMSC exosomes,inhibits the degeneration and apoptosis of nucleus pulposus cells,thereby improving the degree of IDD;while mi R-155 is knocked out or applied from The phagocytic inhibitor 3-MA can reverse the above process.4.After the autophagy level of BMSC itself is induced by RAPA,the level of NPC autophagy-related protein in the co-culture system increases,and the proliferation ability of nucleus pulposus cells is enhanced,and the degree is higher than that of the normal co-culture group.The treatment of degenerative NPC The effect is stronger.5.In the co-culture system of NPC and BMSC after OGD modeling,mi R-21 had a certain effect on the autophagy level of NPC,but its effect was smaller than mi R-155. |
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