| Objective: The autoimmune response and inflammatory stimulation after intervertebral disc herniation are considered to be the main causes of low back pain,lower limb pain and limited movement in patients with lumbar disc herniation.At the same time,miRNA-146 a has been proved to be effective in inhibiting inflammatory response in vivo.In this study,the stable expression of miRNA-146 a gene in BMSCs was obtained by transfecting miRNA-146 a gene into BMSCs,which was the next step to study miR-146 a gene modification,and lay the experimental foundation for the treatment of low back pain caused by intervertebral disc herniation.Methods: The SD rat BMSCs were isolated and purified by adhering to the culture glassware wall and surface marker CD90,CD106,CD45 was detected by flow cytometry.The third generation of BMSCs was transfected with lentiviral vector carrying miR-146 a gene.The expression of EGFP was observed after transfection,and the expression of miR-146 a gene and its target proteins IRAK-1,TRAF6 were detected by qRT-PCR and Western Blot.Results: Flow cytometry showed that BMSCs were positive for CD90,CD106,and negative for CD45.The optimum MOI of BMSCs was 50 after transfected with Lentiviral vector carrying miRNA-146 a,and the transfection rate was up to 80%.The fluorescence expression of EGFP began to increase gradually after 48 hours;qRT-PCR shows that the expression of miR-146 a gene in transfected cells was higher than that in non-transfected group and negative control group(P<0.05);Western Blot indicated that the target proteins IRAK-1 and TRAF6 were lower in transfected BMSCs carrying the miRNA-146 a gene.Conclusion:⑴ BMSCs with high purity can be obtained by whole bone marrow adherence screening method.⑵ The lentiviral vector carrying miR-146 a gene was successfully transfected into BMSCs and stably expressed,which provided a theoretical foundation for gene therapy for lumbar back pain caused by lumbar disc herniation. |
PDF Full Text Request