| Objective: Spinal cord injury refers to the injury of the spinal cord caused by external direct or indirect factors,resulting in various motor,sensory and sphincter disorders,abnormal muscle tone and dysfunction of autonomic nerve function below the corresponding segment of the injury.Spinal cord injury often leads to significant bone mineral loss in lower limbs,which is very easy to lead to osteoporosis.The bone microstructure of the lower limb was significantly damaged,and the bone strength and bone density were significantly reduced,which greatly increased the risk of fracture.However,most of the current clinical treatment programs are mainly to resist bone resorption,and there is no clear and effective treatment program to promote bone regeneration.It is gratifying that bone marrow mesenchymal stem cells are an important source of osteoblasts.As the key cells in bone regeneration,they have important significance in the occurrence and development of osteoporosis.Although some studies have pointed out that bone marrow mesenchymal stem cells have a certain relationship with the occurrence and development of osteoporosis,under the pathological state of osteoporosis after spinal cord injury,the specific changes in the biological behavior of bone marrow mesenchymal stem cells and the corresponding potential mechanism are still unclear.In recent years,the research of lncRNA in bone marrow mesenchymal stem cells has made considerable progress,and the regulation of cell biological behavior at the gene level through its targeted competition with miRNA has been understood by many researchers.According to this direction,gene level regulation of bone marrow mesenchymal stem cells in osteoporosis after spinal cord injury can improve the occurrence and development of osteoporosis after spinal cord injury,which not only helps to reveal the specific reasons for massive loss of bone mass in osteoporosis after spinal cord injury,but also provides a more effective theoretical basis for its treatment.Methods:Rat models of osteoporosis group and sham operation group after spinal cord injury were constructed,and bone marrow mesenchymal stem cells were extracted from the lower limbs of rats by the method of whole bone marrow adherence.HE staining was used to analyze whether the establishment of osteoporosis model of distal femur in rats was successful.Bone marrow mesenchymal stem cells were extracted according to morphology,cell phenotype identification and differentiation ability identification.When primary bone marrow mesenchymal stem cells were cultured to the third generation,the cells were collected for subsequent experiments.PCR was used to verify the differential expression of lncRNA H19 and miR-29b-3p in osteoporosis group and sham operation group after spinal cord injury.The lncRNA H19 small interfering RNA was constructed.The effects of lncRNA H19 knockdown on bone marrow mesenchymal stem cells’ osteogenic differentiation ability and Wnt/β-catenin signal pathway were studied by RT-q PCR,Western blot,alizarin red staining,alkaline phosphatase staining,quantitative and immunofluorescence experiments.Similarly,miR-29b-3p mimic and inhibitor were constructed,and the effects of miR-29b-3p on the osteogenic differentiation ability and Wnt/β-catenin signal pathway of bone marrow mesenchymal stem cells were studied according to RT-q PCR,Western blot,alizarin red staining and alkaline phosphatase staining,quantitative experiment,immunofluorescence,etc.The wild-type and mutant luciferase vectors of Lnc RNA H19 and Dkk1 were constructed,and the targeted binding between lncRNA H19 and miR-29b-3p,miR-29b-3p and Dkk1 was verified by double luciferase gene report experiment.At the same time,the co-transfection technology was used to knock down lncRNA H19 and miR-29b-3p at the same time to verify the effect of miR-29b-3p knockdown on the low expression of lncRNA H19 and the promotion of bone marrow mesenchymal stem cell differentiation.Finally,bone marrow mesenchymal stem cells were infected with lentivirus knockdown lncRNA H19,and bone marrow mesenchymal stem cells were transplanted to rats in the osteoporosis group after spinal cord injury.The therapeutic effect of bone marrow mesenchymal stem cells transplantation was measured by morphometry according to Mirco-CT.Results: 1.The HE staining results of the distal femur of rats showed that the bone trabeculae of the osteoporosis group were significantly reduced and the fat vacuoles were significantly increased after spinal cord injury,which proved that the rat models of the osteoporosis group and the sham operation group after spinal cord injury were successfully established.2.Flow cytometry phenotype identification showed that bone marrow mesenchymal stem cells expressed CD29 and CD90 positively,and CD45 negatively.The cell morphology and osteogenic differentiation ability are in line with the identification criteria.3.RT-q PCR technology confirmed that the expression of lncRNA H19 increased in osteoporosis group after spinal cord injury,while the expression of miR-29b-3p decreased.4.Biological experiments have verified that knockdown of lncRNA H19 can promote bone marrow mesenchymal stem cells to differentiate into osteoblasts.The low expression of miR-29b-3p is not conducive to the osteogenic differentiation of bone marrow mesenchymal stem cells,while the high expression of miR-29b-3p can effectively promote its osteogenic differentiation.5.The double luciferase gene report experiment verified that there is a certain targeted binding relationship between lncRNA H19 and miR-29b-3p,miR-29b-3p and Dkk1.6.The results of co-transfection experiment also showed that the low expression of miR-29b-3p could inhibit the promotion of the low expression of lncRNA H19 on the osteogenic differentiation of bone marrow mesenchymal stem cells.7.In vivo experimental results showed that bone mass loss after spinal cord injury in rats was alleviated after transplantation of bone marrow mesenchymal stem cells infected with lentivirus and knockdown lncRNA H19.Conclusion: 1.lncRNA H19 was highly expressed in osteoporotic bone marrow mesenchymal stem cells after spinal cord injury,while miR-29b-3p showed low expression.2.Low expression of lncRNA H19 and high expression of miR-29b-3p can effectively promote the differentiation of bone marrow mesenchymal stem cells into osteoblasts,and this role is played by Dkk1.3.There is a certain targeted binding relationship between lncRNA H19 and miR-29b-3p,miR-29b-3p and Dkk1.4.After transplantation of bone marrow mesenchymal stem cells with lncRNA H19 knockdown,bone loss after spinal cord injury in rats was relieved to some extent. |
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