Effect Of Lentivirus-mediated RNA Interference Silencing CX43 Expression On Extracellular Matrix And Cell Proliferation Of Nucleus Pulposus

Background: Lumbar intervertebral disc degeneration is a common disease in spinal surgery and also one of the main causes of low back pain.Multiple factors work together to promote lumbar intervertebral disc degeneration.Many studies have proved that the degeneration of the nucleus pulposus,especially the reduction of nucleus pulposus cells and the anabolic imbalance of extracellular matrix play a key role in the degeneration of intervertebral disc.Constant mechanical stress stimulation and inflammatory response promote the expression of CX43 in chondrocytes,thus promoting the degradation of cartilage extracellular matrix and the increase of apoptosis.Some scholars have detected the expression of connexin 43 in intervertebral disc,but the molecular mechanism of nucleus pulposus degeneration and how connexin 43 regulates nucleus pulposus degeneration remains to be further studied.Objective: The expression of CX43 in the nucleus pulposus of intervertebral disc was demonstrated,and the role of CX43 in the proliferation of nucleus pulposus cells and the metabolism of extracellular matrix was clarified.Methods: From September 2018 to January 2020,intervertebral disc tissues removed during lumbar discectomy in the department of spinal surgery of our hospital were taken,and there mean age were 46.6±5.2 years old.Tumors,severe immunity and infectious diseases were excluded,and all patients were informed and signed informed consent before surgery.The tissue was graded according to the Pfirrmann grading criteria.Change the pH and serum concentration,use the CCK-8 method to draw the cell growth curve,and clearly define the environment suitable for the growth of nucleus pulposus cells.2.Validate the expression of CX43 in nucleus pulposus tissues of different grades using Western blot and immunohistochemical techniques.3.Primary nucleus pulpoides were isolated and cultured in vitro by searching literature records and combining with the laboratory conditions,and the cell growth was observed.Polyproteoglycan was identified by toluidine blue staining,and type Ⅱ collagen was identified by immunofluorescence.4.Lentivirus-mediated RNA interference technology was used to transfer small interfering RNA of CX43 into nucleus pulporeal cells andverify its infection efficiency.qRT-PCR was used to detect the relative expression levels of CX43,COL Ⅱ and Aggrecan mRNA.5.CCK-8 was used to investigate the effect of CX43 silencing on cell proliferation,and Cyclin D1 mRNA,which affects cell cycle,was detected by qRT-PCR.Results: 1.Western-Blotting results showed that the expression of CX43 was low in grade Ⅱ and grade Ⅲ nucleus pulposus tissues,and the expression of CX43 was relatively high in grade IV and grade V nucleus pulposus tissues with severe degeneration.It can be seen that with the increase of intervertebral disc degeneration,the expression of CX43 in the nucleus pulposus is increasing.Immunohistochemical examination showed that the intensity of staining in grade IV and grade V degenerative tissues was higher,while the intensity of staining in grade Ⅱ and grade Ⅲ tissues was lower.The results suggest that the expression of CX43 in the nucleus pulposus increased with the increase of intervertebral disc degeneration.2.The nucleus pulposus specimens collected during the operation were successfully isolated by enzyme digestion,and toluidine blue staining was performed,he cytoplasm of the nucleus pulposus cells is slightly lighter and the cytoplasm darker as it gets closer to the nucleus.Immunofluorescence staining showed type Ⅱ collagen protein positive expression in the cell,the cell is also visible around the uneven dyeing,it conforms to the characteristics of nucleus pulposus cells.After passage,P1,P2 and P3 cells were in good growth state and could be used in subsequent cell experiments.3.Lentivirus carrying small interfering RNA of CX43 was used to infect the nucleus pulposus cells and cause their expression in the nucleus pulposus cells.It was empirically shown that the expression of CX43 decreased,and the expression levels of COL Ⅱ and Aggrecan mRNA increased.4.After the expression of CX3 was silenced,cells in the interference group grew faster than those in the control group,and Cyclin D1 mRNA in the interference group was also higher than that in the control group.Conclusion: 1.There is a correlation between CX43 and the degree of degeneration of the nucleus pulposus.With the aggravation of disc degeneration,the expression of CX43 in the nucleus pulposus increased.2.The expression of silent CX43 promoted the proliferation of nucleus pulposus cells and the synthesis of COL Ⅱ and Aggrecan mRNA.