Biological Effects Of MMP-3shRNA Transfection In Human Degenerative Nucleus Pulposus Mediated By Lentiviral

Objective: To screen the most effective MMP-3sh RNA sequence,and observe the biological effects human degenerative intervertebral disc nucleus pulposus cells after MMP-3sh RNA transfection by lentivirus.Methods:The nucleus pulposus tissues from surgery patients in vitro were isolated under sterile circumstance and digested by trypsase and type Ⅱ collagenase. Nucleus pulposus cells were continuous monolayer cultured and identificated by the protein expression of collagen typeⅡby cell immunohistochemistry and immunofluorescence.According to MMP-3m RNA sequence,four pairs of h MMP-3 sh RNA were designed and synthesized and were inserted the LV3 vector digested by enzyme,then it was transfected into the competent cells.The positive clones were identified by PCR,and analyzed by sequencing.The packaging and titer of lentivirus were determined after transfecting 293 T cells. The reconstructed virus was transfected into human degenerative nucleus pulposus cells, and then the expression level of MMP-3、Aggrecan and collagenⅡm RNA and the protein were examined with Real-Time PCR and Western blot respectively.Results:The primary NP cells took nearly 25 days to reach 80% confluence, which showed polygonal and short spindle.It had the expression of collagen type Ⅱ by cell immunohistochemistry and immunofluorescence. Lentiviral vecto was constructed successfully by the identification of restriction enzyme digestion and gene sequencing.The high titer of lentiviral vector was obtained. The m RNA and protein of MMP-3 were significantly reduce in MMP-3sh RNA3 transfected cells(P<0.05).The interfering efficiency of it at 72 h and 96 h resulted in suppression of MMP-3 m RNA expression by 98% and 72%, respectively and at 96 h the interfering efficiency of protein expression was 57.2%. After 5 days transfecting human degenerative nucleus pulposus cells by MMP-3sh RNA 3 lentiviral vector, the m RNA and protein expression level of aggrecan and collagen Ⅱ were improved,respectively(P<0.01).Conclusions:The recombination lentivirus vector with interference gene targeting human MMP-3 is successfully constructed and screened.Lentivirus mediated RNAi can significantly inhibit MMP-3 m RNA expression in human degenerative nucleus pulposus cells,so as to increase the expression of extracellular matrix.Control of the proteolytic activity of MMP-3 by RNAi may inhibitie the degradation of intervertebral disc extracellular matrix and may be against development or progression of intervertebral disc degeneration.