The Experimental Study Of Low-Intensity Pulsed Ultrasound Stimulating Synthsis Of Extracellular Matrix Functional Components In Human Nucleus Pulposus Cell In Vitro

PartⅠThe screening for optimal intensity parameter of low-intensity pulsed ultrasound in stimulating synthesis of COLL2 and Aggrecan in Human Nucleus Pulposus CellObjective Low-intensity pulsed ultrasound (LIPUS) has been reported to stimulate synthesis of COLL2 and Proteoglycan, which forming the capital components of Extracellular Matrix (ECM). Human Nucleus Pulposus Cell (HNPC) Line was used firstly in China and underwent LIPUS to get the optimal intensity parameter which can result in the most production of COLL2 and Aggrecan. (Aggrecan roles as the represent of Proteoglycan family members in study)Method Five groups of intensity were established as 0、15、30、60、120 mW/cm2 and the other important parameters refered to 1.0 MHz frequency with a 20-us tone burst repeated at 1.0 kHz. The cells were stimulated for 20 min each day using a LIPUS generator. The effects of LIPUS were evaluated by measuring mRNA expression of COLL2 and Aggrecan by Real-time PCR, the protein synthesis of COLL2 and Aggrecan by Western Blotting. The direct-viewing of related protein expression in HNPC was showed by cellular immunohistochemisty.Results The mRNA expression trends of COLL2 and Aggrecan were similar to the protein synthesis trends of them respectively. The mRNA expression and protein synthesis of COLL2 were significantly increased in the groups of 30 mW/cm2 and 60 mW/cm2 compared with the other groups (P<0.05). The mRNA expression and protein synthesis of Aggrecan were significantly increased in Groups of 60 mW/cm2 and 120 mW/cm2 compared with the other groups (P<0.05). Yet the mRNA expression and protein synthesis of COLL2 were lower in Group of 120 mW/cm2 compared with Group of 60 mW/cm2.Conclusions The intensity of 60 mW/cm2 serves as the optimal intensity parameter of LIPUS in stimulating the most synthesis of COLL2 and Aggrecan in HNPCPartⅡThe effects of LIPUS on ECM synthesis in IL-1βinduced degenerative HNPCObjective IL-1βhas already been reported to decrease the synthesis of ECM by nucleus pulposus cells, so IL-1βinduced degenerative HNPC can serve as a model partially imitating the real degenerative HNPC in vivo. This part will focus on the effects of LIPUS on ECM synthesis in IL-1βinduced degenerative HNPC.Method The IL-1βinduced degenerative HNPC model was established according to the related literatures. LIPUS parameters includes 60mW/cm2 Intensity,1.0MHz frequency with a 20-us tone burst repeated at 1.0 kHz. The cells were stimulated for 20 min each day using a LIPUS generator. The cells were cultured under three different sets of conditions: IL-1βgroup, IL-1β+LIPUS(+) group, LIPUS(+)+IL-1βgroup. The effects of LIPUS were evaluated by measuring mRNA expression of COLL2, Aggrecan, MMP-3, MMP-13 by Real-time PCR, the protein synthesis of COLL2 and Aggrecan by Western Blotting. The direct-viewings of related protein expression in HNPC were showed by cellular immunofluorescence.Results The mRNA expression and protein synthesis trends of COLL2 and Aggrecan significantly increased in IL-1βinduced degenerative HNPC by LIPUS (P<0.05). Yet in LIPUS(+)+IL-1βgroup the mRNA expression of COLL2 and Aggrecan decreased to the level of IL-1βgroup when LIPUS was removed. And Aggrecan was degraded more quickly than COLL2 and the reason may be IL-1βupregulated more MMP-3 than MMP-13, MMP-3 has the main function of degrading Aggrecan. The accordant results of protein synthesis appeared by cellular immunofluorescence.Conclusions 1L-1βcan effectively induce the decreasing of ECM protein to meet the degenerative condition. LIPUS stimulate IL-1βinduced degenerative HNPC to synthesize ECM functional protein, so it has potential value as one kind of therapy for disc degeneration. Yet, LIPUS lacks continuous downstream effects when it stops working for degenerative HNPC which are still sensitive to inflammatory enviroment.. Keywords Nucleus Pulposus Cell, Degeneration, IL-1β, MMPsPartⅢThe mechanism study for LIPUS stimulating synthesis of COLL2 and Aggrecan in HNPCObjective Low-intensity pulsed ultrasound (LIPUS) has been reported to stimulate synthesis of COLL2 and Aggrecan, but the real mechanism is not absolutely clear. LIPUS has already been demonstrated to upregulate the function of beagles nucleus pulposus cells. HNPC used here will more closer to the reality of human and more suitable for LIPUS study.Method The HNPC were cultured under three different sets of conditions:LIPUS group, LIPUS+TGF-βgroup, LIPUS+TGF-β1+TGF-β1 neutralizing antibody group. The effects of LIPUS were evaluated by measuring mRNA expression of COLL2, Aggrecan by Real-time PCR. TGFβR1 mRNA expression was assessed in LIPUS(-) group, LIPUS(+) group. TGF-β1 group and LIPUS+TGF-β1 group. The correlation of Integrinα5β1 and cytoskeleton was showed by cellular immunofluorescence.Results The mRNA expression of COLL2 and Aggrecan have the top in LIPUS+TGF-β1 group (P<0.05). yet the reverse effect appeared in LIPUS+TGF-β1+TGF-β1 neutralizing antibody group with the lowest production level of mRNA expression. The mRNA expression of TGFβR1 was significantly increased by LIPUS. LIPUS stimulated the expression of Integrinα5β1 in cell membrane accompanied with the inverse proportion of cytoskeleton. The accordant results of protein synthesis appeared by cellular immunofluorescence.Conclusions LIPUS mainly stimulate the synthesis of ECM functional protein by TGF signal pathway and demonstrated a synergistic effect with TGF-β1. LIPUS enhances the cell sensitivity to TGF-β1 by upregulating TGFβR1 mRNA expression in HNPC. The classic mechanotransduction signal pathway "ECM-Integrins-Cytoskeleton" was involved in the effects of LIPUS on HNPC.Part IV The effects for LIPUS stimulating synthesis of COLL2 and Aggrecan in surgical-source degenerative nucleus pulposus cellsObjective A initial study on effects for LIPUS stimulating synthesis of COLL2 and Aggrecan in surgical-source degenerative nucleus pulposus cells.Method The isolating, culturing and characterizing of surgical-source degenerative nucleus pulposus cells:Three experimental groups were established including LIPUS(+) group, LIPUS(-) group and the fresh DMEM/F12 as negative control group. The effects of LIPUS were evaluated by measuring production of COLL2, Aggrecan in supernatant liquid at every LIPUS work point for 5 days by ELISA assay. And the productions of COLL2, Aggrecan in another group system including the initial cells group, LIPUS(+) group and LIPUS(-) group were detected by Western Blotting.Results In LIPUS(+) group the production of COLL2 and Aggrecan were significantly increased after 2-day treatment (P<0.05) and then gradually decreased to the level of the initial cells group (P>0.05); the production of COLL2, Aggrecan in LIPUS(-) group continue to descend. At the day 5, LIPUS(+) group had similar amount of proteins compared with the initial cells, which were significantly higher than those in LIPUS(-) group (P<0.05). Conclusions LIPUS stimulate the synthesis of ECM functional protein surgical-source degenerative nucleus pulposus cells and delay the development of degeneration.