The regulation of cartilage intermediate layer protein expression by tensile loading and the mechanism underlying the process

English Keywords:Nucleus pulposus cells  Cartilage intermediate layer protein  Cyclic tensile stretch  Smad pathway  Intervertebral disc degeneration

English Abstract:

  【Abstract】 Objectives: To investigate the mechanic-mediated regulation of cartilage intermediate layer protein(CILP) by tensile loading and the mechanism underlying the process. Methods: Nucleus pulposus(NP) tissues were collected from a patient who was 38 years old and undergoing a L4/5 minimally invasive transforaminal lumbar interbody fusion(Mis-TLIF) for traumatic fracture in Xinqiao Hospital in June 2016. MRI prior to the operation showed the degeneration of the intervertebral disc was graded at Pfirrmann Ⅱ. NP cells were isolated and cultured, cells(passage=3) were prepared for subsequent mechanic-related experiments. By using Flexcell 5000 system, cyclic tensile stimuli(CTS) of various strength(5%, 10%, 20%) at 1.0HZ were delivered to the silicone membranes within the Bioflex culture plates and NP cells adhered to the membranes via computer-controlled negative pressure for different durations(6h, 12h, 24h, 48h), the group without any treatment was regarded as blank contrrol. RT-qPCR was used to analyze the gene expression of CILP in groups. Lastly, the strength and duration were chosen in which CTS displayed its maximal effect as the stimulus condition, and the gene level of CILP was detected with or without the pre-treatment of special inhibitor(SIS3) to confirm whether the smad siganling pathway mediated the up-regulation of CILP by CTS. Statistical comparisons were performed by using a one-way ANOVA test and student t test. Results: RT-qPCR showed that in all groups treated with 10% CTS, the CILP gene expression was significantly suppressed compared with blank control group(P<0.05) while reached the bottom at 48h group(P<0.05). In groups treated with CTS for 48h, 5% CTS exerted an insignificant effect on CILP expression compared with the control(P>0.05), while 10% CTS significantly down-regulated CILP expression(P<0.05), but 20% CTS markedly increased CILP expression(P<0.05), in contrary with the effect by 10% CTS. In addition, compared with the control, 20% CTS led to an increased phosphorylation of smad3. Further, when the smad3 phosphorylation was inhibited by SIS3, the increase of CILP expression by 20% CTS was significantly suppressed(P<0.05). Conclusions: Tension loading can regulate CILP expression of NP cells, which is relied on the duration and strength. Further, smad signaling pathway mediates the up-regulation of CILP expression by excessive tensional loading.