张力性刺激对人髓核细胞表达软骨层间蛋白的调控及机制

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

中文关键词:  髓核细胞  软骨层间蛋白  张力  Smad信号通路  椎间盘退变

英文关键词:Nucleus pulposus cells  Cartilage intermediate layer protein  Cyclic tensile stretch  Smad pathway  Intervertebral disc degeneration

中文摘要:

  【摘要】 目的：探索张力性刺激对人椎间盘髓核细胞分泌软骨层间蛋白（cartilage intermediate layer protein，CILP）的影响及其机制。方法：选取2016年6月在我院因创伤性骨折行L4/5椎间盘摘除及相邻椎体融合手术的1例38岁男性患者的髓核组织作为细胞来源，术前MRI示该节段椎间盘组织改良Pfirrmann分级为Ⅱ级，分离培养其中的髓核细胞并传代至第3代，进行力学实验。使用Flexcell 5000系统通过电脑控制气压变化来使载有细胞的塑料膜发生形变，进而对膜载体上的细胞给予不同强度、时长的张力性力学加载。实验组分为4组，均以10%的拉伸量为刺激强度，给予不同时长（6h、12h、24h 、48h）的刺激，空白对照组不给予任何刺激。利用RT-qPCR技术检测各组CILP的基因表达量，选出表达变化最为明显的一组，以该组的刺激时长为限，再将实验组分为3组，分别给予不同刺激强度（5%、10%、20%）的张力刺激，空白对照组不给予任何刺激，利用RT-qPCR技术检测各组CILP的基因表达。选取相比较于对照组CILP增长效应最明显的力学强度和时长作为刺激条件，测定髓核细胞在该实验条件下smad3的磷酸化程度，并对髓核细胞预先给予smad3磷酸化的特异性抑制剂SIS3处理后再置于同一力学条件下，以RT-qPCR检测CILP的基因表达量，以此观察smad信号通路是否介导了张力性刺激对于髓核细胞CILP表达的调控。实验结果使用配对t检验以及单因素方差分析进行组间比较。结果：RT-qPCR结果显示，相较于空白对照组，不同刺激时长的10%张力性刺激均明显下调了CILP的表达，其中在刺激48h后对CILP的抑制效应达到最大（P<0.05）；5%的张力性刺激48h后髓核细胞的CILP基因相对表达量与对照组比较无统计学差异（P＞0.05），10%的张力刺激48h明显降低CILP的表达（P<0.05），20%的张力刺激48h显著提高CILP表达量（P<0.05）且smad3的磷酸化水平相对于空白对照组明显增高；相对于只给予20%张力刺激的对照组，先给予SIS3预处理再给予相同力学刺激的实验组的CILP表达水平显著下调（P<0.05）。结论：张力性刺激调控髓核细胞的CILP表达，调控效应随着刺激时长和刺激强度的变化而不同；smad信号通路参与高强度的张力性刺激对CILP的上调效应。

英文摘要:

  【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.