牵张微应变影响髓核细胞生物学功能和退变的研究

Effects of cyclic tensile stress on biological function and degeneration of nucleus pulposus cells

目的探讨周期性牵张微应变对人髓核细胞生物学功能和退变的影响。方法收集人椎间盘髓核组织,酶消化法分离、培养髓核原代细胞,显微镜下观察细胞生长状态。Mechano Culture FX2加载周期性牵张微应变,应力10万μ?,10%拉伸应变,频率0.1 Hz,8640个循环周期。MTT法检测牵张微应变对髓核细胞增殖的影响;流式细胞仪检测牵张微应变对髓核细胞周期和凋亡的影响。基因表达谱芯片检测牵张微应变组和对照组表达差异的基因,生物信息学分析表达差异基因在髓核细胞的功能。荧光定量PCR法检测牵张微应变对髓核细胞表达炎症相关因子、TGF-β、基质降解酶类、ECM分子等的影响。结果牵张微应变组髓核细胞生长状态较好。牵张微应变促进髓核细胞增殖和周期进程,两组细胞S期(t=5.336,P<0.05)和G2/M期(t=7.288,P<0.01)百分比的差异均有统计学意义。牵张微应变抑制髓核细胞凋亡(牵张微应变组8.56%±0.48%,对照组10.63%±0.32%,t=4.474,P<0.05)。基因表达谱芯片检测到866个差异表达基因,基因本体(gene ontology)分析其在细胞中的作用,主要包括focal adhesion、extracellular matrix,membrane raft、condensed chromosome kinetochore、cytoskeleton等。牵张微应变影响髓核细胞表达炎症相关因子、TGF-β基因、基质降解酶、细胞外基质分子等的表达。与对照组相比,牵张微应变组炎症相关因子IL15(t=5.379,P<0.05)、IGF1(t=5.454,P<0.05)、IGFBP7(t=13.57,P<0.01)的表达降低;TGF-β相关基因TGFB1(t=6.931,P<0.05)、TGFB2(t=15.56,P<0.01)、TGFB3(t=7.744,P<0.05)的表达增高;基质降解酶类ADAMTS3(t=5.241,P<0.05)和MMP19(t=24.72,P<0.01)的表达降低,TIMP3(t=8.472,P<0.01)的表达增高;ECM分子COL2A1(t=5.871,P<0.05)、FLRT2(t=5.216,P<0.05)、FN1(t=4.289,P<0.05)的表达增高。结论周期性牵张微应变促进髓核细胞周期和增殖,抑制髓核细胞凋亡,可能通过调节髓核细胞表达炎症相关因子、TGF-β、基质降解酶类、ECM分子等改善髓核细胞退变。

Objective To investigate the effects of cyclic tensile stress on the function and degeneration of nucleus pulp⁃osus cells.Methods The human primary nucleus pulposus cells were isolated and cultured.The cyclic tensile stress(100000μɛ,10%tensile strain,0.1 Hz,8640 cycles)was loaded on the cells for 24 h.The proliferation of the cells was examined by MTT method.The cell cycle and apoptosis were detected through flow cytometry.Gene expression profile chip was used to detect the dif⁃ferentially expressed genes between the tensile stress group and control group.The function of these gene was analyzed by bioinformatics.The expression of inflammatory related factors,TGF⁃β,matrix degrading enzymes and extracellular matrix molecules were examined by qRT⁃PCR.Results The cyclic tensile stress significantly promoted proliferation and cell cycle of nucleus pulposus cells.The cell percentage of S phase(t=5.336,P<0.05)and G2/M phase(t=7.288,P<0.01)was significantly different between the tensile stress group and control group.The cyclic tensile stress inhibited apoptosis of nucleus pulposus cells(8.56%±0.48%vs 10.63%±0.32%,t=4.474,P<0.05).A total of 866 differentially expressed genes were detected.Gene ontology analysis showed the roles of these genes in cells including focal adhesion,extractable matrix,membrane raft,condensed chrome kinetochore,cytoskele⁃ton,etc.The cyclic tensile stress significantly affected the mRNA expression of inflammatory related factors,TGF⁃βgenes,matrix proteinase and extracellular matrix molecules.Compared with the control group,the mRNA expression of inflammatory related fac⁃tors IL15(t=5.379,P<0.05),IGF1(t=5.454,P<0.05)and IGFBP7(t=13.57,P<0.01)were significantly decreased in the tensile stress group;The mRNA expression of TGF⁃βgenes TGFB1(t=6.931,P<0.05),TGFB2(t=15.56,P<0.01)and TGFB3(t=7.744,P<0.05)were significantly increased in the tensile stress group;The mRNA expression of matrix proteinase ADAMTS3(t=5.241,P<0.05)and MMP19(t=24.72,P<0.01)were significantly decreased,and TIMP3(t=8.472,P<0.01)increased in the tensile stress group;The mRNA expression of extracellular matrix molecules COL2A1(t=5.871,P<0.05),FLRT2(t=5.216,P<0.05)and FN1(t=4.289,P<0.05)were significantly increased.Conclusion The cyclic tensile stress promoted cell cycle and proliferation and in⁃hibited apoptosis of nucleus pulposus cells.The cyclic tensile stress may affect the function and degeneration of nucleus pulposus cells by regulating the expression of inflammatory related factors,TGF⁃β,matrix degradation enzymes and ECM molecules.