小干扰 RNA 对椎间盘炎症大鼠神经生长因子的影响研究

摘要

Objective To investigate the inhibition effect of small interfering RNA(siRNA)on nerve growth factor (NGF)induced by inflammatory factor IL - 6,IL - 1β,to provide novel targets for clinical treatment of discogenic low back pain(DLBP), Methods Nucleus pulposus and annulus fibrosus cells isolated from 60 SPF SD rates were divided into groups control(n = 12),study(n = 48), IL - 6 and IL - 1β were added into group study,cultured 48 h,and divided into subgroups A(10 nmol/ L),B(20 nmol/ L),C(50 nmol/ L),D(100 nmol/ L),12 in each, NGF - siRNA was introduced into cul-tured cells,cell conversion rate was determined and NGF - siRNA conversion rate calculated by flow cytometer,NGF mRNA ex-pression by real - time Q - PCR,NGF mRNA relative expression calculated,NGF concentration detected by enzyme - linked im-munosorbent assay(ELISA)before and after the introduction, Results By the results of flow cytometry,the conversion rate of NGF - siRNA cells was 99, 8% , NGF mRNA relative expressions of subgroups A,B,C,D were higher by times 3, 4,3, 7, 4, 7,8, 0 than that of control group before NGF - siRNA introduction(P < 0, 05),and lower by 39, 5% ,45, 5% ,45, 3% , 39, 9% ,47, 8% after introduction than before(P < 0, 05), NGF concentration of subgroups A,B,C,D were higher by times 2, 9,3, 3,4, 5,7, 4 than that of control group before NGF - siRNA introduction(P < 0, 05),and lower by 47, 2% ,33, 8% , 35, 4% ,43, 0% ,54, 9% after introduction than before(P < 0, 05), Conclusion Inflammatory cytokines IL - 6 and IL - 1βcan induce the NGF expression of rats intervertebral disc cells cultured in vitro,and the inductive effect has dose - dependence, NGF - siRNA interference,which can inhibit the inductive effects of inflammatory cytokines on NGF,provides a new target for DLBP treatment.%目的：探讨小干扰 RNA（siRNA）对炎性因子 IL -6、IL -1β诱导椎间盘大鼠神经生长因子（ NGF）的抑制效应，为椎间盘源性腰痛（DLBP）的治疗提供新靶点。方法选择 SPF 级 Sprague - Dawley（SD）大鼠60只，分离椎间盘髓核和纤维环细胞，将大鼠分为对照组（ n =12）和实验组（n =48），实验组中分别加入10 nmol/ L、20 nmol/ L、50 nmol/ L、100 nmol/ L 的 IL -6及 IL -1β共培养48 h，每个亚组12只。将NGF - siRNA导入共培养细胞，导入NGF - siRNA前后以流式细胞仪测定细胞转化率，计算 NGF - siRNA 细胞转化率；采用实时 Q - PCR 检测导入NGF - siRNA前后的 NGF mRNA 表达，计算 NGF mRNA 相对表达量；采用酶联免疫吸附试验（ ELISA）检测导入NGF - siRNA前后细胞液 NGF 浓度。结果流式细胞仪检测结果显示，NGF - siRNA细胞转化率为99,8%。导入NGF - siRNA前，10 nmol/ L、20 nmol/ L、50 nmol/ L、100 nmol/ L 组 NGF mRNA 相对表达量均较对照组上调，分别上调3,4、3,7、4,7、8,0倍（P <0,05）；导入NGF - siRNA后，各组 NGF mRNA 相对表达量均较导入前下调，分别下调39,5%、45,5%、45,3%、39,9%、47,8%（P <0,05）。导入NGF - siRNA前，10 nmol/ L、20 nmol/ L、50 nmol/ L、100 nmol/ L 组细胞液 NGF 浓度均较对照组升高，分别升高2,9、3,3、4,5、7,4倍（P <0,05）；导入NGF - siRNA后，各组细胞液 NGF 浓度均较导入前降低，分别降低47,2%、33,8%、35,4%、43,0%、54,9%（P <0,05）。结论炎性因子IL -6及 IL -1β可诱导体外培养的大鼠椎间盘细胞表达 NGF，且其诱导效应与 IL -6及 IL -1β呈剂量依赖性；NGF - siRNA能够抑制炎性因子 IL -6及 IL -1β对椎间盘细胞 NGF 的诱导效应，为 DLBP 的治疗提供了新靶点。