p38丝裂原活化蛋白激酶通路介导TGF-β1/结缔组织生长因子调控人腰椎黄韧带增生肥厚的机制研究  

Mechanism of p38 mitogen activated protein kinase signaling pathway on promoting the hypertrophy of human lumbar ligamentum flavum via transforming growth factorβ1/connective tissue growth factor

作　　者：卢昌怀 刘志军 张宏波 段扬[2] 曹延林[2] LU Changhuai;LIU Zhijun;ZHANG Hongbo;DUAN Yang;CAO Yanlin(Department of Spine Disease Area of Orthopedics and Traumatology,No.l Traditional Chinese Medicine Hospital of Changde,ChangdeHospital Affiliated to Hunan University of Traditional Chinese Medicine,Changde Hunan,415000,P.R.China;Department of Spinal Surgery,Zhujiang Hospital of Southern Medical University,Guangzhou Guangdong,510282,P.R.China)

机构地区：[1]湖南中医药大学附属常德医院常德市第一中医医院骨伤科脊柱病区,湖南常德415000 [2]南方医科大学珠江医院脊柱外科,广州510282

出　　处：《中国修复重建外科杂志》2019年第6期730-735,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：湖南省中医药管理局科研计划项目(201530);湖南省教育厅一般项目(15C1028)~~

摘　　要：目的探讨p38丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)通路在TGF-β1/结缔组织生长因子(connective tissue growth factor,CTGF)调控人腰椎黄韧带增生肥厚中的作用机制。方法取腰椎间盘突出髓核摘除术中获得的黄韧带组织,采用胶原酶预消化组织块培养法分离培养黄韧带细胞。分别用细胞外调节蛋白激酶通路阻断剂PD98059、c-Jun氨基末端激酶通路阻断剂SP600125、p38通路阻断剂SB203580处理黄韧带细胞,实时荧光定量PCR(real-time fluorescence quantitative PCR,qRT-PCR)检测CTGF、Ⅰ型胶原和Ⅲ型胶原mRNA相对表达量。然后取黄韧带细胞分为A、B、C、D组,分别以小干扰RNA(small interfering RNA,siRNA)、p38 siRNA、siRNA+3 ng/mL TGF-β1、p38 siRNA+3 ng/mL TGF-β1转染细胞,转染24 h后行免疫荧光染色观察p38和磷酸化p38(phosphorylation p38,p-p38)表达,qRT-PCR检测各组CTGF、Ⅰ型胶原和Ⅲ型胶原mRNA相对表达量,Western blot检测各组CTGF蛋白表达。结果 p38通路阻断剂SB203580可明显降低黄韧带细胞CTGF、Ⅰ型胶原和Ⅲ型胶原mRNA相对表达量(P<0.05)。转染24 h后,免疫荧光染色显示A、C、D组细胞呈阳性反应,有p38、p-p38表达,且C、D组强于A组;B组细胞呈阴性反应,无p38、p-p38表达。与A组相比,B组CTGF、Ⅰ型胶原和Ⅲ型胶原mRNA相对表达量以及CTGF蛋白相对表达量显著减少,C、D组显著增加,C组较D组进一步增加,差异均有统计学意义(P<0.05)。结论 p38 MAPK通路介导TGF-β1/CTGF表达,在人腰椎黄韧带细胞增生肥厚过程中具有重要作用。Objective To investigate the mechanism of p38 mitogen activated protein kinase(MAPK) signaling pathway in regulating the hyperplasia and hypertrophy of human lumbar ligamentum flavum via transforming growth factor β1(TGF-β1)/connective tissue growth factor(CTGF). Methods The lumbar ligamentum flavum tissue taken from patient with lumbar intervertebral disc herniation was isolated by collagenase-predigested explant cultures. The ligamentum flavum cells were treated with the extracellular regulated protein kinase pathway blocker PD98059, c-Jun Nterminal kinase pathway blocker SP600125, and p38 pathway blocker SB203580, and then the mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ were detected by real-time fluorescence quantitative PCR(qRT-PCR). The ligamentum flavum cells were divided into 4 groups, and transfected with small interfering RNA(siRNA), p38 siRNA,siRNA+3 ng/mL TGF-β1, and p38 siRNA+3 ng/mL TGF-β1 in groups A, B, C, and D, respectively. After 24 hours of transfection, immunofluorescence staining was performed to observe the expressions of p38 and phosphorylation p38(pp38);the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ in each group were detected by qRTPCR;the protein expression of CTGF in each group was detected by Western blot. Results p38 pathway blocker SB203580 could significantly reduce the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ(P<0.05). After 24 hours of transfection, immunofluorescence staining showed positive staining with p38 and p-p38 expressions in groups A, C, and D and negative staining in group B. Compared with group A, the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ and relative protein expression of CTGF in group B decreased significantly(P<0.05), while those in groups C and D increased significantly(P<0.05);and those indicators significantly increased in group C than in group D(P<0.05). Conclusion TGF-β1/CTGF based on the p38 MAPK signaling pathway play an import

关 键 词：丝裂原活化蛋白激酶 TGF-Β1 结缔组织生长因子 黄韧带 

分 类 号：R681.5[医药卫生—骨科学]