ERK阻滞剂PD98059对去分化软骨细胞逆分化的影响

Effect of ERK inhibitor PD98059 on retro-differentiation of dedifferentiated chondrocytes

目的探讨胞外调节蛋白激酶(ERK)阻制剂PD98059促使去分化软骨细胞逆分化的作用,为解决软骨组织工程种子细胞来源问题提供依据。方法将猪耳廓软骨细胞体外扩增至第5代,实验组加入PD98059(20mmol/L)含10%胎牛血清的F12培养液培养,每3天换液1次,未加PD98059培养为对照组。培养7天后,观察细胞阻断后单层与三维培养细胞表型变化,RT-PCR法检测两组Ⅱ型胶原、蛋白聚糖(Aggrecan)和Sox9基因表达。将经过三维环境培养的细胞植入裸鼠皮下后于第6周、8周组织学检测软骨组织形成情况。结果实验组老化软骨细胞形态与阳性对照组相比无明显变化。Ⅱ型胶原、Aggrecan、Sox9 mRNA均表达阳性。特殊染色显示裸鼠体内6周、8周均有软骨组织形成,Ⅱ型胶原免疫组织化学检测示6周、8周基质中Ⅱ型胶原表达均为阳性,对照组则未见阳性表达。结论 PD98059可部分逆转软骨细胞老化,可作为一种解决软骨组织工程种子细胞问题的新方法。

Objective To investigate the retro-differentiation effect of extracellular signal-regulated protein kinase （ERK） inhibitor PD98059 in promoting aging chondrocytes to retro-differentiate, and to provide evidence for solving the source problem of cartilage tissue engineering seeds .Methods Pig auricle chondrocytes of passage 5 were cultivated in F-12 plus PD98059 （20 mM） and 10% fetal bovine serum （ FBS）, and the culture medium was changed every 3 days, which were taken as the experimental group , and normal chondrocytes of passage 5 was set up as the control group .After 7 days, the cell phenotype change was observed after cell blocking-up in both monolayer culture and 3-D culture, and RT-PCR was applied to detect the expression of collagen type II , Aggrecan and Sox9 mRNA.On week 6 and 8 after 3-D group being implanted into nude mice skin , the immunohistochemistry was used to detect the formation of cartilage .Results There was no significant change in the cell morphology of aging chondrocyte between the experimental and control groups . After PD98059 induction, the expression of collagen type II , Aggrecan and sox9 was detected positive by RT-PCR.Special stain showed there was cartilage tissue forming in nude mice on week 6 and 8.Meanwhile, the immunohistochemistry showed that collagen type II expression was positive , and no positive expression was found in the control group .Conclu-sions PD98059 may promote the aging chondrocyte to retro-differentiate partly , which provides a new method for solving the seed cell problem of cartilage tissue engineering .