IGF-I延缓大鼠终板软骨细胞衰老与凋亡的机制研究

发布时间：2018-08-05 11:36

【摘要】：目的:研究IGF-I是否可以延缓体外培养的大鼠终板软骨细胞衰老与凋亡。方法:大鼠腰椎终板软骨细胞离体培养并传代,取P2代细胞进行相关实验。以COL-2A的mRNA相对表达含量为观察对象,探索IGF-I的作用浓度与时间。IGF-I(20ng/ml)培养终板软骨细胞,观察细胞表型及信号通路变化;同时加入PI3K/AKT信号通路阻断剂(LY2940020)或MAPK/ERK信号通路阻断剂(U0126),观察信号通路蛋白变化。然后取青年,中年和老年三个年龄段大鼠椎间盘,组织学染色观察不同年龄段大鼠椎间盘的形态。分离青年大鼠腰椎脊柱终板软骨原代细胞培养并进行传代,从P2代细胞开始,每三天传代一次,直到P6代,传代同时设置IGF-I刺激细胞实验组;HE、甲苯胺蓝和细胞骨架染色来观察细胞形态变化;western blotting和real-time qPCR分别检测软骨细胞中COL-2A,Sox9,MMP13的蛋白和mRNA表达水平,同时western blotting检测active caspase3的表达变化;多种实验方法来检测IGF-I刺激细胞后的ERK和AKT信号通路的激活。结果:时间与浓度梯度实验中,IGF-I(20ng/ml)作用24h时可增加大约2.5倍COL-2A的mRNA表达,MMP-13的蛋白表达含量明显减少,SOX9蛋白表达明显增加。AKT和ERK1/2都于15min时磷酸化最强,ERK1/2于30min时磷酸化明显减弱,AKT于60min时磷酸化明显减弱,p-ERK核内与细胞质都磷酸化增加,而p-AKT主要表现为细胞核内含量增加。软骨终板表现出与年龄相关的退行性改变。随着体外培养传代次数增加,单层培养细胞也表现出明显的表型和形态学的变化。同时,作为凋亡的标志基因,active caspase3在体外传代的P6代也可以检测出来。IGF-I可以明显提高COL-2A,Sox9,P-ERK,P-AKT的表达,减少MMP13,active caspase3的表达。更重要的是,pERK和pAKT主要表现在细胞核内含量的增加。结论:PI3K/AKT可参与IGF-I引起的COL-2A的表达,而MAPK/ERK调节Sox9、MMP13表达。从某种程度上说,体外培养引起的细胞衰老和凋亡可以在一定程度上模仿体内因为年龄而引起的细胞衰老和凋亡。IGF-I处理后可以延缓终板软骨细胞的衰老和凋亡,且可能通过ERK和AKT信号通路。
[Abstract]:Aim: to study whether IGF-I can delay the aging and apoptosis of rat endplate chondrocytes in vitro. Methods: rat lumbar endplate chondrocytes were cultured and subcultured in vitro. The relative expression of mRNA in COL-2A was studied to explore the effect of IGF-I concentration and time. IGF-I (20ng/ml) cultured endplate chondrocytes and observed the changes of phenotype and signal pathway. PI3K/AKT signaling pathway blockers (LY2940020) or MAPK/ERK signaling pathway blockers (U0126) were added to observe the changes of signal pathway proteins. Then the intervertebral discs of young, middle and aged rats were taken and the morphology of intervertebral discs in different age groups were observed by histological staining. The primary cells of lumbar endplate cartilage were isolated from the lumbar spine of young rats and were subcultured from P2 cells every three days until P6 generation. The morphological changes of the cells were observed by IGF-I stimulated cell test group, toluidine blue and cytoskeleton staining. Western blotting and real-time qPCR were used to detect the protein and mRNA expression of COL-2AnSox9 MMP13 in chondrocytes, and western blotting was used to detect the expression of active caspase3 in chondrocytes. A variety of experimental methods were used to detect the activation of ERK and AKT signaling pathways after IGF-I stimulation. Results: in the time and concentration gradient experiment, IGF-I (20ng/ml) increased the expression of MMP-13 protein in mRNA of about 2.5-fold COL-2A at 24 h. The protein expression of mussox9 decreased significantly. AKT and ERK1/2 were both the most phosphorylated at 15min. ERK1 / 2 was phosphorylated at 30min. The phosphorylation of AK T in the nucleus and cytoplasm of p-ERK was significantly decreased during 60min, and the phosphorylation in the nucleus and the cytoplasm of p-ERK was increased. However, the main manifestation of p-AKT was the increase of nuclear content. Cartilage endplate showed age-related degenerative changes. With the increase of passage times in vitro, monolayer cells also showed obvious phenotypic and morphological changes. At the same time, as a marker of apoptosis, active caspase3 can also be detected in P6 passage in vitro. IGF-I can significantly increase the expression of COL-2Agna Sox9 P-ERKN P-AKT and decrease the expression of MMP13active caspase3. More importantly, PERK and pAKT mainly manifested in the increase of nuclear content. Conclusion: PI3K / AKT may be involved in the expression of COL-2A induced by IGF-I, while MAPK/ERK regulates the expression of Sox9 MMP13. To some extent, the senescence and apoptosis induced by culture in vitro can mimic the aging and apoptosis induced by age in vivo. IGF-I treatment can delay the senescence and apoptosis of endplate chondrocytes. And probably through ERK and AKT signaling pathway.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R681.53

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