IGF-I延緩大鼠終板軟骨細(xì)胞衰老與凋亡的機(jī)制研究
發(fā)布時(shí)間：2018-08-05 11:36
【摘要】：目的:研究IGF-I是否可以延緩體外培養(yǎng)的大鼠終板軟骨細(xì)胞衰老與凋亡。方法:大鼠腰椎終板軟骨細(xì)胞離體培養(yǎng)并傳代,取P2代細(xì)胞進(jìn)行相關(guān)實(shí)驗(yàn)。以COL-2A的mRNA相對表達(dá)含量為觀察對象,探索IGF-I的作用濃度與時(shí)間。IGF-I(20ng/ml)培養(yǎng)終板軟骨細(xì)胞,觀察細(xì)胞表型及信號(hào)通路變化;同時(shí)加入PI3K/AKT信號(hào)通路阻斷劑(LY2940020)或MAPK/ERK信號(hào)通路阻斷劑(U0126),觀察信號(hào)通路蛋白變化。然后取青年,中年和老年三個(gè)年齡段大鼠椎間盤,組織學(xué)染色觀察不同年齡段大鼠椎間盤的形態(tài)。分離青年大鼠腰椎脊柱終板軟骨原代細(xì)胞培養(yǎng)并進(jìn)行傳代,從P2代細(xì)胞開始,每三天傳代一次,直到P6代,傳代同時(shí)設(shè)置IGF-I刺激細(xì)胞實(shí)驗(yàn)組;HE、甲苯胺藍(lán)和細(xì)胞骨架染色來觀察細(xì)胞形態(tài)變化;western blotting和real-time qPCR分別檢測軟骨細(xì)胞中COL-2A,Sox9,MMP13的蛋白和mRNA表達(dá)水平,同時(shí)western blotting檢測active caspase3的表達(dá)變化;多種實(shí)驗(yàn)方法來檢測IGF-I刺激細(xì)胞后的ERK和AKT信號(hào)通路的激活。結(jié)果:時(shí)間與濃度梯度實(shí)驗(yàn)中,IGF-I(20ng/ml)作用24h時(shí)可增加大約2.5倍COL-2A的mRNA表達(dá),MMP-13的蛋白表達(dá)含量明顯減少,SOX9蛋白表達(dá)明顯增加。AKT和ERK1/2都于15min時(shí)磷酸化最強(qiáng),ERK1/2于30min時(shí)磷酸化明顯減弱,AKT于60min時(shí)磷酸化明顯減弱,p-ERK核內(nèi)與細(xì)胞質(zhì)都磷酸化增加,而p-AKT主要表現(xiàn)為細(xì)胞核內(nèi)含量增加。軟骨終板表現(xiàn)出與年齡相關(guān)的退行性改變。隨著體外培養(yǎng)傳代次數(shù)增加,單層培養(yǎng)細(xì)胞也表現(xiàn)出明顯的表型和形態(tài)學(xué)的變化。同時(shí),作為凋亡的標(biāo)志基因,active caspase3在體外傳代的P6代也可以檢測出來。IGF-I可以明顯提高COL-2A,Sox9,P-ERK,P-AKT的表達(dá),減少M(fèi)MP13,active caspase3的表達(dá)。更重要的是,pERK和pAKT主要表現(xiàn)在細(xì)胞核內(nèi)含量的增加。結(jié)論:PI3K/AKT可參與IGF-I引起的COL-2A的表達(dá),而MAPK/ERK調(diào)節(jié)Sox9、MMP13表達(dá)。從某種程度上說,體外培養(yǎng)引起的細(xì)胞衰老和凋亡可以在一定程度上模仿體內(nèi)因?yàn)槟挲g而引起的細(xì)胞衰老和凋亡。IGF-I處理后可以延緩終板軟骨細(xì)胞的衰老和凋亡,且可能通過ERK和AKT信號(hào)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.
【學(xué)位授予單位】：皖南醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R681.53
本文編號(hào)：2165678
[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.
【學(xué)位授予單位】：皖南醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R681.53
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相關(guān)期刊論文 前3條
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，本文編號(hào)：2165678
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