瘦素上调mTORC1信号通路加速大鼠跟腱异位骨化
发布时间：2018-11-11 13:42
【摘要】：目的:探讨瘦素在大鼠跟腱干细胞(TDSCs)成骨分化及跟腱异位骨化形成过程中的作用及机制,为异位骨化的预防和治疗提供理论支持。方法:体外实验,提取大鼠TDSCs进行成骨诱导培养,用不同浓度的瘦素(1、10、100 ng/ml)或100ng/ml瘦素+10nM雷帕霉素处理,3天后通过CCK-8实验检测细胞增殖情况,14天后采用碱性磷酸酶(ALP)染色/活性实验检测ALP表达,茜红素染色检测矿化结节形成,荧光定量PCR检测ALP、Runt相关转录因子2(Runx2)、成骨相关转录因子(OSX)及骨钙素(OCN),蛋白免疫印迹检测Runx2、OSX以及哺乳动物雷帕霉素靶蛋白复合物1(mTORC1)信号通路下游的磷酸化核糖体S6蛋白激酶(PS6K1)和磷酸化核糖体S6蛋白(PS6)的表达量。体内实验,选取6周龄雄性Sprague-Dawley大鼠建立跟腱中段切断术模型,随机分为对照组(control)、异位骨化组(HO)、异位骨化+雷帕霉素组(HO+RA)、异位骨化+瘦素组(HO+LEP)、异位骨化+瘦素+雷帕霉素组(HO+LEP+RA),采用瘦素及雷帕霉素处理10周,跟腱取材行伊红-苏木素染色检测钙化面积,micro-CT检测异位骨化体积,免疫组织化学检测瘦素、Runx2、OSX及PS6的表达量。结果:体外实验:瘦素在1-1OOng/ml浓度范围内对TDSCs的增殖没有显著的毒性作用,差异无统计学意义(p0.05);不同浓度的瘦素(1-1OOng/ml)促进TDSCs成骨分化过程中ALP表达及矿化结节形成,并呈浓度依赖性增加,在1OOng/ml瘦素时达到最大作用,差异具有统计学意义(p0.05);不同浓度的瘦素(1-100ng/ml)促进ALP、Runx2、OSX及OCN的mRNA表达,并随着瘦素浓度的增加而增加,在100ng/ml瘦素时表达最强,差异具有统计学意义(p0.05);不同浓度的瘦素(1-100ng/ml)促进 Runx2、OSX、PS6K1 及 PS6蛋白的表达,并随着瘦素浓度的增加而增加,在1OOng/ml瘦素时表达最强,差异具有统计学意义(p0.05);瘦素+雷帕霉素组较瘦素组的Runx2、OSX、PS6K1及PS6蛋白表达量均显著降低,差异具有统计学意义(p0.05)。体内试验:HO组较contro1组的瘦素表达明显增高,差异具有统计学意义(p0.05);HO+LEP组较HO组和HO+LEP+RA组的钙化面积、异位骨化体积显著增多,差异具有统计学意义(p0.05);HO+RA组较HO组的钙化面积和异位骨化体积显著降低,差异具有统计学意义(p0.05);HO+LEP组较HO组和HO+LEP+RA组的Runx2、OSX及PS6表达显著增强,差异具有统计学意义(p0.05);HO+RA组较HO组的Runx2、OSX及PS6表达显著降低,差异具有统计学意义(p0.05)。结论:瘦素上调mTORC1信号通路加速TDSCs成骨分化和跟腱异位骨化的形成,mTORC1信号通路抑制剂雷帕霉素可有效抑制瘦素诱导的TDSCs成骨分化和跟腱异位骨化形成,为跟腱异位骨化的预防及治疗提供了新思路。
[Abstract]:Aim: to investigate the role and mechanism of leptin in the process of (TDSCs) osteogenic differentiation and heterotopic ossification of Achilles tendon stem cells in rats, and to provide theoretical support for the prevention and treatment of ectopic ossification. Methods: rat TDSCs was extracted for osteoblast induction culture in vitro and treated with different concentrations of leptin (1 10 100 ng/ml) or 100ng/ml leptin 10nM rapamycin. After 3 days, cell proliferation was detected by CCK-8 assay. After 14 days, ALP expression was detected by alkaline phosphatase (ALP) staining / activity assay, mineralized nodule formation was detected by hematoxylin staining, and ALP,Runt related transcription factor 2 (Runx2) was detected by fluorescence quantitative PCR. Detection of Runx2, by Western blotting of osteoblast-associated transcription factor (OSX) and osteocalcin (OCN), Expression of phosphorylated ribosomal S6 protein kinase (PS6K1) and phosphorylated ribosomal S6 protein (PS6) in OSX and mammalian rapamycin target protein complex 1 (mTORC1) signaling pathway. In vivo experiment, 6-week-old male Sprague-Dawley rats were selected to establish the model of amputation of Achilles tendon. They were randomly divided into control group, (control), ectopic ossification group, (HO), ectopic ossification group, rapamycin group, (HO RA), ectopic ossification leptin group, (HO LEP), group. In the ectopic ossification leptin group, (HO LEP RA), was treated with leptin and rapamycin for 10 weeks. The calcareous area was detected by eosinolin-hematoxylin staining, the volume of ectopic ossification was detected by micro-CT and leptin was detected by immunohistochemistry. The expression of Runx2,OSX and PS6. Results: in vitro, leptin had no significant toxic effect on the proliferation of TDSCs in the range of 1-1OOng/ml concentration, and the difference was not statistically significant (p0.05). Different concentrations of leptin (1-1OOng/ml) promoted the expression of ALP and the formation of mineralized nodules in the process of osteogenic differentiation of TDSCs, and increased in a dose-dependent manner, reaching the maximum effect at the time of 1OOng/ml leptin. The difference was statistically significant (p0.05). Different concentrations of leptin (1-100ng/ml) promoted the expression of mRNA in ALP,Runx2,OSX and OCN, and increased with the increase of leptin concentration. The expression of leptin in 100ng/ml was the strongest (p0.05). Different concentrations of leptin (1-100ng/ml) promoted the expression of Runx2,OSX,PS6K1 and PS6 protein, and increased with the increase of leptin concentration. The expression of leptin in 1OOng/ml was the strongest (p0.05). The expression of Runx2,OSX,PS6K1 and PS6 in leptin group was significantly lower than that in leptin group (p0.05). In vivo test: the expression of leptin in HO group was significantly higher than that in contro1 group (p0.05). The calcification area and ectopic ossification volume in HO LEP group were significantly larger than those in HO group and HO LEP RA group (p0.05). The calcification area and ectopic ossification volume in HO RA group were significantly lower than those in HO group, and the difference was statistically significant (p0.05); HO LEP group was significantly higher than HO group and HO LEP RA group in Runx2,OSX and PS6 expression, the difference was statistically significant (p0.05). The expression of Runx2,OSX and PS6 in HO RA group was significantly lower than that in HO group (p0.05). Conclusion: leptin upregulated mTORC1 signaling pathway to accelerate the formation of TDSCs osteogenic differentiation and heterotopic calcaneal ossification. Rapamycin, an inhibitor of mTORC1 signaling pathway, can effectively inhibit leptin induced TDSCs osteogenic differentiation and Achilles tendon ectopic ossification. It provides a new idea for the prevention and treatment of heterotopic ossification of Achilles tendon.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R681
本文编号：2324999
[Abstract]:Aim: to investigate the role and mechanism of leptin in the process of (TDSCs) osteogenic differentiation and heterotopic ossification of Achilles tendon stem cells in rats, and to provide theoretical support for the prevention and treatment of ectopic ossification. Methods: rat TDSCs was extracted for osteoblast induction culture in vitro and treated with different concentrations of leptin (1 10 100 ng/ml) or 100ng/ml leptin 10nM rapamycin. After 3 days, cell proliferation was detected by CCK-8 assay. After 14 days, ALP expression was detected by alkaline phosphatase (ALP) staining / activity assay, mineralized nodule formation was detected by hematoxylin staining, and ALP,Runt related transcription factor 2 (Runx2) was detected by fluorescence quantitative PCR. Detection of Runx2, by Western blotting of osteoblast-associated transcription factor (OSX) and osteocalcin (OCN), Expression of phosphorylated ribosomal S6 protein kinase (PS6K1) and phosphorylated ribosomal S6 protein (PS6) in OSX and mammalian rapamycin target protein complex 1 (mTORC1) signaling pathway. In vivo experiment, 6-week-old male Sprague-Dawley rats were selected to establish the model of amputation of Achilles tendon. They were randomly divided into control group, (control), ectopic ossification group, (HO), ectopic ossification group, rapamycin group, (HO RA), ectopic ossification leptin group, (HO LEP), group. In the ectopic ossification leptin group, (HO LEP RA), was treated with leptin and rapamycin for 10 weeks. The calcareous area was detected by eosinolin-hematoxylin staining, the volume of ectopic ossification was detected by micro-CT and leptin was detected by immunohistochemistry. The expression of Runx2,OSX and PS6. Results: in vitro, leptin had no significant toxic effect on the proliferation of TDSCs in the range of 1-1OOng/ml concentration, and the difference was not statistically significant (p0.05). Different concentrations of leptin (1-1OOng/ml) promoted the expression of ALP and the formation of mineralized nodules in the process of osteogenic differentiation of TDSCs, and increased in a dose-dependent manner, reaching the maximum effect at the time of 1OOng/ml leptin. The difference was statistically significant (p0.05). Different concentrations of leptin (1-100ng/ml) promoted the expression of mRNA in ALP,Runx2,OSX and OCN, and increased with the increase of leptin concentration. The expression of leptin in 100ng/ml was the strongest (p0.05). Different concentrations of leptin (1-100ng/ml) promoted the expression of Runx2,OSX,PS6K1 and PS6 protein, and increased with the increase of leptin concentration. The expression of leptin in 1OOng/ml was the strongest (p0.05). The expression of Runx2,OSX,PS6K1 and PS6 in leptin group was significantly lower than that in leptin group (p0.05). In vivo test: the expression of leptin in HO group was significantly higher than that in contro1 group (p0.05). The calcification area and ectopic ossification volume in HO LEP group were significantly larger than those in HO group and HO LEP RA group (p0.05). The calcification area and ectopic ossification volume in HO RA group were significantly lower than those in HO group, and the difference was statistically significant (p0.05); HO LEP group was significantly higher than HO group and HO LEP RA group in Runx2,OSX and PS6 expression, the difference was statistically significant (p0.05). The expression of Runx2,OSX and PS6 in HO RA group was significantly lower than that in HO group (p0.05). Conclusion: leptin upregulated mTORC1 signaling pathway to accelerate the formation of TDSCs osteogenic differentiation and heterotopic calcaneal ossification. Rapamycin, an inhibitor of mTORC1 signaling pathway, can effectively inhibit leptin induced TDSCs osteogenic differentiation and Achilles tendon ectopic ossification. It provides a new idea for the prevention and treatment of heterotopic ossification of Achilles tendon.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R681
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