目的 通过给予外源性SHH激动剂或抑制剂从而激活或抑制SHH信号通路,观察其在兔急性心肌梗死(AMI)模型中对血管生成的影响。方法 通过用丝线结扎新西兰大白兔左冠状动脉前降支来构建兔急性心肌梗死模型。将兔随机分为对照组(假手术组)、...目的 通过给予外源性SHH激动剂或抑制剂从而激活或抑制SHH信号通路,观察其在兔急性心肌梗死(AMI)模型中对血管生成的影响。方法 通过用丝线结扎新西兰大白兔左冠状动脉前降支来构建兔急性心肌梗死模型。将兔随机分为对照组(假手术组)、模型组(结扎左前降支急性心肌梗死模型)、激动剂组(结扎左前降支,通过使用Purmorphamine激活SHH通路)、抑制剂组(结扎左前降支,通过使用Cyclopamine抑制SHH通路),术后24 h处死动物并留取静脉血液及心肌组织。采用ELISA方法检测动物模型术后24 h血清中血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF),使用免疫组化检测各组心肌组织中VEGF、bFGF蛋白的表达,实时荧光定量PCR(RT-PCP)检测各组心肌组织Hedgehog通路SHH、SMO、Gli-1 mRNA表达情况。结果 与对照组相比,急性心肌梗死模型组用ELISA方法测得的血清中VEGF、bFGF均升高(228.03±9.62pg/mL vs 306.59±12.42pg/mL,78.25±8.35pg/mL vs 141.80±5.58pg/mL),差异有统计学意义(P<0.05),与模型组相比,激动剂组VEGF、bFGF水平升高(306.59±12.42pg/mL vs 374.68±17.52pg/mL,141.80±5.58pg/mL vs 167.00±9.57pg/mL),抑制剂组VEGF、bFGF水平降低(306.59±12.42pg/mL vs 266.12±13.69pg/mL,141.80±5.58pg/mL vs 106.53±7.23pg/mL),差异有统计学意义(P<0.05);与对照组比较,模型组兔梗死心肌组织中VEGF、bFGF蛋白表达升高(0.0951±0.0080 vs 0.1732±0.0068,0.0636±0.0047 vs 0.1921±0.0063),差异有统计学意义(P<0.05),与模型组比较,激动剂组的心肌组织中VEGF、bFGF表达升高(0.1732±0.0068 vs 0.1963±0.0127,0.1921±0.0063 vs 0.2320±0.0120),抑制剂组的VEGF、bFGF表达减少(0.1732±0.0068 vs 0.1472±0.0416,0.1921±0.0063 vs 0.1645±0.0048),差异有统计学意义(P<0.05);与对照组相比,模型组SHH、SMO、Gli-1基因表达水平显著升高(1±0.058 vs 1.376±0.171,1±0.067 vs 1.202±0.877,1±0.105 vs 1.028±0.083),差异有统计学意义(P<0.05),与模型组相比,激动剂组SHH、SMO、Gli-1基因表达水平显著增高(1.376±0.171 vs 2.165±0.267,1.202±0.877 vs 1.990±0.152,1.028±0.083 vs 1.619±0.174),抑制剂组SHH、SMO、Gli-1基因表达显著下降(1.376±0.171 vs 0.911±0.15,1.202±0.877 vs 0.536±0.389,1.028±0.083 vs 0.583±0.053),差异有统计学意义(P<0.05)。结论 激活SHH信号通路,血管生成因子的表达相应增加,从而促进心肌梗死后血管生成,提示SHH信号通路激活与AMI血管再生有关。展开更多
目的:研究O-GlcNAcylation调节蛋白激酶C受体1(receptor for activated C kinase 1,Rack1)的稳定性在SHH型髓母细胞瘤(SHH type medulloblastoma,SHH-MB)形成中的功能作用。方法:选取中国人民解放军西部战区总医院临床肿瘤标本库中分子...目的:研究O-GlcNAcylation调节蛋白激酶C受体1(receptor for activated C kinase 1,Rack1)的稳定性在SHH型髓母细胞瘤(SHH type medulloblastoma,SHH-MB)形成中的功能作用。方法:选取中国人民解放军西部战区总医院临床肿瘤标本库中分子分型所确定的SHH-MB肿瘤及癌旁组织,分析样本中Rack1和O-GlcNAcylation(O-Glc NAc)的表达水平差异。对于人源髓母细胞瘤细胞系Daoy使用糖基化转移酶(OGT)抑制剂(OSMI-1)和去糖基化转移酶(OGA)抑制剂(TM-G)进行处理,通过Cell Counting Kit-8(CCK-8)法和免疫荧光染色检测肿瘤细胞增殖能力。采用O-Glc NAc酶标记系统、免疫共沉淀(Co-IP)和Western blot法判断Rack1有无发生O-Glc NAc,而后通过环己酰亚胺(CHX)实验和泛素化修饰实验证实O-GlcNAcylation对Rack1蛋白水平的影响。构建敲低Rack1的髓母细胞瘤模型,通过Cell Counting Kit-8(CCK-8)法、免疫荧光染色和划痕实验检测肿瘤细胞增殖能力。同时通过在免疫缺陷型小鼠进行异种原位肿瘤移植进行验证,在所得组织样本中(sh-NC和shRack1)使用Western blot检测下游SHH信号通路变化。结果:Rack1和O-GlcNAcylation在SHH-MB中表达水平显著增高,且Rack1表达水平和患者生存率呈负相关关系。对Daoy细胞系使用OSMI-1、TM-G处理后,发现O-Glc NAc能明显促进Daoy细胞增殖,而抑制细胞O-GlcNAc则抑制细胞增殖。分子实验证实Rack1蛋白O-GlcNAcylation可以调节其蛋白稳定性,进而促进肿瘤细胞增殖。在Daoy细胞系敲低Rack1表达,其细胞增殖能力明显低于对照组;在动物水平方面,相较于对照组,Rack1蛋白敲低的肿瘤组织增殖受到显著抑制。并且Rack1可通过调节SHH信号通路参与SHH-MB形成。结论:O-GlcNAcylation可通过调节Rack1蛋白的稳定性进而参与SHH-MB形成。展开更多
Plp1-lineage Schwann cells(SCs)of peripheral nerve play a critical role in vascular remodeling and osteogenic differentiation during the early stage of bone healing,and the abnormal plasticity of SCs would jeopardize ...Plp1-lineage Schwann cells(SCs)of peripheral nerve play a critical role in vascular remodeling and osteogenic differentiation during the early stage of bone healing,and the abnormal plasticity of SCs would jeopardize the bone regeneration.However,how Plp1-lineage cells respond to injury and initiate the vascularized osteogenesis remains incompletely understood.Here,by employing single-cell transcriptional profiling combined with lineage-specific tracing models,we uncover that Plp1-lineage cells undergoing injury-induced glia-to-MSCs transition contributed to osteogenesis and revascularization in the initial stage of bone injury.Importantly,our data demonstrated that the Sonic hedgehog(Shh)signaling was responsible for the transition process initiation,which was strongly activated by c-Jun/SIRT6/BAF170 complex-driven Shh enhancers.Collectively,these findings depict an injuryspecific niche signal-mediated Plp1-lineage cells transition towards Gli1+MSCs and may be instructive for approaches to promote bone regeneration during aging or other bone diseases.展开更多
文摘目的 通过给予外源性SHH激动剂或抑制剂从而激活或抑制SHH信号通路,观察其在兔急性心肌梗死(AMI)模型中对血管生成的影响。方法 通过用丝线结扎新西兰大白兔左冠状动脉前降支来构建兔急性心肌梗死模型。将兔随机分为对照组(假手术组)、模型组(结扎左前降支急性心肌梗死模型)、激动剂组(结扎左前降支,通过使用Purmorphamine激活SHH通路)、抑制剂组(结扎左前降支,通过使用Cyclopamine抑制SHH通路),术后24 h处死动物并留取静脉血液及心肌组织。采用ELISA方法检测动物模型术后24 h血清中血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF),使用免疫组化检测各组心肌组织中VEGF、bFGF蛋白的表达,实时荧光定量PCR(RT-PCP)检测各组心肌组织Hedgehog通路SHH、SMO、Gli-1 mRNA表达情况。结果 与对照组相比,急性心肌梗死模型组用ELISA方法测得的血清中VEGF、bFGF均升高(228.03±9.62pg/mL vs 306.59±12.42pg/mL,78.25±8.35pg/mL vs 141.80±5.58pg/mL),差异有统计学意义(P<0.05),与模型组相比,激动剂组VEGF、bFGF水平升高(306.59±12.42pg/mL vs 374.68±17.52pg/mL,141.80±5.58pg/mL vs 167.00±9.57pg/mL),抑制剂组VEGF、bFGF水平降低(306.59±12.42pg/mL vs 266.12±13.69pg/mL,141.80±5.58pg/mL vs 106.53±7.23pg/mL),差异有统计学意义(P<0.05);与对照组比较,模型组兔梗死心肌组织中VEGF、bFGF蛋白表达升高(0.0951±0.0080 vs 0.1732±0.0068,0.0636±0.0047 vs 0.1921±0.0063),差异有统计学意义(P<0.05),与模型组比较,激动剂组的心肌组织中VEGF、bFGF表达升高(0.1732±0.0068 vs 0.1963±0.0127,0.1921±0.0063 vs 0.2320±0.0120),抑制剂组的VEGF、bFGF表达减少(0.1732±0.0068 vs 0.1472±0.0416,0.1921±0.0063 vs 0.1645±0.0048),差异有统计学意义(P<0.05);与对照组相比,模型组SHH、SMO、Gli-1基因表达水平显著升高(1±0.058 vs 1.376±0.171,1±0.067 vs 1.202±0.877,1±0.105 vs 1.028±0.083),差异有统计学意义(P<0.05),与模型组相比,激动剂组SHH、SMO、Gli-1基因表达水平显著增高(1.376±0.171 vs 2.165±0.267,1.202±0.877 vs 1.990±0.152,1.028±0.083 vs 1.619±0.174),抑制剂组SHH、SMO、Gli-1基因表达显著下降(1.376±0.171 vs 0.911±0.15,1.202±0.877 vs 0.536±0.389,1.028±0.083 vs 0.583±0.053),差异有统计学意义(P<0.05)。结论 激活SHH信号通路,血管生成因子的表达相应增加,从而促进心肌梗死后血管生成,提示SHH信号通路激活与AMI血管再生有关。
基金supported by the National Natural Science Foundation of China(grants 81970910 and 82370931)Jiangsu Province Capability Improvement Project through Science,Technology and Education-Jiangsu Provincial Research Hospital Cultivation Unit(YJXYYJSDW4)Jiangsu Provincial Medical Innovation Center(CXZX202227).
文摘Plp1-lineage Schwann cells(SCs)of peripheral nerve play a critical role in vascular remodeling and osteogenic differentiation during the early stage of bone healing,and the abnormal plasticity of SCs would jeopardize the bone regeneration.However,how Plp1-lineage cells respond to injury and initiate the vascularized osteogenesis remains incompletely understood.Here,by employing single-cell transcriptional profiling combined with lineage-specific tracing models,we uncover that Plp1-lineage cells undergoing injury-induced glia-to-MSCs transition contributed to osteogenesis and revascularization in the initial stage of bone injury.Importantly,our data demonstrated that the Sonic hedgehog(Shh)signaling was responsible for the transition process initiation,which was strongly activated by c-Jun/SIRT6/BAF170 complex-driven Shh enhancers.Collectively,these findings depict an injuryspecific niche signal-mediated Plp1-lineage cells transition towards Gli1+MSCs and may be instructive for approaches to promote bone regeneration during aging or other bone diseases.
