旨在利用HIF-1α激动剂二甲基乙二酰甘氨酸(DMOG)上调HIF-1α的表达水平,研究HIF-1α对猪肠道上皮细胞糖酵解相关酶基因及β防御素表达的调节作用.以猪空肠上皮细胞系IPEC-J2细胞为研究对象,分为对照组和DMOG处理组,试验设置6个处理浓...旨在利用HIF-1α激动剂二甲基乙二酰甘氨酸(DMOG)上调HIF-1α的表达水平,研究HIF-1α对猪肠道上皮细胞糖酵解相关酶基因及β防御素表达的调节作用.以猪空肠上皮细胞系IPEC-J2细胞为研究对象,分为对照组和DMOG处理组,试验设置6个处理浓度(0、0.25、0.5、1、2、4 mM)和2个处理时间(4 h和24 h),采用CCK8检测细胞活力确定后续试验的培养基是否含血清及DMOG的浓度范围,基于上述试验结果,CCK8检测不同作用时间(0、1、2、4、8、24 h)下,低剂量(0.25 mM)和高剂量(2 mM)DMOG对细胞活力的影响,酶联免疫吸附实验(ELISA)和蛋白免疫印迹(WB)检测HIF-1α的蛋白表达水平,实时荧光定量PCR(qPCR)检测HIF-1α及其下游靶基因mRNA的表达情况,比色法检测糖酵解途径关键酶活性的变化.结果发现,2 mM的DMOG处理4、8和24 h时能显著抑制IPEC-J2细胞活力(P<0.05).WB结果表明,2 mM DMOG能显著上调细胞内总HIF-1α的蛋白表达(P<0.05).2 mM DMOG处理4 h能显著上调HIF-1α下游靶基因葡萄糖转运蛋白1(GLUT-1)、乳酸脱氢酶A(LDHA)、6-磷酸果糖-2-激酶(PFK-3)、己糖激酶2(HK2)的mRNA表达水平(P<0.05),同时上调糖酵解关键限速酶丙酮酸激酶(PK)、磷酸果糖激酶(PFK)、己糖激酶(HK)的活性(P<0.05),2 mM DMOG处理24 h则显著上调细胞内PK活性(P<0.05),表明DMOG可能通过上调HIF-1α途径,促进IPEC-J2细胞的代谢类型转向糖酵解.ELISA结果发现,2 mM DMOG处理4 h能够促进猪β防御素1(PBD1)和猪β防御素2(PBD2)的蛋白表达水平(P<0.05).综上所述,DMOG能够上调猪IPEC-J2细胞中HIF-1α及其下游糖酵解相关酶基因和β防御素的表达,初步表明低氧诱导因子HIF-1α可能对猪IPEC-J2细胞β防御素表达具有调节作用.展开更多
Vascularization and bone regeneration are closely related in the process of bone remodeling,and designing a bioactive scaffold with pro-angiogenic and osteogenic properties may accelerate the repair of bone defects.In...Vascularization and bone regeneration are closely related in the process of bone remodeling,and designing a bioactive scaffold with pro-angiogenic and osteogenic properties may accelerate the repair of bone defects.In this work,an iron-based metal-organic framework(MIL-88)was developed as a carrier for loading a pro-angiogenic small molecular drug(dimethyloxallyl glycine,DMOG),and then embedded into the PLGA nanofibrous scaffolds to repair cranial defects in rats.Imaging and histological evaluation indicated that PLGA/MIL@D scaffold markedly enhanced vascularization and bone regeneration in vivo.Moreover,in vitro assay showed that co-delivery system significantly promoted angiogenesis by stimulating endothelial cell migration,tube formation,and enhanced osteogenesis by promoting expression of osteoblast related proteins.In addition,PLGA/MIL@D scaffold promotes angiogenesis by activating the hypoxia-inducible factor-1(HIF-1)/vascular endothelial growth factor(VEGF)signaling pathway.Altogether,this bioactive PLGA/MIL@D scaffold can combine angiogenesis with osteogenesis,and will be a bright strategy for the repair of bone defects.展开更多
The coupled process of osteogenesis-angiogenesis plays a crucial role in periodontal tissue regeneration.Although various cytokines or chemokines have been widely applied in periodontal in situ tissue engineering,most...The coupled process of osteogenesis-angiogenesis plays a crucial role in periodontal tissue regeneration.Although various cytokines or chemokines have been widely applied in periodontal in situ tissue engineering,most of them are macromolecular proteins with the drawbacks of short effective half-life,poor stability and high cost,which constrain their clinical translation.Our study aimed to develop a difunctional structure for periodontal tissue regeneration by incorporating an angiogenic small molecule,dimethyloxalylglycine(DMOG),and an osteoinductive inorganic nanomaterial,nanosilicate(nSi)into poly(lactic-co-glycolic acid)(PLGA)fibers by electrospinning.The physiochemical properties of DMOG/nSi-PLGA fibrous membranes were characterized.Thereafter,the effect of DMOG/nSi-PLGA membranes on periodontal tissue regeneration was evaluated by detecting osteogenic and angiogenic differentiation potential of periodontal ligament stem cells(PDLSCs)in vitro.Additionally,the fibrous membranes were transplanted into rat periodontal defects,and tissue regeneration was assessed with histological evaluation,micro-computed tomography(micro-CT),and immunohistochemical analysis.DMOG/nSi-PLGA membranes possessed preferable mechanical property and biocompatibility.PDLSCs seeded on the DMOG/nSi-PLGA membranes showed up-regulated expression of osteogenic and angiogenic markers,higher alkaline phosphatase(ALP)activity,and more tube formation in comparison with single application.Further,in vivo study showed that the DMOG/nSi-PLGA membranes promoted recruitment of CD90+/CD34stromal cells,induced angiogenesis and osteogenesis,and regenerated cementum-ligament-bone complex in periodontal defects.Consequently,the combination of DMOG and nSi exerted admirable effects on periodontal tissue regeneration.DMOG/nSi-PLGA fibrous membranes could enhance and orchestrate osteogenesis-angiogenesis,and may have the potential to be translated as an effective scaffold in periodontal tissue engineering.展开更多
文摘目的探讨在缺氧缺血清环境下,脯氨酸羟化酶抑制剂——二甲基乙二酰甘氨酸(DMOG)促进骨髓间充质干细胞(MSCs)血管新生的作用及其机制。方法 MSCs从大鼠骨髓中分离得到,分为:常氧条件下溶剂对照组(N-DMSO)、缺氧条件下溶剂对照组(H-DMSO)、20μM DMOG加药组(D-20μM)、100μM DMOG加药组(D-100μM)及500μM DMOG加药组(D-500μM),观察以下指标:(1)通过缺氧条件下CCK-8检测DMOG在20μM、100μM及500μM剂量下对MSCs的保护作用,确定DMOG的最佳剂量;(2)通过Matrigel实验观察DMOG促进MSCs血管新生的作用;(3)通过Western blot法检测血管新生通路相关蛋白表达。结果 (1)不同浓度组DMOG对缺氧损伤MSCs的影响:N-DMSO组OD值3.25±0.05,H-DMSO组2.23±0.14,D-20μM组2.68±0.43,D-100μM组3.11±0.25,D-500μM组3.23±0.04。与N-DMSO组比较,H-DMSO组OD值降低(P<0.01),与H-DMSO组比较,D-20μM组、D-100μM组、D-500μM OD值均升高(P<0.05或0.01)。(2)不同浓度组DMOG对正常MSCs的影响:D-20μM组3.19±0.02,D-100μM组3.15±0.06,D-500μM组2.51±0.08。与N-DMSO组比较,D-500μM组OD值降低(P<0.01),D-20μM组、D-100μM组与N-DMSO组比较差异均无统计学意义(均P>0.05),提示DMOG在20μM及100μM对细胞无毒性。(3)血管新生相关蛋白的表达:与H-DMSO组相比,D-100μM组缺氧3、6及24h HIF-1α均增加(1.44±0.32 vs 7.79±0.23,2.4±0.28 vs 3.51±0.79,0.93±0.37 vs2.46±0.07,P<0.05或0.01),p AKT则在缺氧6h增加(0.47±0.15 vs 0.71±0.03,P<0.05),VEGF在缺氧6、24h均增加(0.63±0.10 vs 0.87±0.14,0.42±0.06 vs 0.70±0.06,P<0.05或0.01),以缺氧24h最为显著。pm TOR/m TOR及p ERK/ERK蛋白两组比较差异均无统计学意义(均P>0.05)。结论 DMOG通过抑制HIF-1α降解及促进AKT磷酸化提高缺氧环境下MSCs的血管新生能力。
文摘旨在利用HIF-1α激动剂二甲基乙二酰甘氨酸(DMOG)上调HIF-1α的表达水平,研究HIF-1α对猪肠道上皮细胞糖酵解相关酶基因及β防御素表达的调节作用.以猪空肠上皮细胞系IPEC-J2细胞为研究对象,分为对照组和DMOG处理组,试验设置6个处理浓度(0、0.25、0.5、1、2、4 mM)和2个处理时间(4 h和24 h),采用CCK8检测细胞活力确定后续试验的培养基是否含血清及DMOG的浓度范围,基于上述试验结果,CCK8检测不同作用时间(0、1、2、4、8、24 h)下,低剂量(0.25 mM)和高剂量(2 mM)DMOG对细胞活力的影响,酶联免疫吸附实验(ELISA)和蛋白免疫印迹(WB)检测HIF-1α的蛋白表达水平,实时荧光定量PCR(qPCR)检测HIF-1α及其下游靶基因mRNA的表达情况,比色法检测糖酵解途径关键酶活性的变化.结果发现,2 mM的DMOG处理4、8和24 h时能显著抑制IPEC-J2细胞活力(P<0.05).WB结果表明,2 mM DMOG能显著上调细胞内总HIF-1α的蛋白表达(P<0.05).2 mM DMOG处理4 h能显著上调HIF-1α下游靶基因葡萄糖转运蛋白1(GLUT-1)、乳酸脱氢酶A(LDHA)、6-磷酸果糖-2-激酶(PFK-3)、己糖激酶2(HK2)的mRNA表达水平(P<0.05),同时上调糖酵解关键限速酶丙酮酸激酶(PK)、磷酸果糖激酶(PFK)、己糖激酶(HK)的活性(P<0.05),2 mM DMOG处理24 h则显著上调细胞内PK活性(P<0.05),表明DMOG可能通过上调HIF-1α途径,促进IPEC-J2细胞的代谢类型转向糖酵解.ELISA结果发现,2 mM DMOG处理4 h能够促进猪β防御素1(PBD1)和猪β防御素2(PBD2)的蛋白表达水平(P<0.05).综上所述,DMOG能够上调猪IPEC-J2细胞中HIF-1α及其下游糖酵解相关酶基因和β防御素的表达,初步表明低氧诱导因子HIF-1α可能对猪IPEC-J2细胞β防御素表达具有调节作用.
基金supported by the Fundamental Research Funds for the Central Universities of China(No.DUT22YG201)。
文摘Vascularization and bone regeneration are closely related in the process of bone remodeling,and designing a bioactive scaffold with pro-angiogenic and osteogenic properties may accelerate the repair of bone defects.In this work,an iron-based metal-organic framework(MIL-88)was developed as a carrier for loading a pro-angiogenic small molecular drug(dimethyloxallyl glycine,DMOG),and then embedded into the PLGA nanofibrous scaffolds to repair cranial defects in rats.Imaging and histological evaluation indicated that PLGA/MIL@D scaffold markedly enhanced vascularization and bone regeneration in vivo.Moreover,in vitro assay showed that co-delivery system significantly promoted angiogenesis by stimulating endothelial cell migration,tube formation,and enhanced osteogenesis by promoting expression of osteoblast related proteins.In addition,PLGA/MIL@D scaffold promotes angiogenesis by activating the hypoxia-inducible factor-1(HIF-1)/vascular endothelial growth factor(VEGF)signaling pathway.Altogether,this bioactive PLGA/MIL@D scaffold can combine angiogenesis with osteogenesis,and will be a bright strategy for the repair of bone defects.
基金the National Natural Science Foundation of China(No.81670993,81873716,and 81901009)The Construction Engineering Special Fund of“Taishan Scholars”of Shandong Province(No.ts20190975 and tsqn201909180)+1 种基金National Key R&D Program of China(No.2017YFB0405400)Collaborative Innovation Center of Technology and Equipment for Biological Diagnosis and Therapy in Universities of Shandong,The National Key Research and Development Program of China(No.2017YFA0104604),Open Foundation of Shandong Provincial Key Laboratory of Oral Tissue Regeneration(No.SDKQ201901,SDKQ201904).The funders had no role in study design,data collection and analysis,decision to publish or preparation of the manuscript.The authors also thank Prof.Hongyu Zhang and Dr.Yi Wang from Tsinghua University for technical guidance.The authors declare that no financial or other potential competing interests exist with regard to this study.
文摘The coupled process of osteogenesis-angiogenesis plays a crucial role in periodontal tissue regeneration.Although various cytokines or chemokines have been widely applied in periodontal in situ tissue engineering,most of them are macromolecular proteins with the drawbacks of short effective half-life,poor stability and high cost,which constrain their clinical translation.Our study aimed to develop a difunctional structure for periodontal tissue regeneration by incorporating an angiogenic small molecule,dimethyloxalylglycine(DMOG),and an osteoinductive inorganic nanomaterial,nanosilicate(nSi)into poly(lactic-co-glycolic acid)(PLGA)fibers by electrospinning.The physiochemical properties of DMOG/nSi-PLGA fibrous membranes were characterized.Thereafter,the effect of DMOG/nSi-PLGA membranes on periodontal tissue regeneration was evaluated by detecting osteogenic and angiogenic differentiation potential of periodontal ligament stem cells(PDLSCs)in vitro.Additionally,the fibrous membranes were transplanted into rat periodontal defects,and tissue regeneration was assessed with histological evaluation,micro-computed tomography(micro-CT),and immunohistochemical analysis.DMOG/nSi-PLGA membranes possessed preferable mechanical property and biocompatibility.PDLSCs seeded on the DMOG/nSi-PLGA membranes showed up-regulated expression of osteogenic and angiogenic markers,higher alkaline phosphatase(ALP)activity,and more tube formation in comparison with single application.Further,in vivo study showed that the DMOG/nSi-PLGA membranes promoted recruitment of CD90+/CD34stromal cells,induced angiogenesis and osteogenesis,and regenerated cementum-ligament-bone complex in periodontal defects.Consequently,the combination of DMOG and nSi exerted admirable effects on periodontal tissue regeneration.DMOG/nSi-PLGA fibrous membranes could enhance and orchestrate osteogenesis-angiogenesis,and may have the potential to be translated as an effective scaffold in periodontal tissue engineering.
