背景:鼻黏膜纤毛上皮细胞受损后导致鼻黏膜生物功能严重创伤。相对于其他成体干细胞,人脐血干细胞具有更好的诱导分化潜能。目的:观察人脐血干细胞通过体外培养、诱导分化为鼻黏膜纤毛上皮细胞的可行性。方法:收集正常健康人脐血,分离...背景:鼻黏膜纤毛上皮细胞受损后导致鼻黏膜生物功能严重创伤。相对于其他成体干细胞,人脐血干细胞具有更好的诱导分化潜能。目的:观察人脐血干细胞通过体外培养、诱导分化为鼻黏膜纤毛上皮细胞的可行性。方法:收集正常健康人脐血,分离人脐血干细胞,通过体外培养,鉴定干细胞表面标记物后进行传代培养;取第3代脐血干细胞,用携带增强型绿色荧光蛋白重组腺相关病毒进行感染,采用气液界面培养法,分别于诱导感染1,2周后对干细胞进行MUC8基因PCR检测。在脐血干细胞培养3周后进行FOXJ1免疫荧光染色。结果与结论:(1)培养鉴定结果:原代人脐血干细胞传代至第3代时,细胞形态较均一,折光性良好,可表达干细胞表面标记物;(2)转染鉴定结果:细胞转染3 h后,可见第3代干细胞呈现出绿色荧光,培养48 h后,流式细胞仪检测细胞阳性率达96.2%,说明干细胞转染效果很好;(3)RT-PCR检测:人脐血干细胞MUC8 m RNA无表达,而鼻黏膜上皮细胞MUC8 m RNA呈现出强表达,培养1周时MUC8mRNA呈现弱表达,培养2周后有一定的增强;(4)免疫荧光染色:在转染的绿色荧光蛋白背景下可观测到FOXJ1红色荧光呈现阳性表达结果;(5)结果说明:人脐血干细胞在适宜培养条件下可分化为鼻黏膜纤毛上皮细胞。展开更多
African swine fever(ASF)is a highly pathogenic swine infectious disease that affects domestic pigs and wild boar,which is caused by the African swine fever virus(ASFV).ASF has caused huge economic losses to the pig in...African swine fever(ASF)is a highly pathogenic swine infectious disease that affects domestic pigs and wild boar,which is caused by the African swine fever virus(ASFV).ASF has caused huge economic losses to the pig industry and seriously threatens global food security and livestock health.To date,there is no safe and effective commercial vaccine against ASF.Unveiling the underlying mechanisms of ASFV-host interplay is critical for developing effective vaccines and drugs against ASFV.In the present study,RNA-sequencing,RT-qPCR and Western blotting analysis revealed that the transcriptional and protein levels of the host factor FoxJ1 were significantly down-regulated in primary porcine alveolar macrophages(PAMs)infected by ASFV.RT-qPCR analysis showed that overexpression of FoxJ1 upregulated the transcription of type I interferon and interferon stimulating genes(ISGs)induced by poly(dA:dT).FoxJ1 revealed a function to positively regulate innate immune response,therefore,suppressing the replication of ASFV.In addition,Western blotting analysis indicated that FoxJ1 degraded ASFV MGF505-2R and E165R proteins through autophagy pathway.Meanwhile,RT-qPCR and Western blotting analysis showed that ASFV S273R inhibited the expression of FoxJ1.Altogether,we determined that FoxJ1 plays an antiviral role against ASFV replication,and ASFV protein impairs FoxJ1-mediated antiviral effect by degradation of FoxJ1.Our findings provide new insights into the antiviral function of FoxJ1,which might help design antiviral drugs or vaccines against ASFV infection.展开更多
Foxj1 has been found to play an important role in cilia formation and function in vertebrates. The zebrafish or Xenopus genome expresses two Foxjl genes, foxjlalFoxJ1 and foxjlb/FoxJ1.2. In this study, we have generat...Foxj1 has been found to play an important role in cilia formation and function in vertebrates. The zebrafish or Xenopus genome expresses two Foxjl genes, foxjlalFoxJ1 and foxjlb/FoxJ1.2. In this study, we have generated a zebrafish transgenic line T2BGSZIO by Tol2 transposon-based gene trapping approach. T2BGSZ10 transgenic fish carry an insertion of the transposon genome into the first intron of thefoxj1b locus. This insertion results in GFP expression in the forebrain, otic vesicles, floorplate, pronephric ducts and other domains during embryogenesis, which recaptures the expression pattern offoxj1b. Although normal expression offoxj1b is dramatically reduced, T2BGSZ10 homozygous embryos develop normally and grow to adulthood without detectable defects, which may be due to the incomplete interruption of foxjlb expression. Nevertheless, this transgenic line may serve as a useful model for dynamic observation of GFP-labeled tissues and organs and for isolation of GFP-labeled cells.展开更多
文摘背景:鼻黏膜纤毛上皮细胞受损后导致鼻黏膜生物功能严重创伤。相对于其他成体干细胞,人脐血干细胞具有更好的诱导分化潜能。目的:观察人脐血干细胞通过体外培养、诱导分化为鼻黏膜纤毛上皮细胞的可行性。方法:收集正常健康人脐血,分离人脐血干细胞,通过体外培养,鉴定干细胞表面标记物后进行传代培养;取第3代脐血干细胞,用携带增强型绿色荧光蛋白重组腺相关病毒进行感染,采用气液界面培养法,分别于诱导感染1,2周后对干细胞进行MUC8基因PCR检测。在脐血干细胞培养3周后进行FOXJ1免疫荧光染色。结果与结论:(1)培养鉴定结果:原代人脐血干细胞传代至第3代时,细胞形态较均一,折光性良好,可表达干细胞表面标记物;(2)转染鉴定结果:细胞转染3 h后,可见第3代干细胞呈现出绿色荧光,培养48 h后,流式细胞仪检测细胞阳性率达96.2%,说明干细胞转染效果很好;(3)RT-PCR检测:人脐血干细胞MUC8 m RNA无表达,而鼻黏膜上皮细胞MUC8 m RNA呈现出强表达,培养1周时MUC8mRNA呈现弱表达,培养2周后有一定的增强;(4)免疫荧光染色:在转染的绿色荧光蛋白背景下可观测到FOXJ1红色荧光呈现阳性表达结果;(5)结果说明:人脐血干细胞在适宜培养条件下可分化为鼻黏膜纤毛上皮细胞。
基金supported by grants from the National Key R&D Program of China(2021YFD1800100 and 2021YFD1801300)National Natural Science Foundation of China(31941002)+2 种基金Technology Major Project of Gansu Province(20ZD7A006,21ZD3NA001 and NCC0006)the Chinese Academy of Agricultural Science and Technology Innovation Project(CAAS-ZDRW202006 and CAAS-ASTIP-2022-LVRI)the Research funding from Lanzhou Veterinary Research Institute(CAASASTIP-JBGS-20210101)。
文摘African swine fever(ASF)is a highly pathogenic swine infectious disease that affects domestic pigs and wild boar,which is caused by the African swine fever virus(ASFV).ASF has caused huge economic losses to the pig industry and seriously threatens global food security and livestock health.To date,there is no safe and effective commercial vaccine against ASF.Unveiling the underlying mechanisms of ASFV-host interplay is critical for developing effective vaccines and drugs against ASFV.In the present study,RNA-sequencing,RT-qPCR and Western blotting analysis revealed that the transcriptional and protein levels of the host factor FoxJ1 were significantly down-regulated in primary porcine alveolar macrophages(PAMs)infected by ASFV.RT-qPCR analysis showed that overexpression of FoxJ1 upregulated the transcription of type I interferon and interferon stimulating genes(ISGs)induced by poly(dA:dT).FoxJ1 revealed a function to positively regulate innate immune response,therefore,suppressing the replication of ASFV.In addition,Western blotting analysis indicated that FoxJ1 degraded ASFV MGF505-2R and E165R proteins through autophagy pathway.Meanwhile,RT-qPCR and Western blotting analysis showed that ASFV S273R inhibited the expression of FoxJ1.Altogether,we determined that FoxJ1 plays an antiviral role against ASFV replication,and ASFV protein impairs FoxJ1-mediated antiviral effect by degradation of FoxJ1.Our findings provide new insights into the antiviral function of FoxJ1,which might help design antiviral drugs or vaccines against ASFV infection.
基金supported by the National High-tech R&D Program (863 Program) (No. 2006AA02Z167)the Major Science Programs of China (No. 2006CB943401)the National Basic Research Program of China (No. 2005CB522502)
文摘Foxj1 has been found to play an important role in cilia formation and function in vertebrates. The zebrafish or Xenopus genome expresses two Foxjl genes, foxjlalFoxJ1 and foxjlb/FoxJ1.2. In this study, we have generated a zebrafish transgenic line T2BGSZIO by Tol2 transposon-based gene trapping approach. T2BGSZ10 transgenic fish carry an insertion of the transposon genome into the first intron of thefoxj1b locus. This insertion results in GFP expression in the forebrain, otic vesicles, floorplate, pronephric ducts and other domains during embryogenesis, which recaptures the expression pattern offoxj1b. Although normal expression offoxj1b is dramatically reduced, T2BGSZ10 homozygous embryos develop normally and grow to adulthood without detectable defects, which may be due to the incomplete interruption of foxjlb expression. Nevertheless, this transgenic line may serve as a useful model for dynamic observation of GFP-labeled tissues and organs and for isolation of GFP-labeled cells.
