Objective To investigate the possibility of the transfection of glial-cell line derived neurotro-phic factor (GDNF) gene into Schwann cells (SCs). Methods SCs cultures from sciatic nerves of neonatal rats were establi...Objective To investigate the possibility of the transfection of glial-cell line derived neurotro-phic factor (GDNF) gene into Schwann cells (SCs). Methods SCs cultures from sciatic nerves of neonatal rats were established. A recombinant retrovirus vector containing GDNF gene was constructed and transferred into SCs. Expression levels of GDNF mRNA and protein were respectively identified with reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry. Determination of GDNF synthesis rates from Retro. pLNCX2-GDNF-transduced SCs (GDNF-SCs) in vitro by enzyme-linked immunoassay sensitive assay (ELISA). Biololgical activity of conditioned medium from GENF-SCs was analysed by co-culture with rat motoneurons. Results Transfection of GDNF gene into SCs lead to significantly enhanced expression of GDNF mRNA and protein. The rate of GDNF secreted by GDNF-SCs was also enhanced(5. 1-fold) ,and more motoneurons survived co-cultured with conditioned medium of GNDF-SCs than with that of normal SCs. Conclusion GNDF gene transfection may be a better way to graft SCs promoting regeneration and repairing demyelination in PNS and CNS.展开更多
Long non-coding RNAs(lncRNAs),which represent a new frontier in molecular biology,play important roles in regulating gene expression at epigenetic,transcriptional and post-transcriptional levels.More and more lncRNAs ...Long non-coding RNAs(lncRNAs),which represent a new frontier in molecular biology,play important roles in regulating gene expression at epigenetic,transcriptional and post-transcriptional levels.More and more lncRNAs have been found to play important roles in normal cell physiological activities,and participate in the development of varieties of tumors and other diseases.Previously,we have only been able to determine the function of lncRNAs through multiple mechanisms,including genetic imprinting,chromatin remodeling,splicing regulation,mRNA decay,and translational regulation.Application of technological advances to research into the function of lncRNAs is extremely important.The major tools for exploring lncRNAs include microarrays,RNA sequencing(RNA-seq),Northern blotting,real-time quantitative reverse transcription-polymerase chain reaction(qRT-PCR),fluorescence in situ hybridization(FISH),RNA interference(RNAi),RNA-binding protein immunoprecipitation(RIP),chromatin isolation by RNA purification(ChIRP),crosslinking-immunopurification(CLIP),and bioinformatic prediction.In this review,we highlight the functions of lncRNAs,and advanced methods to research lncRNA-protein interactions.展开更多
Objective: To evaluate the osteogenic potential of bone morphogenetic protein (BMP)-2 gene transfected goat bone marrow-derived mesenchymal stem cells (MSCs). Methods: Goat bone marrow- derived MSCs were transfected b...Objective: To evaluate the osteogenic potential of bone morphogenetic protein (BMP)-2 gene transfected goat bone marrow-derived mesenchymal stem cells (MSCs). Methods: Goat bone marrow- derived MSCs were transfected by Adv-human bone morphogenetic protein (hBMP)-2 gene(Group 1), Adv-beta gal transfected MSCs (Group 2)and uninfected MSCs(Group 3). Western blot analysis, alkaline phosphatase staining, Von Kossa staining and transmission electron microscopy were adopted to determine the phenotype of MSCs. Then the cells were injected into thigh muscles of the nude mice. Radiographical and histological evaluations were performed at different intervals. Results: Only Adv-hBMP-2 transfected MSCs produced hBMP-2. These cells were positive for alkaline phosphatase staining at the 12th day and were positive for Von Kossa staining at the 16th day after gene transfer. Electron microscopic observation showed that there were more rough endoplasmic reticulum, mitochondria and lysosomes in Adv-hBMP-2 transfected MSCs compared to MSCs of other two groups. At the 3rd and 6th weeks after cell injection, ectopic bones were observed in muscles of nude mice of Group 1. Only fibrous tissue or a little bone was found in other two groups. Conclusions: BMP-2 gene transfected MSCs can differentiate into osteoblasts in vitro and induce bone formation in vivo.展开更多
文摘Objective To investigate the possibility of the transfection of glial-cell line derived neurotro-phic factor (GDNF) gene into Schwann cells (SCs). Methods SCs cultures from sciatic nerves of neonatal rats were established. A recombinant retrovirus vector containing GDNF gene was constructed and transferred into SCs. Expression levels of GDNF mRNA and protein were respectively identified with reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry. Determination of GDNF synthesis rates from Retro. pLNCX2-GDNF-transduced SCs (GDNF-SCs) in vitro by enzyme-linked immunoassay sensitive assay (ELISA). Biololgical activity of conditioned medium from GENF-SCs was analysed by co-culture with rat motoneurons. Results Transfection of GDNF gene into SCs lead to significantly enhanced expression of GDNF mRNA and protein. The rate of GDNF secreted by GDNF-SCs was also enhanced(5. 1-fold) ,and more motoneurons survived co-cultured with conditioned medium of GNDF-SCs than with that of normal SCs. Conclusion GNDF gene transfection may be a better way to graft SCs promoting regeneration and repairing demyelination in PNS and CNS.
基金supported by the National Basic Research Program of China(2010CB912801,2013CB910801)National High Technology Research and Development Program of China(2012AA022501)National Natural Science Foundation of China(31070702,31270836)
文摘Long non-coding RNAs(lncRNAs),which represent a new frontier in molecular biology,play important roles in regulating gene expression at epigenetic,transcriptional and post-transcriptional levels.More and more lncRNAs have been found to play important roles in normal cell physiological activities,and participate in the development of varieties of tumors and other diseases.Previously,we have only been able to determine the function of lncRNAs through multiple mechanisms,including genetic imprinting,chromatin remodeling,splicing regulation,mRNA decay,and translational regulation.Application of technological advances to research into the function of lncRNAs is extremely important.The major tools for exploring lncRNAs include microarrays,RNA sequencing(RNA-seq),Northern blotting,real-time quantitative reverse transcription-polymerase chain reaction(qRT-PCR),fluorescence in situ hybridization(FISH),RNA interference(RNAi),RNA-binding protein immunoprecipitation(RIP),chromatin isolation by RNA purification(ChIRP),crosslinking-immunopurification(CLIP),and bioinformatic prediction.In this review,we highlight the functions of lncRNAs,and advanced methods to research lncRNA-protein interactions.
文摘Objective: To evaluate the osteogenic potential of bone morphogenetic protein (BMP)-2 gene transfected goat bone marrow-derived mesenchymal stem cells (MSCs). Methods: Goat bone marrow- derived MSCs were transfected by Adv-human bone morphogenetic protein (hBMP)-2 gene(Group 1), Adv-beta gal transfected MSCs (Group 2)and uninfected MSCs(Group 3). Western blot analysis, alkaline phosphatase staining, Von Kossa staining and transmission electron microscopy were adopted to determine the phenotype of MSCs. Then the cells were injected into thigh muscles of the nude mice. Radiographical and histological evaluations were performed at different intervals. Results: Only Adv-hBMP-2 transfected MSCs produced hBMP-2. These cells were positive for alkaline phosphatase staining at the 12th day and were positive for Von Kossa staining at the 16th day after gene transfer. Electron microscopic observation showed that there were more rough endoplasmic reticulum, mitochondria and lysosomes in Adv-hBMP-2 transfected MSCs compared to MSCs of other two groups. At the 3rd and 6th weeks after cell injection, ectopic bones were observed in muscles of nude mice of Group 1. Only fibrous tissue or a little bone was found in other two groups. Conclusions: BMP-2 gene transfected MSCs can differentiate into osteoblasts in vitro and induce bone formation in vivo.
