Objective To establish C57BL/6J embryonic stem （ES） cell lines with potential germ- line contribution Methods ES cells were isolated from blastocyst inner cell mass of C5 7BL/6J mice, and cultured for 15 passages, a...Objective To establish C57BL/6J embryonic stem （ES） cell lines with potential germ- line contribution Methods ES cells were isolated from blastocyst inner cell mass of C5 7BL/6J mice, and cultured for 15 passages, and then injected into blastococels of ICR mice blastocysts to establish chimeric mice. Results Three ES cell lines （mC57ES1,mC57ES3, mC57ES7） derived from the inner cell mass of C57BL/6J mice blastocysts were established. They were characteristic of undifferentiated state, including normal XY karyotype, expression of a specific cell surface marker “stage-specific embryonic antigen-I” and alkaline phosphatase in continuous passage. When injected into immunodeficient mice, mC57ES1 cells consistently differentiated into derivatives of all three embryonic germ layers. When mC57ES1 cells were transferred into ICR mice blastocysts, 4 chimeric mice have been obtained. One male of them revealed successful germ-line transmission. Conclussion We have obtained C57BL/6J ES cell lines with a potential germ-line contribution, which can be used to generate transgenic and gene knock-out mice.展开更多
Since stem cell therapy is the most effective treatment in the field of tissue reparation and reconstitution,the present study aimed to explore the different sources of mesenchymal stem cells(MSCs)on the different eff...Since stem cell therapy is the most effective treatment in the field of tissue reparation and reconstitution,the present study aimed to explore the different sources of mesenchymal stem cells(MSCs)on the different effects of pulmonary fibrosis-related cytokines in C57BL/6 mice.For reaching this goal,we isolated MSCs from umbilical cord blood and placenta and used for stem cell therapy in a mouse model of pulmonary fibrosis model.The pulmonary fibrosis model was done by injecting bleomycin into the trachea of C57BL/6 mice.Then we assessed the degree of pulmonary fibrosis in each mouse lung tissue at weeks 1,2,3,and 4.In addition,flow cytometry was used to evaluate the frequency of CD73,CD90,CD106,CD34,CD45,CD14 cells at the mononuclear cell level;and western blotting assays revealed the expression of IκB-α.Our results showed that stem cell therapy by placenta-derived MSC had a lower level of CD34,CD45,CD14 cells at the mononuclear cell level,and that improved pulmonary fibrosis at both molecular and pathological levels.In addition,western blotting assays revealed that the expression of IκB-αwas down-regulated in MSC-treated animals.In addition,placenta-derived MSC was the most effective in improving pulmonary fibrosis in comparison to other sources.This study suggests that MSC might be a novel therapeutic approach in pulmonary fibrosis due to an enhanced anti-inflammatory effect.Also,MSC modification by gene editing could enhance their therapeutic effect in mouse pulmonary fibrosis.展开更多
Objective To labele MESPU35, a embryonic stem (ES) cell line derived from C57BL/6j mouse, with enhanced green fluorescent protein (EGFP) for further application.Methods The EGFP gene was controlled by the hybrid CA ...Objective To labele MESPU35, a embryonic stem (ES) cell line derived from C57BL/6j mouse, with enhanced green fluorescent protein (EGFP) for further application.Methods The EGFP gene was controlled by the hybrid CA promoter/enhancer (CMV enhancer/ chicken beta-actin promoter/ beta-actin intron) to construct the vector of the transgene, pCA-EGFP. The vector was transfected into MESPU35 by electroporation.Results We generated EGFP expressing ES cells demonstrating normal properties. The green fluorescence of EGFP expressing cells was maintained in propagation of the ES cells for more than 30 passages as well as in differentiated cells. Cultured in suspension, the 'green' ES cells aggregated, and formed embryoid bodies maintaining the green fluorescence at varying developmental stages. The 'green' embryoid bodies could expand and differentiate into various types of cells, exhibiting ubiquitous green fluorescence. Conclusions The hybrid CA promoter/enhancer used to control the EGFP expressing ES cells, resulted in more intense and ubiquitous activity. The EGFP transfected cells yield bright green fluorescence, which can be visualized in real time and in situ. In addition, the ES cells, MESPU35, are derived from C57BL/6j mice, which are the most widely used in oncology, physiology and genetics. Compared to 129 substrains, C57BL/6j mice avoid a number of potential problems apparent in the other strains.展开更多
文摘Objective To establish C57BL/6J embryonic stem （ES） cell lines with potential germ- line contribution Methods ES cells were isolated from blastocyst inner cell mass of C5 7BL/6J mice, and cultured for 15 passages, and then injected into blastococels of ICR mice blastocysts to establish chimeric mice. Results Three ES cell lines （mC57ES1,mC57ES3, mC57ES7） derived from the inner cell mass of C57BL/6J mice blastocysts were established. They were characteristic of undifferentiated state, including normal XY karyotype, expression of a specific cell surface marker “stage-specific embryonic antigen-I” and alkaline phosphatase in continuous passage. When injected into immunodeficient mice, mC57ES1 cells consistently differentiated into derivatives of all three embryonic germ layers. When mC57ES1 cells were transferred into ICR mice blastocysts, 4 chimeric mice have been obtained. One male of them revealed successful germ-line transmission. Conclussion We have obtained C57BL/6J ES cell lines with a potential germ-line contribution, which can be used to generate transgenic and gene knock-out mice.
基金This work was financially supported by the National Natural Science Foundation of China(31001638&30870602)This project was supported by the key medical research plan of the Hebei province of China(No.ZD2013063)+1 种基金Tsing-hua University(2009THZ02122)National Heart,Lung,and Blood Institute Research Grant HL 104402 from the National Institutes of Health of the US Public Health Service.
文摘Since stem cell therapy is the most effective treatment in the field of tissue reparation and reconstitution,the present study aimed to explore the different sources of mesenchymal stem cells(MSCs)on the different effects of pulmonary fibrosis-related cytokines in C57BL/6 mice.For reaching this goal,we isolated MSCs from umbilical cord blood and placenta and used for stem cell therapy in a mouse model of pulmonary fibrosis model.The pulmonary fibrosis model was done by injecting bleomycin into the trachea of C57BL/6 mice.Then we assessed the degree of pulmonary fibrosis in each mouse lung tissue at weeks 1,2,3,and 4.In addition,flow cytometry was used to evaluate the frequency of CD73,CD90,CD106,CD34,CD45,CD14 cells at the mononuclear cell level;and western blotting assays revealed the expression of IκB-α.Our results showed that stem cell therapy by placenta-derived MSC had a lower level of CD34,CD45,CD14 cells at the mononuclear cell level,and that improved pulmonary fibrosis at both molecular and pathological levels.In addition,western blotting assays revealed that the expression of IκB-αwas down-regulated in MSC-treated animals.In addition,placenta-derived MSC was the most effective in improving pulmonary fibrosis in comparison to other sources.This study suggests that MSC might be a novel therapeutic approach in pulmonary fibrosis due to an enhanced anti-inflammatory effect.Also,MSC modification by gene editing could enhance their therapeutic effect in mouse pulmonary fibrosis.
文摘Objective To labele MESPU35, a embryonic stem (ES) cell line derived from C57BL/6j mouse, with enhanced green fluorescent protein (EGFP) for further application.Methods The EGFP gene was controlled by the hybrid CA promoter/enhancer (CMV enhancer/ chicken beta-actin promoter/ beta-actin intron) to construct the vector of the transgene, pCA-EGFP. The vector was transfected into MESPU35 by electroporation.Results We generated EGFP expressing ES cells demonstrating normal properties. The green fluorescence of EGFP expressing cells was maintained in propagation of the ES cells for more than 30 passages as well as in differentiated cells. Cultured in suspension, the 'green' ES cells aggregated, and formed embryoid bodies maintaining the green fluorescence at varying developmental stages. The 'green' embryoid bodies could expand and differentiate into various types of cells, exhibiting ubiquitous green fluorescence. Conclusions The hybrid CA promoter/enhancer used to control the EGFP expressing ES cells, resulted in more intense and ubiquitous activity. The EGFP transfected cells yield bright green fluorescence, which can be visualized in real time and in situ. In addition, the ES cells, MESPU35, are derived from C57BL/6j mice, which are the most widely used in oncology, physiology and genetics. Compared to 129 substrains, C57BL/6j mice avoid a number of potential problems apparent in the other strains.
