BACKGROUND： Cell culture in vitro trials have demonstrated that neurotrophin-3 （NT-3） can enhance the survival of sensory neurons and sympathetic neurons, and can also support embryo-derived motor neurons. This eff...BACKGROUND： Cell culture in vitro trials have demonstrated that neurotrophin-3 （NT-3） can enhance the survival of sensory neurons and sympathetic neurons, and can also support embryo-derived motor neurons. This effect is dependent on nerve growth factor on the surface of cells. Understanding the role of NT-3 and its receptor in the early development of human embryonic brains will help to investigate the correlation between early survival of nerve cells and the microenvironment of neural regeneration. OBJECTIVE： To observe the proliferation of cerebral neurons in the development of human embryonic brain, and to investigate the location, expression and distribution of NT-3 and its receptor TrkC during human brain development. DESIGN, TIME AND SETTING： An observation study on cells was performed in the Department of ttuman Anatomy, Histology and Embryology, Chengdu Medical College in September 2007. MATERIALS： Fifteen specimens of flesh human embryo, aged 6 weeks, were used in this study. METHODS： The proliferation of cerebral neurons was detected using proliferating cell nuclear antigen, and the immunocytochemistry ABC technique was applied to observe the location, expression and distribution of NT-3 and its receptor TrkC in the brain of the human embryo. MAIN OUTCOME MEASURES： Location, expression and distribution of NT-3 and its receptor in the brain of the human embryo. RESULTS： In the early period （aged 6 weeks） of human embryonic development, proliferating cell nuclear antigen-positive reactive substances were mainly observed in the nucleus of the forebrain ventricular zone and subventricular zone, and the intensity was stronger in the subventricular zone than the forebrain ventricle. NT-3 positive reactive substance was mainly distributed in the cytoblastema of the forebrain neuroepithelial layer and nerve cell process, while TrkC was mainly distributed in the cell membrane of the forebrain ventricular zone and subventricular zone. During embryonic development, NT-3 and TrkC showed a positive immune reaction to a greater or lesser extent in ependymal epithelium. CONCLUSION： During early human embryonic development, cerebral nerve cells proliferate in the ventricular zone and subventricular zone, and NT-3 is expressed in the neural axon. The results show that the highly expressed NT-3 could promote the proliferation of neural axons and maintain the neuron body＇s survival.展开更多
β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 （A〉G） mutations is one of the three most common mutations in China and Southeast Asia patients with β-th...β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 （A〉G） mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor （BE） system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB -28 （A〉G） mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB -28 （A〉G） homozygous mutation. Data showed that base editor could precisely correct HBB -28 （A〉G） mutation in the patient＇s primary cells. To model homozygous mutation disease embryos, we consb＇ucted nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured （IVM） oocytes.Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB -28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.展开更多
Objective To investigate the effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocyte L-02 cells. Methods Lipid peroxide (LPO) level, reduced glutathione (GSH) content, DNA damage...Objective To investigate the effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocyte L-02 cells. Methods Lipid peroxide (LPO) level, reduced glutathione (GSH) content, DNA damage, apoptosis, and cell cycle analysis were measured after in vitro cultured L-02 cells were exposed to sodium fluoride at different doses (40 μg/mL, 80 μg/mL, and 160 μg/mL) for 24 hours. Results Fluoride caused an increase of LPO levels and a decrease of GSH content in L-02 cells. There appeared to be an obvious dose-effect relationship between the fluoride concentration and the observed changes. Fluoride also caused DNA damage and apoptosis and increased the cell number in S phase of cell cycle in the cells tested. There was a statistically significant difference in DNA damage and apoptosis when comparing the high dose of fluoride treated cells with the low dose of fluoride treated cells. Conclusion Fluoride can cause lipid peroxidation, DNA damage, and apoptosis in the L-02 cell experimental model and there is a significant positive correlation between fluoride concentration and these pathological changes.展开更多
BACKGROUND： Total saponins of Panax ginseng （TSPG） exhibits neuroprotection against Parkinson＇s disease in the substantia nigra. OBJECTIVE： To investigate the effects of TSPG on human embryonic neural stem cells ...BACKGROUND： Total saponins of Panax ginseng （TSPG） exhibits neuroprotection against Parkinson＇s disease in the substantia nigra. OBJECTIVE： To investigate the effects of TSPG on human embryonic neural stem cells （NSCs） proliferation and differentiation into dopaminergic neurons using in vitro studies, and to observe NSC differentiation in a mouse model of Parkinson＇s disease, as well as behavioral changes before and after transplantation. DESIGN, TIME AND SETTING： In vitro neural cell biology trial and in vivo randomized, controlled animal trial were performed at the Institute of Basic Medical Sciences, Chongqing Medical University between September 2004 and December 2007. MATERIALS： TSPG （purity 〉 95%） was isolated, extracted, and identified by Chongqing Academy of Chinese Materia Medica. Recombinant human basic fibroblast growth factor （bFGF） and recombinant human epidermal growth factor （EGF） were purchased from PeproTech, USA. A total of 25 C57/BL6J mice, aged 18-20 weeks were included. Twenty were used to establish a Parkinson＇s disease model with i.p. injection of MPTP （1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine） and TSPG alone or combined with interleukin-1 （IL-1）-treated NSCs prior to transplantation into the corpus striatum. The remaining five mice were pretreated for 3 days with TSPG prior to MPTP injection, serving as the TSPG prevention group. METHODS： Primary NSCs were isolated, cultured and purified from embryonic cerebral cortex. Immunocytochemistry was employed to detect specific antigen expression in the NSCs. In vitro experiment： （1） to induce proliferation, NSCs were treated with TSPG, EGF＋bFGF, or TSPG＋EGF＋bFGF, respectively; （2） to induce dopaminergic neuronal differentiation, NSCs were treated with TSPG, IL-1, or TSPG＋IL-1, respectively. MAIN OUTCOME MEASURES： In vitro experiment： the effects of TSPG on NSCs proliferation were evaluated with flow cytometry and MTT assay. Tyrosine hydroxylase expression was determined by immunocytochemistry assay to observe effects of TSPG on dopaminergic neuronal differentiation. In vivo experiment： differentiation of grafted NSCs in the mouse brain was determined by immunohistochemical staining. Behavioral changes were evaluated by spontaneous activity frequency, memory function, and score of paralysis agitans. RESULTS： （1） NSCs were cultured and passaged for more than three passages. Immunocytochemistry revealed positive nestin staining, as well as neurofilament protein and glial fibrillary acidic protein. （2） TSPG significantly increased NSC proliferation, in particular when combined with EGF and bFGF, which was twice as effective as FGF or bFGF alone. TSPG also induced dopaminergic differentiation in NSCs, in particular when TSPG was added together with IL-1, resulting in an effect five times greater than that of IL-1 alone. （3） At day 30 following transplantation, most NSCs in the TSPG prevention group differentiated into dopaminergic neurons, and the scores of paralysis agitans, spontaneous activity, and memory function were significantly increased compared with TSPG alone or TSPG＋IL-1 groups （P 〈 0.05）. CONCLUSION： TSPG stimulated NSC proliferation, in particular when combined with FGF and bFGF. TSPG significantly induced dopaminergic neuronal differentiation of NSCs, and the effect was greater when combined with IL-1. In addition, TSPG greatly improved behavior in the Parkinson＇s disease mouse model following NSC transplantation. Following NSC transplantation, TSPG pretreatment exhibited superior efficacy over either TSPG alone or TSPG in combination with IL-1, in terms of behavioral improvements in the Parkinson＇s disease mouse model.展开更多
BACKGROUND： Numerous current studies have suggested that human telomerase reverse transcriptase （hTERT） gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses; however, the...BACKGROUND： Numerous current studies have suggested that human telomerase reverse transcriptase （hTERT） gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses; however, the mechanism of action remains unknown. OBJECTIVE： To evaluate the neuroprotective effects and possible mechanism of action of hTERT gene transfection in human embryonic cortical neurons treated with beta-amyloid fragment 25-35 （AI325-35）. DESIGN, TIME AND SETTING： The randomized, controlled and molecular biological studies were performed at the Department of Anatomy and Brain Research, Zhongshan School of Medicine, Sun Yat-sen University, China, from September 2005 to June 2008. MATERIALS： AdEasy-1 Expression System was gifted by Professor Guoquan Gao from Sun Yat-Sen University, China. Human cortical neurons were derived from 12-20 week old aborted fetuses, obtained from the Guangzhou Maternal and Child Health Hospital, China. Mouse anti-Odk5 and mouse anti-p16 monoclonal antibodies （Lab Vision, USA）, and mouse anti-hTERT monoclonal antibody （Epitomics, USA）, were used in this study. METHODS： （1） Recombinant adenovirus vectors, encoding hTERT （Ad-hTERT） and green fluorescent protein （Ad-GFP）, were constructed using the AdEasy-1 Expression System. Human embryonic cortical neurons in the Ad-hTERT group were transfected with Ad-hTERT for 1-21 days. Likewise, human embryonic cortical neurons in the Ad-GFP group were transfected with Ad-GFP for 1-21 days. Human embryonic cortical neurons in the control group were cultured as normal. （2） Human embryonic cortical neurons in the Ad-hTERT group were treated with 10 pmol/L Aβ25-35 for 24 hours. Normal human embryonic cortical neurons treated with 10 pmol/Lβ25.35 for 24 hours served as a model group. Human embryonic cortical neurons in the Ad-GFP and control groups were not treated with Aβ25-35. MAIN OUTCOME MEASURES： Expression of hTERT in human embryonic cortical neurons was evaluated by immunocytochemical staining and Western blot assay. Telomerase activity was measured using a PCR-based telomeric repeat amplification protocol （TRAP） ELISA kit. Neural activity in human embryonic cortical neurons was examined by MTT assay; apoptosis was measured using TUNEL assay; and Cdk5 and p16 protein expressions were measured by Western blot. RESULTS： Expression of hTERT protein was significantly increased and peaked at day 3 post-transfection in the Ad-hTERT group. No hTERT expression was detected in the Ad-GFP and control groups. Telomerase activity was significantly greater in the Ad-hTERT group compared with the Ad-GFP and control groups （P 〈 0.01）. Compared with the control group, cell activity was significantly decreased （P 〈 0.05）, and cell apoptotic rate, Cdk5 and p16 expression were significantly increased （P 〈 0.01） in the model group. Compared with the model group, cell activity was increased in the Ad-hTERT group, and peaked at day 3 post-transfection （P 〈 0.05）. Neuroprotective effects also peaked at day 3 post-transfection; and the apoptotic rate, Cdk5 and p16 expression significantly decreased （P 〈 0.01）. CONCLUSION： Expression of hTERT in human embryonic cortical neurons can relieve Aβ25-35-induced neuronal apoptosis. The possible mechanism by which hTERT produces these neuroprotective effects may be associated with inhibition of Cdk5 and p16 expression.展开更多
Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection ...Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV.RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time-and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.展开更多
Neural tube development comprises neural induction, neural epithelial cell proliferation, and apoptosis, as well as migration of nerve cells. Too much or too little apoptosis leads to abnormal nervous system developme...Neural tube development comprises neural induction, neural epithelial cell proliferation, and apoptosis, as well as migration of nerve cells. Too much or too little apoptosis leads to abnormal nervous system development. The present study analyzed expression and distribution of apoptotic-related factors, including Fas, FasL, and caspase-3, during human embryonic neural tube development. Experimental results showed that increased caspase-3 expression promoted neural apoptosis via a mitochondrial-mediated intrinsic pathway at 4 weeks during early human embryonic neural tube development. Subsequently, Fas and FasL expression increased during embryonic development. The results suggest that neural cells influence neural apoptosis through synergistic effects of extrinsic pathways. Therefore, neural apoptosis during the early period of neural tube development in the human embryo might be regulated by the death receptor induced apoptotic extrinsic pathways.展开更多
Researchers from all around the world emphasize on the enormous possible benefits that stem cells may have for the treatment of diseases. However, this technology is considered morally problematic when the source of t...Researchers from all around the world emphasize on the enormous possible benefits that stem cells may have for the treatment of diseases. However, this technology is considered morally problematic when the source of the stem cell is from a human embryo. Nonetheless, there is a consensus that of all the types of stem cells, hESC （human embryonic stem ceils） are the most promising for particular and important research and therapies. Yet, there are controversial issues regarding the ＂killing＂ of the human embryo for stem cell derivation. There are two general ethical conditions that should govern the instrumental use of embryo. One of them, the principle of subsidiarity, which is defined as ＂a state we have that we have to choose the less contentious means of achieving the intended goal＂. Based on this principle, we ought only to use hESC when there are no other alternatives, which are less morally controversially. Subsidiarity is based on the assumption that there is something ethically unsound about the use ofhESC. However, this principle only makes sense if it is based on consistently upheld views of the moral status of embryo, moreover, the law should also not limit or prohibit hESC research based on this principle. In this paper, I argue---using the South African law for hESC technology--that criterion for deciding which type of stem cells to use should be based on their potential and suitability for advancing scientific knowledge and development of new therapies which will be greatly beneficial in alleviating human suffering.展开更多
The embryonic mesoderm comprises heterogeneous cell subpopulations with distinct lineage biases.It is unclear whether a bias for the human hematopoietic lineage emerges at this early developmental stage.In this study,...The embryonic mesoderm comprises heterogeneous cell subpopulations with distinct lineage biases.It is unclear whether a bias for the human hematopoietic lineage emerges at this early developmental stage.In this study,we integrated single-cell transcriptomic analyses of human mesoderm cells from embryonic stem cells and embryos,enabling us to identify and define the molecular features of human hematopoietic mesoderm(HM)cells biased towards hematopoietic lineages.We discovered that BMP4 plays an essential role in HM specification and can serve as a marker for HM cells.Mechanistically,BMP4 acts as a downstream target of HDAC1,which modulates the expression of BMP4 by deacetylating its enhancer.Inhibition of HDAC significantly enhances HM specification and promotes subsequent hematopoietic cell differentiation.In conclusion,our study identifies human HM cells and describes new mechanisms for human hematopoietic development.展开更多
Approximately 140 million people worldwide are homozygous carriers of APOE4(ε4),a strong genetic risk factor for late onset familial and sporadic Alzheimer’s disease(AD),91%of whom will develop AD at earlier age tha...Approximately 140 million people worldwide are homozygous carriers of APOE4(ε4),a strong genetic risk factor for late onset familial and sporadic Alzheimer’s disease(AD),91%of whom will develop AD at earlier age than heterozygous carriers and noncarriers.Susceptibility to AD could be reduced by targeted editing of APOE4,but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies.Here,we first screened eight cytosine base editor variants at four injection stages(from 1-to 8-cell stage),and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate(up to 100%)with the lowest bystander effects.In particular,80%of AD-susceptibleε4 allele copies were converted to the AD-neutralε3 allele in humanε4-carrying embryos.Stringent control measures combined with targeted deep sequencing,whole genome sequencing,and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells.Furthermore,base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage.Finally,we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia.Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos,a potential approach for reducing human susceptibility to AD or other genetic diseases.展开更多
Objective To quantify proline 4-hydroxylase, alpha polypeptide ii （ P4HA2 ） mRNA of human embryo lung fibroblast （HELF） with SYBR green based reversed transcript PCR （RT-PCR） for correcting cytomegalovirus （CMV...Objective To quantify proline 4-hydroxylase, alpha polypeptide ii （ P4HA2 ） mRNA of human embryo lung fibroblast （HELF） with SYBR green based reversed transcript PCR （RT-PCR） for correcting cytomegalovirus （CMV） inactivation or clearance efficiency in donor blood. Methods A pair of specific primers of exon 12a of P4HA2 was designed, and the related PCR-reaction system and condition were optimized. Then the recombinant plasmid containing the target fragment was constructed for making standard curve with SYBR green based real-time RT-PCR. Finally, the sensitivity, reproducibility, and specificity of this method were fully estimated. Results The sensitivity of the method was 1.5E ＋ 04 copies/mL of P4HA2 mRNA, corresponding to 10^3 fibroblasts. In addition, existence of 8. 67E ＋ 06 leukocytes could not interfere with the accurate quantification of HELF in the large dynamic range. The intra-assay variability and inter-assay variability both varied in different concentrations, being higher in low concentrations and lower in high concentrations. But all of them were below 13. 76% in variation, which showed acceptable stability of this method. Conclusion SYBR green and specific primer based real-time RT-PCR show up a good quality for quantifying HELF P4HA2 mRNA with good specificity, stability, and high sensitivity. Approximate 10 copies of P4HA2 mRNA per cell in average can be detected by the method. Therefore, this method can be used to deduct fibroblast-associated CMV for correcting CMV inactivation efficiency in leukocytes.展开更多
Early human embryogenesis is a very sophisticated process due to its unique gene regulatory network.Autophagy has been suggested to play an important role in mediating the development of early embryonic cells in mamma...Early human embryogenesis is a very sophisticated process due to its unique gene regulatory network.Autophagy has been suggested to play an important role in mediating the development of early embryonic cells in mammals.However,evidence showing how autophagy regulates early human embryogenesis remains to be further explored.In this study,we systematically investigated the human transcriptome and methylome patterns of autophagy-related(ATG)genes in early embryonic cells at single-cell resolution.We analyzed the transcriptomic data of 365 cells and methylome data of 265 cells.The results showed that most ATG genes remained epigenetically active and were expressed stably throughout early embryogenesis,whereas the dynamics varied among different developmental stages.This evidence indicated that the autophagy pathway was constitutively activated and exerted a fundamental role in early human embryo development.Our work,for the first time,comprehensively reveals the features of autophagy during early human embryo development.展开更多
文摘BACKGROUND： Cell culture in vitro trials have demonstrated that neurotrophin-3 （NT-3） can enhance the survival of sensory neurons and sympathetic neurons, and can also support embryo-derived motor neurons. This effect is dependent on nerve growth factor on the surface of cells. Understanding the role of NT-3 and its receptor in the early development of human embryonic brains will help to investigate the correlation between early survival of nerve cells and the microenvironment of neural regeneration. OBJECTIVE： To observe the proliferation of cerebral neurons in the development of human embryonic brain, and to investigate the location, expression and distribution of NT-3 and its receptor TrkC during human brain development. DESIGN, TIME AND SETTING： An observation study on cells was performed in the Department of ttuman Anatomy, Histology and Embryology, Chengdu Medical College in September 2007. MATERIALS： Fifteen specimens of flesh human embryo, aged 6 weeks, were used in this study. METHODS： The proliferation of cerebral neurons was detected using proliferating cell nuclear antigen, and the immunocytochemistry ABC technique was applied to observe the location, expression and distribution of NT-3 and its receptor TrkC in the brain of the human embryo. MAIN OUTCOME MEASURES： Location, expression and distribution of NT-3 and its receptor in the brain of the human embryo. RESULTS： In the early period （aged 6 weeks） of human embryonic development, proliferating cell nuclear antigen-positive reactive substances were mainly observed in the nucleus of the forebrain ventricular zone and subventricular zone, and the intensity was stronger in the subventricular zone than the forebrain ventricle. NT-3 positive reactive substance was mainly distributed in the cytoblastema of the forebrain neuroepithelial layer and nerve cell process, while TrkC was mainly distributed in the cell membrane of the forebrain ventricular zone and subventricular zone. During embryonic development, NT-3 and TrkC showed a positive immune reaction to a greater or lesser extent in ependymal epithelium. CONCLUSION： During early human embryonic development, cerebral nerve cells proliferate in the ventricular zone and subventricular zone, and NT-3 is expressed in the neural axon. The results show that the highly expressed NT-3 could promote the proliferation of neural axons and maintain the neuron body＇s survival.
基金We are grateful to Dr. Qi Zhou for helpful suggestions. This work was supported by National Key R＆D Program of China （2017YFC1001901 and 2017YFC1001600）, the Science and Technology Planning Project of Guangdong Province （2015B020228002）, the Guangzhou Science and Technology Project （201707010085） and the National Natural Science Foundation of China （Grant No. 81771579）.
文摘β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 （A〉G） mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor （BE） system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB -28 （A〉G） mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB -28 （A〉G） homozygous mutation. Data showed that base editor could precisely correct HBB -28 （A〉G） mutation in the patient＇s primary cells. To model homozygous mutation disease embryos, we consb＇ucted nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured （IVM） oocytes.Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB -28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.
基金The work was supported by grants from the National Nature Science Foundation of China (No. 30271155) China national key basic research and development program (No. 2022CB512908).
文摘Objective To investigate the effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocyte L-02 cells. Methods Lipid peroxide (LPO) level, reduced glutathione (GSH) content, DNA damage, apoptosis, and cell cycle analysis were measured after in vitro cultured L-02 cells were exposed to sodium fluoride at different doses (40 μg/mL, 80 μg/mL, and 160 μg/mL) for 24 hours. Results Fluoride caused an increase of LPO levels and a decrease of GSH content in L-02 cells. There appeared to be an obvious dose-effect relationship between the fluoride concentration and the observed changes. Fluoride also caused DNA damage and apoptosis and increased the cell number in S phase of cell cycle in the cells tested. There was a statistically significant difference in DNA damage and apoptosis when comparing the high dose of fluoride treated cells with the low dose of fluoride treated cells. Conclusion Fluoride can cause lipid peroxidation, DNA damage, and apoptosis in the L-02 cell experimental model and there is a significant positive correlation between fluoride concentration and these pathological changes.
文摘BACKGROUND： Total saponins of Panax ginseng （TSPG） exhibits neuroprotection against Parkinson＇s disease in the substantia nigra. OBJECTIVE： To investigate the effects of TSPG on human embryonic neural stem cells （NSCs） proliferation and differentiation into dopaminergic neurons using in vitro studies, and to observe NSC differentiation in a mouse model of Parkinson＇s disease, as well as behavioral changes before and after transplantation. DESIGN, TIME AND SETTING： In vitro neural cell biology trial and in vivo randomized, controlled animal trial were performed at the Institute of Basic Medical Sciences, Chongqing Medical University between September 2004 and December 2007. MATERIALS： TSPG （purity 〉 95%） was isolated, extracted, and identified by Chongqing Academy of Chinese Materia Medica. Recombinant human basic fibroblast growth factor （bFGF） and recombinant human epidermal growth factor （EGF） were purchased from PeproTech, USA. A total of 25 C57/BL6J mice, aged 18-20 weeks were included. Twenty were used to establish a Parkinson＇s disease model with i.p. injection of MPTP （1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine） and TSPG alone or combined with interleukin-1 （IL-1）-treated NSCs prior to transplantation into the corpus striatum. The remaining five mice were pretreated for 3 days with TSPG prior to MPTP injection, serving as the TSPG prevention group. METHODS： Primary NSCs were isolated, cultured and purified from embryonic cerebral cortex. Immunocytochemistry was employed to detect specific antigen expression in the NSCs. In vitro experiment： （1） to induce proliferation, NSCs were treated with TSPG, EGF＋bFGF, or TSPG＋EGF＋bFGF, respectively; （2） to induce dopaminergic neuronal differentiation, NSCs were treated with TSPG, IL-1, or TSPG＋IL-1, respectively. MAIN OUTCOME MEASURES： In vitro experiment： the effects of TSPG on NSCs proliferation were evaluated with flow cytometry and MTT assay. Tyrosine hydroxylase expression was determined by immunocytochemistry assay to observe effects of TSPG on dopaminergic neuronal differentiation. In vivo experiment： differentiation of grafted NSCs in the mouse brain was determined by immunohistochemical staining. Behavioral changes were evaluated by spontaneous activity frequency, memory function, and score of paralysis agitans. RESULTS： （1） NSCs were cultured and passaged for more than three passages. Immunocytochemistry revealed positive nestin staining, as well as neurofilament protein and glial fibrillary acidic protein. （2） TSPG significantly increased NSC proliferation, in particular when combined with EGF and bFGF, which was twice as effective as FGF or bFGF alone. TSPG also induced dopaminergic differentiation in NSCs, in particular when TSPG was added together with IL-1, resulting in an effect five times greater than that of IL-1 alone. （3） At day 30 following transplantation, most NSCs in the TSPG prevention group differentiated into dopaminergic neurons, and the scores of paralysis agitans, spontaneous activity, and memory function were significantly increased compared with TSPG alone or TSPG＋IL-1 groups （P 〈 0.05）. CONCLUSION： TSPG stimulated NSC proliferation, in particular when combined with FGF and bFGF. TSPG significantly induced dopaminergic neuronal differentiation of NSCs, and the effect was greater when combined with IL-1. In addition, TSPG greatly improved behavior in the Parkinson＇s disease mouse model following NSC transplantation. Following NSC transplantation, TSPG pretreatment exhibited superior efficacy over either TSPG alone or TSPG in combination with IL-1, in terms of behavioral improvements in the Parkinson＇s disease mouse model.
基金the National Key Basic Research Program of China,No. 2006cb500700the National Natural Science Foundation of China,No.30470904the Natural Science and Technology Foundation of Guangdong Province,No. 04009356, 2008B030301320
文摘BACKGROUND： Numerous current studies have suggested that human telomerase reverse transcriptase （hTERT） gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses; however, the mechanism of action remains unknown. OBJECTIVE： To evaluate the neuroprotective effects and possible mechanism of action of hTERT gene transfection in human embryonic cortical neurons treated with beta-amyloid fragment 25-35 （AI325-35）. DESIGN, TIME AND SETTING： The randomized, controlled and molecular biological studies were performed at the Department of Anatomy and Brain Research, Zhongshan School of Medicine, Sun Yat-sen University, China, from September 2005 to June 2008. MATERIALS： AdEasy-1 Expression System was gifted by Professor Guoquan Gao from Sun Yat-Sen University, China. Human cortical neurons were derived from 12-20 week old aborted fetuses, obtained from the Guangzhou Maternal and Child Health Hospital, China. Mouse anti-Odk5 and mouse anti-p16 monoclonal antibodies （Lab Vision, USA）, and mouse anti-hTERT monoclonal antibody （Epitomics, USA）, were used in this study. METHODS： （1） Recombinant adenovirus vectors, encoding hTERT （Ad-hTERT） and green fluorescent protein （Ad-GFP）, were constructed using the AdEasy-1 Expression System. Human embryonic cortical neurons in the Ad-hTERT group were transfected with Ad-hTERT for 1-21 days. Likewise, human embryonic cortical neurons in the Ad-GFP group were transfected with Ad-GFP for 1-21 days. Human embryonic cortical neurons in the control group were cultured as normal. （2） Human embryonic cortical neurons in the Ad-hTERT group were treated with 10 pmol/L Aβ25-35 for 24 hours. Normal human embryonic cortical neurons treated with 10 pmol/Lβ25.35 for 24 hours served as a model group. Human embryonic cortical neurons in the Ad-GFP and control groups were not treated with Aβ25-35. MAIN OUTCOME MEASURES： Expression of hTERT in human embryonic cortical neurons was evaluated by immunocytochemical staining and Western blot assay. Telomerase activity was measured using a PCR-based telomeric repeat amplification protocol （TRAP） ELISA kit. Neural activity in human embryonic cortical neurons was examined by MTT assay; apoptosis was measured using TUNEL assay; and Cdk5 and p16 protein expressions were measured by Western blot. RESULTS： Expression of hTERT protein was significantly increased and peaked at day 3 post-transfection in the Ad-hTERT group. No hTERT expression was detected in the Ad-GFP and control groups. Telomerase activity was significantly greater in the Ad-hTERT group compared with the Ad-GFP and control groups （P 〈 0.01）. Compared with the control group, cell activity was significantly decreased （P 〈 0.05）, and cell apoptotic rate, Cdk5 and p16 expression were significantly increased （P 〈 0.01） in the model group. Compared with the model group, cell activity was increased in the Ad-hTERT group, and peaked at day 3 post-transfection （P 〈 0.05）. Neuroprotective effects also peaked at day 3 post-transfection; and the apoptotic rate, Cdk5 and p16 expression significantly decreased （P 〈 0.01）. CONCLUSION： Expression of hTERT in human embryonic cortical neurons can relieve Aβ25-35-induced neuronal apoptosis. The possible mechanism by which hTERT produces these neuroprotective effects may be associated with inhibition of Cdk5 and p16 expression.
文摘Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV.RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time-and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.
文摘Neural tube development comprises neural induction, neural epithelial cell proliferation, and apoptosis, as well as migration of nerve cells. Too much or too little apoptosis leads to abnormal nervous system development. The present study analyzed expression and distribution of apoptotic-related factors, including Fas, FasL, and caspase-3, during human embryonic neural tube development. Experimental results showed that increased caspase-3 expression promoted neural apoptosis via a mitochondrial-mediated intrinsic pathway at 4 weeks during early human embryonic neural tube development. Subsequently, Fas and FasL expression increased during embryonic development. The results suggest that neural cells influence neural apoptosis through synergistic effects of extrinsic pathways. Therefore, neural apoptosis during the early period of neural tube development in the human embryo might be regulated by the death receptor induced apoptotic extrinsic pathways.
文摘Researchers from all around the world emphasize on the enormous possible benefits that stem cells may have for the treatment of diseases. However, this technology is considered morally problematic when the source of the stem cell is from a human embryo. Nonetheless, there is a consensus that of all the types of stem cells, hESC （human embryonic stem ceils） are the most promising for particular and important research and therapies. Yet, there are controversial issues regarding the ＂killing＂ of the human embryo for stem cell derivation. There are two general ethical conditions that should govern the instrumental use of embryo. One of them, the principle of subsidiarity, which is defined as ＂a state we have that we have to choose the less contentious means of achieving the intended goal＂. Based on this principle, we ought only to use hESC when there are no other alternatives, which are less morally controversially. Subsidiarity is based on the assumption that there is something ethically unsound about the use ofhESC. However, this principle only makes sense if it is based on consistently upheld views of the moral status of embryo, moreover, the law should also not limit or prohibit hESC research based on this principle. In this paper, I argue---using the South African law for hESC technology--that criterion for deciding which type of stem cells to use should be based on their potential and suitability for advancing scientific knowledge and development of new therapies which will be greatly beneficial in alleviating human suffering.
基金supported by the CAMS Innovation Fund for Medical Sciences(2021-I2M-1-073,2021-I2M-1-040,2022-I2M-JB-015)the National Key Research and Development Program of China(2021YFA1100703,2021YFA1103000)+2 种基金Haihe Laboratory of Cell Ecosystem Innovation Fund(22HHXBSS00031)the National Natural Science Foundation of China(82125003,32271161,82200141)Tianjin Municipal Science and Technology Commission Grant(20JCYBJC00240,22ZXSYSY00010,22JCQNJC01270)。
文摘The embryonic mesoderm comprises heterogeneous cell subpopulations with distinct lineage biases.It is unclear whether a bias for the human hematopoietic lineage emerges at this early developmental stage.In this study,we integrated single-cell transcriptomic analyses of human mesoderm cells from embryonic stem cells and embryos,enabling us to identify and define the molecular features of human hematopoietic mesoderm(HM)cells biased towards hematopoietic lineages.We discovered that BMP4 plays an essential role in HM specification and can serve as a marker for HM cells.Mechanistically,BMP4 acts as a downstream target of HDAC1,which modulates the expression of BMP4 by deacetylating its enhancer.Inhibition of HDAC significantly enhances HM specification and promotes subsequent hematopoietic cell differentiation.In conclusion,our study identifies human HM cells and describes new mechanisms for human hematopoietic development.
基金supported by Chinese National Science and Technology major project R&D Program of China(2018YFC2000101)Strategic Priority Research Program of Chinese Academy of Science(XDB32060000)+7 种基金National Natural Science Foundation of China(Grant Nos.31871502,31901047,31925016,91957122,82021001,and 31922048)Basic Frontier Scientific Research Program of Chinese Academy of Sciences From 0 to 1 original innovation project(ZDBS-LYSM001)Shanghai Municipal Science and Technology Major Project(2018SHZDZX05)Shanghai City Committee of Science and Technology Project(18411953700,18JC1410100,19XD1424400 and 19YF1455100)Innovative Research Team of High-Level Local Universities in Shanghai(SHSMU-ZDCX20212200 and SHSMU-ZLCX20210200)International Partnership Program of Chinese Academy of Sciences(153D31KYSB20170059)Postdoctoral Science Foundation of China(2020M681417 and 2021T140684)Sailing Program of Shanghai(21YF1453000)(to J.H.).
文摘Approximately 140 million people worldwide are homozygous carriers of APOE4(ε4),a strong genetic risk factor for late onset familial and sporadic Alzheimer’s disease(AD),91%of whom will develop AD at earlier age than heterozygous carriers and noncarriers.Susceptibility to AD could be reduced by targeted editing of APOE4,but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies.Here,we first screened eight cytosine base editor variants at four injection stages(from 1-to 8-cell stage),and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate(up to 100%)with the lowest bystander effects.In particular,80%of AD-susceptibleε4 allele copies were converted to the AD-neutralε3 allele in humanε4-carrying embryos.Stringent control measures combined with targeted deep sequencing,whole genome sequencing,and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells.Furthermore,base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage.Finally,we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia.Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos,a potential approach for reducing human susceptibility to AD or other genetic diseases.
基金Supported by Science and Technology Commission of Shanghai, China (No.074119521)
文摘Objective To quantify proline 4-hydroxylase, alpha polypeptide ii （ P4HA2 ） mRNA of human embryo lung fibroblast （HELF） with SYBR green based reversed transcript PCR （RT-PCR） for correcting cytomegalovirus （CMV） inactivation or clearance efficiency in donor blood. Methods A pair of specific primers of exon 12a of P4HA2 was designed, and the related PCR-reaction system and condition were optimized. Then the recombinant plasmid containing the target fragment was constructed for making standard curve with SYBR green based real-time RT-PCR. Finally, the sensitivity, reproducibility, and specificity of this method were fully estimated. Results The sensitivity of the method was 1.5E ＋ 04 copies/mL of P4HA2 mRNA, corresponding to 10^3 fibroblasts. In addition, existence of 8. 67E ＋ 06 leukocytes could not interfere with the accurate quantification of HELF in the large dynamic range. The intra-assay variability and inter-assay variability both varied in different concentrations, being higher in low concentrations and lower in high concentrations. But all of them were below 13. 76% in variation, which showed acceptable stability of this method. Conclusion SYBR green and specific primer based real-time RT-PCR show up a good quality for quantifying HELF P4HA2 mRNA with good specificity, stability, and high sensitivity. Approximate 10 copies of P4HA2 mRNA per cell in average can be detected by the method. Therefore, this method can be used to deduct fibroblast-associated CMV for correcting CMV inactivation efficiency in leukocytes.
基金supported by the National Key Research and Development Program(2019YFA0801400,2017YFA01038012018YFC1004000)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDA16020703)。
文摘Early human embryogenesis is a very sophisticated process due to its unique gene regulatory network.Autophagy has been suggested to play an important role in mediating the development of early embryonic cells in mammals.However,evidence showing how autophagy regulates early human embryogenesis remains to be further explored.In this study,we systematically investigated the human transcriptome and methylome patterns of autophagy-related(ATG)genes in early embryonic cells at single-cell resolution.We analyzed the transcriptomic data of 365 cells and methylome data of 265 cells.The results showed that most ATG genes remained epigenetically active and were expressed stably throughout early embryogenesis,whereas the dynamics varied among different developmental stages.This evidence indicated that the autophagy pathway was constitutively activated and exerted a fundamental role in early human embryo development.Our work,for the first time,comprehensively reveals the features of autophagy during early human embryo development.
