[Objective]The aim of this study was to explore the technical system of induced expression in vitro of goat mammary gland epithelial cell,and evaluate expression efficiency of mammary gland specific vector and foreign...[Objective]The aim of this study was to explore the technical system of induced expression in vitro of goat mammary gland epithelial cell,and evaluate expression efficiency of mammary gland specific vector and foreign protein at the cell level.[Method]Goat mammary gland epithelial cell transfected by human lactoferrin gene was inducted by culturing in DMEM/F12 medium supplemented with 5 mg/L insulin,5 mg/L prolactin and 1 mg/L hydrocortisone.Supernatant was collected per 6 hours and concentrated.Expression situation of foreign protein were detected by SDS-PAGE and Western blotting.[Result]There was target protein expression in the induced culture medium,which molecular weight was about 42 kD.[Conclusion]The method used in this study can induce goat mammary gland epithelial cell to express foreign gene,it lays a foundation for researching heterologous expression of foreign gene and producing mammary gland bioreactor.展开更多
[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer techn...[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer technology,the single goat fetal fibroblasts(GFF)and mammary gland epithelial cells(GMGE)harboring human lactoferrin(hLF)gene were transferred to the enucleated oocyte.Reconstructed karyoplast-cytoplast couplets were fused,activated,and cultured in vitro.Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients,and blastocysts were transferred into uterine horn.[Result] The pregnancy rate was similar between GFF and GMGE(oviduct transfer:26.47% vs.20.00%),and between oviduct transfer and uterine horn transfer(26.47% vs.25.00%)for GFF group;pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group(40.00% vs.26.67% and 21.43%).[Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type,embryo stage and transfer position but be done by the number of embryo transferred per recipient.In addition,the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.展开更多
Intercellular communication via gap junctions allows cells within multicellular organisms to share small molecules. The effect of such interactions has been elucidated using mouse gene knockout strategies. Although se...Intercellular communication via gap junctions allows cells within multicellular organisms to share small molecules. The effect of such interactions has been elucidated using mouse gene knockout strategies. Although several mutations in human gap junction-encoding connexin(Cx) have been described, Cx mutants in mice do not always recapitulate the human disease. Among the 20 mouse Cxs, Cx26, Cx43, and Cx45 play roles in early cardiac or placental development, and disruption of the genes results in lethality that hampers further analyses. Embryonic stem cells(ESCs) that lack Cx43 or Cx45 have made analysis feasible in both in vitro differentiated cell cultures and in vivo chimeric tissues. The success of mouse ESCs studies is leading to the use of induced pluripotent stem cells to learn more about the pathogenesis of human Cx diseases. This review summarizes the current status of mouse Cx disruption models and ESC differentiation studies, and discusses their implication for understanding human Cx diseases.展开更多
AIM： TO accurately and realistically elucidate human stem cell behaviors in vivo and the fundamental mechanisms controlling human stem cell fates in vivo, which is urgently required in regenerative medicine and treat...AIM： TO accurately and realistically elucidate human stem cell behaviors in vivo and the fundamental mechanisms controlling human stem cell fates in vivo, which is urgently required in regenerative medicine and treatments for some human diseases, a surrogate human-rat chimera model was developed. METHODS： Human-rat chimeras were achieved by in utero transplanting low-density mononuclear cells from human umbilical cord blood into the fetal rats at 9-11 d of gestation, and subsequently, a variety of methods, including flow cytometry, PCR as well as immunohistochemical assay, were used to test the human donor contribution in the recipients. RESULTS： Of 29 live-born recipients, 19 had the presence of human CD45^＋ cells in peripheral blood （PB） detected by flow cytometry, while PCR analysis on genomic DNA from 11 different adult tissues showed that 14 selected from flow cytometry-positive 19 animals possessed of donor-derived human cell engraftment in multiple tissues （i.e. liver, spleen, thymus, heart, kidney, blood, lung, muscle, gut and skin） examined at the time of tissue collection, as confirmed by detecting human 132- microglobulin expression using immunohistochemistry. Tn this xenogeneic system, the engrafted donor-derived human cells persisted in multiple tissues for at least 6 mo after birth. Moreover, transplanted human donor cells underwent site-specific differentiation into CK18-positive human cells in chimeric liver and CEHS-positive human cells in chimeric spleen and thymus of recipients. CONCLUSION： Taken together, these findings suggest that we successfully developed human-rat chimeras, in which xenogeneic human cells exist up to 6 mo later. This humanized small animal model, which offers an in vivo environment more closely resembling to the situations in human, provides an invaluable and effective approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the future. The potential for new advances in our better understanding the living biological systems in human provided by investigators in humanized animals will remain promising.展开更多
Since first identified in 2010, Piezo proteins have been found to perform as poreforming mechanosensitive ion channels across a wide range of animals. As a Piezo ortholog primarily expressed in mammalian systems, Piez...Since first identified in 2010, Piezo proteins have been found to perform as poreforming mechanosensitive ion channels across a wide range of animals. As a Piezo ortholog primarily expressed in mammalian systems, Piezo1 has been observed to distribute mainly in nonsensory tissues, regulating osmotic homeostasis, proprioception, and light touch. With previous studies on the putative structure of Piezo1, the gating system and several mechanotransduction mechanisms have been proposed. Besides, mutations of specific amino acid sequences in Piezo1 have been linked to several human diseases such as dehydrated hereditary xerocytosis (DHS) and congenital lymphatic dysplasia (CLD). However, most of these mutations have not been well characterized. To further elucidate the relations between these mutations and diseases, UCSF Chimera is used as the tool to visualize the structural importance of each of these mutated amino acids. With the aid from UCSF Chimera, this study has recorded and interpreted clashes and contacts originated from each of the mutations. Accordingly, specific mechanisms between mutations and human diseases are proposed, which pave the way for healing.展开更多
AIM： To examine the human hepatic parenchymal and stromal components in rat liver and the phenotypic changes of human cells in liver of human-rat chimera （HRC） generated by in utero transplantation of human cells d...AIM： To examine the human hepatic parenchymal and stromal components in rat liver and the phenotypic changes of human cells in liver of human-rat chimera （HRC） generated by in utero transplantation of human cells during partial hepatectomy （PHx）-induced liver regeneration. METHODS： Human hepatic parenchymal and stromal components and phenotypic changes of human cells during liver regeneration were examined by flow oytometry, in situ hybridization and immunohistochemistry. RESULTS： ISH analysis positive cells in hepatic demonstrated human Aluparenchyma and stroma of recipient liver. Functional human hepatocytes generated in this model potentially constituted human hepatic functional units with the presence of donor-derived human endothelial and biliary duct cells in host liver. Alpha fetoprotein （AFP）^＋, CD34^＋ and CD45^＋ cells were observed in the chimeric liver on day 10 after PHxinduced liver regeneration and then disappeared in PHx group, but not in non-PHx group, suggesting that dynamic phenotypic changes of human cells expressing AFP, CD34 and CD45 cells may occur during the chimeric liver regeneration. Additionally, immunostaining for human proliferating cell nuclear antigen （PCNA） showed that the number of PCNA-positive cells in the chimeric liver of PHx group was markedly increased, as compared to that of control group, indicating that donor-derived human cells are actively proliferated during PHx-induced regeneration of HRC liver. CONCLUSION： HRC liver provides a tool for investigating human liver regeneration in a humanized animal model.展开更多
Human pluripotent stem cell(hPSC)models provide unprecedented opportunities to study human neurological disorders by recapitulating human-specific disease mechanisms.In particular,hPSC-based human–animal brain chimer...Human pluripotent stem cell(hPSC)models provide unprecedented opportunities to study human neurological disorders by recapitulating human-specific disease mechanisms.In particular,hPSC-based human–animal brain chimeras enable the study of human cell pathophysiology in vivo.In chimeric brains,human neural and immune cells can maintain human-specific features,undergo maturation,and functionally integrate into host brains,allowing scientists to study how human cells impact neural circuits and animal behaviors.The emerging human–animal brain chimeras hold promise for modeling human brain cells and their interactions in health and disease,elucidating the disease mechanism from molecular and cellular to circuit and behavioral levels,and testing the efficacy of cell therapy interventions.Here,we discuss recent advances in the generation and applications of using human–animal chimeric brain models for the study of neurological disorders,including disease modeling and cell therapy.展开更多
基金Supported by Doctoral Start Fund of Henan University of Science and Technology.
文摘[Objective]The aim of this study was to explore the technical system of induced expression in vitro of goat mammary gland epithelial cell,and evaluate expression efficiency of mammary gland specific vector and foreign protein at the cell level.[Method]Goat mammary gland epithelial cell transfected by human lactoferrin gene was inducted by culturing in DMEM/F12 medium supplemented with 5 mg/L insulin,5 mg/L prolactin and 1 mg/L hydrocortisone.Supernatant was collected per 6 hours and concentrated.Expression situation of foreign protein were detected by SDS-PAGE and Western blotting.[Result]There was target protein expression in the induced culture medium,which molecular weight was about 42 kD.[Conclusion]The method used in this study can induce goat mammary gland epithelial cell to express foreign gene,it lays a foundation for researching heterologous expression of foreign gene and producing mammary gland bioreactor.
基金Supported by the National High-tech R&D Program(2004AA213072)the Doctor Fund of Henan University of Science and Technology~~
文摘[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer technology,the single goat fetal fibroblasts(GFF)and mammary gland epithelial cells(GMGE)harboring human lactoferrin(hLF)gene were transferred to the enucleated oocyte.Reconstructed karyoplast-cytoplast couplets were fused,activated,and cultured in vitro.Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients,and blastocysts were transferred into uterine horn.[Result] The pregnancy rate was similar between GFF and GMGE(oviduct transfer:26.47% vs.20.00%),and between oviduct transfer and uterine horn transfer(26.47% vs.25.00%)for GFF group;pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group(40.00% vs.26.67% and 21.43%).[Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type,embryo stage and transfer position but be done by the number of embryo transferred per recipient.In addition,the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.
文摘Intercellular communication via gap junctions allows cells within multicellular organisms to share small molecules. The effect of such interactions has been elucidated using mouse gene knockout strategies. Although several mutations in human gap junction-encoding connexin(Cx) have been described, Cx mutants in mice do not always recapitulate the human disease. Among the 20 mouse Cxs, Cx26, Cx43, and Cx45 play roles in early cardiac or placental development, and disruption of the genes results in lethality that hampers further analyses. Embryonic stem cells(ESCs) that lack Cx43 or Cx45 have made analysis feasible in both in vitro differentiated cell cultures and in vivo chimeric tissues. The success of mouse ESCs studies is leading to the use of induced pluripotent stem cells to learn more about the pathogenesis of human Cx diseases. This review summarizes the current status of mouse Cx disruption models and ESC differentiation studies, and discusses their implication for understanding human Cx diseases.
基金Supported by The National Natural Science Foundation of China, No. 30271177 and No. 39870676 the National 9th Five-year Program, No. 101033+3 种基金 The Major Science and Technology Projects of Guangdong Province, No. B602 Natural Science Foundation of Guangdong Province, No. 021903 The Postdoctoral Fellowship Foundation of China (Series 29)The Special Fund of Scientifi c Instrument Collaborative Share-net in Guangzhou, No. 2006176
文摘AIM： TO accurately and realistically elucidate human stem cell behaviors in vivo and the fundamental mechanisms controlling human stem cell fates in vivo, which is urgently required in regenerative medicine and treatments for some human diseases, a surrogate human-rat chimera model was developed. METHODS： Human-rat chimeras were achieved by in utero transplanting low-density mononuclear cells from human umbilical cord blood into the fetal rats at 9-11 d of gestation, and subsequently, a variety of methods, including flow cytometry, PCR as well as immunohistochemical assay, were used to test the human donor contribution in the recipients. RESULTS： Of 29 live-born recipients, 19 had the presence of human CD45^＋ cells in peripheral blood （PB） detected by flow cytometry, while PCR analysis on genomic DNA from 11 different adult tissues showed that 14 selected from flow cytometry-positive 19 animals possessed of donor-derived human cell engraftment in multiple tissues （i.e. liver, spleen, thymus, heart, kidney, blood, lung, muscle, gut and skin） examined at the time of tissue collection, as confirmed by detecting human 132- microglobulin expression using immunohistochemistry. Tn this xenogeneic system, the engrafted donor-derived human cells persisted in multiple tissues for at least 6 mo after birth. Moreover, transplanted human donor cells underwent site-specific differentiation into CK18-positive human cells in chimeric liver and CEHS-positive human cells in chimeric spleen and thymus of recipients. CONCLUSION： Taken together, these findings suggest that we successfully developed human-rat chimeras, in which xenogeneic human cells exist up to 6 mo later. This humanized small animal model, which offers an in vivo environment more closely resembling to the situations in human, provides an invaluable and effective approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the future. The potential for new advances in our better understanding the living biological systems in human provided by investigators in humanized animals will remain promising.
文摘Since first identified in 2010, Piezo proteins have been found to perform as poreforming mechanosensitive ion channels across a wide range of animals. As a Piezo ortholog primarily expressed in mammalian systems, Piezo1 has been observed to distribute mainly in nonsensory tissues, regulating osmotic homeostasis, proprioception, and light touch. With previous studies on the putative structure of Piezo1, the gating system and several mechanotransduction mechanisms have been proposed. Besides, mutations of specific amino acid sequences in Piezo1 have been linked to several human diseases such as dehydrated hereditary xerocytosis (DHS) and congenital lymphatic dysplasia (CLD). However, most of these mutations have not been well characterized. To further elucidate the relations between these mutations and diseases, UCSF Chimera is used as the tool to visualize the structural importance of each of these mutated amino acids. With the aid from UCSF Chimera, this study has recorded and interpreted clashes and contacts originated from each of the mutations. Accordingly, specific mechanisms between mutations and human diseases are proposed, which pave the way for healing.
基金Supported by The National Natural Science Foundation of China, No. 30271177 and No. 39870676the Major Scienceand Technology Projects of Guangdong Province, No. B602+4 种基金the Natural Science Foundation of Guangdong Province, No.021903the Science and Technology Planning Project of Guangdong Province, No. 2009B060300008the Science and Technology Projects of Guangzhou City, No. 2002Z2E0121the Medical Scientific Research Foundation of Guangdong Province, No. A2007359the Science and Technology Talented Man Foundation of Outstanding Young and Middle-aged People of Southern Medical University,the Special Fund of Scientific Instrument Collaborative Share-net in Guangzhou, No. 2006176
文摘AIM： To examine the human hepatic parenchymal and stromal components in rat liver and the phenotypic changes of human cells in liver of human-rat chimera （HRC） generated by in utero transplantation of human cells during partial hepatectomy （PHx）-induced liver regeneration. METHODS： Human hepatic parenchymal and stromal components and phenotypic changes of human cells during liver regeneration were examined by flow oytometry, in situ hybridization and immunohistochemistry. RESULTS： ISH analysis positive cells in hepatic demonstrated human Aluparenchyma and stroma of recipient liver. Functional human hepatocytes generated in this model potentially constituted human hepatic functional units with the presence of donor-derived human endothelial and biliary duct cells in host liver. Alpha fetoprotein （AFP）^＋, CD34^＋ and CD45^＋ cells were observed in the chimeric liver on day 10 after PHxinduced liver regeneration and then disappeared in PHx group, but not in non-PHx group, suggesting that dynamic phenotypic changes of human cells expressing AFP, CD34 and CD45 cells may occur during the chimeric liver regeneration. Additionally, immunostaining for human proliferating cell nuclear antigen （PCNA） showed that the number of PCNA-positive cells in the chimeric liver of PHx group was markedly increased, as compared to that of control group, indicating that donor-derived human cells are actively proliferated during PHx-induced regeneration of HRC liver. CONCLUSION： HRC liver provides a tool for investigating human liver regeneration in a humanized animal model.
文摘Human pluripotent stem cell(hPSC)models provide unprecedented opportunities to study human neurological disorders by recapitulating human-specific disease mechanisms.In particular,hPSC-based human–animal brain chimeras enable the study of human cell pathophysiology in vivo.In chimeric brains,human neural and immune cells can maintain human-specific features,undergo maturation,and functionally integrate into host brains,allowing scientists to study how human cells impact neural circuits and animal behaviors.The emerging human–animal brain chimeras hold promise for modeling human brain cells and their interactions in health and disease,elucidating the disease mechanism from molecular and cellular to circuit and behavioral levels,and testing the efficacy of cell therapy interventions.Here,we discuss recent advances in the generation and applications of using human–animal chimeric brain models for the study of neurological disorders,including disease modeling and cell therapy.
