AIM： To use the tyrosinase minigene as a visual marker to perform microinjection training and improve the techniques related with transgene to greatly elevate the effidency of gene transfer. METHODS： A mouse tyrosin...AIM： To use the tyrosinase minigene as a visual marker to perform microinjection training and improve the techniques related with transgene to greatly elevate the effidency of gene transfer. METHODS： A mouse tyrosinase minigene, i.e., TyBS, in which the 2.25-kb authentic genomic 5＇ non-coding flanking sequence of mouse tyrosinase was fused to a mouse tyrosinase cDNA, was introduced into the fertilized eggs of outbred Kunming albino mice. RESULTS： Of the 11 animals that developed from the injected eggs, two mice （P1 and #8） exhibited pigmented hair （P1） and eyes （P1 and #8）, as confirmed by PCR analysis for the tyrosinase minigene integrated into the genome. When founder P1 was bred to Kunming male mouse, six progeny out of 11 offspring inherited the transgene and the pigmented-eye phenotype. CONCLUSION： Taken together, these results suggest that this minigene encodes the active tyrosinase protein and that its 5＇ flanking region contains the sequences regulating the expression of mouse tyrosinase gene as expected. We have rescued the albino phenotype by introduction and expression of a functional tyrosinase minigene in the Kunming albino mouse and the transgene can be passed to subsequent generation. These findings also indicate that TyBS can be a useful visual marker gene in the co-transgenic experiments.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 adv展开更多
Objective To investigate whether germ like cells isolated from embryoid body formed by mouse embryonic stem cells could survive and initiate spermatogenesis in seminiferous tubules of adult mice. Methods SSEA-1＋ cell...Objective To investigate whether germ like cells isolated from embryoid body formed by mouse embryonic stem cells could survive and initiate spermatogenesis in seminiferous tubules of adult mice. Methods SSEA-1＋ cells were isolated from embryoid bodies prepared from mouse EGFP-ES cells, after retinoic acid treatment, the cells were detected for the expression of alkaline phosphatase, Rnh2, stella, fragilis, Tex14, Sry, Hsp90-a, Stra8 and integrin a6, and then, the cells were transplanted into seminiferous tubules of busulfan-treated adult mice. Results Six days after retinoic acid treatment, alkaline phosphatase expressing cells could still be found in embryoid body （EB） derived cells, indicating the existence of retinoic acid-resistant primordial germ cells. When the SSEA-1＋ cells isolated from embryoid bodies were stimulated with retinoic acid for 6 days, some of these cells expressed cell markers of Hsp90-a, Stra8 and integrin a6, resembling the expression profile of spermatogonial stem cells. Forty-five days after cell transplantation, a little amount of GFP-expressing cells attached to the basement membrane of seminiferous tubule and formed small colonies; Three months later, these cells started amplification in the form of cell chains with varied length, and moving towards the lumen of the seminiferous tubules. Five months after the transplantation, multilayered cell mass was found in seminiferous tubules of two, out of four recipient mice. There was no GFP-expressing cells existed in non-cell-transplanted seminiferous tubules. Conclusion In our study, although full-termed spermatogenesis was not observed in all of the recipients, the results did indicate that the embryoid body contains germ like cells, and these cells can survive and initiate amplification in seminiferous tubules of adult mouse.展开更多
AIM: To construct a hepatitis B virus (HBV)-based vector with a reporter gene and to establish an HBV infection system to evaluate the availability of the vector. METHODS: The HBV-based vectors with green fluorescence...AIM: To construct a hepatitis B virus (HBV)-based vector with a reporter gene and to establish an HBV infection system to evaluate the availability of the vector. METHODS: The HBV-based vectors with green fluorescence protein (GFP) were packaged into the liver of immunodeficient mice through transfer and helper plasmid using hydrodynamic technology. Wild type HBV (wt HBV) was provided by plasmid MC2009. Primary human hepatocytes (PHH) were isolated and infected with recombinant HBV (rHBV) or wt HBV. GFP expression was monitored by confocal and flow cytometry. HBV DNA and HBV surface antigen (HBSAg) were analyzed by PCR and ELISA. RESULTS: 3 × 107 wt HBV copies/mL and 5 × 106 rHBV copies/mL were collected from mice serum. In the wt HBV infected group, HBV progeny was 2 × 107 copies/mL and HBSAg was 770 ng/mL. In the rHBV infected group, GFP fluorescence was detected on d 3 post-infection and over 85% of the parenchymal cells expressed green fluorescence on d 12 post-infection. Compared with wt HBV in the PHH infection system, no rHBV DNA or HBSAg were detected in PHH culture media. CONCLUSION: An effective HBV based vector was developed, which proved to be a useful HBV infection system. This vector and infection system can be applied to develop a therapeutic vector and study the HBV life cycle and viral pathogenesis.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
Background: Hand, foot and mouth disease (HFMD) remains an important public health problem in China. Many studies on the epidemiological characteristics of HFMD have been reported, but studies in North Sichuan region ...Background: Hand, foot and mouth disease (HFMD) remains an important public health problem in China. Many studies on the epidemiological characteristics of HFMD have been reported, but studies in North Sichuan region have been neglected. Methods: HFMD-related enterovirus infected cases were clinically confirmed and underwent real-time RT-PCR (rRT-PCR) from May 2018 to October 2023 in Guangyuan Central Hospital. Results: During 2018-2023, other EV (437 cases, 81.08%) was the most predominant serotype followed by CV-A16 (94 cases, 17.44%), EV-A71 (8 cases, 1.48%) was the least predominant serotype. Peak infections occurred in July and October. There were no significant differences in gender, age and serotypes. HFMD was concentrated in children under 47 months of age, with the highest incidence in children aged 12 - 23 months and the highest proportion of other EV infections in the whole age group. COVID-19 did not cause significant changes in gender, age and serotype. Overall, there was a significant increase in the proportion of children aged 12 - 23 months infected with CV-A16, and an increase in the proportion of children aged over 36 months infected with other EVs. Conclusions: The incidence of HFMD caused by EV-A71 has decreased significantly, and other EVs have become the main pathogens of HFMD in North Sichuan region in recent years. In the prevention and control of CV-A16, more attention should be paid to children aged 12 - 23 months and the dominant serotype should be closely monitored. Our study highlights the importance of developing of new diagnostic reagents and vaccines for the prevention and control of enterovirus infection. This study for the first time provides insights into district interventions to local conditions.展开更多
基金Supported by the National Natural Science Foundation of China, No. 30271177 and No. 39870676 National 9th Five-year Program, No. 101033+3 种基金 Major Science and Technology Projects of Guangdong Province, No. B602 Natural Science Foundation of Guangdong Province, No. 021903 Postdoctoral Fellowship Foundation of China (Series 29) Special Fund of Scientific Instrument Collaborative Share-net in Guangzhou. No. 2006176
文摘AIM： To use the tyrosinase minigene as a visual marker to perform microinjection training and improve the techniques related with transgene to greatly elevate the effidency of gene transfer. METHODS： A mouse tyrosinase minigene, i.e., TyBS, in which the 2.25-kb authentic genomic 5＇ non-coding flanking sequence of mouse tyrosinase was fused to a mouse tyrosinase cDNA, was introduced into the fertilized eggs of outbred Kunming albino mice. RESULTS： Of the 11 animals that developed from the injected eggs, two mice （P1 and #8） exhibited pigmented hair （P1） and eyes （P1 and #8）, as confirmed by PCR analysis for the tyrosinase minigene integrated into the genome. When founder P1 was bred to Kunming male mouse, six progeny out of 11 offspring inherited the transgene and the pigmented-eye phenotype. CONCLUSION： Taken together, these results suggest that this minigene encodes the active tyrosinase protein and that its 5＇ flanking region contains the sequences regulating the expression of mouse tyrosinase gene as expected. We have rescued the albino phenotype by introduction and expression of a functional tyrosinase minigene in the Kunming albino mouse and the transgene can be passed to subsequent generation. These findings also indicate that TyBS can be a useful visual marker gene in the co-transgenic experiments.
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 adv
基金supported by grants from the Natural Science Foundation of Guangdong Province (No. 5001351)
文摘Objective To investigate whether germ like cells isolated from embryoid body formed by mouse embryonic stem cells could survive and initiate spermatogenesis in seminiferous tubules of adult mice. Methods SSEA-1＋ cells were isolated from embryoid bodies prepared from mouse EGFP-ES cells, after retinoic acid treatment, the cells were detected for the expression of alkaline phosphatase, Rnh2, stella, fragilis, Tex14, Sry, Hsp90-a, Stra8 and integrin a6, and then, the cells were transplanted into seminiferous tubules of busulfan-treated adult mice. Results Six days after retinoic acid treatment, alkaline phosphatase expressing cells could still be found in embryoid body （EB） derived cells, indicating the existence of retinoic acid-resistant primordial germ cells. When the SSEA-1＋ cells isolated from embryoid bodies were stimulated with retinoic acid for 6 days, some of these cells expressed cell markers of Hsp90-a, Stra8 and integrin a6, resembling the expression profile of spermatogonial stem cells. Forty-five days after cell transplantation, a little amount of GFP-expressing cells attached to the basement membrane of seminiferous tubule and formed small colonies; Three months later, these cells started amplification in the form of cell chains with varied length, and moving towards the lumen of the seminiferous tubules. Five months after the transplantation, multilayered cell mass was found in seminiferous tubules of two, out of four recipient mice. There was no GFP-expressing cells existed in non-cell-transplanted seminiferous tubules. Conclusion In our study, although full-termed spermatogenesis was not observed in all of the recipients, the results did indicate that the embryoid body contains germ like cells, and these cells can survive and initiate amplification in seminiferous tubules of adult mouse.
基金the grants from the National Science Foundation of China, No. 30271177 and No. 39870676the Natural Science Foundation of Guangdong Province, No. 021903
文摘AIM: To construct a hepatitis B virus (HBV)-based vector with a reporter gene and to establish an HBV infection system to evaluate the availability of the vector. METHODS: The HBV-based vectors with green fluorescence protein (GFP) were packaged into the liver of immunodeficient mice through transfer and helper plasmid using hydrodynamic technology. Wild type HBV (wt HBV) was provided by plasmid MC2009. Primary human hepatocytes (PHH) were isolated and infected with recombinant HBV (rHBV) or wt HBV. GFP expression was monitored by confocal and flow cytometry. HBV DNA and HBV surface antigen (HBSAg) were analyzed by PCR and ELISA. RESULTS: 3 × 107 wt HBV copies/mL and 5 × 106 rHBV copies/mL were collected from mice serum. In the wt HBV infected group, HBV progeny was 2 × 107 copies/mL and HBSAg was 770 ng/mL. In the rHBV infected group, GFP fluorescence was detected on d 3 post-infection and over 85% of the parenchymal cells expressed green fluorescence on d 12 post-infection. Compared with wt HBV in the PHH infection system, no rHBV DNA or HBSAg were detected in PHH culture media. CONCLUSION: An effective HBV based vector was developed, which proved to be a useful HBV infection system. This vector and infection system can be applied to develop a therapeutic vector and study the HBV life cycle and viral pathogenesis.
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Background: Hand, foot and mouth disease (HFMD) remains an important public health problem in China. Many studies on the epidemiological characteristics of HFMD have been reported, but studies in North Sichuan region have been neglected. Methods: HFMD-related enterovirus infected cases were clinically confirmed and underwent real-time RT-PCR (rRT-PCR) from May 2018 to October 2023 in Guangyuan Central Hospital. Results: During 2018-2023, other EV (437 cases, 81.08%) was the most predominant serotype followed by CV-A16 (94 cases, 17.44%), EV-A71 (8 cases, 1.48%) was the least predominant serotype. Peak infections occurred in July and October. There were no significant differences in gender, age and serotypes. HFMD was concentrated in children under 47 months of age, with the highest incidence in children aged 12 - 23 months and the highest proportion of other EV infections in the whole age group. COVID-19 did not cause significant changes in gender, age and serotype. Overall, there was a significant increase in the proportion of children aged 12 - 23 months infected with CV-A16, and an increase in the proportion of children aged over 36 months infected with other EVs. Conclusions: The incidence of HFMD caused by EV-A71 has decreased significantly, and other EVs have become the main pathogens of HFMD in North Sichuan region in recent years. In the prevention and control of CV-A16, more attention should be paid to children aged 12 - 23 months and the dominant serotype should be closely monitored. Our study highlights the importance of developing of new diagnostic reagents and vaccines for the prevention and control of enterovirus infection. This study for the first time provides insights into district interventions to local conditions.
