The existing treatment modalities for skin injuries mainly include dressings, negative-pressure w... more The existing treatment modalities for skin injuries mainly include dressings, negative-pressure wound treatment, autologous skin grafting, and high-pressure wound treatment. All of these therapies have limitations such as high time cost, the inability to remove inactivated tissue in a timely manner, surgical debridement, and oxygen toxicity. Mesenchymal stem cells have a unique self-renewal ability and wide differentiation potential, and they are one of the most promising stem cell types in cell therapy and have great application prospects in the field of regenerative medicine. Collagen exerts structural roles by promoting the molecular structure, shape, and mechanical properties of cells, and adding it to cell cultures can also promote cell proliferation and shorten the cell doubling time. The effects of collagen on MSCs were examined using Giemsa staining, EdU staining, and growth curves. Mice were subjected to allogeneic experiments and autologous experiments to reduce individual...
Pregnancy loss (PL) is one of the common complications that women can experience during pregnancy... more Pregnancy loss (PL) is one of the common complications that women can experience during pregnancy, with an occurrence rate of 1 to 5%. The potential causes of pregnancy loss are unclear, with no effective treatment modalities being available. It has been previously reported that the level of miR-125b was significantly increased in placentas of PL patients. However, the role of miR-125b in the development of PL still remains unknown. In the current study, an miR-125b placenta-specific over-expression model was constructed by lentiviral transfecting zona-free mouse embryos followed by embryo transfer. On gestation day 15, it was observed that the placenta was significantly smaller in the miR-125b placenta-specific overexpression group than the control group. Additionally, the abortion rate of the miR-125b placenta-specific overexpression group was markedly higher than in the control group. The blood vessel diameter was larger in the miR-125b-overexpressing specific placenta. In additi...
Porcine-induced pluripotent stem cells (piPSCs) could serve as a great model system for human ste... more Porcine-induced pluripotent stem cells (piPSCs) could serve as a great model system for human stem cell preclinical research. However, the pluripotency gene network of piPSCs, especially the function for the core transcription factor estrogen-related receptor beta (ESRRB), was poorly understood. Here, we constructed ESRRB-overexpressing piPSCs (ESRRB-piPSCs). Compared with the control piPSCs (CON-piPSCs), the ESRRB-piPSCs showed flat, monolayered colony morphology. Moreover, the ESRRB-piPSCs showed greater chimeric capacity into trophectoderm than CON-piPSCs. We found that ESRRB could directly regulate the expressions of trophoblast stem cell (TSC)-specific markers, including KRT8, KRT18 and CDX2, through binding to their promoter regions. Mutational analysis proved that the N-terminus zinc finger domain is indispensable for ESRRB to regulate the TSC markers. Furthermore, this regulation needs the participation of OCT4. Accordingly, the cooperation between ESRRB and OCT4 facilitates...
Previous studies have demonstrated that transcription factor Etv5 plays an important role in the ... more Previous studies have demonstrated that transcription factor Etv5 plays an important role in the segregation between epiblast and primitive endoderm at the second fate decision of early embryo. However, it remains elusive whether Etv5 functions in the segregation between inner cell mass and trophectoderm at the first cell fate decision. In this study, we firstly generated Etv5 knockout mouse embryonic stem cells (mESCs) by CRISPR/Cas9, then converted them into extended potential stem cells (EPSCs) by culturing the cells in small molecule cocktail medium LCDM (LIF, CHIR99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride), and finally investigated their differentiation efficiency of trophoblast stem cells (TSCs). The results showed that Etv5 knockout significantly decreased the efficiency of TSCs (CDX2 +) differentiated from EPSCs. In addition, Etv5 knockout resulted in higher incidence of the differentiated cells with tetraploid and octoploid than that from wild type. Mechanistically, Etv5 was activated by extracellular-signal-regulated kinase (ERK) signaling pathway; in turn, Etv5 had a positive feedback on the expression of fibroblast growth factor receptor 2 (FGFR2) which lies upstream of ERK. Etv5 knockout decreased the expression of FGFR2, whose binding with fibroblast growth factor 4 was essentially needed for TSCs differentiation. Collectively, the findings in this study suggest that Etv5 is required to safeguard the TSCs differentiation by regulating FGFR2 and provide new clues to understand the specification of trophectoderm in vivo. Keywords Etv5 • Mouse embryonic stem cells • Extended potential stem cells • Trophoblast stem cells • Fibroblast growth factor receptor 2 Kui Zhu and Yuan Liu contributed equally to this work.
Global human health has been compromised by high-fat diets. This study aimed to investigate the r... more Global human health has been compromised by high-fat diets. This study aimed to investigate the relationship between a high-fat diet and parthenogenetic embryo quality. Mice fed a high-fat or a normal diet was used as treated or control groups, respectively. Estradiol (E), total cholesterol (TC) and total triglyceride (TG) were detected by Enzyme-Linked ImmunoSorbent Assay (ELISA). Cumulus-oocyte complexes (COCs) were collected from the mice in the treated and control groups. The ultrastructure of COCs, the expression level of genes involved in mitochondrial and nuclear functions in cumulus cells and oocytes quality were evaluated with transmission electron microscopy, real-time quantitative polymerase chain reaction (RT-PCR) and artificial parthenogenesis, respectively. The results showed that the efficiency of parthenogenetic embryonic development in vitro was significantly higher in the treated group than in the control group ( < .05). The expression level of genes involved in...
Self-renewal and differentiation of male germline stem cells (mGSCs) provide the basic function f... more Self-renewal and differentiation of male germline stem cells (mGSCs) provide the basic function for continual spermatogenesis. Studies of in vitro culture of germline stem cells are important and meaningful for basic biological research and practical application. Growth factors, such as GDNF, bFGF, CSF1, and EGF, could maintain the self-renewal of mGSCs. Insulin-like growth factor 1 (IGF-1), an important growth factor, and its pathway have been reported to maintain the survival of several types of stem cells and play important roles in male reproduction. However, the mechanism through which the IGF-1 pathway acts to regulate the self-renewal of mGSCs remains unclear. We analyzed the effect of IGF-1 on the proliferation and apoptosis of bovine mGSCs. We evaluated the expression profile of long noncoding RNA (LncRNA) H19 in bovine and mouse tissues. Moreover, we investigated whether LncRNA H19 could regulate the IGF-1 pathway. Results showed that IGF-1 could activate the phosphorylati...
Methylation is an important issue in gene expression regulation and also in the fields of genetic... more Methylation is an important issue in gene expression regulation and also in the fields of genetics and reproduction. In this study, we created fat-1 transgenic sheep, investigated the fine-mapping and the modulatory mechanisms of promoter methylation. Sheep fetal fibroblasts were transfected by pCAG-fat1-IRES-EGFP. Monoclonal cell line was screened as nuclear donor and carried out nuclear transfer (441 transgenic cloned embryos, 52 synchronism recipient sheep). Six offsprings were obtained. Expressions of exogenous genes fat-1 and EGFP were detectable in 10 examined tissues and upregulated omega-3 fatty acid content. Interestingly, more or less EGFP negative cells were detectable in the positive transgenic fetal skin cells. EGFP negative and positive cells were sorted by flow cytometry, and their methylation status in the whole promoter region (1701 nt) were investigated by bisulphate sequencing. The fine-mapping of methylation in CAG promoter were proposed. The results suggested th...
Many men endure immunosuppressive or anticancer treatments that contain alkylating agents before ... more Many men endure immunosuppressive or anticancer treatments that contain alkylating agents before the age of sexual maturity, especially the increasing number of preadolescent males who undergo busulfan treatment for myeloablative conditioning before hematopoietic stem cell transplantation. Before sperm production, there are no sperm available for cryopreservation. Thus, it is necessary to identify a solution to ameliorate the busulfan-induced damage of spermatogonial stem cells (SSCs). In this study, we demonstrated that melatonin relieved the previously described SSC loss and apoptosis in mouse testes. Melatonin increased the expression of manganese superoxide dismutase (MnSOD), which regulated the production of busulfan-induced reactive oxygen species (ROS). Moreover, melatonin promoted sirtuin type 1 (SIRT1) expression. SIRT1 participated in the deacetylation of p53, which promotes p53 ubiquitin degradation. Decreased concentrations of deacetylated p53 resulted in spermatogonial ...
Precision genetic engineering accelerates the genetic improvement of livestock for agriculture an... more Precision genetic engineering accelerates the genetic improvement of livestock for agriculture and biomedicine. We have recently reported our success in producing gene-modified goats using the CRISPR/Cas9 system through microinjection of Cas9 mRNA and sgRNAs targeting the MSTN and FGF5 genes in goat embryos. By investigating the influence of gene modification on the phenotypes of Cas9-mediated goats, we herein demonstrate that the utility of this approach involving the disruption of FGF5 results in increased number of second hair follicles and enhanced fiber length in Cas9-mediated goats, suggesting more cashmere will be produced. The effects of genome modifications were characterized using H&E and immunohistochemistry staining, quantitative PCR, and western blotting techniques. These results indicated that the gene modifications induced by the disruption of FGF5 had occurred at the morphological and genetic levels. We further show that the knockout alleles were likely capable of ge...
After partial hepatectomy, hepatocytes proliferate to restore mass and function of the liver. Mac... more After partial hepatectomy, hepatocytes proliferate to restore mass and function of the liver. Macrophages, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells (DC), eosinophils, gamma delta T (γδT) cells, and conventional T cells, as well as other subsets of the immune cells residing in the liver control liver regeneration, either through direct interactions with hepatocytes or indirectly by releasing inflammatory cytokines. Here, we review recent progress regarding the immune cells in the liver and their functions during liver regeneration.
Biochemical and biophysical research communications, Jan 21, 2016
Porcine pancreatic stem cells (PSCs) are one kind of the potential cells for treatment of human d... more Porcine pancreatic stem cells (PSCs) are one kind of the potential cells for treatment of human diabetes. Autophagy is a highly conserved cellular degradation process in which it helps to maintain the balance between the synthesis, degradation and subsequent recycling of cellular components. However, how autophagy contributes to PSCs has not yet been investigated. Here, we established GFP-LC3 transfected porcine PSC lines in which the accumulation of autophagosomes can be efficiently visualized to evaluate the autophagic activity. Moreover, we observed that starved PSCs which showed increased autophagic activity exhibited an increased tendency to proliferate through the results of BrdU, flow cytometry and western blotting. Furthermore, increased expression of active β-catenin after inducing autophagy indicated that it might be the canonical Wnt signaling that autophagy activated to exert the function on the stimulation of PSCs proliferation. Collectively, these results demonstrated ...
The CRISPR/Cas9 system provides a flexible approach for genome engineering of genetic loci. Here,... more The CRISPR/Cas9 system provides a flexible approach for genome engineering of genetic loci. Here, we successfully achieved precise gene targeting in sheep by co-injecting one-cell-stage embryos with Cas9 mRNA and RNA guides targeting three genes (MSTN, ASIP, and BCO2). We carefully examined the sgRNAs:Cas9-mediated targeting effects in injected embryos, somatic tissues, as well as gonads via cloning and sequencing. The targeting efficiencies in these three genes were within the range of 27-33% in generated lambs, and that of simultaneously targeting the three genes was 5.6%, which demonstrated that micro-injection of zygotes is an efficient approach for generating gene-modified sheep. Interestingly, we observed that disruption of the MSTN gene resulted in the desired muscle hypertrophy that is characterized by enlarged myofibers, thereby providing the first detailed evidence supporting that gene modifications had occurred at both the genetic and morphological levels. In addition, pr...
2010 3rd International Conference on Biomedical Engineering and Informatics, 2010
In vitro derivation of oocytes from mouse embryonic stem cells (ESCs) would benefit the study of ... more In vitro derivation of oocytes from mouse embryonic stem cells (ESCs) would benefit the study of oogenesis. Here, we induced mouse ESCs to differentiate into oocyte-like cells by culturing adherent embryoid bodies (EBs) in different medium, including retinoic acid (RA) and follicular fluid. The extent of differentiation was evaluated through morphology and the expression of certain specific germ cell markers
Porcine pancreatic stem cells (PSCs) are highly valuable in transplantation applications for type... more Porcine pancreatic stem cells (PSCs) are highly valuable in transplantation applications for type II diabetes. However, there are still many problems to be solved before they can be used in the clinic, such as insufficient cell number availability and low secretion level of insulin. It has been reported that Wnt3a plays pivotal roles during cell proliferation and differentiation. Here, we have aimed to establish an ideal research platform using the T-REx(™) system, to study mechanisms of Wnt3a during PSC proliferation and differentiation. Construction of the recombinant plasmid and cell transfection were used for establishment of a porcine PSC line. Related gene expressions were examined using quantitative real-time PCR (QRT-PCR), western blotting, immunostaining and flow cytometry. BrdU incorporation assay and cell cycle analysis were used to investigate Wnt3a roles in PSCs. Wnt3a-expressing clones regulated by T-REx(™) were successfully obtained. Wnt3a and GFP expression were stri...
Sheng wu gong cheng xue bao = Chinese journal of biotechnology, 2007
Retinoic acid plays an important role in maintaining the structure and function in male testis. R... more Retinoic acid plays an important role in maintaining the structure and function in male testis. Recent studies showed that there is a group of genes that can be specially activated by retinoic acid during the development of male reproductive gland. The gene Stra 8 (Stimulated by Retinoic Acid) was one of the gene in this group. In mouse, Stra 8 is restrictively expressed in male germ line cells, and its function is related to the development of sperm. In order to investigate the feature of Stra 8 gene expression,the 1.4 kb (-1407 - +7) promoter region of Stra 8 gene was amplified from mouse genomic DNA. The DNA fragment was then cloned into a promoter less vector to form the construct that contained the 1.4 kb promoter region, and the reporter gene of EGFP that was regulated by 1.4kb Stra 8 promoter. To investigate the specificity of Stra 8 promoter,the vector pStro-EGFP was transfected into undifferentiated mouse stem cells such as ES-129, bone marrow mesenchymal stem cell (mMSC) a...
2010 3rd International Conference on Biomedical Engineering and Informatics, 2010
... [1] AM Shapiro, JR Lakey, EA Ryan, GS Korbutt, E Toth, GL Warnock, NM Kneteman and RV Rajotte... more ... [1] AM Shapiro, JR Lakey, EA Ryan, GS Korbutt, E Toth, GL Warnock, NM Kneteman and RV Rajotte. ... Reprod Fertil Dev, 1994, 6： 569-575. [13] S Mueller, K Prelle, N Rieger, H Petznek , C Lassnig, U Luksch, B Aigner, M Baetscher, E Wolf , M Mueller and B Gottfried. ...
Zhonghua nan ke xue = National journal of andrology, 2010
To investigate the effects of carnitine on human sperm motility and its potential role in the tre... more To investigate the effects of carnitine on human sperm motility and its potential role in the treatment of male infertility diseases. We obtained sperm by testis puncture from obstructive azoospermia patients and cultured them in vitro with normal culture solution (the control group) and the solution with L-carnitine at the concentration of 100 and 250 mmol/L, respectively. We observed the changes in sperm motility and morphology before and after the treatment, detected the expressions of the germ-specific genes, Vasa, Dazl, Acr, Prm1 and ATPase 6.0 by RT-PCR, and investigated the relationship between L-carnitine and the genes associated with sperm development and maturation. After 24 -72 hours of treatment, the percentage of motile sperm was significantly higher in the 100 mmol/L L-carnitine group than in the control and 250 mmol/L L-carnitine groups (P < 0.01); the number of forward moving sperm was obviously increased and sperm morphology remained normal in the 100 mmol/L L-ca...
Integrative biology : quantitative biosciences from nano to macro, 2014
Recent studies have found that uncontrolled diabetes and consequential hyperglycemic conditions c... more Recent studies have found that uncontrolled diabetes and consequential hyperglycemic conditions can lead to an increased incidence of osteoporosis. Osteoblasts, adipocytes, and mesenchymal stem cells (MSCs) are all components of the bone marrow microenvironment and thus may have an effect on diabetes-related osteoporosis. However, few studies have investigated the influence of these three cell types on each other, especially in the context of hyperglycemia. Thus, we developed a hydrogel-based 3D culture platform engineered to allow live-cell retrieval in order to investigate the interactions between MSCs, osteoblasts, and adipocytes in mono-, co-, and tri-culture configurations under hyperglycemic conditions for 7 days of culture. Gene expression, histochemical analysis of differentiation markers, and cell viability were measured for all cell types, and MSC-laden hydrogels were degraded to retrieve cells to assess their colony-forming capacity. Multivariate models of gene expression...
Sheng wu gong cheng xue bao = Chinese journal of biotechnology, 2010
The integrity and transparency of cornea plays a key role in vision. Limbal Stem Cells (LSCs) are... more The integrity and transparency of cornea plays a key role in vision. Limbal Stem Cells (LSCs) are precursors of cornea, which are responsible for self-renewal and replenishing corneal epithelium. Though it is successful to cell replacement therapy for impairing ocular surface by Limbal Stem Cell Transplantation (LSCT), the mechanism of renew is unclear after LSCT. To real time follow-up the migration and differentiation of corneal transplanted epithelial cells after transplanting, we transfected venus (a fluorescent protein gene) into goat LSCs, selected with G418 and established a stable transfected cell line, named GLSC-V. These cells showed green fluorescence, and which could maintain for at least 3 months. GLSC-V also were positive for anti-P63 and anti-Integrinbeta1 antibody by immunofluorescent staining. We founded neither GLSC-V nor GLSCs expressed keratin3 (k3) and keratinl2 (k12). However, GLSC-V had higher levels in expression of p63, pcna and venus compared with GLSCs. Fu...
Male germline stem cells (mGSCs) are stem cells present in male testis responsible for spermatoge... more Male germline stem cells (mGSCs) are stem cells present in male testis responsible for spermatogenesis during their whole life. Studies have shown that mGSCs can be derived in vitro and resemble embryonic stem cells (ESCs) properties both in the mouse and humans. However, little is know about these cells in domestic animals. Here we report the first successful establishment of goat GSCs derived from 2-5-month fetal testis, and developmental potential assay of these cells both in vitro and in vivo. These cells express pluripotent markers such as Oct4, Sox2, C-myc, and Tert when cultured as human ESCs conditions. Embryoid bodies (EBs) formed by goat mGSCs were induced with 2 × 10(-6) M retinoic acid (RA). Immunofluorescence analysis showed that some cells inside of the EBs were positive for meiosis marker-SCP3, STRA8, and germ cell marker-VASA, and haploid marker-FE-J1, PRM1, indicating their germ cell lineage differentiation. Some cells become elongated sperm-like cells after inducti...
The existing treatment modalities for skin injuries mainly include dressings, negative-pressure w... more The existing treatment modalities for skin injuries mainly include dressings, negative-pressure wound treatment, autologous skin grafting, and high-pressure wound treatment. All of these therapies have limitations such as high time cost, the inability to remove inactivated tissue in a timely manner, surgical debridement, and oxygen toxicity. Mesenchymal stem cells have a unique self-renewal ability and wide differentiation potential, and they are one of the most promising stem cell types in cell therapy and have great application prospects in the field of regenerative medicine. Collagen exerts structural roles by promoting the molecular structure, shape, and mechanical properties of cells, and adding it to cell cultures can also promote cell proliferation and shorten the cell doubling time. The effects of collagen on MSCs were examined using Giemsa staining, EdU staining, and growth curves. Mice were subjected to allogeneic experiments and autologous experiments to reduce individual...
Pregnancy loss (PL) is one of the common complications that women can experience during pregnancy... more Pregnancy loss (PL) is one of the common complications that women can experience during pregnancy, with an occurrence rate of 1 to 5%. The potential causes of pregnancy loss are unclear, with no effective treatment modalities being available. It has been previously reported that the level of miR-125b was significantly increased in placentas of PL patients. However, the role of miR-125b in the development of PL still remains unknown. In the current study, an miR-125b placenta-specific over-expression model was constructed by lentiviral transfecting zona-free mouse embryos followed by embryo transfer. On gestation day 15, it was observed that the placenta was significantly smaller in the miR-125b placenta-specific overexpression group than the control group. Additionally, the abortion rate of the miR-125b placenta-specific overexpression group was markedly higher than in the control group. The blood vessel diameter was larger in the miR-125b-overexpressing specific placenta. In additi...
Porcine-induced pluripotent stem cells (piPSCs) could serve as a great model system for human ste... more Porcine-induced pluripotent stem cells (piPSCs) could serve as a great model system for human stem cell preclinical research. However, the pluripotency gene network of piPSCs, especially the function for the core transcription factor estrogen-related receptor beta (ESRRB), was poorly understood. Here, we constructed ESRRB-overexpressing piPSCs (ESRRB-piPSCs). Compared with the control piPSCs (CON-piPSCs), the ESRRB-piPSCs showed flat, monolayered colony morphology. Moreover, the ESRRB-piPSCs showed greater chimeric capacity into trophectoderm than CON-piPSCs. We found that ESRRB could directly regulate the expressions of trophoblast stem cell (TSC)-specific markers, including KRT8, KRT18 and CDX2, through binding to their promoter regions. Mutational analysis proved that the N-terminus zinc finger domain is indispensable for ESRRB to regulate the TSC markers. Furthermore, this regulation needs the participation of OCT4. Accordingly, the cooperation between ESRRB and OCT4 facilitates...
Previous studies have demonstrated that transcription factor Etv5 plays an important role in the ... more Previous studies have demonstrated that transcription factor Etv5 plays an important role in the segregation between epiblast and primitive endoderm at the second fate decision of early embryo. However, it remains elusive whether Etv5 functions in the segregation between inner cell mass and trophectoderm at the first cell fate decision. In this study, we firstly generated Etv5 knockout mouse embryonic stem cells (mESCs) by CRISPR/Cas9, then converted them into extended potential stem cells (EPSCs) by culturing the cells in small molecule cocktail medium LCDM (LIF, CHIR99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride), and finally investigated their differentiation efficiency of trophoblast stem cells (TSCs). The results showed that Etv5 knockout significantly decreased the efficiency of TSCs (CDX2 +) differentiated from EPSCs. In addition, Etv5 knockout resulted in higher incidence of the differentiated cells with tetraploid and octoploid than that from wild type. Mechanistically, Etv5 was activated by extracellular-signal-regulated kinase (ERK) signaling pathway; in turn, Etv5 had a positive feedback on the expression of fibroblast growth factor receptor 2 (FGFR2) which lies upstream of ERK. Etv5 knockout decreased the expression of FGFR2, whose binding with fibroblast growth factor 4 was essentially needed for TSCs differentiation. Collectively, the findings in this study suggest that Etv5 is required to safeguard the TSCs differentiation by regulating FGFR2 and provide new clues to understand the specification of trophectoderm in vivo. Keywords Etv5 • Mouse embryonic stem cells • Extended potential stem cells • Trophoblast stem cells • Fibroblast growth factor receptor 2 Kui Zhu and Yuan Liu contributed equally to this work.
Global human health has been compromised by high-fat diets. This study aimed to investigate the r... more Global human health has been compromised by high-fat diets. This study aimed to investigate the relationship between a high-fat diet and parthenogenetic embryo quality. Mice fed a high-fat or a normal diet was used as treated or control groups, respectively. Estradiol (E), total cholesterol (TC) and total triglyceride (TG) were detected by Enzyme-Linked ImmunoSorbent Assay (ELISA). Cumulus-oocyte complexes (COCs) were collected from the mice in the treated and control groups. The ultrastructure of COCs, the expression level of genes involved in mitochondrial and nuclear functions in cumulus cells and oocytes quality were evaluated with transmission electron microscopy, real-time quantitative polymerase chain reaction (RT-PCR) and artificial parthenogenesis, respectively. The results showed that the efficiency of parthenogenetic embryonic development in vitro was significantly higher in the treated group than in the control group ( < .05). The expression level of genes involved in...
Self-renewal and differentiation of male germline stem cells (mGSCs) provide the basic function f... more Self-renewal and differentiation of male germline stem cells (mGSCs) provide the basic function for continual spermatogenesis. Studies of in vitro culture of germline stem cells are important and meaningful for basic biological research and practical application. Growth factors, such as GDNF, bFGF, CSF1, and EGF, could maintain the self-renewal of mGSCs. Insulin-like growth factor 1 (IGF-1), an important growth factor, and its pathway have been reported to maintain the survival of several types of stem cells and play important roles in male reproduction. However, the mechanism through which the IGF-1 pathway acts to regulate the self-renewal of mGSCs remains unclear. We analyzed the effect of IGF-1 on the proliferation and apoptosis of bovine mGSCs. We evaluated the expression profile of long noncoding RNA (LncRNA) H19 in bovine and mouse tissues. Moreover, we investigated whether LncRNA H19 could regulate the IGF-1 pathway. Results showed that IGF-1 could activate the phosphorylati...
Methylation is an important issue in gene expression regulation and also in the fields of genetic... more Methylation is an important issue in gene expression regulation and also in the fields of genetics and reproduction. In this study, we created fat-1 transgenic sheep, investigated the fine-mapping and the modulatory mechanisms of promoter methylation. Sheep fetal fibroblasts were transfected by pCAG-fat1-IRES-EGFP. Monoclonal cell line was screened as nuclear donor and carried out nuclear transfer (441 transgenic cloned embryos, 52 synchronism recipient sheep). Six offsprings were obtained. Expressions of exogenous genes fat-1 and EGFP were detectable in 10 examined tissues and upregulated omega-3 fatty acid content. Interestingly, more or less EGFP negative cells were detectable in the positive transgenic fetal skin cells. EGFP negative and positive cells were sorted by flow cytometry, and their methylation status in the whole promoter region (1701 nt) were investigated by bisulphate sequencing. The fine-mapping of methylation in CAG promoter were proposed. The results suggested th...
Many men endure immunosuppressive or anticancer treatments that contain alkylating agents before ... more Many men endure immunosuppressive or anticancer treatments that contain alkylating agents before the age of sexual maturity, especially the increasing number of preadolescent males who undergo busulfan treatment for myeloablative conditioning before hematopoietic stem cell transplantation. Before sperm production, there are no sperm available for cryopreservation. Thus, it is necessary to identify a solution to ameliorate the busulfan-induced damage of spermatogonial stem cells (SSCs). In this study, we demonstrated that melatonin relieved the previously described SSC loss and apoptosis in mouse testes. Melatonin increased the expression of manganese superoxide dismutase (MnSOD), which regulated the production of busulfan-induced reactive oxygen species (ROS). Moreover, melatonin promoted sirtuin type 1 (SIRT1) expression. SIRT1 participated in the deacetylation of p53, which promotes p53 ubiquitin degradation. Decreased concentrations of deacetylated p53 resulted in spermatogonial ...
Precision genetic engineering accelerates the genetic improvement of livestock for agriculture an... more Precision genetic engineering accelerates the genetic improvement of livestock for agriculture and biomedicine. We have recently reported our success in producing gene-modified goats using the CRISPR/Cas9 system through microinjection of Cas9 mRNA and sgRNAs targeting the MSTN and FGF5 genes in goat embryos. By investigating the influence of gene modification on the phenotypes of Cas9-mediated goats, we herein demonstrate that the utility of this approach involving the disruption of FGF5 results in increased number of second hair follicles and enhanced fiber length in Cas9-mediated goats, suggesting more cashmere will be produced. The effects of genome modifications were characterized using H&E and immunohistochemistry staining, quantitative PCR, and western blotting techniques. These results indicated that the gene modifications induced by the disruption of FGF5 had occurred at the morphological and genetic levels. We further show that the knockout alleles were likely capable of ge...
After partial hepatectomy, hepatocytes proliferate to restore mass and function of the liver. Mac... more After partial hepatectomy, hepatocytes proliferate to restore mass and function of the liver. Macrophages, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells (DC), eosinophils, gamma delta T (γδT) cells, and conventional T cells, as well as other subsets of the immune cells residing in the liver control liver regeneration, either through direct interactions with hepatocytes or indirectly by releasing inflammatory cytokines. Here, we review recent progress regarding the immune cells in the liver and their functions during liver regeneration.
Biochemical and biophysical research communications, Jan 21, 2016
Porcine pancreatic stem cells (PSCs) are one kind of the potential cells for treatment of human d... more Porcine pancreatic stem cells (PSCs) are one kind of the potential cells for treatment of human diabetes. Autophagy is a highly conserved cellular degradation process in which it helps to maintain the balance between the synthesis, degradation and subsequent recycling of cellular components. However, how autophagy contributes to PSCs has not yet been investigated. Here, we established GFP-LC3 transfected porcine PSC lines in which the accumulation of autophagosomes can be efficiently visualized to evaluate the autophagic activity. Moreover, we observed that starved PSCs which showed increased autophagic activity exhibited an increased tendency to proliferate through the results of BrdU, flow cytometry and western blotting. Furthermore, increased expression of active β-catenin after inducing autophagy indicated that it might be the canonical Wnt signaling that autophagy activated to exert the function on the stimulation of PSCs proliferation. Collectively, these results demonstrated ...
The CRISPR/Cas9 system provides a flexible approach for genome engineering of genetic loci. Here,... more The CRISPR/Cas9 system provides a flexible approach for genome engineering of genetic loci. Here, we successfully achieved precise gene targeting in sheep by co-injecting one-cell-stage embryos with Cas9 mRNA and RNA guides targeting three genes (MSTN, ASIP, and BCO2). We carefully examined the sgRNAs:Cas9-mediated targeting effects in injected embryos, somatic tissues, as well as gonads via cloning and sequencing. The targeting efficiencies in these three genes were within the range of 27-33% in generated lambs, and that of simultaneously targeting the three genes was 5.6%, which demonstrated that micro-injection of zygotes is an efficient approach for generating gene-modified sheep. Interestingly, we observed that disruption of the MSTN gene resulted in the desired muscle hypertrophy that is characterized by enlarged myofibers, thereby providing the first detailed evidence supporting that gene modifications had occurred at both the genetic and morphological levels. In addition, pr...
2010 3rd International Conference on Biomedical Engineering and Informatics, 2010
In vitro derivation of oocytes from mouse embryonic stem cells (ESCs) would benefit the study of ... more In vitro derivation of oocytes from mouse embryonic stem cells (ESCs) would benefit the study of oogenesis. Here, we induced mouse ESCs to differentiate into oocyte-like cells by culturing adherent embryoid bodies (EBs) in different medium, including retinoic acid (RA) and follicular fluid. The extent of differentiation was evaluated through morphology and the expression of certain specific germ cell markers
Porcine pancreatic stem cells (PSCs) are highly valuable in transplantation applications for type... more Porcine pancreatic stem cells (PSCs) are highly valuable in transplantation applications for type II diabetes. However, there are still many problems to be solved before they can be used in the clinic, such as insufficient cell number availability and low secretion level of insulin. It has been reported that Wnt3a plays pivotal roles during cell proliferation and differentiation. Here, we have aimed to establish an ideal research platform using the T-REx(™) system, to study mechanisms of Wnt3a during PSC proliferation and differentiation. Construction of the recombinant plasmid and cell transfection were used for establishment of a porcine PSC line. Related gene expressions were examined using quantitative real-time PCR (QRT-PCR), western blotting, immunostaining and flow cytometry. BrdU incorporation assay and cell cycle analysis were used to investigate Wnt3a roles in PSCs. Wnt3a-expressing clones regulated by T-REx(™) were successfully obtained. Wnt3a and GFP expression were stri...
Sheng wu gong cheng xue bao = Chinese journal of biotechnology, 2007
Retinoic acid plays an important role in maintaining the structure and function in male testis. R... more Retinoic acid plays an important role in maintaining the structure and function in male testis. Recent studies showed that there is a group of genes that can be specially activated by retinoic acid during the development of male reproductive gland. The gene Stra 8 (Stimulated by Retinoic Acid) was one of the gene in this group. In mouse, Stra 8 is restrictively expressed in male germ line cells, and its function is related to the development of sperm. In order to investigate the feature of Stra 8 gene expression,the 1.4 kb (-1407 - +7) promoter region of Stra 8 gene was amplified from mouse genomic DNA. The DNA fragment was then cloned into a promoter less vector to form the construct that contained the 1.4 kb promoter region, and the reporter gene of EGFP that was regulated by 1.4kb Stra 8 promoter. To investigate the specificity of Stra 8 promoter,the vector pStro-EGFP was transfected into undifferentiated mouse stem cells such as ES-129, bone marrow mesenchymal stem cell (mMSC) a...
2010 3rd International Conference on Biomedical Engineering and Informatics, 2010
... [1] AM Shapiro, JR Lakey, EA Ryan, GS Korbutt, E Toth, GL Warnock, NM Kneteman and RV Rajotte... more ... [1] AM Shapiro, JR Lakey, EA Ryan, GS Korbutt, E Toth, GL Warnock, NM Kneteman and RV Rajotte. ... Reprod Fertil Dev, 1994, 6： 569-575. [13] S Mueller, K Prelle, N Rieger, H Petznek , C Lassnig, U Luksch, B Aigner, M Baetscher, E Wolf , M Mueller and B Gottfried. ...
Zhonghua nan ke xue = National journal of andrology, 2010
To investigate the effects of carnitine on human sperm motility and its potential role in the tre... more To investigate the effects of carnitine on human sperm motility and its potential role in the treatment of male infertility diseases. We obtained sperm by testis puncture from obstructive azoospermia patients and cultured them in vitro with normal culture solution (the control group) and the solution with L-carnitine at the concentration of 100 and 250 mmol/L, respectively. We observed the changes in sperm motility and morphology before and after the treatment, detected the expressions of the germ-specific genes, Vasa, Dazl, Acr, Prm1 and ATPase 6.0 by RT-PCR, and investigated the relationship between L-carnitine and the genes associated with sperm development and maturation. After 24 -72 hours of treatment, the percentage of motile sperm was significantly higher in the 100 mmol/L L-carnitine group than in the control and 250 mmol/L L-carnitine groups (P < 0.01); the number of forward moving sperm was obviously increased and sperm morphology remained normal in the 100 mmol/L L-ca...
Integrative biology : quantitative biosciences from nano to macro, 2014
Recent studies have found that uncontrolled diabetes and consequential hyperglycemic conditions c... more Recent studies have found that uncontrolled diabetes and consequential hyperglycemic conditions can lead to an increased incidence of osteoporosis. Osteoblasts, adipocytes, and mesenchymal stem cells (MSCs) are all components of the bone marrow microenvironment and thus may have an effect on diabetes-related osteoporosis. However, few studies have investigated the influence of these three cell types on each other, especially in the context of hyperglycemia. Thus, we developed a hydrogel-based 3D culture platform engineered to allow live-cell retrieval in order to investigate the interactions between MSCs, osteoblasts, and adipocytes in mono-, co-, and tri-culture configurations under hyperglycemic conditions for 7 days of culture. Gene expression, histochemical analysis of differentiation markers, and cell viability were measured for all cell types, and MSC-laden hydrogels were degraded to retrieve cells to assess their colony-forming capacity. Multivariate models of gene expression...
Sheng wu gong cheng xue bao = Chinese journal of biotechnology, 2010
The integrity and transparency of cornea plays a key role in vision. Limbal Stem Cells (LSCs) are... more The integrity and transparency of cornea plays a key role in vision. Limbal Stem Cells (LSCs) are precursors of cornea, which are responsible for self-renewal and replenishing corneal epithelium. Though it is successful to cell replacement therapy for impairing ocular surface by Limbal Stem Cell Transplantation (LSCT), the mechanism of renew is unclear after LSCT. To real time follow-up the migration and differentiation of corneal transplanted epithelial cells after transplanting, we transfected venus (a fluorescent protein gene) into goat LSCs, selected with G418 and established a stable transfected cell line, named GLSC-V. These cells showed green fluorescence, and which could maintain for at least 3 months. GLSC-V also were positive for anti-P63 and anti-Integrinbeta1 antibody by immunofluorescent staining. We founded neither GLSC-V nor GLSCs expressed keratin3 (k3) and keratinl2 (k12). However, GLSC-V had higher levels in expression of p63, pcna and venus compared with GLSCs. Fu...
Male germline stem cells (mGSCs) are stem cells present in male testis responsible for spermatoge... more Male germline stem cells (mGSCs) are stem cells present in male testis responsible for spermatogenesis during their whole life. Studies have shown that mGSCs can be derived in vitro and resemble embryonic stem cells (ESCs) properties both in the mouse and humans. However, little is know about these cells in domestic animals. Here we report the first successful establishment of goat GSCs derived from 2-5-month fetal testis, and developmental potential assay of these cells both in vitro and in vivo. These cells express pluripotent markers such as Oct4, Sox2, C-myc, and Tert when cultured as human ESCs conditions. Embryoid bodies (EBs) formed by goat mGSCs were induced with 2 × 10(-6) M retinoic acid (RA). Immunofluorescence analysis showed that some cells inside of the EBs were positive for meiosis marker-SCP3, STRA8, and germ cell marker-VASA, and haploid marker-FE-J1, PRM1, indicating their germ cell lineage differentiation. Some cells become elongated sperm-like cells after inducti...
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