目的:探讨苯妥英钠(PHT)对血管内皮细胞转分化作用的影响及其作用机制,为提高牙周组织再生能力的治疗提供思路。方法:通过transwell建立RAOEC与大鼠巨噬细胞共培养体系。研究设为空白组:基础培养基;P0组:基础培养基+100 ng/m L LPS;P25...目的:探讨苯妥英钠(PHT)对血管内皮细胞转分化作用的影响及其作用机制,为提高牙周组织再生能力的治疗提供思路。方法:通过transwell建立RAOEC与大鼠巨噬细胞共培养体系。研究设为空白组:基础培养基;P0组:基础培养基+100 ng/m L LPS;P25组:基础培养基+100 ng/m L LPS+25μg/m L PHT;P50组:基础培养基+100 ng/m L LPS+50μg/m L PHT,每组设3个复孔。T组:利用8 cm2培养皿单独接种RAOEC细胞,添加含有10μg/m L TGF-β1的培养基培养。Elisa检测不同处理组中TGF-β1的分泌量。RT-PCR及Western-blot,鉴定RAOEC转分化的标志CD31、α-SMA、SCF的mRNA及蛋白表达量。结果:一定浓度范围内,随着PHT浓度的升高,大鼠血管内皮细胞表型发生明显转变;ELISA检测结果显示,P0组、P25组、P50组细胞上清液TGF-β1含量分别为1.87±1.01、5.33±0.94、8.96±1.25,差异具有统计学意义(P<0.05);RT-PCR检测结果显示,P0、P25、P50组间比较,CD31 mRNA表达量逐步降低,α-SMA与SCF mRNA表达量逐步升高,差异具有统计学意义(P<0.05);Western-blot灰度分析显示,P0、P25、P50组间比较,CD31灰度值逐步降低,α-SMA与SCF灰度值逐步增高,差异具有统计学意义(P<0.05)。结论:PHT具有促进大鼠血管内皮细胞转分化的作用,为PHT应用于牙周组织再生提供了新思路。展开更多
Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by ...Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX （F IX） gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay.Results The cell line bore a missense mutation in the 6th coding exon （c.676 C〉T） of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% （10/45） and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.展开更多
Strategies to fill the huge gap in supply versus demand of human organs include bioartificial organs, growing humanized organs in animals, cell therapy, and implantable bioengineered constructs. Reproducing the comple...Strategies to fill the huge gap in supply versus demand of human organs include bioartificial organs, growing humanized organs in animals, cell therapy, and implantable bioengineered constructs. Reproducing the complex relations between different cell types, generation of adequate vasculature, and immunological complications are road blocks in generation of bioengineered organs, while immunological complications limit the use of humanized organs produced in animals. Recent developments in induced pluripotent stem cell (iPSC) biology offer a possibility of generating human, patient-specific organs in non-human primates (NHP) using patient-derived iPSC and NHP-derived iPSC lacking the critical developmental genes for the organ of interest complementing a NHP tetraploid embryo. The organ derived in this way will have the same human leukocyte antigen (HLA) profile as the patient. This approach can be curative in genetic disorders as this offers the possibility of gene manipulation and correction of the patient's genome at the iPSC stage before tetraploid complementation. The process of generation of patient-specific organs such as the liver in this way has the great advantage of making use of the natural signaling cascades in the natural milieu probably resulting in organs of great quality for transplantation. However, the inexorable scientific developments in this direction involve several social issues and hence we need to educate and prepare society in advance to accept the revolutionary consequences, good, bad and ugly.展开更多
Induced pluripotent stem (iPS) cell technology demonstrates that somatic cells can be reprogrammed to a pluripotent state by over-expressing four reprogramming factors.This technology has created an interest in derivi...Induced pluripotent stem (iPS) cell technology demonstrates that somatic cells can be reprogrammed to a pluripotent state by over-expressing four reprogramming factors.This technology has created an interest in deriving iPS cells from domesticated animals such as pigs,sheep and cattle.Moloney murine leukemia retrovirus vectors have been widely used to generate and study mouse iPS cells.However,this retrovirus system infects only mouse and rat cells,which limits its use in establishing iPS cells from other mammals.In our study,we demonstrate a novel retrovirus strategy to efficiently generate porcine iPS cells from embryonic fibroblasts.We transfected four human reprogramming factors (Oct4,Sox2,Klf4 and Myc) into fibroblasts in one step by using a VSV-G envelope-coated pantropic retrovirus that was easily packaged by GP2-293 cells.We established six embryonic stem (ES)-like cell lines in human ES cell medium supplemented with bFGF.Colonies showed a similar morphology to human ES cells with a high nuclei-cytoplasm ratio and phase-bright flat colonies.Porcine iPS cells could form embryoid bodies in vitro and differentiate into the three germ layers in vivo by forming teratomas in immunodeficient mice.展开更多
文摘目的:探讨苯妥英钠(PHT)对血管内皮细胞转分化作用的影响及其作用机制,为提高牙周组织再生能力的治疗提供思路。方法:通过transwell建立RAOEC与大鼠巨噬细胞共培养体系。研究设为空白组:基础培养基;P0组:基础培养基+100 ng/m L LPS;P25组:基础培养基+100 ng/m L LPS+25μg/m L PHT;P50组:基础培养基+100 ng/m L LPS+50μg/m L PHT,每组设3个复孔。T组:利用8 cm2培养皿单独接种RAOEC细胞,添加含有10μg/m L TGF-β1的培养基培养。Elisa检测不同处理组中TGF-β1的分泌量。RT-PCR及Western-blot,鉴定RAOEC转分化的标志CD31、α-SMA、SCF的mRNA及蛋白表达量。结果:一定浓度范围内,随着PHT浓度的升高,大鼠血管内皮细胞表型发生明显转变;ELISA检测结果显示,P0组、P25组、P50组细胞上清液TGF-β1含量分别为1.87±1.01、5.33±0.94、8.96±1.25,差异具有统计学意义(P<0.05);RT-PCR检测结果显示,P0、P25、P50组间比较,CD31 mRNA表达量逐步降低,α-SMA与SCF mRNA表达量逐步升高,差异具有统计学意义(P<0.05);Western-blot灰度分析显示,P0、P25、P50组间比较,CD31灰度值逐步降低,α-SMA与SCF灰度值逐步增高,差异具有统计学意义(P<0.05)。结论:PHT具有促进大鼠血管内皮细胞转分化的作用,为PHT应用于牙周组织再生提供了新思路。
基金Supported by the National Science and Technology Major Project(2011ZX09102-010-04)
文摘Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX （F IX） gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay.Results The cell line bore a missense mutation in the 6th coding exon （c.676 C〉T） of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% （10/45） and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.
文摘Strategies to fill the huge gap in supply versus demand of human organs include bioartificial organs, growing humanized organs in animals, cell therapy, and implantable bioengineered constructs. Reproducing the complex relations between different cell types, generation of adequate vasculature, and immunological complications are road blocks in generation of bioengineered organs, while immunological complications limit the use of humanized organs produced in animals. Recent developments in induced pluripotent stem cell (iPSC) biology offer a possibility of generating human, patient-specific organs in non-human primates (NHP) using patient-derived iPSC and NHP-derived iPSC lacking the critical developmental genes for the organ of interest complementing a NHP tetraploid embryo. The organ derived in this way will have the same human leukocyte antigen (HLA) profile as the patient. This approach can be curative in genetic disorders as this offers the possibility of gene manipulation and correction of the patient's genome at the iPSC stage before tetraploid complementation. The process of generation of patient-specific organs such as the liver in this way has the great advantage of making use of the natural signaling cascades in the natural milieu probably resulting in organs of great quality for transplantation. However, the inexorable scientific developments in this direction involve several social issues and hence we need to educate and prepare society in advance to accept the revolutionary consequences, good, bad and ugly.
基金supported by the National Basic Research Program of China (Grant Nos. 2009CB941003, 2011CBA0110 and 2011CBA01000)
文摘Induced pluripotent stem (iPS) cell technology demonstrates that somatic cells can be reprogrammed to a pluripotent state by over-expressing four reprogramming factors.This technology has created an interest in deriving iPS cells from domesticated animals such as pigs,sheep and cattle.Moloney murine leukemia retrovirus vectors have been widely used to generate and study mouse iPS cells.However,this retrovirus system infects only mouse and rat cells,which limits its use in establishing iPS cells from other mammals.In our study,we demonstrate a novel retrovirus strategy to efficiently generate porcine iPS cells from embryonic fibroblasts.We transfected four human reprogramming factors (Oct4,Sox2,Klf4 and Myc) into fibroblasts in one step by using a VSV-G envelope-coated pantropic retrovirus that was easily packaged by GP2-293 cells.We established six embryonic stem (ES)-like cell lines in human ES cell medium supplemented with bFGF.Colonies showed a similar morphology to human ES cells with a high nuclei-cytoplasm ratio and phase-bright flat colonies.Porcine iPS cells could form embryoid bodies in vitro and differentiate into the three germ layers in vivo by forming teratomas in immunodeficient mice.
