AIM： To investigate whether the function of hepatocytes co-cultured with bone marrow mesenchymal stem cells （MSCs） could be maintained in serum from acute-on- chronic liver failure （ACLF） patients.METHODS： Hepat...AIM： To investigate whether the function of hepatocytes co-cultured with bone marrow mesenchymal stem cells （MSCs） could be maintained in serum from acute-on- chronic liver failure （ACLF） patients.METHODS： Hepatocyte supportive functions and cy- totoxicity of sera from 18 patients with viral hepatitis B-induced ACLF and 18 healthy volunteers were evalu- ated for porcine hepatocytes co-cultured with MSCs and hepatocyte mono-layered culture, respectively. Chemo- kine profile was also examined for the normal serum and liver failure serum.RESULTS： Hepatocyte growth factor （HGF） and Tumor necrosis factor; tumor necrosis factor （TNF）-a were re- markably elevated in response to ACLF while epidermal growth factor （EGF） and VEGF levels were significantly decreased. Liver failure serum samples induced a higher detachment rate, lower viability and decreased liver sup- port functions in the homo-hepatocyte culture. Hepato-cytes co-cultured with MSCs could tolerate the cytotoxic- ity of the serum from ACLF patients and had similar liver support functions compared with the hepatocytes cul- tured with healthy human serum in vitro. In addition, co- cultured hepatocytes maintained a proliferative capability despite of the insult from liver failure serum.CONCLUSION： ACLF serum does not impair the cell morphology, viability, proliferation and overall metabolic capacities of hepatocyte co-cultured with MSCs in vitro.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
Recent studies have revealed that the γ-chain of theIL-2 receptor is shared by the receptors for IL-4, IL7, IL-9, IL-13, and IL-15, and it is therefore also referred toas the common γ-chain (γc). Mutations of γc r...Recent studies have revealed that the γ-chain of theIL-2 receptor is shared by the receptors for IL-4, IL7, IL-9, IL-13, and IL-15, and it is therefore also referred toas the common γ-chain (γc). Mutations of γc result inX-linked severe combined immunodeficiency syndrome inhumans, indicating that rye is essential for normal development and function of the immune system. We demonstratethat human hematopoietic cells express two γc transcriptsdiffering in their carboxyl terminal coding region. Onetranscript is the previously reported sequence (γc-long),whereas the newly identified sequence exhibits a deletion of72 nucleotides close to the 3’-end of the open reading frame(γc-short). This alteration predicts a loss of 24 amino acidsincluding a conserved tyrosine residue which is shared byseveral members of the cytokine receptor family. Thepresence of these two distinct forms of rye transcripts wasdemonstrated by sequencing of reversely transcribed andpolymerase chain reaction (RT-PCR) amplified mRNA, restriction digestion of the RT-PCR products, RNAse protection, and Northern blotting from human cell lines andhuman peripheral blood lymphocytes. Furthermore, thetwo variants were present in peripheral blood lymphocytesfrom both female and male donors, which rules out allelicvariants since rye is a single copy gene located on the Xchromosome. A truncation mutant at a site near the observed changes in γc-short has been reported by othersto alter biochemical events activated by cytokines. Thiscombined with the loss of a potential SH2 "docking" sitein γc-short suggests that γc-long and γc-short may link todifferent signaling pathways and may play an importantrole in determining the cellular response to IL-2, IL-4, IL-7, IL-9, IL-13, IL-15.展开更多
The paper describes the expression of human protein VEGF165 in Escherichia coli and its purification. This growth factor isoform contains exon 7, which is essential for binding to extracellular domain of VEGF receptor...The paper describes the expression of human protein VEGF165 in Escherichia coli and its purification. This growth factor isoform contains exon 7, which is essential for binding to extracellular domain of VEGF receptor 2, located on endothelial cells lining the surface of blood vessels. This binding stimulates the cascade of downstream signalling events leading to process known as angiogenesis, hVEGF165 overexpressed with His-tag in BL21 E. coli cells forms inclusion bodies （insoluble protein）, so the research found the procedure for its solubilization and purification on a Nickel based affinity chromatography. Although this eukaryotic signal protein needs posttranslational processing for its full function as a homodimer, author verified the biological activity of our hVEGF165 protein, obtained as monomer, by wound healing test.展开更多
OBJECTIVE: To investigate the direct effects of the Flt3 ligand (FL) on hematopoiesis, such as the stimulation of the formation of hematopoietic colonies and the proliferation of dendritic cells, as well as the indire...OBJECTIVE: To investigate the direct effects of the Flt3 ligand (FL) on hematopoiesis, such as the stimulation of the formation of hematopoietic colonies and the proliferation of dendritic cells, as well as the indirect stimulation of hematopoiesis, especially via the proliferation of endothelial cells. METHODS: Mononuclear cells from human cord blood were plated in methylcellulose medium containing different cytokines to induce hematopoietic colony formation. Dendritic cells (DCs) were induced from the mononuclear cells with a cytokine cocktail with or without recombinant human soluble FL (rhFL; 100 ng/ml). The Flt3 receptors on the surface of a human microvascular endothelial cell line (ECV) were analyzed by flow cytometry. The proliferation of ECV stimulated by rhFL was measured with the microculture tetrazolium assay. The levels of FL, IL-6, IL-8, G-CSF and GM-CSF in the supernatant of ECV cultures were measured by enzyme linked immunoabsorbent assay (ELISA). RESULTS: rhFL stimulates colony formation from cord blood when used as a sole stimulant. FL in combination with other cytokines increased colony formation significantly. The number of DCs was approximately 2.5 times higher when rhFL was used. rhFL stimulates the proliferation of ECV on which Flt3 receptors are expressed. Furthermore, ECV secretes FL, IL-6, IL-8, G-CSF and GM-CSF, which were augmented by tumor necrosis factor-alpha and rhFL. CONCLUSIONS: rhFL enhances hematopoietic colony formation and DC proliferation from human cord blood cells. FL not only stimulates the proliferation of ECV, but is also secreted by ECV. FL may exert direct and indirect effects on hematopoiesis.展开更多
基金Supported by the National Natural Science Foundation of China,No.30772129Jiangsu Provincial Key Medical Center for Hepatobiliary Disease,No.ZX200605
文摘AIM： To investigate whether the function of hepatocytes co-cultured with bone marrow mesenchymal stem cells （MSCs） could be maintained in serum from acute-on- chronic liver failure （ACLF） patients.METHODS： Hepatocyte supportive functions and cy- totoxicity of sera from 18 patients with viral hepatitis B-induced ACLF and 18 healthy volunteers were evalu- ated for porcine hepatocytes co-cultured with MSCs and hepatocyte mono-layered culture, respectively. Chemo- kine profile was also examined for the normal serum and liver failure serum.RESULTS： Hepatocyte growth factor （HGF） and Tumor necrosis factor; tumor necrosis factor （TNF）-a were re- markably elevated in response to ACLF while epidermal growth factor （EGF） and VEGF levels were significantly decreased. Liver failure serum samples induced a higher detachment rate, lower viability and decreased liver sup- port functions in the homo-hepatocyte culture. Hepato-cytes co-cultured with MSCs could tolerate the cytotoxic- ity of the serum from ACLF patients and had similar liver support functions compared with the hepatocytes cul- tured with healthy human serum in vitro. In addition, co- cultured hepatocytes maintained a proliferative capability despite of the insult from liver failure serum.CONCLUSION： ACLF serum does not impair the cell morphology, viability, proliferation and overall metabolic capacities of hepatocyte co-cultured with MSCs in vitro.
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Recent studies have revealed that the γ-chain of theIL-2 receptor is shared by the receptors for IL-4, IL7, IL-9, IL-13, and IL-15, and it is therefore also referred toas the common γ-chain (γc). Mutations of γc result inX-linked severe combined immunodeficiency syndrome inhumans, indicating that rye is essential for normal development and function of the immune system. We demonstratethat human hematopoietic cells express two γc transcriptsdiffering in their carboxyl terminal coding region. Onetranscript is the previously reported sequence (γc-long),whereas the newly identified sequence exhibits a deletion of72 nucleotides close to the 3’-end of the open reading frame(γc-short). This alteration predicts a loss of 24 amino acidsincluding a conserved tyrosine residue which is shared byseveral members of the cytokine receptor family. Thepresence of these two distinct forms of rye transcripts wasdemonstrated by sequencing of reversely transcribed andpolymerase chain reaction (RT-PCR) amplified mRNA, restriction digestion of the RT-PCR products, RNAse protection, and Northern blotting from human cell lines andhuman peripheral blood lymphocytes. Furthermore, thetwo variants were present in peripheral blood lymphocytesfrom both female and male donors, which rules out allelicvariants since rye is a single copy gene located on the Xchromosome. A truncation mutant at a site near the observed changes in γc-short has been reported by othersto alter biochemical events activated by cytokines. Thiscombined with the loss of a potential SH2 "docking" sitein γc-short suggests that γc-long and γc-short may link todifferent signaling pathways and may play an importantrole in determining the cellular response to IL-2, IL-4, IL-7, IL-9, IL-13, IL-15.
文摘The paper describes the expression of human protein VEGF165 in Escherichia coli and its purification. This growth factor isoform contains exon 7, which is essential for binding to extracellular domain of VEGF receptor 2, located on endothelial cells lining the surface of blood vessels. This binding stimulates the cascade of downstream signalling events leading to process known as angiogenesis, hVEGF165 overexpressed with His-tag in BL21 E. coli cells forms inclusion bodies （insoluble protein）, so the research found the procedure for its solubilization and purification on a Nickel based affinity chromatography. Although this eukaryotic signal protein needs posttranslational processing for its full function as a homodimer, author verified the biological activity of our hVEGF165 protein, obtained as monomer, by wound healing test.
基金agrantfromtheIAEAFoundationfortheProtectionofAcuteIrradiationInjury (No CPR/9/0 2 5 )
文摘OBJECTIVE: To investigate the direct effects of the Flt3 ligand (FL) on hematopoiesis, such as the stimulation of the formation of hematopoietic colonies and the proliferation of dendritic cells, as well as the indirect stimulation of hematopoiesis, especially via the proliferation of endothelial cells. METHODS: Mononuclear cells from human cord blood were plated in methylcellulose medium containing different cytokines to induce hematopoietic colony formation. Dendritic cells (DCs) were induced from the mononuclear cells with a cytokine cocktail with or without recombinant human soluble FL (rhFL; 100 ng/ml). The Flt3 receptors on the surface of a human microvascular endothelial cell line (ECV) were analyzed by flow cytometry. The proliferation of ECV stimulated by rhFL was measured with the microculture tetrazolium assay. The levels of FL, IL-6, IL-8, G-CSF and GM-CSF in the supernatant of ECV cultures were measured by enzyme linked immunoabsorbent assay (ELISA). RESULTS: rhFL stimulates colony formation from cord blood when used as a sole stimulant. FL in combination with other cytokines increased colony formation significantly. The number of DCs was approximately 2.5 times higher when rhFL was used. rhFL stimulates the proliferation of ECV on which Flt3 receptors are expressed. Furthermore, ECV secretes FL, IL-6, IL-8, G-CSF and GM-CSF, which were augmented by tumor necrosis factor-alpha and rhFL. CONCLUSIONS: rhFL enhances hematopoietic colony formation and DC proliferation from human cord blood cells. FL not only stimulates the proliferation of ECV, but is also secreted by ECV. FL may exert direct and indirect effects on hematopoiesis.
