Biomedical subjects

Robert Chunhua Zhao

Publications and source records attributed to Robert Chunhua Zhao.

At least 19 recordsLinked to original sources

PubMed2006

Proliferation and differentiation of bone marrow stromal cells under hypoxic conditions.

Low oxygen tension is a potent differentiation inducer of numerous cell types and an effective stimulus of many gene expressions. Here, we described that under 8% O(2), bone marrow stromal cells (MSCs) exhibited proliferative and morphologic changes. The level of differentiated antigen H-2Dd and the number of G(2)/S/M phase cells increased evidently under 8% O(2) condition. Also, the proportion of wide, flattened, and epithelial-like cells (which were alkaline phosphatase staining positive) in MSCs increased significantly. When cultured in adipogenic medium, there was a 5- to 6-fold increase in the number of lipid droplets under hypoxic conditions compared with that in normoxic culture. We also demonstrated the existence of MSC differentiation under hypoxic conditions by electron microscopy. Expression of Oct4 was inhibited under 8% O(2) condition, but after adipocyte differentiation in normoxic culture and hypoxia-mimicking agents cobalt chloride (CoCl(2)) and deferoxamine mesylate (DFX) treatments, Oct4 was still expressed in MSCs. These results indicate hypoxia accelerates MSC differentiation and hypoxia and hypoxia-mimicking agents exert different effects on MSC differentiation.

Animals↗

PubMed2006

Knockdown of hypoxia-inducible factor-1alpha in breast carcinoma MCF-7 cells results in reduced tumor growth and increased sensitivity to methotrexate.

The hypoxia-inducible factor (HIF) 1alpha is a key regulator of the cellular response to oxygen deprivation. Specific disruption of the HIF-1 pathway is important for exploring its role in tumor biology and developing more efficient weapons to treat cancer. In this study, we stably transfected human breast tumor MCF-7 cells with short hairpin RNA expression vectors targeting HIF-1alpha. After knockdown of HIF-1alpha, hypoxia-induced expression of its target genes such as vascular endothelial growth factor, Glut-1, phosphoglycerate kinase, and P-glycoprotein were markedly attenuated. Moreover, HIF-1alpha knockdown was found to suppress the shift from S-phase to G(1) induced by hypoxia and increase drug sensitivity to methotrexate. The growth rates of HIF1alpha-knockdown tumors were drastically retarded in both subcutaneous and orthotopic xenograft models, which were accompanied by decreased angiogenesis and reduced expression of glucose transporter in tissue sections. These data demonstrate that HIF-1alpha knockdown reduces tumorigenicity of MCF-7 cells and suggest a promising combination of both anti-HIF-1 strategy and traditional chemotherapy to improve cancer treatment.

Antineoplastic Agents↗

PubMed2006

Ex vivo expansion and in vivo infusion of bone marrow-derived Flk-1+CD31-CD34- mesenchymal stem cells: feasibility and safety from monkey to human.

Flk-1(+)CD31(-)CD34(-) mesenchymal stem cells (MSCs) are multipotent cells with extensive proliferation ability and immunomodulatory function. On the basis of our previous work showing their potential application in tissue engineering and immune diseases, we designed a preclinical and Phase I trial to evaluate the feasibility and safety of intravenous infusion of these cells after ex vivo expansion. Rhesus monkey and human Flk-1(+)CD31(-)CD34(-) MSCs were isolated from bone marrow and passaged a maximum of six passages. Karyotype analysis showed Flk-1(+)CD31(-)CD34(-) MSCs remained normally diploid within six passages of expansion. Expanded cells were transplanted into rhesus monkeys and human volunteers, respectively. During the infusion of these cells, the life signs of the recipients were normal. Laboratory tests for blood, bone marrow, kidney, and liver function were conducted and no significant changes were observed before and after cell infusion. Identification of the sry sequence from the male donor in female recipients showed that allogeneic rhesus Flk- 1(+)CD31(-)CD34(-) MSCs were capable of homing to and establishing residence within the recipients' bone marrow. No significant changes were observed in lymphocyte cell subpopulation before and after infusion. These result showed that Flk-1(+)CD31(-)CD34(-) MSCs have no obvious side effects after intravenous administration, encouraging investigators to perform further studies in stem cell therapy.

Adult↗

PubMed2005

Human adipose tissue-derived stem cells differentiate into endothelial cells in vitro and improve postnatal neovascularization in vivo.

In this study, we isolated CD31(-), CD34(-), CD106(-) (VCAM-1(-)), and fetal liver kinase(+) (Flk1(+)) cells from adipose tissue. These cells can be induced to differentiate into cells of osteogenic and adipogenic lineages in vitro and were termed adipose derived adult stem cells (ADAS cells). We also showed that they have characteristics of endothelial progenitor cells. In vitro, ADAS cells expressed endothelial markers when cultured with VEGF. In vivo, ADAS cells can differentiate in response to local cues into endothelial cells that contributed to neoangiogenesis in hindlimb ischemia models. PI3 kinase inhibitor LY294002 blocked the differentiation of ADAS cells into endothelial cells in vitro. Because ADAS cells can be expanded in culture without obvious senescence for more than 20 population doublings, they may be a potential source of endothelial cells for cellular pro-angiogenic therapies.

Adipocytes↗

PubMed2005

Molecular cloning and characterization of a novel human C4orf13 gene, tentatively a member of the sodium bile acid cotransporter family.

By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a novel human cDNA (C4orf13). This cDNA is 2706 bp in length, encoding a 340-amino-acid polypeptide that contains a typical SBF (sodium bile acid cotransporter family) domain and ten possible transmembrane segments. The putative protein C4orf13 shows high similarity with its orthologs in Mus musculus and Xenopus laevis. Human C4orf13 is mapped to chromosome 4q31.2 and contains 12 exons. RT-PCR analysis shows that human C4orf13 is widely expressed in human tissues, and the expression levels in liver and lung are relatively high, expression levels in placenta, kidney, spleen, and thymus are moderate, low levels of expression are detected in heart, prostate, and testis.

Amino Acid Sequence↗

PubMed2005

Small intestinal organoid-derived SP cells contribute to repair of irradiation-induced skin injury.

Side population (SP) cells, characterized by their ability to efflux the fluorescent dye Hoechst 33342, were isolated from the small intestine of mice. In the abdominal irradiation model, small intestinal organoid-derived SP (sioSP) cells from ROSA 26 mice were submucosally injected into the small intestinal of the irradiated C57BL/6 mice. In contrast to the control mice, mice receiving sioSP cell transplantation demonstrated far less skin injury. Most importantly, hairs in the irradiated body part of the transplanted mice almost remained black, whereas the counterpart in the control mice almost turned white. Histochemistry studies showed the donor cells gave rise to skin cells in the irradiated skin. Thus, our study demonstrated for the first time that stem cells from the small intestine can differentiate into skin cells under local cues and thus supports the theory of stem cell plasticity.

Animals↗

PubMed2004

Systemic infusion of FLK1(+) mesenchymal stem cells ameliorate carbon tetrachloride-induced liver fibrosis in mice.

BACKGROUND: Fibrosis is the common end stage of most liver diseases, for which, unfortunately, there is no effective treatment available currently. It has been shown that mesenchymal stem cells (MSCs) from bone marrow (BM) could engraft in the lung after bleomycin exposure and ameliorate its fibrotic effects. This study was designed to evaluate the effect of Flk1 MSCs from murine BM (termed here Flk1 mMSCs) on fibrosis formation induced by carbon tetrachloride (CCl4). METHODS: A CCl(4)-induced hepatic fibrosis model was used. Flk1 mMSCs were systemically infused immediately or 1 week after mice were challenged with CCl(4). Control mice received only saline infusion. Fibrosis index and donor-cell engraftment were assessed 2 or 5 weeks after CCl(4) challenge. RESULTS: We found that Flk1 mMSCs transplantation immediately, but not 1 week after exposure to CCl(4), significantly reduced CCl(4)-induced liver damage and collagen deposition. In addition, levels of hepatic hydroxyproline and serum fibrosis markers in mice receiving immediate Flk1 mMSCs transplantation after CCl(4) challenge were significantly lower compared with those of control mice. More importantly, histologic examination suggested that hepatic damage recovery was much better in these immediately Flk1 mMSCs-treated mice. Immunofluorescence, polymerase chain reaction, and fluorescence in situ hybridization analysis revealed that donor cells engrafted into host liver, had epithelium-like morphology, and expressed albumin, although at low frequency. CONCLUSION: These results suggest that Flk1 mMSCs might initiate endogenous hepatic tissue regeneration, engraft into host liver in response to CCl(4) injury, and ameliorate its fibrogenic effects.

Actins↗

PubMed2004

Multipotency of Flk1CD34 progenitors derived from human fetal bone marrow.

We report that a cell population derived from human fetal bone marrow, termed Flk1+CD34- multipotent stem cells, can differentiate not only into osteogenic, adipogenic, and endothelial lineages but also into hepatocyte-like cells and neural and erythroid cells at the single-cell level. We depleted mononuclear cells from fetal bone marrow of CD45+, GlyA+, and CD34+ cells with the use of micromagnetic beads, then cultured them by limiting dilution. Three single colonies were harvested, expanded, and characterized. The clones have been expanded for more than 50 cell doublings, and cell-doubling time was about 30 hours. About 90% cells were in the G(0)/G(1) phase of the cell cycle, and the cells from the single colony maintained Flk1+ and CD34-. Because fetal bone marrow-derived Flk1+CD34-multipotent stem cells have the capacity for self-renewal and multilineage differentiation even after being expanded for more than 50 cell doublings, they may be an ideal source of stem cells for the treatment of inherited or degenerative diseases.

Antigens, CD34↗

PubMed2004

Mechanisms of and perspectives on the mesenchymal stem cell in immunotherapy.

Mesenchymal stem cells (MSCs) are an important cell population in the bone-marrow microenvironment and are considered to be engaged mainly in the support of hematopoiesis. Recent work has shown that MSCs also have profound immunomodulatory function, both in vitro and in vivo. Because MSCs can be expanded rapidly to the numbers required for clinical application, several preclinical and clinical studies have been performed in the areas of immune diseases and bone-marrow transplantation. In this review we discuss the mechanisms underlying the MSC's immunomodulating properties and its potential applications.

Animals↗

PubMed2004

Characterization of a human Sec14-like protein cDNA SEC14L3 highly homologous to human SPF/TAP.

Supernatant protein factor (SPF) and alpha-tocopherol-associated protein (TAP) both belong to a widespread lipid-binding Sec 14-like protein family. All the members of the family have the lipid-binding motif called CRAL_TRIO. SPF is showed to stimulate the conversion of squalene to lanosterol and enhance cholesterol biosynthesis. TAP is identified to be involved in the intracellular distribution of alpha-tocopherol. Recently TAP is identified as SPF though they have very different functions. Here we report a human SPF/TAP homology SEC14L3 with 2082 base pairs in length and contains an open reading frame encoding a 400 amino acids protein. Analysis shows that SEC14L3 is mapped to 22q12 and expresses only in the liver among the used sixteen tissues in the test.

ATP-Binding Cassette Transporters↗

PubMed2004

Cloning and expression of human GTDC1 gene (glycosyltransferase-like domain containing 1) from human fetal library.

The glycosyltransferases (GTs) catalyze the synthesis of the carbohydrate portions of glycoproteins, glycolipids, and proteoglycans. Here we report the cloning and characterization of a novel human GTDC1 (glycosyltransferase-like domain containing 1) gene, which locates on human chromosome 2q22. The GTDC1 cDNA is 2954 bp in length, encoding a putative protein of 458 amino acids. At protein level human GTDC1 has 75 and 37% identity with its homologous counterparts in the mouse and fruitfly, respectively. RT-PCR analysis revealed its relatively high expression level in the adult lung, spleen, testis, and peripheral blood leukocyte.

Amino Acid Sequence↗

PubMed2003

Isolation and identification of mesenchymal stem cells from human fetal pancreas.

Mesenchymal stem cells (MSCs) have been cultured from many sources, including bone marrow and liver. To further support our hypothesis that MSCs exist in most postnatal tissues, we isolated a clonogenic, multipotent, rapidly proliferating population of cells from a fetal pancreas and termed them "pancreas-derived mesenchymal stem cells" (PMSCs). They withstood being passaged as many as 30 times without sustaining significant structural changes. In this study, we showed that PMSCs are positive for CD44, CD29, and CDI3 but negative for CD34 and HLA-DR and that they stained with collagen I and III but not with von Willebrand factor antibody. During the log phase of growth, PMSCs proliferated, doubling in population in about 30 hours. Cell-cycle analysis showed that more than 90% of cells were in the G0 and G1 phases, whereas a small subpopulation of cells were actively engaged in proliferation (S + G2 + M = 3.55%). Under differentiation culture conditions, PMSCs differentiated into cells of osteogenic, chondrogenic, and adipogenic lineages. These results demonstrate that PMSCs can be isolated from human fetal pancreas by means of their adherent ability and that they are capable of self-renewal, propagation, and multipotent differentiation.

Base Sequence↗

PubMed2003

Molecular cloning and characterization of a novel human hydroxysteroid dehydrogenase-like 2 (HSDL2) cDNA from fetal brain.

Hydroxysteroid dehydrogenases (HSDs) are responsible for the biosynthesis of steroid hormones and play a crucial role in mammalian physiology and development. By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a novel human hydroxysteroid dehydrogenase-like cDNA (HSDL2). This cDNA is 3211 bp in length, encoding a 418-amino-acid polypeptide, which contains a typical motif for NAD(P)+-binding (TGxxxGxG), an SDR active site motif (S-Y-K) and a sterol carrier protein domain. HSDL2 shows high similarity with the homologues in the mouse and fruit fly. The HSDL2 gene is mapped to chromosome 9q32 and contains 11 exons. RT-PCR analysis shows that the HSDL2 gene is widely expressed in human tissues and the expression levels in liver, kidney, prostate, testis, and ovary are relatively high.

Amino Acid Motifs↗

PubMed2003

Cloning and characterization of the human IFT20 gene.

Intraflagellar transport (IFT), in which molecular motors and IFT particle proteins participate, is very important in assembling and maintaining many cilia/flagella, such as the motile cilia that drive the swimming of cells and embryos, the nodal cilia that generate left-right asymmetry in vertebrate embryos, and the sensory cilia that detect sensory stimuli in some animals. Here we report the molecular cloning and characterization of a novel human cDNA that is 1060 base pair in length, encoding a putative protein of 158 amino acid residues which shares high homology to Mus musculus intraflagellar transport protein mIFT20 (Accession number AAL99202, identity 82.3%, similarity 82.9%) at protein level. Bioinformatics analysis revealed that the gene is composed of 6 exons and is located on human chromosome 17p 11.1. RT-PCR showed that this gene is expressed in human brain, lung, kidney and pancreas, and lower expression were also detected in human placenta, liver, thymus, prostate and testis.

Amino Acid Sequence↗

PubMed2003

Cloning and identification of a novel human RNPC3 gene that encodes a protein with two RRM domains and is expressed in the cell nucleus.

The RNA recognition motifs (RRM) domain is one of the most common eukaryotic protein folds. Proteins containing RRM domains function in important steps of posttranscriptional regulation of gene expression and are involved in processing and transport of mRNA precursors. Here we describe the cloning and characterization of a novel human RNPC3 gene containing two RNA recognition motifs. The 1870 bp cDNA encodes a protein with 517 amino acids. It also contains two bipartite nuclear targeting sequences, which is important for nuclear targeting for proteins, especially those functioning in the cell nucleus. The GFP location of the RNPC3 gene product shows that this protein is located in the cell nucleus. RT-PCR reveals that it is abundantly expressed in kidney and pancreas.

Amino Acid Motifs↗

PubMed2003

Cloning and characterization of human synaptotagmin 10 gene.

Synaptotagmin (Syt) is a membrane protein family of secretory vesicles, abundant in neural and some endocrine cells. All the members of this family contain one transmembrane region and two conserved C2 domains. Here we reported a new human Syt 10 gene isolated when screening a human fetal brain cDNA library. This cDNA clone is 3287 bp and contains an open reading frame from 299 to 1870 encoding a putative protein of 523 amino acids. It shares 94.6 and 94.8% homology to rat Syt 10 and mouse Syt 10 at protein level, respectively. RT-PCR result showed that it is expressed only in pancreas, lung and kidney.

Amino Acid Sequence↗

PubMed2003

Multiorgan engraftment and multilineage differentiation by human fetal bone marrow Flk1+/CD31-/CD34- Progenitors.

We previously reported that Flk1(+)/CD31(-)/CD34(-) cells isolated from human fetal bone marrow can differentiate at the single cell level into endothelial and hematopoietic cells in vitro. Here we report that within this cell population reside cells that can differentiate into the epithelium of liver, lung, gut, as well as the cells of both hematopoietic and endothelial system after primary or secondary transplantation into irradiated nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Hence, Flk1(+)/CD31(-)/CD34(-) cells possess remarkable differentiation potential and may thereby provide an alternative to hematopoietic stem cells for transplantation. In addition, our results show this stem cell population effectively accelerated wound healing in NOD/SCID mice and thus holds therapeutic promise for treatment of genetic disorders, organ dysfunction, and tissue repair in humans.

Animals↗

PubMed2003

Cloning and characterization of a novel splice variant of the brain-specific protein densin-180.

Densin-180 is a brain-specific synaptic protein of the o-sialoglycoprotein family. It functions in specific adhesion between presynaptic and postsynaptic membranes at glutamatergic synapses. We have cloned a novel splice variant of densin-180 from a fetal brain cDNA library and termed it LRRC7. It is located on 1p31 and consists of 8 exons. It encodes a putative protein of 216 amino acids which contains a nuclear localization signal and four leucine-rich repeats (LRRs). LRRs are 20-29 residue sequence motifs present in a number of proteins with diverse functions. Expression analysis of LRRC7 shows it is expressed ubiquitously while its splice variant densin-180 is brain-specific.

Alternative Splicing↗