Biomedical subjects

Wei Ge

Publications and source records attributed to Wei Ge.

49 records · Page 3Linked to original sources

PubMed2004

[Inhibition effect of bone marrow mesenchymal stem cells on T-lymphocyte proliferation through up-regulation of CD8+CD28- T cells].

The aim of this study was to explore effect of CD8+CD28- T-lymphocyte in the inhibition of mesenchymal stem cells (MSC) on T-lymphocyte proliferation. T cells were harvested by using nylon column and CD8+ T cells were sorted by magnetic beads; the T-lymphocyte proliferation in the presence of PHA was evaluated by MTT; the proportion of CD8+CD28- T cells was assayed by fluorescence-activated cell sorter (FACS). The results showed that MSC inhibited T-lymphocyte proliferation and the inhibitory effect depended on the amount of MSC; the data of FACS indicated that in the CD8+ T cells co-cultured with MSC, CD8+CD28- T cells were up-regulated significantly, compared with the non-treated CD8+ T cells. In conclusion, MSC perform their immunosuppressive function by up-regulation of CD8+CD28- T cells.

Bone Marrow Cells↗

PubMed2003

Spatial expression patterns of activin and its signaling system in the zebrafish ovarian follicle: evidence for paracrine action of activin on the oocytes.

We have previously demonstrated that activin is likely an ovarian mediator of pituitary gonadotropin(s) and local epidermal growth factor in their stimulating oocyte maturation and maturational competence in the zebrafish. However, the downstream events controlled by activin remain unknown. One possible mechanism is that activin may directly work on the oocytes to promote the development of oocyte maturational competence. To substantiate this hypothesis, we performed the present study to demonstrate the expression of the activin system in different compartments of zebrafish follicles, namely, the follicle cells and oocytes. The proteins examined include activin subunits (betaA and betaB), activin-binding protein (follistatin), activin type II receptors (type IIA and IIB), the type I activin receptor-like kinases (ALK1-like, ALK2-like, and ALK4-like), and the intracellular activin signaling molecules (Smad2, Smad3, Smad4, and Smad7). The results showed that the entire activin signaling system is expressed by the full-grown immature zebrafish oocytes ( approximately 0.65 mm in diameter), including ALK4-like (ActRIB), ALK2-like (ActRIA), ActRIIA, ActRIIB, Smad2, Smad3, Smad4, and Smad7, therefore supporting our hypothesis that the oocytes are one of the direct targets of activin actions in the zebrafish ovary. In contrast, activin itself (betaA and betaB) and ALK1-like type I receptor are predominantly expressed in the follicle cells surrounding the oocytes. Interestingly, although follistatin is expressed in both the follicle cells and oocytes, its level of expression is significantly higher in the oocytes than the follicle cells, implying that follistatin may serve as a signal from the oocytes to modulate the activity of activin produced by the follicle cells. Taken together, the present study provides convincing evidence that although all members of the activin system are expressed in the whole follicle, they exhibit distinct spatial patterns of expression among different compartments of the follicle. It is likely that activin works directly on the oocytes in a paracrine manner to promote oocyte maturation and maturational competence. On the other hand, instead of being controlled passively by the follicle cells, the oocytes may actively participate in the regulation of follicle development by releasing various modulating molecules such as follistatin.

Activin Receptors↗

PubMed2003

Cloning, regulation of messenger ribonucleic acid expression, and function of a new isoform of pituitary adenylate cyclase-activating polypeptide in the zebrafish ovary.

Increasing evidence suggests that pituitary adenylate cyclase-activating polypeptide (PACAP) acts as a local factor in the ovary of mammals. In nonmammalian vertebrates, although the expression of PACAP has also been demonstrated in the ovary, the information on its functions and regulation is limited. In the present study, we identified a new type of PACAP, zebrafish (zf)PACAP38-2, from the zebrafish ovary. The precursor of GHRH-zfPACAP38-2 consists of 175 amino acids with only 64% homology with another type of zebrafish PACAP, zfPACAP38-1. RT-PCR analysis detected two messengers of zfPACAP38-2 in the zebrafish ovary. The short product was more abundant, and it encodes zfPACAP38-2 only, whereas the long form codes for both zfPACAP38-2 and GHRH. Using a primary culture of zebrafish follicle cells, we demonstrated that gonadotropin (human chorionic gonadotropin and goldfish pituitary extract) significantly stimulated zfPACAP38-2 expression within 2 h; however, the effect decreased to the control level after 8 h of treatment. The stimulation of zfPACAP38-2 expression by gonadotropin could be mimicked by cAMP analogs and forskolin but suppressed by H89 (10 mum), suggesting the involvement of the cAMP-protein kinase A signaling pathway. We also examined the expression of PACAP receptor VPAC2-R in the zebrafish ovary. Unlike zfPACAP38-2, which showed a trend of increase during follicle development, the expression of VPAC2-R mRNA in the follicles showed no significant stage-dependent variation, and its expression in the follicle cells did not respond to gonadotropin treatment. Our studies further demonstrated that synthetic zfPACAP38-2 stimulated oocyte maturation and increased the expression of follistatin in zebrafish ovarian follicle cells. These results suggest that zfPACAP38-2 is a potential ovarian factor that mediates gonadotropin actions in paracrine/autocrine manners, and its functional roles are likely, to some extent, related to the ovarian activin/follistatin system.

Amino Acid Sequence↗

PubMed2003

Induction of laccase activity in the edible straw mushroom, Volvariella volvacea.

Volvariella volvacea, strain V14, produces multiple forms of extracellular laccase when grown in submerged culture in a defined medium with glucose as sole carbon source, and on cotton waste 'compost' representative of the conditions used for industrial-scale mushroom cultivation. In liquid culture, enzyme synthesis is associated with the onset of secondary growth, and is positively regulated by copper (up to 200 microM CuSO(4)) and by various aromatic compounds. In solid-state systems, only low levels of laccase are detectable during the vegetative growth phase but enzyme activity increases sharply at the onset of fruiting and during sporophore development.

Agaricales↗

PubMed2003

Gonadotropin regulation of follistatin expression in the cultured ovarian follicle cells of zebrafish, Danio rerio.

Follistatin is a single-chain glycoprotein initially identified in the mammalian ovary. As a specific binding protein of activin, it effectively modifies the paracrine/autocrine roles of activin in a variety of tissues including the ovary. In the zebrafish, we have demonstrated that the human chorionic gonadotropin (hCG)-induced oocyte maturation and oocyte maturational competence can be blocked by follistatin, suggesting a role for ovarian activin in the signaling pathway of gonadotropin in the zebrafish ovary. The up-regulation of zebrafish ovarian activin betaA subunit by gonadotropin further supports this hypothesis. Since follistatin has extremely high affinity for activin, its expression level in various tissues is critical in fine-tuning local activin activities. In the present study, we investigated the regulation of follistatin expression by gonadotropin in a primary culture of zebrafish ovarian follicle cells using semi-quantitative RT-PCR. Both hCG and goldfish pituitary extract strongly increased the expression of follistatin in the cultured follicle cells in clear time- and dose-dependant manners. The effect of hCG (15IU/ml) reached the maximal level at 2h of treatment and longer treatment (4-8h) led to decreased response. The up-regulation of follistatin expression by hCG could be mimicked by all the drugs that increase the intracellular cAMP level including 8-Br-cAMP, db-cAMP, forskolin, and 3-isobutyl-1-methylxanthine (IBMX). The hCG (15IU/ml)- and forskolin (10microM)-induced follistatin expression could be blocked by H89 (10microM), a specific protein kinase A (PKA) inhibitor. These results strongly suggest that the regulation of follistatin expression in the zebrafish ovary by gonadotropin is primarily mediated by cAMP-PKA signaling pathway. The up-regulation of follistatin mRNA by gonadotropin in cultured zebrafish ovarian follicle cells, together with our previous studies on gonadotropin regulation of activin beta subunits, suggests that the ovarian activin-follistatin system is tightly controlled by gonadotropin in fish ovary.

Activins↗

PubMed2003

Involvement of cyclic adenosine 3',5'-monophosphate in the differential regulation of activin betaA and betaB expression by gonadotropin in the zebrafish ovarian follicle cells.

Activin is a dimeric protein consisting of two similar but distinct beta-subunits, betaA and betaB. In our previous studies, both activin A (betaAbetaA) and activin B (betaBbetaB) have been demonstrated to stimulate oocyte maturation and promote oocyte maturational competence in the zebrafish. Follistatin, a specific activin-binding protein, can block both activin- and gonadotropin-induced final oocyte maturation in vitro, suggesting that activin is likely a downstream mediator of gonadotropin actions in the zebrafish ovary. In the present study, a full-length cDNA encoding zebrafish ovarian activin betaA was cloned and sequenced. The precursor of zebrafish activin betaA consists of 395 amino acids and its mature region exhibits about 78% homology with that of mammals. Using an in vitro primary culture of the ovarian follicle cells and semiquantitative RT-PCR assays, we examined the regulation of activin betaA and betaB expression by human chorionic gonadotropin (hCG) and its intracellular signal transduction mechanisms. hCG (15 IU/ml) increased the mRNA level of activin betaA-subunit; however, it significantly down-regulated the steady-state expression level of activin betaB in a time- and dose-dependent manner. The differential regulation of the two beta-subunits by hCG could be mimicked by 3-isobutyl-1-methylxanthine, forskolin, and dibutyryl-cAMP, suggesting involvement of the intracellular cAMP pathway. Interestingly, H89 (a specific inhibitor of protein kinase A, PKA) could effectively block hCG- and forskolin-stimulated activin betaA expression at 10 micro M, but it was unable to reverse the inhibitory effects of hCG and forskolin on betaB expression. This suggests that the hCG-stimulated activin betaA expression is dependent on the activation of the cAMP-PKA pathway, whereas the inhibitory effect of hCG on activin betaB expression is likely mediated by PKA-independent pathway(s).

1-Methyl-3-isobutylxanthine↗

PubMed2003

[Dendritic cells (DC) induced from acute myeloid leukemia (AML) cells with cytokine cocktails].

OBJECTIVES: To explore the feasibility of DC being in vitro induced from AML cells with cytokine cocktails and their biological properties. METHODS: AML cells were cultured in either presence or absence of cytokine cocktails. DC were studied for morphology, and cytochemical and immunofluorescent staining. Functions of DC were examined by MLC, FITC-conjugated dextran uptake test, and LDH release assay. RT-PCR and FISH were used to analyze the specific fusion genes of culture-derived DC. RESULTS: Classical DC morphological changes occurred in all 15 cultured AML cells. DC-associated surface molecules such as CD(1a), CD(80), CD(86), CD(106), CD(83) and HLA-DR were upregulated (P < 0.05). The allostimulatory abilities of culture-derived DC were significantly higher than those of AML cells uncultured or cultured in the absence of cytokines (P < 0.05). Culture-derived DC only in the presence of GM-CSF + IL-4 have phagocytotic activities. CTL assay was performed in 5 of the 15 samples. At effector/target ratio of 20:1, auto-T lymphocytes primed with the culture-derived DC exhibited no more killing activity to auto-AML cells than those stimulated by IL-2 or uncultured AML cells. Culture-derived DC presenced the native AML-specific aberrant karyotype and related fusion gene. CONCLUSIONS: Cytokine cocktails could in vitro induce AML cells into DC with classical morphology, immunophenotype and function. DC maturity induced by different cytokine cocktails could be variable. Culture-derived DC were originated from the native AML cells. AML cells could make the auto-T lymphocyte anergy.

Adolescent↗

PubMed2003

Induction of T-cell immunity against leukemia by dendritic cells pulsed with total RNA isolated from leukemia cells.

OBJECTIVES: To assess the feasibility and efficacy of eliciting leukemia-specific T-cell responses in syngeneic mice in vitro and in vivo using dendritic cells (DCs) pulsed with total RNA from leukemia cells. METHODS: DCs generated from bone marrow culture in vitro in the presence of combined cytokines were pulsed with cellular total RNA isolated from cultured L615 cells by cationic lipid 1,2-dioleoyloxy-3-(trimethylammonium) propane (DOTAP). T-cell responses were evaluated by in vitro proliferation, and cytotoxicity assay. And in vivo immune protection and prognosis of mice with leukemia were studied. RESULTS: DCs pulsed with total RNA isolated from cultured L615 cells (DCs/RNA) were remarkably effective in stimulating L615-specific T-cell response in vitro, but did not cross-react with other leukemia cells from syngeneic mice. Vaccination of naive mice with viable DCs/RNA vaccine was able to partly protect from challenge with a lethal dose of live L615 cells, leading to low leukemia incidence and overall survival prolongation. Statistically significant survival was also observed in a low lethal dose of L615-bearing mice that received treatment using viable DCs/RNA vaccine alone, suggesting that systemic administration of IL-2 could enhance the anti-tumor efficacy of leukemia RNA/DCs vaccine. CONCLUSIONS: These data support the use of DCs/RNA vaccine as a feasible and effective route to elicit leukemia immunity against unidentified leukemia-associated antigens for treatment of leukemia-bearing animals.

Animals↗

PubMed2002

Gonadotropin regulation of activin betaA and activin type IIA receptor expression in the ovarian follicle cells of the zebrafish, Danio rerio.

We have previously demonstrated that both activin and its receptors are expressed in the zebrafish ovary, suggesting paracrine roles for activin in the ovarian functions. Activin significantly stimulated zebrafish oocyte maturation in vitro, and this effect could be blocked by follistatin, an activin-binding protein. Interestingly, follistatin also blocked the stimulatory effect of gonadotropin (hCG) on the oocyte maturation. Taken together, these results have led to a hypothesis that the ovarian activin system may play a role in mediating the actions of gonadotropin in the ovary. To test this hypothesis, the present study was undertaken to investigate if gonadotropin has any effect on the expression of activin betaA subunit and activin type IIA (ActRIIA) receptor in the zebrafish ovary. A primary culture of zebrafish ovarian follicle cells was established in the present study, and the cultured cells expressed both activin betaA and ActRIIA receptor when assayed with RT-PCR. The primary culture consisted of three major types of cells, presumably the fibroblasts, the thecal cells and the granulosa cells, according to the morphological features, histochemical staining for 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and RT-PCR for aromatase. Using a semi-quantitative RT-PCR with beta-actin as the internal control, we demonstrated that hCG significantly stimulated mRNA expression of both activin betaA and ActRIIA receptor in the cultured follicle cells in a time- and dose-dependent manner. Treatment of the cells with hCG quickly increased the steady-state mRNA levels of activin betaA and ActRIIA receptor, and the effect peaked at 2 h of treatment. The stimulatory effect of gonadotropin diminished with longer treatment and no effect was observed at 8 h of treatment. The effect of hCG also exhibited strong dose dependence when assayed at 2 h of treatment. The levels of activin betaA and ActRIIA receptor mRNA elevated with increasing dose of hCG; however, the effect significantly decreased at dosage higher than 15 IU/ml. Consistent with the stimulatory effect of gonadotropin on the expression of activin betaA and ActRIIA receptor, IBMX, forskolin and 8-Br-cAMP all significantly increased the mRNA levels of activin betaA and ActRIIA receptor. These results suggest that gonadotropin activates the activin system in the zebrafish ovary by increasing the expression of both activin and its receptors.

1-Methyl-3-isobutylxanthine↗

PubMed2002

Cloning of zebrafish ovarian carbonyl reductase-like 20 beta-hydroxysteroid dehydrogenase and characterization of its spatial and temporal expression.

20 beta-Hydroxysteroid dehydrogenase (20 beta-HSD) is a crucial enzyme that converts 17 alpha-hydroxyprogesterone to 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP), which triggers oocyte maturation in most teleost fish. A full-length cDNA for a carbonyl reductase-like 20 beta-HSD (CR/20 beta-HSD) has been cloned from the zebrafish ovary. Although the zebrafish CR/20 beta-HSD is expressed in all of the tissues tested, it is predominantly expressed in the ovary, testis, kidney, and gill. In the ovary, the enzyme was shown to be expressed in the follicle cells and its expression appeared to be constitutive. No significant difference was noticed in the level of CR/20 beta-HSD expression among follicles of different stages. Furthermore, analysis of the ovarian samples taken at different times before spawning showed no significant change of the enzyme expression. In agreement with these results, treatment of the cultured zebrafish ovarian follicle cells with gonadotropin and activin had little effect on the expression of the enzyme. Taken together, these results point to the possibility that the gonadotropin-induced DHP production and final oocyte maturation in the zebrafish may not involve significant change of CR/20 beta-HSD expression as evidenced in the salmonids, or that there might be other isoforms of 20 beta-HSD whose expression is tightly controlled by endocrine and paracrine factors.

Alcohol Oxidoreductases↗

PubMed2002

Gonadotropin and activin enhance maturational competence of oocytes in the zebrafish (Danio rerio).

In most teleosts, 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) serves as the most potent maturation-inducing steroid (MIS) to initiate final oocyte maturation. The maturational competence or the responsiveness of oocytes to DHP increases when the ovarian follicles approach the final stage of growth. In the zebrafish, we demonstrated in the present study that full-grown oocytes (approximately 0.7 mm) exhibited the highest maturational competence, which diminished progressively with decreasing size of the follicles. Using midvitellogenic follicles (0.49-0.56 mm), which had little response to DHP, as the material, the present study aimed at investigating the endocrine and paracrine mechanisms that regulate maturational competence of the oocytes. In agreement with the results of studies in other teleost fish, pretreatment of follicles with gonadotropin (hCG) significantly enhanced the responsiveness of midvitellogenic oocytes to DHP in a clear time- and dose-dependent manner. Interestingly, activin, an ovarian growth factor, also had a potent stimulatory effect on the acquisition of oocyte maturational competence. Pretreatment with either recombinant human activin A or goldfish activin B significantly increased the rate of DHP-induced oocyte maturation from approximately 3% to approximately 70%, also in a clear dose-dependent manner. Similar to the results with hCG, pretreatment with activin alone had no effect in inducing maturation of midvitellogenic oocytes without subsequent DHP treatment, although both exhibited a strong effect in promoting maturation of full-grown oocytes. The effect of activin on maturational competence of oocytes could be reduced by cotreatment with follistatin, a potent activin-binding protein. Interestingly, follistatin treatment also significantly reduced the effect of hCG on maturational competence of oocytes, suggesting a mediating role for endogenous activin or activin-related molecules in the action of gonadotropin. The effects of hCG and activin on maturational competence of oocytes could be significantly inhibited by actinomycin D (1 microg/ml) and completely blocked by cycloheximide (1 microg/ml), suggesting that the hCG and activin-induced acquisition of oocyte maturational competence involves de novo protein synthesis at both the transcriptional and translational levels.

Activins↗

PubMed2002

Epidermal growth factor and TGFalpha promote zebrafish oocyte maturation in vitro: potential role of the ovarian activin regulatory system.

Epidermal growth factor (EGF) and TGFalpha are well known for their activities in the ovary. Both factors initiate signal transduction by binding to the common EGF receptor that has been demonstrated in the ovary across vertebrates from fish to humans. Using zebrafish as the model, we demonstrated in the present study that recombinant human EGF and TGFalpha significantly enhanced final maturation of the fully grown, follicle-enclosed oocytes (0.58-0.65 mm) in vitro in a clear time- and dose-dependent manner. The effect of EGF/TGFalpha was additive to that of hCG at low concentrations, but the additivity diminished when the concentration increased. Both actinomycin D and cycloheximide completely blocked the effect of EGF/TGFalpha, indicating that the promotion of oocyte maturation by EGF/TGFalpha requires de novo mRNA transcription and protein synthesis. Interestingly, the effect of EGF/TGFalpha could be blocked by cotreatment with follistatin, a potent binding protein for activin, an ovarian growth factor belonging to the TGFbeta superfamily. Semiquantitative RT-PCR assays showed that both EGF and TGFalpha significantly stimulated the expression of activin betaA and activin type II receptor in the cultured zebrafish ovarian follicle cells in a clear time- and dose-dependent manner. This together with our previous report that activin had a potent stimulatory effect on zebrafish oocyte maturation strongly suggests that the intrinsic ovarian activin system is probably a downstream mediator of EGF/TGFalpha actions in the zebrafish ovary.

Activin Receptors, Type II↗

PubMedDate not supplied

Engrafted bone marrow-derived flk-(1+) mesenchymal stem cells regenerate skin tissue.

Stem cell plasticity has created great interest because of its potential therapeutic application in degenerative or inherited diseases. Transplantation of bone marrow-derived stem cells was shown to give rise to cells of muscle, liver, nerve, endothelium, epithelium, and so on. But there are still disputes about stem cell plasticity, especially concerning the contribution of bone marrow-derived cells to skin cells. In this study, CM-DiI fluorescence-labeled Flk-(1+) bone marrow mesenchymal stem cells (bMSCs) of BALB/c mice (H-2Kd, white) were transplanted into lethally irradiated C57BL/6 mice (H-2Kb, black). By fluorescence tracing, we found that donor cells could migrate and take residency at the skin, which was confirmed by Y chromosome-specific PCR and Southern blot. The recipient mice grew white hairs about 40 days later and white hairs could spread over the body. Immunochemistry staining and RT-PCR demonstrated that skin tissue within the white hair regions was largely composed of donor-derived H-2Kd cells, including stem cells and committed cells. Furthermore, most skin cells cultured from white hair skin originated from the donor. Thus, our findings provide direct evidence that bone marrow-derived cells can give rise to functional skin cells and regenerate skin tissue. These may have important scientific implications in stem cell biology and transplantation therapy for skin tissue injury.

Animals↗