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1.
Parasitol Res ; 123(9): 318, 2024 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-39249568

RESUMO

Several miRNA-based studies on Theileria-transformed bovine cells have been conducted; however, the mechanism by which transformed cells exhibit uncontrolled proliferation is not yet fully understood. Therefore, it is necessary to screen more microRNAs that may play a role in the transformation process of host cells infected with Theileria annulata to better understand the transformation mechanisms of Theileria-infected cells. RNA sequencing was used to analyze miRNAs expression in the host bovine lymphocytes infected with T. annulata at different time points after buparvaquone (BW720) treatment and DMSO treatment (control groups). Differential miRNAs related to cell proliferation and apoptosis were identified through comparison with gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and a regulatory network of miRNA-mRNA was constructed. In total, 272 differentially expressed miRNAs were found at 36, 60 and 72 h. The miRNAs change of bta-miR-2285t, novel-miR-622, bta-miR-2478, and novel-miR-584 were significant. Analysis of 27 of these co-differential expressed miRNAs revealed that 15 miRNAs were down-regulated and 12 miRNAs were up-regulated. A further analysis of the changes in the expression of each of these 27 miRNAs in the three datasets suggested that bta-miR-2285t, bta-miR-345-5p, bta-miR-34a, bta-miR-150, and the novel-miR-1372 had significantly changed. Predicted target genes for these 27 miRNAs were analyzed by KEGG and the results demonstrated that EZR, RASSF, SOCS1 were mainly enriched in the signaling pathway microRNAs in cancer. MAPKAPK2, RELB, FLT3LG, and GADD45B were mainly enriched in the MAPK signaling pathway, and some genes were enriched in Axon guidance. This study has provided valuable information to further the understanding of the regulatory function of miRNAs in the host microenvironment and host-parasite interaction mechanisms.


Assuntos
Linfócitos , MicroRNAs , Naftoquinonas , Theileria annulata , Animais , Theileria annulata/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Bovinos , Naftoquinonas/farmacologia , Linfócitos/metabolismo , Theileriose/parasitologia , Theileriose/tratamento farmacológico , Perfilação da Expressão Gênica , Redes Reguladoras de Genes
2.
Steroids ; 202: 109349, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38072091

RESUMO

RFRP-3 is a functional ortholog of avian GnIH and regulates reproductive activities in the gonads of animals. However, the role of RFRP-3 in the function of ovarian granulosa cells in mice remains unclear. First, we detected the expression of the RFRP-3 receptor (GPR147) in the ovarian granulosa cells of mice. Second, the effect of RFRP-3 treatment on estradiol and progesterone secretions from granulosa cells was tested by ELISA. Meanwhile, the expression of genes and proteins regulating steroid hormone synthesis was respectively examined by qPCR and western blot. Furthermore, the effect of RFRP-3 treatment on the apoptosis of granulosa cells was analyzed. The results revealed that the GPR147 protein (a RFRP-3 receptor) was expressed in the ovarian granulosa cells of mice. Low and medium doses RFRP-3 treatment significantly reduced progesterone secretion in the granulosa cells (P < 0.05), while RFRP-3 suppressed p450scc, 3ß-HSD, StAR, and FSHR expression in a non-dose-dependent manner. Moreover, RFRP-3 treatment might induce the apoptosis of granulosa cells. Additionally, low doses RFRP-3 significantly reduced p-ERK1/2 protein expression (P < 0.05) in the ovarian granulosa cells. We here, for the first time, confirmed that GPR147 was expressed in the ovarian granulosa cells of mice. Our findings suggested that and RFRP-3 regulates the granulosa cell function through the ERK signaling pathway, which will lay the foundation for uncovering molecular mechanisms by which RFRP-3 regulates follicle development in future.


Assuntos
Neuropeptídeos , Progesterona , Receptores de Neuropeptídeos , Feminino , Camundongos , Animais , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Progesterona/farmacologia , Células da Granulosa , Apoptose
3.
Fertil Steril ; 121(2): 323-333, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-37995798

RESUMO

OBJECTIVE: To study biomarkers to develop a novel diagnosis model for endometriosis and validate it using clinical samples. DESIGN: We used publicly available data sets and weighted gene coexpression network analysis to identify differentially expressed genes. Ten machine learning algorithms were used to develop an integrative model for predicting endometriosis. The accuracy and robustness of the model were validated using data sets and clinical samples. SETTING: Department of Obstetrics and Gynecology, Tangdu Hospital, Air Force Medical University, Xi'an, Shaanxi, China. PATIENT(S): The study included clinical patients between the ages of 20 and 40 years who required laparoscopic surgery and who had not undergone hormone therapy within the previous 3 months. All the healthy individuals had given birth to a child at least once in their lives. Patients with inflammatory conditions, malignant diseases, immune diseases, myoma, or adenomyosis were excluded. Paraffin blocks of the samples were collected (case, n = 5; control, n = 5). Blood samples of 58 individuals were collected (case, n = 28; control, n = 30). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The areas under the receiver operator characteristic curve of our diagnostic model were measured for data sets and clinical samples. Multiplex immunohistochemical staining and real-time quantitative polymerase chain reaction assays were used for the validation of the model from tissue slides and peripheral blood samples. RESULT(S): A nine-gene panel endometriosis messenger RNA score (EMScore), was constructed to distinguish the patients with endometriosis from healthy individuals using algorithms. The EMScore accurately predicted endometriosis, and the areas under the receiver operator characteristic curve of our diagnostic model were 0.920, and 0.942 for tissue and blood samples, respectively. Moreover, the EMScore outperformed other acknowledged signatures for predicting endometriosis across seven clinical cohorts. Overall, the EMScore constitutes a sensitive and specific noninvasive diagnostic method for endometriosis. CONCLUSION(S): We developed the EMScore, a novel model that can aid in the diagnosis of endometriosis using peripheral blood samples. This study will contribute to the development of improved clinical noninvasive and sensitive diagnostic tools for endometriosis. These nine genes might be potential target molecules for treating endometriosis.


Assuntos
Endometriose , Laparoscopia , Feminino , Humanos , Biomarcadores , China , Endometriose/diagnóstico , Endometriose/genética , Adulto Jovem , Adulto
4.
Front Vet Sci ; 9: 997294, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36337204

RESUMO

Theileria annulata schizont-infected host cells in culture in vitro show unlimited proliferation similar to tumor cells; thus far, T. annulata and T. parva are the only eukaryotes that have been found to transform mammalian cells (immortalized). The transformation of these cells is reversible; when the parasite is eliminated in transformed cells by buparvaquone (BW720c), the host cells show normal growth and apoptosis. TFG is a tropomyosin-receptor kinase fused gene that is conserved among many species and is an important proto-oncogene. In this study, the bovine TFG gene was amplified by PCR from the cDNA of T. annulata schizont-transformed cells, cloned into the pGEX-4T-1 vector and expressed in Escherichia coli BL21 (DE3). After purification, the fusion protein was injected into rabbits to produce polyclonal antibodies. Using T. annulata-transformed cells together with BW720c treatment to kill the parasite, we aimed to identify changes in TFG gene expression by real-time PCR and Western blotting. The results showed that the bovine TFG gene was ~582 bp in size; SDS-PAGE analysis showed that the fusion protein was expressed in BL21 (DE3) cells with a molecular mass of 48 kD, and Western blotting indicated that the polyclonal antibodies could react with bovine TFG proteins from T. annulata-transformed cells and showed high specificity. Compared with that in the control group, the transcription level of the host TFG gene decreased significantly in the BW720c test group, and the expression of host tumor-related TFG protein decreased sharply after 72 h of drug treatment, suggesting that the TFG protein expression in transformed cells was directly related to T. annulata. This finding laid a foundation for further study on the interaction between T. annulata and host cells.

5.
Parasit Vectors ; 15(1): 356, 2022 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-36199104

RESUMO

BACKGROUND: When Theileria annulata infects host cells, it undertakes unlimited proliferation as tumor cells. Although the transformed cells will recover their limited reproductive characteristics and enter the apoptosis process after treatment with buparvaquone (BW720c), the metabolites and metabolic pathways involved are not clear. METHODS: The transformed cells of T. annulata were used as experimental materials, and the buparvaquone treatment group and DMSO control group were used. Qualitative and quantitative analysis was undertaken of 36 cell samples based on the LC-QTOF platform in positive and negative ion modes. The metabolites of the cell samples after 72 h of drug treatment were analyzed, as were the different metabolites and metabolic pathways involved in the BW720c treatment. Finally, the differential metabolites and metabolic pathways in the transformed cells were found. RESULTS: A total of 1425 metabolites were detected in the negative ion mode and 1298 metabolites were detected in the positive ion mode. After drug treatment for 24 h, 48 h, and 72 h, there were 56, 162, and 243 differential metabolites in negative ion mode, and 35, 121, and 177 differential metabolites in positive ion mode, respectively. These differential metabolites are mainly concentrated on various essential amino acids. CONCLUSION: BW720c treatment induces metabolic disturbances in T. annulata-infected cells by regulating the metabolism of leucine, arginine, and L-carnitine, and induces host cell apoptosis.


Assuntos
Theileria annulata , Theileria , Theileriose , Animais , Arginina/uso terapêutico , Carnitina/uso terapêutico , Bovinos , Dimetil Sulfóxido/uso terapêutico , Leucina/uso terapêutico , Naftoquinonas , Theileriose/tratamento farmacológico
6.
Front Oncol ; 12: 988578, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36203450

RESUMO

Purpose: High-grade serous ovarian cancer (HGSOC) remains the most lethal female cancer due to metastasis. CircRNAs are recently identified to be modified by N6-methyladenosine (m6A) in many cells. However, the significance of m6A-modified circular RNAs (circRNAs) has not been elucidated in HGSOC peritoneal metastasis. Here, we aimed to investigate the participation and potential functions of m6A-modified circRNAs in HGSCO peritoneal metastasis. Methods: Cancerous tissues were collected from the in situ and the peritoneal metastasis lesions of HGSCO patients. M6A-tagged circRNAs were identified by m6A-modified RNA immunoprecipitation sequencing (m6A-RIP-seq). Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to predict the potential functions of the m6A-modified circRNAs. Results: For the m6A-modified circRNAs, 259 were upregulated and 227 were downregulated in the peritoneal metastasis than in the situ lesions of HGSCO patients. For the m6A peaks, 1541 were upregulated and 1293 were downregulated in the peritoneal metastasis than in the in situ lesions of HGSCO patients. For the differential expressed circRNAs, 1911(19.6%) were upregulated and 2883(29.6%) were downregulated in the peritoneal metastasis than in the in situ lesions of HGSCO patients. The upregulated m6A-modified circRNAs were associated with the HIF-1 signaling. The downregulated m6A-modified circRNAs were associated with the MAPK signaling. Conclusions: This work firstly identified the transcriptome-wide map of m6A-modified circRNAs in peritoneal metastasis of HGSCO. Our findings provided novel evidences about the participation of m6A-modified circRNAs via HIF-1 and MAPK signaling and a new insight in molecular target of HGSCO peritoneal metastasis.

7.
Dis Markers ; 2022: 2751696, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35371343

RESUMO

Objective: To detect the expression levels of microRNA-200a/b (miR-200a/b) in tumor tissues and serum of patients with epithelial ovarian cancer (EOC) and to explore its clinical significance. Methods: A retrospective selection of 30 cases of benign ovarian disease or healthy physical examination (control group) and 55 cases of EOC patients. Real-time quantitative PCR was used to detect the expression level of miR-200a/b in tumor tissues and serum, and the miR-200a/b expresses relevance in the two types of samples were evaluated at the same time. Receiver operating characteristic curve (ROC) and Kaplan-Meier survival analysis were used to evaluate the diagnostic value of miR-200a/b expression and its influence on prognosis, respectively. Results: The serum and tissue miR-200a/b expression levels in EOC patients were higher than those in the control group (P < 0.001), and there was a significant positive correlation between serum and tissue miR-200a/b expression (R 2 = 0.9419, P < 0.001 and R 2 = 0.9605, P < 0.001). ROC analysis showed that the expression of serum miR-200a/b can distinguish EOC patients from the control group. In addition, there were significant differences in the TNM stage, tumor differentiation, and lymph node metastasis between the miR-200a/b high- and low-expression groups (P < 0.05). Kaplan-Meier survival analysis found that the overall survival and disease-free survival of patients with high miR-200a/b expression were shorter than those of patients with low miR-200a/b expression (P < 0.05). Conclusion: Upregulation of miR-200a/b expression is a common molecular event in EOC patients, and miR-200a/b can be used as a noninvasive biomarker for the diagnosis and prognosis of EOC.


Assuntos
Carcinoma Epitelial do Ovário , MicroRNAs , Neoplasias Epiteliais e Glandulares , Neoplasias Ovarianas , Biomarcadores Tumorais/genética , Carcinoma Epitelial do Ovário/diagnóstico , Carcinoma Epitelial do Ovário/genética , Feminino , Humanos , MicroRNAs/genética , Neoplasias Epiteliais e Glandulares/diagnóstico , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/genética , Prognóstico , Estudos Retrospectivos
8.
Reprod Sci ; 27(7): 1465-1476, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-31997258

RESUMO

The objective of this study was to determine if mouse bone marrow-derived mesenchymal stem cells (BMMSCs) ameliorate preterm birth and perinatal brain injury induced by intrauterine inflammation (IUI). A mouse model of IUI-induced perinatal brain injury at embryonic (E) day 17 was utilized. BMMSCs were derived from GFP-transgenic mice and phenotypically confirmed to be CD44+, Sca-1+, CD45-, CD34-, CD11b-, and CD11c- by flow cytometry and sorted by fluorescence-activated cell sorting (FACS). Dams were assigned to four groups: phosphate-buffered saline (PBS) + PBS, PBS + BMMSCs, lipopolysaccharide (LPS) + PBS, and LPS + BMMSCs. Following maternal IUI, there was a significant increase in CD8+ T cells in the placentas. Maternally administered BMMSCs trafficked to the fetal side of the placenta and resulted in significantly decreased placental CD8+ T cells. Furthermore, fetal trafficking of maternally administered BMMSCs correlated with an improved performance on offspring neurobehavioral testing in LPS + BMMSC group compared with LPS + PBS group. Our data support that maternal administration of BMMSCs can alleviate perinatal inflammation-induced brain injury and improve neurobehavioral outcomes in the offspring via CD8+ T cell immunomodulation at the feto-placental interface.


Assuntos
Lesões Encefálicas/metabolismo , Lesões Encefálicas/prevenção & controle , Linfócitos T CD8-Positivos/metabolismo , Modelos Animais de Doenças , Transplante de Células-Tronco Mesenquimais/métodos , Útero/metabolismo , Animais , Animais Recém-Nascidos , Medula Óssea/fisiologia , Lesões Encefálicas/etiologia , Células Cultivadas , Feminino , Inflamação/complicações , Inflamação/metabolismo , Inflamação/patologia , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Transgênicos , Gravidez , Nascimento Prematuro/etiologia , Nascimento Prematuro/metabolismo , Nascimento Prematuro/prevenção & controle
9.
Acta Biomater ; 78: 274-284, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-30071352

RESUMO

Fluorogenic labeling is a potential technique in biology that allows for direct detection and tracking of cells undergoing various biological processes. Compared to traditional genetic modification approaches, labeling cells with nanoparticles has advantages, especially for the additional safety they provide by avoiding genomic integration. However, it remains a challenge to determine whether nanoparticles interfere with cell traits and provide long-lasting signals in living cells. We employed an amphiphilic fluorophore-derived nanoparticle (denoted by TPE-11) bearing a tetraphenylethene (TPE) moiety and two ionic heads; this nanoparticle has an aggregation-induced emission (AIE) effect and the ability to self-assemble. TPE-11 exhibited the property of higher or longer fluorescence intensities in cell imaging than the other two nanomaterials under the same conditions. We used this nanomaterial to label human embryonic stem (hES) cells and monitor their differentiation. Treatment with low concentrations of TPE-11 (8.0 µg/mL) resulted in high-intensity labeling of hES cells, and immunostaining analysis and teratoma formation assays showed that at this concentration, their pluripotency remained unaltered. TPE-11 nanoparticles allowed for long-term monitoring of hES cell differentiation into neuron-like cells; remarkably, strong nanoparticle signals were detected throughout the nearly 40-day differentiation process. Thus, these results demonstrate that the TPE-11 nanoparticle has excellent biocompatibility for hES cells and is a potential fluorogen for labeling and tracking the differentiation of human pluripotent stem cells. STATEMENT OF SIGNIFICANCE: This study uses a nanoparticle-based approach to label human embryonic stem (hES) cells and monitor their differentiation. hES cells are distinguished by two distinctive properties: the state of their pluripotency and the potential to differentiate into various cell types. Thus, these cells will be useful as a source of cells for transplantation or tissue engineering applications. We noticed the effect of aggregation-induced emission, and the ability to self-assemble could enhance the persistence of signals. Treatment with low concentrations of TPE-11 nanoparticles showed high-intensity labeling of hES cells, and immunostaining analysis and teratoma formation assays showed that at this concentration, their pluripotency remained unaltered. Additionally, these nanoparticles allowed for long-term monitoring of hES cell differentiation into neuron-like cells lasting for 40 days.


Assuntos
Diferenciação Celular , Corantes Fluorescentes/química , Células-Tronco Embrionárias Humanas/citologia , Nanopartículas/química , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Tensoativos/química , Biomarcadores/metabolismo , Morte Celular , Fluorescência , Células HeLa , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Imageamento Tridimensional , Neurônios/metabolismo , Células-Tronco Pluripotentes/metabolismo , Teratoma/patologia
10.
Int J Clin Exp Pathol ; 11(12): 5901-5908, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-31949677

RESUMO

The aberrant expression of microRNAs (miRNAs) underlies a series of human diseases, including ovarian cancers. In our previous study, we found that miR-129-1-3p and miR-129-2-3p levels were significantly decreased in serous ovarian cancer via a microarray and quantitative PCR. In this study, we investigated the pathological role of miR-129-3p in an ovarian cancer cell line, SKOV3 cells. The results demonstrated that miR-129-3p overexpression distinctively inhibited the proliferation, migration, and invasion of ovarian cancer cells. The regulator of cell cycling, BZW1 (basic leucine zipper and W2 domains 1), was validated as a novel direct target of miR-129-3p. Specifically, miR-129-3p bound directly to the 3' untranslated region of BZW1 and suppressed its expression. Our results indicate that miR-129-3p serves as a tumor suppressor by targeting BZW1 in ovarian cancer cells and highlight that the restoration of miR-129-3p might be a novel therapeutic strategy for ovarian cancer.

11.
Reprod Sci ; 25(8): 1175-1185, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29017418

RESUMO

Fetal brain injury induced by intrauterine inflammation is a major risk factor for adverse neurological outcomes, including cerebral palsy, cognitive dysfunction, and behavioral disabilities. There are no adequate therapies for neuronal protection to reduce fetal brain injury, especially new strategies that may apply promptly and conveniently. In this study, we explored the effect of maternal glucose administration in a mouse model of intrauterine inflammation at term. Our results demonstrated that maternal glucose supplementation significantly increased survival birth rate and improved the neurobehavioral performance of pups exposed to intrauterine inflammation. Furthermore, we demonstrated that maternal glucose administration improved myelination and oligodendrocyte development in offspring exposed to intrauterine inflammation. Though the maternal blood glucose concentration was temporally prevented from decrease induced by intrauterine inflammation, the glucose concentration in fetal brain was not recovered by maternal glucose supplementation. The adenosine triphosphate (ATP) level and autophagy in fetal brain were regulated by maternal glucose supplementation, which may prevent dysregulation of cellular metabolism. Our study is the first to provide evidence for the role of maternal glucose supplementation in the cell survival of fetal brain during intrauterine inflammation and further support the possible medication with maternal glucose treatment.


Assuntos
Autofagia , Lesões Encefálicas/embriologia , Lesões Encefálicas/prevenção & controle , Encéfalo/efeitos dos fármacos , Encéfalo/embriologia , Corioamnionite/prevenção & controle , Glucose/administração & dosagem , Trifosfato de Adenosina/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/fisiopatologia , Lesões Encefálicas/induzido quimicamente , Corioamnionite/induzido quimicamente , Modelos Animais de Doenças , Feminino , Hipoglicemia/tratamento farmacológico , Lipopolissacarídeos/administração & dosagem , Bainha de Mielina/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Gravidez
12.
Cell Physiol Biochem ; 44(4): 1435-1444, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29186714

RESUMO

BACKGROUND: Despite the great potential of utilizing human embryonic stem cells (hESCs)-derived cells as cell source for transplantation, these cells were often rejected during engraftment by the immune system due to adaptive immune response. METHODS: We first evaluated HLA-G expression level in both hESCs and differentiated progenitor cells. After that, we generated modified hESC lines that over-express HLA-G1 using lentiviral infection with the construct contains both HLA-G1 and GFP tag. The lentivirus was first produced by co-transfecting HLA-G1 expressing lentiviral vector together with packaging vectors into packaging cell line 293T. Then the produced virus was used for the infection of selected hESC lines. We characterized the generated cell lines phenotype, including pluripotency and self-renewal abilities, as well as immune tolerance ability by mixed lymphocyte reaction (MLR) and cytotoxicity assays. RESULTS: Although the hESCs do not express high levels of HLA-G1, over-expression of HLA-G1 in hESCs still retains their stem cell characteristics as determined by retaining the expression levels of OCT4 and SOX2, two critical transcriptional factors for stem cell function. Furthermore, the HLA-G1 overexpressing hESCs retain the self-renewal and pluripotency characteristics of stem cells, which can differentiate into different types of cells, including pigment cells, smooth muscle cells, epithelia-like cells, and NPCs. After differentiation, the differentiated cells including NPCs retain the high levels of HLA-G1 protein. In comparison with conventional NPCs, these HLA-G1 positive NPCs have enhanced immune tolerance ability. CONCLUSIONS: Ectopic expression of HLA-G1, a non-classical major histocompatibility complex class I (MHC I) antigen that was originally discovered involving in engraftment tolerance during pregnancy, can enhance the immunological tolerance in differentiated neural progenitor cells (NPCs). Our study shows that stably overexpressing HLA-G1 in hESCs might be a feasible strategy for enhancing the engraftment of NPCs during transplantation.


Assuntos
Antígenos HLA-G/metabolismo , Tolerância Imunológica/fisiologia , Células-Tronco Neurais/metabolismo , Diferenciação Celular , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Antígenos HLA-G/genética , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Cariótipo , Lentivirus/genética , Células-Tronco Neurais/citologia , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Teratoma/patologia , Transfecção
13.
Korean J Parasitol ; 54(1): 39-46, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26951977

RESUMO

Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (ß-actin), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata.


Assuntos
Células/parasitologia , Interações Hospedeiro-Parasita/genética , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Transdução de Sinais/genética , Theileriose/fisiopatologia , Animais , Linfócitos B/parasitologia , Bovinos , Linhagem Celular , Células Cultivadas , Perfilação da Expressão Gênica , Reprodutibilidade dos Testes , Theileria annulata/fisiologia
14.
Epilepsia ; 54(11): e155-8, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24116395

RESUMO

To search for a method for treatment of bilateral temporal lobe epilepsy (BTLE), we report one patient with BTLE experienced bilateral stereotactic radiofrequency amygdalohippocampectomy (SAHE). Neuropsychological examinations were performed before and 5 days, and 6, 18, and 48 months after operation. No seizure occurred in the follow-up time, and no long-term memory and intelligence deficits were found except for a transient decline of the scores immediately after operation. Because severe damage of memory could be caused by bilateral resection surgery, bilateral SAHE should be considered as a possible approach for the treatment of BTLE. However, further studies with accumulation of cases are needed, especially in the detailed assessment of neuropsychological function.


Assuntos
Tonsila do Cerebelo/cirurgia , Epilepsia do Lobo Temporal/cirurgia , Hipocampo/cirurgia , Memória/fisiologia , Lobo Temporal/cirurgia , Adulto , Eletroencefalografia/métodos , Epilepsia do Lobo Temporal/diagnóstico , Epilepsia do Lobo Temporal/psicologia , Seguimentos , Humanos , Masculino , Testes Neuropsicológicos , Procedimentos Neurocirúrgicos/métodos , Resultado do Tratamento
15.
Biochem Biophys Res Commun ; 434(3): 460-5, 2013 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-23583402

RESUMO

The human leukocyte antigen G (HLA-G) is expressed on the fetal-maternal interface and plays a role in protecting fetal-derived trophoblasts from the maternal immune response, allowing trophoblasts to invade the uterus. However, HLA-G also possesses immune suppressing-independent functions. We found that HLA-G expressing BeWo choriocarcinoma cells increased cell-cell fusion compared to control BeWo cells under forskolin treatment. Regardless of forskolin treatment, the expression of fusogenic gene mRNAs, including syncytin-1, the transcription factor glial cell missing 1 (Gcm1), and beta human chorionic gonadotropin (ß-hCG) were elevated. HLA-G up-regulates ß-hCG production in human choriocarcinoma cells because HLA-G knockdown in JEG-3 cells induces a dramatic decrease in ß-hCG compared with control cells. The defect in ß-hCG production in HLA-G knocked-down cells could not be completely overcome by stimulating hCG production through increasing intracellular cAMP levels. HLA-G expressing cells have increased phosphorylation levels for extracellular signal-regulated kinase1/2 (Erk1/2) in BeWo cells. The Erk1/2 pathway is inactivated after the inhibition of HLA-G expression in JEG-3 cells. Finally, Erk1/2 inhibition was able to suppress the increased hCG production induced by HLA-G expression. Together, these data suggest novel roles for HLA-G in regulating ß-hCG production via the modulation of the Erk1/2 pathway and by inducing trophoblast cell fusion.


Assuntos
Fusão Celular , Coriocarcinoma/imunologia , Gonadotropina Coriônica/biossíntese , Antígenos HLA-G/imunologia , Sistema de Sinalização das MAP Quinases , Trofoblastos/citologia , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Coriocarcinoma/metabolismo , Coriocarcinoma/patologia , Primers do DNA , Imunofluorescência , Técnicas de Silenciamento de Genes , Antígenos HLA-G/genética , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Regulação para Cima
16.
Stem Cells Dev ; 22(7): 1147-58, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23151296

RESUMO

The technology to reprogram human somatic cells back to pluripotency allows the production of patient-specific induced pluripotent stem cells (iPSCs) and holds a great promise for regenerative medicine. Choosing the most suitable cell type for induction and reducing the risk of viral transgene activation, especially oncogene activation, are important for iPSC research. To date, human dermal fibroblasts (HDFs) are the most frequent cell source used for iPSC generation, but they have several limitations. An invasive skin biopsy must be performed to obtain HDFs, and HDFs must be cultured for a prolonged period before they can be used for experiments. Thus, in an effort to develop a suitable source for iPSC studies to avoid the limitations mentioned above, we have here identified stromal cells derived from menstrual blood (MenSCs) as suitable candidates. In the present study, we found that MenSCs can be reprogrammed to pluripotent status by doxycycline-inducible lentiviral transduction of OCT4, SOX2, and KLF4. Additionally, we found that MenSCs have a significantly higher reprogramming efficiency than HDFs. The combination of OCT4 and SOX2 is sufficient to reprogram MenSCs into iPSCs without the use of c-MYC or KLF4. The resulting MenSC-iPSCs showed the same characteristics as human embryonic stem cells with regard to morphology, pluripotent markers, gene expression, and the epigenetic status of pluripotent-cell-specific genes. These cells were able to differentiate into various cell types of all 3 germ layers both in vitro and in vivo. Therefore, MenSCs may be a preferred candidate for generation of iPSCs.


Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas , Ciclo Menstrual/sangue , Células-Tronco Pluripotentes/metabolismo , Adulto , Técnicas de Cultura de Células , Diferenciação Celular/genética , Células Cultivadas , Metilação de DNA , Feminino , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Cariótipo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Regiões Promotoras Genéticas/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
17.
Exp Mol Med ; 44(11): 633-41, 2012 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-22902510

RESUMO

Triptolide, a compound extracted from the traditional Chinese medicine preparation of Tripterygium wilfordii Hook F., has been reported to have anti-inflammatory and anti-cancer activities. However, its effect on ovarian cancer invasion is unknown. We observed that MMP7 and MMP19 expression increased in ovarian cancer tissue. Triptolide treatment inhibited the migration and invasion of ovarian cancer cells SKOV3 and A2780 at the concentration of 15 nM. We also observed that triptolide suppressed MMP7 and MMP19 promoter activity in a dose-dependent manner, down-regulating the expressions of these promoters on mRNA and protein level. Moreover, triptolide enhanced E-cadherin expression in ovarian cancer cells. In vivo, triptolide inhibited tumor formation and metastasis in nude mice, and suppressed MMP7 and MMP19 expression; it also enhanced E-cadherin expression in tumor in a dose-dependent manner. Over expression of MMP7 and MMP19, or suppression of E-cadherin expression partially abolished the inhibitory effect of triptolide on invasion of ovarian cancer cells. To summarize, triptolide significantly inhibited the migration and invasion of ovarian cancer cells by suppression of MMP7 and MMP19 and up-regulation of E-cadherin expression. This study shows that triptolide is a good candidate for the treatment of ovarian cancer and reduction of metastasis.


Assuntos
Antineoplásicos Alquilantes/farmacologia , Caderinas/genética , Cistadenocarcinoma Seroso/tratamento farmacológico , Diterpenos/farmacologia , Metaloproteinase 7 da Matriz/metabolismo , Metaloproteinases da Matriz Secretadas/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Fenantrenos/farmacologia , Animais , Antígenos CD , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cistadenocarcinoma Seroso/patologia , Compostos de Epóxi/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinase 7 da Matriz/genética , Metaloproteinases da Matriz Secretadas/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Paclitaxel/farmacologia , Regiões Promotoras Genéticas , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Reprod Biomed Online ; 25(4): 415-24, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22877943

RESUMO

Human leukocyte antigen (HLA)-G is thought to confer fetal-maternal tolerance and play a crucial role in ensuring a successful pregnancy. There is increasing evidence that HLA-G is regulated at the post-transcriptional level. This study investigated the role of miR-133a in regulating HLA-G expression and the pathogenesis of recurrent spontaneous abortion (RSA). Twelve patients (25-30 years) with RSA at 7 gestational weeks were screened by array-based comparative genome hybridization: 16.7% were found to have an abnormal karyotype and all induced abortion (IA) patients had normal karyotype. The villi of RSA and IA patients with normal karyotype were further screened by miRNA microarrays. Multi-software prediction and real-time PCR confirmed that miR-133a was most likely to bind to HLA-G 3' untranscribed region (UTR). Relevance analysis showed that, compared with IA villi, miR-133a was greatly overexpressed in RSA villi with normal karyotype (P<0.01), but not in abnormal RSA villi. A luciferase reporter assay suggested that miR-133a interacted with HLA-G 3' UTR. Overexpression of miR-133a in JEG-3 cells decreased HLA-G expression at the protein level, with no effect on mRNA. These findings provide strong evidence that miR-133a regulates HLA-G expression by reducing translation and is involved in the pathogenesis of RSA.


Assuntos
Aborto Habitual/metabolismo , Vilosidades Coriônicas/metabolismo , Regulação para Baixo , Antígenos HLA-G/metabolismo , MicroRNAs/metabolismo , Regulação para Cima , Regiões 3' não Traduzidas , Aborto Habitual/genética , Adulto , Linhagem Celular , China , Amostra da Vilosidade Coriônica , Hibridização Genômica Comparativa , Feminino , Genes Reporter , Antígenos HLA-G/genética , Humanos , Cariótipo , MicroRNAs/genética , Proteínas Mutantes/metabolismo , Gravidez , Primeiro Trimestre da Gravidez , Proteínas Recombinantes/metabolismo
19.
Biochem Genet ; 50(9-10): 809-21, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22733180

RESUMO

The goals of this study were to analyze the change in the global gene expression profile of exogenous human leukocyte antigen-G1 (HLA-G1) overexpressed in human embryonic stem (hES) cells and to explore the molecular mechanism by which the overexpression of HLA-G1 modifies immunologic pathways. Microarray and quantitative real-time PCR analyses were performed to quantify the differential expression pattern of HLA-G1 + H1 hES cells. The results showed that HLA-G1 differentially regulated the expression of 425 genes with at least a twofold increase or decrease. These differentially expressed genes were classified into 13 functional groups, including cellular components, biological processes, and molecular functions. The pathways of focal adhesion, the TGF-ß signaling pathway, and the immune response were the most predominantly affected. The synergism of these genes could explain the mechanism of the immunosuppression of HLA-G1 + H1 hES cells. Thus, the expression pattern reflected a broad spectrum of roles of HLA-G1 in hES cells.


Assuntos
Células-Tronco Embrionárias/metabolismo , Antígenos HLA-G/genética , Transcriptoma , Animais , Western Blotting , Diferenciação Celular , Linhagem Celular Tumoral , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/imunologia , Células-Tronco Embrionárias/transplante , Adesões Focais , Regulação da Expressão Gênica , Antígenos HLA-G/imunologia , Antígenos HLA-G/metabolismo , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Camundongos , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Teratoma/metabolismo , Transcrição Gênica , Transfecção , Fator de Crescimento Transformador beta/metabolismo
20.
Brain Res ; 1437: 134-42, 2012 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-22227454

RESUMO

Human embryonic stem (hES) cells have the capacity for self-renewal and exhibit multipotentiality. hES cells have promise for serving as an unlimited source of ideal seed cells for cell transplantation. However, the rejection that occurs between the transplant recipient and the transplanted cell poses a major challenge for therapeutic transplantation. This study was designed to devise methods to enhance immune tolerance in cell therapy. We established an hES cell line that could stably express human leukocyte antigen-G1 (HLA-G1). The established HLA-G1-H1 hES cells still retained all the characteristics of normal human embryonic stem cells. By using the SDIA method, we induced dopaminergic (DA) neurons by coculturing HLA-G1-H1 hES cells with the mouse stromal cell line PA6. Tyrosine hydroxylase (TH)+neurons were detected on the 10th day of differentiation, and 70% of the HLA-G1-H1 hES cells were TH+mature DA neurons because the differentiation time was only 3 weeks. Cells that had been differentiating for different periods of time still expressed HLA-G1, and these differentiated DA neurons released dopamine and other catecholamines in response to K+ depolarization as measured by HPLC. After careful study, we found that HLA-G1-H1 hES cells are capable of inhibiting the proliferation of mixed T-lymphocytes. DA neurons derived from HLA-G1-H1 hES attenuated the release of proinflammatory cytokines IL-1ß and IFN-γ from lipopolysaccharide (LPS)-stimulated BV2 microglia. The efficiency of inhibition was significant and dose-dependent. This method might be used to treat Parkinson's patients via cell transplantation.


Assuntos
Neurônios Dopaminérgicos/imunologia , Células-Tronco Embrionárias/imunologia , Antígenos HLA-G/biossíntese , Tolerância Imunológica , Animais , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Regulação da Expressão Gênica/imunologia , Inibidores do Crescimento/fisiologia , Células HEK293 , Antígenos HLA-G/fisiologia , Humanos , Teste de Cultura Mista de Linfócitos , Camundongos
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