RESUMO
To investigate the effect and mechanism of serum amyloid A (SAA) on the expression of scavenger receptor class B type I (SR-BI) and inflammatory response in THP-1 macrophages, the human THP-1 cells were treated with SAA and p38-MAPK agonist (anisomycin) or p38-MAPK inhibitor (SB203580). Then, the expressions of SR-BI, phosphorylated p38-MAPK and inflammatory factors (MCP-1, TNF-α, IL-1ß) were examined by real-time quantitative PCR, Western blotting and ELISA, respectively. The results showed that, compared with control group, SAA increased the levels of inflammatory factors (MCP-1, TNF-α, IL-1ß), down-regulated the expressions of SR-BI, and up-regulated the expression of phosphorylated p38-MAPK protein in a concentration- and time-dependent manner in THP-1 cells (P < 0.05). After treatment with SAA and p38-MAPK agonist (anisomycin) in THP-1 cells, the expression of SR-BI was down-regulated, and the levels of inflammatory factors and phosphorylated p38-MAPK protein expression were increased, compared with the group only treated by SAA (P < 0.05). In contrast, the SR-BI expression was up-regulated, whereas inflammatory factors and phosphorylated p38-MAPK protein expressions were decreased after the cells were treated with SAA and p38-MAPK inhibitor (SB203580) (P < 0.05). The results suggest that SAA-promoted inflammatory response in THP-1 macrophages may be through the phosphorylation of p38-MAPK and inhibition of SR-BI expression.
Assuntos
Sistema de Sinalização das MAP Quinases , Macrófagos , Linhagem Celular , Quimiocina CCL2 , Humanos , Inflamação , Interleucina-1beta , Fosforilação , Proteína Amiloide A Sérica , Fator de Necrose Tumoral alfa , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
AIMS: The goal of cell transcription for treatment of diabetes is to generate surrogate ß-cells from an appropriate cell line. However, the induced replacement cells have showed less physiological function in producing insulin compared with normal ß-cells. METHODS: Here, we report a procedure for induction of insulin-producing cells (IPCs) from bone marrow murine mesenchymal stem cells (BM-mMSCs). These BM-mMSCs have the potential to differentiate into insulin-producing cells when a combination of PDX-1 (pancreatic and duodenal homeobox-1), NeuroD1 (neurogenic differentiation-1), and MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homolog A) genes are transfected into them and expressed in these cells. RESULTS: Insulin biosynthesis and secretion were induced in mMSCs into which these three genes have been transfected and expressed. The amount of induced insulin in the mMSCs which have been transfected with the three genes together is significantly higher than in those mMSCs that were only transfected with one or two of these three genes. Transplantation of the transfected cells into mice with streptozotocin-induced diabetes results in insulin expression and the reversal of the glucose challenge. CONCLUSIONS: These findings suggest major implications for cell replacement strategies in generation of surrogate ß-cells for the treatment of diabetes.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células da Medula Óssea/citologia , Proteínas de Homeodomínio/metabolismo , Insulina/metabolismo , Fatores de Transcrição Maf Maior/metabolismo , Células-Tronco Mesenquimais/citologia , Proteínas do Tecido Nervoso/metabolismo , Transativadores/metabolismo , Adenoviridae/metabolismo , Animais , Diferenciação Celular , Transplante de Células , Diabetes Mellitus/metabolismo , Células HEK293 , Humanos , Imuno-Histoquímica/métodos , Células Secretoras de Insulina/citologia , Camundongos , Camundongos Endogâmicos C57BL , TransfecçãoRESUMO
OBJECTIVE: To evaluate the effects of insulin gene transcription regulators PDX-1, NeuroD1 and MafA on the differentiation of bone marrow mesenchymal stem cells (mMSCs) into insulin-producing cells. METHODS: Murine mMSCs were isolated, cultured and expanded. The base sequences of transcription factors PDX-1, NeuroD1 and MafA were obtained by total gene synthesis and the recombinant adenovirus vectors harboring target genes constructed and transfected into packaging cell line 293A. mMSCs were infected with adenovirus separately or together, and then differentiated in vitro into insulin-producing cells. Reverse transcription-polymerase chain reaction (RT-PCR) was utilized to detect insulin gene expression, immunofluorescence for identifying the presence of insulin protein and insulin enzyme-linked immunosorbent assay (ELISA) for evaluating the secretory volume of insulin. RESULTS: The differentiation extent of mMSCs into ß-cell was analyzed. The ß-cell-specific transcriptional regulators and insulin gene were expressed in mMSCs after transfection. Immunofluorescent analyses revealed the activated expression of insulin in the cytoplasm of differentiated cells. A significant content of insulin was released in these cells in response to a certain concentrations of glucose stimulation. The insulin content of mMSCs infected with a combination of three transcription factors was significantly higher than that of the control group [(112.84 ± 9.67) mU/L vs (1.60 ± 0.22) mU/L, P < 0.05]. CONCLUSION: After modification by transcriptional factors PDX-1, NeuroD1 and MafA, mMSCs can secrete insulin through starting endogenous insulin gene transcription.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular , Células Secretoras de Insulina/citologia , Células-Tronco Mesenquimais/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Expressão Gênica , Vetores Genéticos , Proteínas de Homeodomínio/genética , Fatores de Transcrição Maf Maior/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Organismos Geneticamente Modificados , Transativadores/genéticaRESUMO
OBJECTIVE: To explore the altered expressions of embryonic stem-related genes Oct4 and Nanog in pancreatic cancer stem cells (CSCs). METHODS: The uni-cell suspension of human pancreatic cancer cell line PANC-1 was prepared and incubated with CD24 and CD44 antibodies. Flow cytometer was used to separate CD24(+)CD44(+) pancreatic cancer stem cells. Tumor cell spheres were observed under light microscope. Then CSCs were induced to differentiate with 10% fetal bovine serum and the expressions of CD24 and CD44 re-evaluated by flow cytometer. Finally the cells were divided into 2 groups, group 1: CD24(+)CD44(+) and group 2: non-separated group. RT-PCR (reverse transcription-polymerase chain reaction) and Q-PCR (quantitative-polymerase chain reaction) were used to examine the transcriptions of Oct4 and Nanog in CSCs. The immunofluorescence was employed to examine the expressions of Oct4 and Nanog. Chemo-sensitivity to gemcitabine was determined by CCK8 assay in each group. RESULTS: About 1%-3% CD24(+)CD44(+) CSCs were separated from cell line PANC-1. The sorted cells were cultured in a stem cell culture medium to observe the spheroid-forming capacity. And they showed a higher colony-forming efficiency than the unsorted cells [(122 ± 6), P < 0.05]. When cultured in medium with serum, these cells gradually returned to the status of parental cells with a low expression of CD24 and CD44. Both Oct4 and Nanog were highly expressed in CD24(+)CD44(+) stem cells. And the CD24(+)CD44(+) subgroup demonstrated a higher resistance to gemcitabine. CONCLUSION: Subpopulation cells CD44(+)CD24(+) have the properties of tumor stem cells. The up-regulated levels of Oct4 and Nanog may be highly correlated with the multi-potency and a higher drug-resistance of pancreatic CSCs.
Assuntos
Proteínas de Homeodomínio/genética , Células-Tronco Neoplásicas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Neoplasias Pancreáticas/metabolismo , Antígeno CD24/metabolismo , Linhagem Celular Tumoral , Citometria de Fluxo , Expressão Gênica , Humanos , Receptores de Hialuronatos/metabolismo , Proteína Homeobox NanogRESUMO
OBJECTIVE: To evaluate the effect of treatment of type 1 diabetes by transplantation of bone-derived stem cells expressing human insulin gene. METHODS: Murine bone marrow-derived stem cells expressing green fluorescent protein (GFP-mMSCs) were isolated from 4/6-week-old GFP mice and transfected with a recombinant retrovirus-murine stem cell virus (MSCV) encoding human insulin gene, thus constructing the GFP-mMSCs-MCV-insulin. 16 C57BL/6J mice were injected with streptozotocin so as to establish models of type 1 diabetes and then randomly divided into 4 equal groups: Group A, undergoing injection into the liver with GFP-mMSC-MCV-insulin 1 week after the establishment of the model, Group B, undergoing intrahepatic transplantation of the GFP-mMSCs transfected with blank vector, Group C, undergoing intrahepatic transplantation of untransfected GFP-mMSCs, and Group D, undergoing intrahepatic transplantation of phosphate-buffered saline (PBS). Another 4 normal mice were used as controls and underwent intrahepatic transplantation of PBS too. After the transplantation the blood glucose, serum insulin, and body weight were detected everyday. 6 weeks later immunohistochemistry was used to detect the expression of human insulin in the mice liver tissues. RESULTS: The body weight of Group A increased by 6% within 6 weeks after treatment, and the average blood glucose level 7 d and 42 d after transplantation were (10.4 +/- 2.8) mmol/L and (6.5 +/- 0.9) mmol/L respectively, both significantly lower than those of Group D [(26.8 +/- 2.5) mmol/L and (25.4 +/- 4.1) mmol/L respectively, both P < 0.05]. Immunohistochemistry showed secretion of human insulin in serum and liver. CONCLUSION: The clinical manifestations of diabetes can be relieved effectively by intrahepatic transplantation of mMSCs expressing human insulin gene. This study implies a novel approach of gene therapy for type 1 diabetes.