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1.
Article in Chinese | WPRIM | ID: wpr-905829

ABSTRACT

Objective:The purpose of this article was to observe the effect of modified Shengjiangsan on podocyte apoptosis in membranous nephropathy (MN) rats, to explore the molecular mechanism of its treatment of MN and to provide experimental basis for its clinical application. Method:The MN rat model was established by injection of cationic bovine serum albumin into the tail vein of rats. The successfully modeled rats were then randomly divided into model group (equal volume of normal saline), modified Shengjiangsan group (27.3 g·kg<sup>-1</sup>) and benazepril group (10 mg·kg<sup>-1</sup>), with corresponding drug dosage once a day for 4 weeks of continuous intervention. After drug administration, the 24-hour urine protein (UTP) was detected. Real time fluorescent quantitative polymerase chain reaction (Real-time PCR) and immunohistochemical (IHC) methods were used to detect Podocalyxin, Nephrin, Podocin, Synaptopodin mRNA and protein expression levels in rat kidney tissue. terninal-deoxynucleoitidyl transferase medsated nick and labeling (TUNEL) method was used to detect cell apoptosis rate in rat kidney tissue, and Western blot was used to detect Notch1, Hes1, B lymphoblastoma-2 (Bcl-2) associated X protein (Bax), and Bcl-2 protein expression levels in rat kidney tissue. Result:Compared with the normal group, UTP in the model group increased significantly, renal tissue cell apoptosis increased significantly, podocyte marker proteins podocalyxin, Nephrin, Podocin, Synaptopodin mRNA and protein expression levels decreased significantly, and Notch1, Hes1, Bax protein expression increased significantly, and Bcl-2 protein expression was significantly reduced(<italic>P</italic><0.05). Compared with the model group, UTP levels in MN rats were significantly reduced in modified Shengjiangsan and benazepril groups, with reduced rate of renal cell apoptosis, increased mRNA and protein expression levels of podocalyxin, Nephrin, Podocin, and Synaptopodin in renal tissue, decreased Notch1, Hes1, Bax protein expression, and increased Bcl-2 protein expression(<italic>P</italic><0.05). Conclusion:Modified Shengjiangsan can inhibit the Notch signaling pathway, reduce the apoptosis of rat kidney tissue podocytes, and reduce the kidney injury of MN rats.

2.
Article in Chinese | WPRIM | ID: wpr-688040

ABSTRACT

<p><b>OBJECTIVE</b>To evaluate the effects of tumor necrosis factor-α (TNF-α) on osteogenic differentiation and Notch signaling pathway of periodontal ligament stem cells (PDLSCs) and to investigate the regulatory role of Notch signaling pathway on the osteogenic differentiation of PDLSCs under the influence of TNF-α.</p><p><b>METHODS</b>PDLSCs were obtained through enzyme digestion and tissue block method. The expression levels of stem cell surface markers CD105, CD90, CD146, CD45, and CD31 were detected by fluorescence activated cell sorter (FACS). PDLSCs were divided into experimental (10 ng·mL⁻¹ TNF-α) and control groups (0 ng·mL⁻¹ TNF-α). The proliferation ability of PDLSCs was detected using cell counting kit-8 (CCK-8). The effect of TNF-α on the osteogenic ability of PDLSCs were tested by measuring alkaline phosphatase (ALP) activity and conducting alizarin red staining and quantitative real-time polymerase chain reaction (PCR). We tested Notch signal pathway receptors Notch1, Notch2, ligand JAG1, JGA2, and downstream gene Hes-1. Changes in DLL1 expression were detected by quantitative real-time PCR.</p><p><b>RESULTS</b>FACS profiling showed that PDLSCs were strongly positive for CD105, CD90, and CD146 but negative for CD45 and CD31. CCK-8 results showed that TNF-α could promote the proliferation of PDLSCs (P<0.05). ALP activity in the experimental group was lower than that in the control group (P<0.05). Alizarin red staining showed that the experimental group had decreased mineralized nodules as compared with the control group. Quantitative real-time PCR results showed that the mRNA expression of osteogenic marker genes cementum attachment protein (CAP), osteopontin (OPN), and Runt-related transcription factor 2 (Runx2) significantly decreased in the experimental group as compared with those in the control group (P<0.05). The expression levels of Notch1, Notch2, JAG1, JGA2 and Hes-1 were significantly decreased (P<0.05), whereas those of Notch3 and DLL1 were increased in Notch signaling pathway-related molecules (P<0.05).</p><p><b>CONCLUSIONS</b>TNF-α can promote PDLSCs proliferation and inhibit bone differentiation and Notch signaling pathway expression, indicating that the Notch signaling pathway regulates PDLSCs osteogenic differentiation.</p>

3.
Chinese Journal of Immunology ; (12): 261-266, 2018.
Article in Chinese | WPRIM | ID: wpr-702713

ABSTRACT

Objective:To evaluate the effect and mechanism of davallia mariesil flavones (DMF) improving osteoporosis via Notch signaling pathway.Methods:(1) 120 cases patients with osteoporosis in our hospital from January 2016 to January 2017 were analyzed,and divided into experiment group (60 cases) and control group (60 cases) using digital random grouping method.Experiment group was treated with DMF combined with D-calfor.Control group was treated with D-calfor only.After treatment, the levels of serum calcium,serum phosphor,TNF-α,MCP-1 and IL-6 in serum were detected to evaluate the clinical efficacy.(2) Bone marrow stromal cells were separated and cultivate.NC group:DMEM(contain 10% FBS).RA group:RA(0.4 mmol/L).DMF+RA group:DMEM(contain DMF)+RA(0.4 mmol/L).Jaggedl+RA group:Jaggedl(1 000 μg/L)+RA(0.4 mmol/L).Jaggedl+DMF+RA group:Jaggedl(1 000 μg/L)+DMEM(contain DMF)+RA(0.4 mmol/L).DAPT+RA group:DAPT(16 μmol/L)+RA(0.4 mmol/L). DAPT+DMF+RA group:DAPT(16 μmol/L)+DMEM(contain DMF)+RA(0.4 mmol/L).Western blotting assays and PCR were performed to assess mRNA and protein levels of Notch-1,Hes-1.Results: (1) In clinical study,the effective rate in treatment group was obviously higher than control group (91.67%>76.67%,P<0.05).The levels of serum calcium and serum phosphor in the experiment group was higher than in the control group (P<0.05).The levels of TNF-α,MCP-1 and IL-6 in the experiment group was lower than in the control group (P<0.05).(2) In experimental study,compared with the RA group,the expressions of Notch-1,Hes-1 mRNA and protein were upregulated in Jaggedl+RA group,but were downregulated in DAPT+RA group,DMF+RA group (P<0.05). Compared with the Jaggedl+RA group,the expressions of Notch-1,Hes-1 mRNA and protein were downregulated in Jaggedl+DMF+RA group (P<0.05).Compared with the DAPT+RA group,the expressions of Notch-1,Hes-1 mRNA and protein were downregulated in DAPT+DMF+RA group (P<0.05).Conclusion:DMF could improve the condition of osteoporosis.The mechanism may be associated with inhibiting the Notch signaling pathway by DMF.

4.
Article in Chinese | WPRIM | ID: wpr-750330

ABSTRACT

@#Objective    To investigate the effect and mechanism of epigallocatechin-3-gallate (EGCG) on restenosis of the vein graft. Methods    Totally 90 Sprague-Dawley rats were randomly divided a the control group, a vein graft group and an EGCG+vein graft group. At week 1, 2 and 4, the intimal and tunica thickness of the venous graft wall was evaluated by hematoxylin-eosin staining, and the expression of Ki-67 was assessed by immunohistochemistry analysis, and then the expression of hairy and enhancer of split-1 (HES1) was measured by Western blot assay. Results    At week 2, the intimal thickness (46.76±4.89 μm vs. 8.93±0.82 μm, 46.76±4.89 μm vs. 34.24±3.57 μm), tunica thickness (47.28±4.37 vs. 16.33±1.52 μm, 47.28±4.37 vs. 36.27±3.29 μm), positive cell rate of Ki-67 (21.59%±2.29% vs. 1.12%±0.22%, 21.59%±2.29%vs. 15.38%±1.30%), expression of HES1 respectively increased in the experimental group than those in the control group and the EGCG+vein graft group (P<0.05, respectively). At week 4, the intimal thickness (66.38±6.23 μm vs. 8.29±0.79 μm,   66.38±6.23 μm vs. 48.39±4.23 μm), tunica thickness (63.27±6.18 μm vs. 15.29±1.49 μm, 63.27±6.18 μm vs. 44.63±4.49 μm), positive cell rate of Ki-67 (33.19%±3.03% vs. 1.09%±0.19%, 33.19%±3.03% vs. 24.37%±2.73%), expression of HES1 increased in the experimental group than those in the control group and EGCG+vein graft group (P<0.05, respectively). Conclusion    EGCG may inhibite restenosis of vein graft by inhibiting Notch signal pathway.

5.
Herald of Medicine ; (12): 856-860, 2015.
Article in Chinese | WPRIM | ID: wpr-467299

ABSTRACT

Objective To investigate the effects of osthole on neural stem cells ( NSCs) differentiation and explore the potential mechanism. Methods Brain-derived NSCs from newborn mice were isolated and cultured in vitro and determined by immunofluorescence. The P5 generations of NSCs were placed in culture solution with osthole at concentrations of (0,10,50, 100 μmol·L-1 ) . The neuron, astrocyte and oligodendroglia cell differentiation were determined by immunofluorescence. The mRNA expression of Notch 1 and its target genes Mash 1 and Neurogenin 2 were assessed by RT-PCR. Results The neurosphere displayed Nestin and Sox 2-postive by immunofluorescence, suggesting that the cultured cells were NSCs. Osthole promoted NSCs differentiating into more neuron(P<0. 01) and oligodendrocyte(P<0. 05), but not astrocyte. Meanwhile, osthole significantly reduced the mRNA expression of Notch 1(P<0. 01) and increased Ngn 2(P<0. 01)at the dose of 100 μmol·L-1. Conclusion Osthole enhances NSCs differentiating into more neuron and oligodendrocyte via probablly inhibiting Notch signal pathway.

6.
Tianjin Medical Journal ; (12): 883-888, 2014.
Article in Chinese | WPRIM | ID: wpr-474034

ABSTRACT

Objective To investigate the effect of construct the Notch1 (NICD) eukaryotic expression vector on the proliferation and differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods Rat BMSCs were experimented as the object. NICD eukaryotic expression vector was constructed. pEGFP-N1-NICD expressing plasmids were used to transfect BMSCs. The study included control group (CON group), empty vector group (VEC group) and the trans-fection group (TRA group). After 48-hour transfection, BMSCs were observed for general morphology. The protein expres-sions of NSE, GFAP and Notch1 were detected by real-time PCR and Western blotting assay respectively. The apoptosis, cy-cle distribution and cell proliferation were evaluated by flow cytometry and MTT assay. Results The DNA sequencing con-firmed that the pEGFP-N1-NICD recombinant plasmid was successfully constructed, and both VEC group and TRA group expressed green fluorescence after 48-hour transfection. The relative expression levels of Notch1 and GFAP mRNA and pro-tein were significantly higher in TRA group than those in VEC group and CON group (P<0.05), and there was no significant difference between VEC group and CON group. After 48-hour transfection, the ratio of living cells was significantly lower in TRA group than that of CON group and VEC group, and the early apoptotic rate and late apoptotic rate were significantly higher in TRA group than those of CON group and VEC group (P<0.05). The late apoptotic rate was significantly higher in VEC group than that of CON group. The proportion of G1/G0 cells was significantly higher in TRA group than that of CON group and VEC group, but S and G2/M cells were significantly lower (P<0.05). The value of growth curve was gradually de-creased in TRA group than that of CON group and VEC group (P<0.05). Conclusion The high expression of NICD gene might induce apoptosis of BMSCs, inhibit the proliferation in part, and induce into glial-like cell differentiation.

7.
Article in Chinese | WPRIM | ID: wpr-443107

ABSTRACT

Objective To observe the effects of exogenous basic fibroblast growth factor (bFGF) on radiation-induced apoptosis of C17.2 neural stem cells (NSCs) with γ-secretase inhibitor (DAPT) condition and explore the relationship between bFGF and Notch signal pathway.Methods The cell viability was detected by using the MTF method.After the cells attached to the flasks,different concentrations of DAPT was added in accordance with the experimental design and cultured cells for 24 h.C17.2 NSCs were subjected to irradiation exposure by linear accelerator and treated with bFGF (40 ng/ml) 5 min after the exposure.After 48 hours,the apoptosis of cells was detected by using Flow Cytometry.Results After adding in DAPT,the cells growth was inhibited and depended on the concentrations of DAPT.Compared with the control group,all groups had statistically significant differences(P<0.05).Flow cytometry showed compared with the control group all groups had significant differences (P<0.05).The apoptosis rate was (11.53±0.81)% in radiation group,(7.18±0.0.94)% in radiation+bFGF group,(9.82±0.77) % in DAPT group,(21.45±0.98) % in Radiation+DAPT group and (10.26+ 1.03) % in Radiation+ DAPT +bFGF group.Between Radiation + bFGF group and Radiation group,it had statistically significant difference(P<0.05).The pairwise comparisons of DAPT group and Radiation + DAPT group which had the same DAPT concentration had statistically significant differences (P<0.05).The pairwise comparisons of Radiation + DAPT+bFGF group and Radiation + DAPT group which had the same DAPT concentration had statistically significant difference(P<0.05).Conclusion Fx ogenous bFGF can inhibit apoptosis of C17.2 NSCs.Notch signaling patbway inhibitor DAPT can promote apoptosis of C17.2 NSCs which are subjected to irradiation exposure by linear accelerator and bFGF can weak apoptosis.bFGF protective effect on radiation-induced neural stem cells may be related to the Notch signaling pathway.

8.
Article in English | WPRIM | ID: wpr-820682

ABSTRACT

OBJECTIVE@#To investigate the effect of the spinal cord extracts (SCE) after spinal cord injuries (SCIs) on the proliferation of rat embryonic neural stem cells (NSCs) and the expressions of mRNA of Notch1 as well as of Hes1 in this process in vitro.@*METHODS@#The experiment was conducted in 4 different mediums: NSCs+PBS (Group A-blank control group), NSCs+SCE with healthy SD rats (Group B-normal control group), NSCs+SCE with SD rats receiving sham-operation treatment (Group C-sham-operation group) and NSCs+ SCE with SCIs rats (Group D-paraplegic group). Proliferative abilities of 4 different groups were analyzed by MTT chromatometry after co-culture for 1, 2, 3, 4 and 5 d, respectively. The expressions of Notch1 and Hes1 mRNA were also detected with RT-PCR after co-culture for 24 and 48 h, respectively.@*RESULTS@#After co-culture for 1, 2, 3, 4 and 5 d respectively, the MTT values of group D were significantly higher than those of group A, group B and group C (P0.05). Both the expressions of Notch1 and Hes1 mRNA of group D were significantly higher than those of other 3 groups after co-culture for 24 h and 48 h as well (P0.05). There was no difference in expressions of Notch1 and Hes1 mRNA between 24 h and 48 h treatment in group D.@*CONCLUSIONS@#SCE could promote the proliferation of NSCs. It is demonstrated that the microenvironment of SCI may promote the proliferation of NSCs. Besides, SCE could increase the expression of Notch1 and Hes1 mRNA of NSC. It can be concluded that the Notch signaling pathway activation is one of the mechanisms that locally injured microenvironment contributes to the proliferation of ENSC after SCIs. This process may be performed by up-regulating the expressions of Notch1 and Hes1 gene.


Subject(s)
Animals , Basic Helix-Loop-Helix Transcription Factors , Genetics , Metabolism , Cell Extracts , Pharmacology , Cell Proliferation , Cells, Cultured , Female , Homeodomain Proteins , Genetics , Metabolism , Male , Neural Stem Cells , Cell Biology , Rats , Rats, Sprague-Dawley , Receptors, Notch , Genetics , Metabolism , Signal Transduction , Spinal Cord , Chemistry , Spinal Cord Injuries , Metabolism , Transcription Factor HES-1
9.
Journal of International Oncology ; (12): 544-546,560, 2012.
Article in Chinese | WPRIM | ID: wpr-598052

ABSTRACT

Notch signal pathway can regulate the morphogenesis,apoptosis and cellular proliferation of normal tissues and organs,which plays an important role in regulating hematopoietic cells proliferation and differentiation in bone marrow microenvironment.The occurrence and development of multiple myeloma(MM)are closely related to the bone marrow microenvironment in which many signal pathways are involved.Recent studies show that Notch signal pathway promotes the development and progression of many cancers including MM,which plays key roles in tumor invasion and drug resistance.This review focus on the recent findings on Notch signal pathway in MM,and reveals that Notch signal pathway will be identified as a potential new therapeutic target in MM.

10.
Indian J Med Sci ; 2010 Sept; 64(9) 402-407
Article in English | IMSEAR | ID: sea-145560

ABSTRACT

Objective: To investigate the molecular mechanism underlying the differentiation of human umbilical cord-derived mesenchymal stem cells (hUCMSCs) into myocardial cells induced by 5-azacytidine (5-aza), and to explore the expression and significance of DLL4-Notch signaling in this process. Materials and Methods: hUCMSCs were isolated and purified from the umbilical cords of normal or cesarean term deliveries under sterile conditions. After treatment with 5-aza for 24 h, hUCMSCs was continued to culture, the expression of GATA4 and NKx2.5 at 4 weeks after induction, DLL4 and Notch1 mRNA at 1d, 3d, 5d, 7d after induction were detected. The expression of cardiac troponin I (cTnI) after 4 weeks was determined by immunocytochemistry. Results: hUCMSCs treated with 5-aza were stained positively for cTnI 4 weeks after induction. The expression of Notch1 and DLL4 mRNA in the 5-aza-induced group was stable and significantly higher than that in the control group (mean Ct value for the Notch1 gene: 0.51 ± 0.21 in the 5-aza-induced group vs. 7.85 ± 0.35 in the control group; mean Ct value for the DLL4 gene: 1.60 ± 0.49 in the 5-aza-induced group vs. 12.42 ± 0.73 in the control group). Similar results were observed for Nkx2.5 and GATA4 genes. The expressions of Nkx2.5 and GATA4 mRNA in the 5-aza group were 4.72 ± 0.58 and 3.76 ± 0.06 times higher than that in the control group, respectively, with statistical significance. Conclusions: hUCMSCs can be differentiated into myocardial cells by 5-aza induction in vitro. 5-Aza may affect this process by regulating the expression of GATA4 and Nkx2.5 genes. The DLL4-Notch signal pathway may be involved in this process.


Subject(s)
Azacitidine/metabolism , Cell Differentiation/drug effects , GATA4 Transcription Factor/metabolism , Homeodomain Proteins/metabolism , Humans , Mesenchymal Stem Cells/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Receptor, Notch1/metabolism , Receptors, Notch/metabolism , Signal Transduction/drug effects , Transcription Factors/metabolism , Umbilical Cord/cytology
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