Effect of triptolide on expressions of Notch receptors and ligands in rats with adjuvant- induced arthritis and reduced pulmonary function / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of triptolide on Notch receptor and ligand expressions in rats with adjuvant-induced arthritis (AA).</p><p><b>METHODS</b>Forty rats were randomly divided into normal control (NC) group, model (MC) group, methotrexate group and triptolide groups. Rat models of AA were established by an intradermal injection of 0.1 mL Freund's complete adjuvant into the right paw. Twelve days after the injection, the rats were treated with corresponding drugs for 30 days; the rats in NC group and MC group were given saline only. Paw edema volume (E), arthritis index (AI), pulmonary function, histomorphologies, and Notch receptor/ ligand expression in the lung tissue were analyzed after the treatments.</p><p><b>RESULTS</b>Compared with the NC group, E, AI, Notch3, Notch4, and Delta1 expressions in the lung tissues significantly increased while pulmonary function and pulmonary expressions of Notch1, Jagged1, and Jagged2 significantly decreased the model rats (P<0.01). Compared with the MC group, triptolide-treated rats showed significantly improved pulmonary functions, increased expressions of Notch1, Jagged1, and Jagged2 and decreased expressions of Notch3, Notch4, and Delta1 in the lungs (P<0.05, P<0.01); the therapeutic effect of triptolide was better than that of methotrexate.</p><p><b>CONCLUSION</b>Triptolide can reduce inflammatory reaction and immune complex deposition to improve joint and pulmonary symptoms in rats with AA possibly by up-regulating the expressions of Notch3, Notch4, and Delta1 and down-regulating the expressions of Jagged1, Jagged2, and Notch1.</p>

mRNA expression of notch ligand-delta-like-1 and jagged-1 in mesenchymal stem cells of MDS patients / 中国实验血液学杂志

This study was aimed to investigated the mRNA expression levels of Notch ligands- Delta-like-1 and Jagged-1 in bone marrow mesenchymal stem cells of patients with myelodysplastic syndrome (MDS), and to explore their relation with onset of MDS. Bone marrow mesenchymal stem cells of 38 patients with MDS and 16 normal subjects as control were collected to detect mRNA expression of Delta-like-1 and Jagged-1 by using real-time quantitative polymerase chain reaction. The results showed that the expression levels of Delta-like-1 and Jagged-1 in mesenchymal stem cells of MDS patients were significantly higher than that in normal controls (P < 0.05). According to WHO criteria, the mRNA expression of Delta-like-1 in RA/RAS, RCMD and RAEB groups were significantly higher than that in normal controls (P < 0.05), the mRNA expression of Jagged-1 in RAEB group was also significantly higher than that in normal controls (P < 0.05). The mRNA expression of Delta-like-1 was significantly correlated with the proportion of blasts in the bone marrow of MDS patients (r = 0.502, P < 0.05). The expression levels of Delta-like-1 and Jagged-1 in MDS patients with abnormal karyotypes were significantly higher than those in MDS patients with normal karyotypes (P < 0.05). The mRNA expression of Delta-like-1 in higher risk group according to International Prognostic Scoring System was significantly higher than that in lower risk group (P < 0.05), there was no significant difference in Jagged-1 expression levels between higher risk group and lower risk group (P > 0.05). It is concluded that the changes of Delta-like-1 and Jagged-1 expression level in MSC may play a role in the pathogenesis of myelodysplastic syndrome.

Construction and expression of fusion protein TRX-hJagged1 in E.coli BL21 / 中国实验血液学杂志

This study was purposed to construct prokaryotic expression vector and to investigate the expression of Notch ligand Jagged1 in E.coli. An expression vector pET-hJagged1 was constructed, which can be inserted in Jagged1 with different lengths, but the DSL domain of human Jagged1 should be contained. Then the recombinant plasmids were transformed into the competent cell of E.coli BL21, and the expression of the fusion protein was induced by IPTG. Fusion protein was purified from the supernatant of cell lysates via the Nickel affinity chromatography. The results showed that prokaryotic expression vectors pET-hJagged1 (Bgl II), pET-hJagged1 (Hind I) and pET-hJagged1 (Stu I) were successfully constructed, but only pET-hJagged1 (Stu I) could express the soluble TRX-hJagged1. The purified TRX-Jagged1 protein could be obtained via the Nickel affinity chromatography, and then confirmed by Western Blot. It is concluded that prokaryotic expression vector pET-hJagged1 is successfully constructed, but only pET-hJagged1 (Stu I) can express the soluble TRX-hJagged1 and the TRX-Jagged1 fusion protein is obtained through the prokaryotic expression system, which laid a solid foundation for further to explore the effects of Jagged1 in hematopoietic and lymphoid system.

Role of Notch-Jagged/Delta signaling pathway in arthritis rats of reduced lung function induced by adjuvant / 中南大学学报(医学版)

OBJECTIVE@#To observe the changes of pulmonary function and Notch signaling pathway of lung tissues in adjuvant-induced arthritis rats, and to investigate the mechanism of reduced lung function.@*METHODS@#A total of 30 rats were randomly divided into a normal group and a model group. Rats in the model group were induced to establish the adjuvant arthritis AA model by intradermally injecting 0.1 mL Freund's complete adjuvant into the right paw. After 30 days, we observed the paw edema volume, arthritis index, pulmonary function, histomorphology, and Notch receptor/ligand of the lung tissue.@*RESULTS@#Compared with the normal group, the paw edema volume, arthritis index, average expiratory flow within 0.3 s (FEV0.3/FVC), and the level of Notch3, Notch4 and Jagged2 of the lung tissue in the model group was significantly increased, while maximum expiratory flow at 50% of vital capacity (FEF50), maximum expiratory flow at 75% of vital capacity (FEF75), forced expiratory flow (PEF) and the expression of Notch1 of Jagged1 and Delta1 in the lung were significantly decreased (P<0.05, P<0.01). There were significant positive correlations between FEV0.3/FVC and Notch4. FEV0.3/FVC, FEF25, FEF50 and Notch3, Delta1 were negatively correlated, respectively (P<0.05, P<0.01).@*CONCLUSION@#While arthritis occurs in AA rats, pulmonary function declines and significantly correlates with the expression of Notch receptor/ligand. The deposition of immune complex in the lung after the injection of CFA activates the Notch signaling pathway, and results in further decline of pulmonary function by signaling cascades.

Paraquat involves differentiation of human neural stem cells via Notch signaling / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate effects of paraquat on the mRNA expression of key elements of Notch signaling (Notch1, Jagged1 and DTX1) during differentiation process of human neural stem cells (hNSCs).</p><p><b>METHODS</b>hNSCs exposed to PQ at the concentrations 0.10, 1.00, 10.00 M. Cell proliferation ability was assessed using MTT assay and mRNA expressions of Notch1, Jagged1 and DTX1 were detected by Real-time RT-PCR at 2, 4, 8, 12 d of differentiation.</p><p><b>RESULTS</b>Compared with control group, NOTCH1, JAG1 mRNA expression levels exposed to PQ at the concentration of 0.10 M significantly reduced at 2, 4, 8 d and significantly went up at 12d (P < 0.01). Compared with control group, NOTCH1, JAG1 and DTX1 mRNA expression levels exposed to PQ at the concentration of 10.00 M significantly reduced at 2, 8, 12 d (P < 0.01). PQ could down-regulate Notch1, Jagged1 and DTX1 mRNA expressions at the early stage of differentiation, then up-regulate Notch1 mRNA expression, and down-regulate Notch1, Jagged1 and DTX1 mRNA expressions at the end of differentiation.</p><p><b>CONCLUSION</b>Notch signaling pathway may be involved in differentiation of neural stem cell exposed to PQ.</p>

A preliminary study on the culture of single hematopoietic stem cell / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the biological behavior including survival and proliferation of CD34 + CD38--Lin--cells when they are cultured at single cell level.</p><p><b>METHODS</b>Purified umbilical cord blood CD34 + CD38--Lin--cells were separated at single cell level in 96-well plates using flow cytometry for four groups: control group (CD34 + CD38--Lin--cell plus stem cell medium) , Shh group (CD34 + CD38--Lin--cell plus stem cell medium and Shh), BMP-4 group (CD34 + CD38--Lin--cell plus stem cell medium and BMP-4), Jagged-1 group (CD34 + CD38--Lin--cell plus stem cell medium and Jagged-1). Methylcellulose medium was used in the colony-forming experiment which was also in four groups as previously. The number of cells and colony-forming units in each well for the four groups was evaluated at different time points (day 1, 3, 7) with fluorescence microscopy counting method.</p><p><b>RESULTS</b>Division of single cell was observed to be amplified in all of these groups from day 3. And meanwhile, after 1-week culture, the survival rates for the treated groups were all higher than the control group (Jagged-1 group > BMP-4 group > Shh group > control), while the cell number in each well was also highest in the Jagged-1 group (Jagged-1 group > BMP-4 group > control). The number of wells with a cell number of zero was significantly fewer in all treated groups (especially the Jagged-1 group) than in the control group; meanwhile, the number of wells with a cell number higher than 17 was evidently higher in all the treated groups (especially the BMP-4 group) more than controls. Colony-forming units for erythroid (BFU-E), granulocyte (CFU-G), macrophage (CFU-M), and granulocyte macrophage (CFU-GM) were observed for all of these experimental groups, and there was no significant difference between the four experimental groups.</p><p><b>CONCLUSIONS</b>CD34 + CD38 - Lin - cell can achieve the survival, self-renewal and proliferation when cultured at single cell level, and the adding of Shh, BMP-4, and Jagged-1 can enhance such capabilities. However, CD34 + CD38 - Lin - cell can only maintain cell totipotency in its niche.</p>

Effect of Notch signaling on the activation of hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate whether Notch signaling is activated in hepatic stellate cells (HSCs), and to determine whether manipulation of the Notch signaling pathway can effect the activation of HSCs.</p><p><b>METHODS</b>The expression of Notch signaling components in unactivated or TGF-b1-activated HSC-T6 cells was detected by Taqman Probe-based gene expression analysis. Differential expression of Notch3 and Jagged1 was detected by immunofluorescence analysis. Notch3-mediated expression of the myofibroblastic markers, a-SMA and collagen I, was detected in HSC-T6 cells transfected with pcDNA3.1-N3ICD or Notch3 siRNA by Western blotting.</p><p><b>RESULTS</b>Notch signaling components were expressed in both unactivated and activated HSC-T6 cells, but the TGF-b1-treated cells showed significantly higher expression levels of Jagged1 (3.9-fold, F = 2543.482), Notch3 (4.2-fold, F = 287.982), and HES1 (3.2-fold, F = 1719.851). Transfection-mediated over-expression of Notch3 led to significantly increased expression of a-SMA (6.8-fold, t = 13.157) and collagen I (5.5-fold, t = 9.810) (both P less than 0.01). Transient knock-down of Notch3 expression by siRNA decreased expression of the myofibroblastic markers (a-SMA by approximately 90%, t = 19.863 and collagen I by 84%, t = 10.376; both, P less than 0.01). Moreover, knock-down of Notch3 antagonized the TGF-b1-induced expression of a-SMA and collagen I.</p><p><b>CONCLUSION</b>Notch signaling may participate in liver fibrogenesis by regulating HSC activation. Selective interruption of Notch3 may represent a new anti-fibrotic strategy to treat liver fibrosis.</p>

Gene expression of Notch1 and Jagged1 in children with acute leukemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the gene expression of Notch1 and Jagged1 in children with acute leukemia (AL) and their possible roles in the pathogenesis of AL.</p><p><b>METHODS</b>Mononuclear cells from bone marrow or peripheral blood of 47 children with AL and 20 controls (normal children or children with nonmalignant hematologic disease) were collected from February 2009 to July 2011. A two-step method to semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the gene expression of Notch1 and Jagged1. Of the 47 children with AL, there were 26 cases of B-ALL, 6 cases of T-ALL and 15 cases of AML.</p><p><b>RESULTS</b>The positive expression rate of Notch1 in the ALL and AML groups was higher than in the control group (P<0.05). The expression level of Notch1 in T-ALL children was higher than in B-ALL children (P<0.01). The positive expression rate of Jagged1 in the ALL and AML groups was not significantly different from the control group, however, the expression level of Jagged1 in the ALL and AML groups was higher than in the control group (P<0.05).</p><p><b>CONCLUSIONS</b>There are significant differences in the gene expression of Notch1 between children with different types of ALL, and a higher expression of Notch1 relates to T-ALL. The activation of Notch1 signal is common in children with AL. The abnormal gene expression of Notch1 in children with AML shows the role of Notch1 in AML. The gene expression of Jagged1 in children with ALL or AML is abnormal, and this needs to be confirmed by further research.</p>

Effect of C-reactive protein on Notch pathway components in human periphery blood endothelial progenitor cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of C-reactive protein (CRP) on the expressions of Notch pathway components in human peripheral blood endothelial progenitor cells (EPC) in vitro.</p><p><b>METHODS</b>Mononuclear cells isolated by density gradient centrifugation of human peripheral blood mixed with 6% hydroxyethyl starch (Hes) were plated on fibronectin-coated 6-well culture dishes. After 7 days, the adherent cells were cultured in the presence of 10 and 20 mg/L CRP for 48 h, and the proliferation, migration, and adhesion abilities of the cells were observed. The mRNA expressions of Notch-1 and its ligand Jagged-1 in the EPCs were measured by RT-PCR, and their protein expressions by Western blotting.</p><p><b>RESULTS</b>CRP at 10 and 20 mg/L caused a significant reduction in the number of viable EPCs (61∓3 and 54∓3, respectively) as compared with PBS (71∓4, P<0.05). CRP also resulted in a significant suppression of the proliferation, migration and adhesion capacities of the EPCs. The mRNA and protein expressions of Jagged-1 and Notch-1 in the EPCs significantly increased following CRP exposure in comparison with PBS treatment.</p><p><b>CONCLUSION</b>CRP can suppress the proliferation, migration and adhesion capacities of the EPCs probably by affecting the expressions of the Notch-1 pathway components.</p>

Expression of JAG1 and DLL1 genes in colorectal cancer and its clinical significance / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To study the expression of JAG1 and DLL1 in colorectal cancer and its clinical significance.</p><p><b>METHODS</b>Patients with colorectal cancer were treated in the Center of Colorectal Surgery of the Third Affiliated Hospital of Nanjing University of TCM were collected prospectively and followed up. A tissue microarray was made and expressions of JAG1 and DLL1 were detected by immunohistochemical staining.</p><p><b>RESULTS</b>A total of 146 cases with colorectal cancer were included. The differences in JAG1 expression were significant among different tumor differentiation types and the differences in DLL1 expression were significant among different tumor locations(all P<0.05). There were no significant differences in the expression of the two genes and microsatellite instability(MSI)(P>0.05). One hundred and thirty-four (91.8%) cases were followed up and the mean follow-up time was (42.3±13.3) months. Tumor-free survival was noticed in 86 patients. The overall survival was 93% at 1 year, 74% in 3 years, and 67% in 5 years. Multivariate analysis showed that long-term survival rate was related to TMN stage, pathology types, MSI status and expression of JAG1. The prognosis of patients with high expression of JAG1 was better than those with low and negative expression(P<0.05).</p><p><b>CONCLUSIONS</b>The expressions of JAG1 and DLL1 are related to tumor differentiation and tumor location. The expression of JAG1 gene is associated with long-term survival.</p>

Inhibitory effect and mechanism of (-)-epigallocatechin-3-gallate on HT29 and HCT-8 colorectal cancer cell lines and expression of HES1 and JAG1 / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To study the inhibitory effect of (-)-epigallocatechin-3-gallate (EGCG) on cancer cells line HCT-8 and HT29 and its influence on the expression of HES1 and JAG1.</p><p><b>METHODS</b>Colorectal cancer cells line HCT-8 and HT29 were cultured in vitro and treated with different concentrations of EGCG(10 mg/L, 20 mg/L, 35 mg/L). The inhibition of proliferation was tested by MTT analysis. Influence of EGCG on the cell apoptosis and cell cycle of HCT-8 and HT29 were detected with flow cytometry, and gene expression of HCT-8 and HT29 after EGCG treatment with real-time polymerase chain reaction.</p><p><b>RESULTS</b>EGCG affected the proliferation and apoptosis of HCT-8 and HT29. The inhibition rates of the three different concentrations of EGCG were(28.894±5.076)%, (34.903±1.794)%, and (39.028±0.105)% on HCT-8, and (14.682±4.244)%, (22.429±3.847)%, and (29.840±5.076)% on HT29. EGCG caused G(2)/M phase arrest and M phase transition in HCT-8 cell line, and S phase arrest and G2 phase transition in HT29 cell line. EGCG down-regulated HES1 gene expression in both cell lines, however, the differences were not statistically significant(both P>0.05). EGCG upregulated JAG1 gene expression in both cell lines, however only the difference in HCT-8 was statistically significant(0.201±0.018 vs. 0.440±0.077, P=0.029).</p><p><b>CONCLUSIONS</b>EGCG can significantly inhibit the proliferation of HT29 cells and HCT-8 cells by changing cell cycle and inducing cell apoptosis. The mechanism may be related to the upregulation of JAG1 gene expression.</p>

Expression of human Jagged-1 protein on eukaryotic cells and establishment of stable transfectant cell line / 中国实验血液学杂志

Jagged-1 protein is one of the ligands belonging to Notch signaling pathway. Notch signaling pathway is one of the major signaling pathways mediated by contact between cells and plays an important role to regulate the process of proliferation and differentiation of hematopoietic cells in the hematopoietic microenvironment. To study the biological effect after the combination of receptor and ligand in Notch signaling pathway and the mechanism of Notch signaling pathway in bone marrow stromal cells mediated-drug resistance, a NIH-3T3 cell line over-expressing Jagged-1 protein was constructed for further research purposes. A full coding region of Jagged-1 gene was cloned and inserted into eukaryotic expression plasmid to construct pEGFP-IRES2-Jagged-1 eukaryotic expression vector, then transfected into NIH-3T3 cell line, a mammalian cells. As a result Western blot analysis confirmed that the transfectant NIH-3T3 cells highly expressed Jagged-1 protein and flow cytometry analysis confirmed that the NIH-3T3-pEGFP-IRES2-Jagged-1 cell line over-expressed Jagged-1 protein was monoclonal after screened by selective medium and limiting dilution analysis. It is concluded that the pEGFP-IRES2-Jagged-1 eukaryotic expression vector and a stable transfectant monoclonal NIH-3T3 cell line are successfully established. The construction of the stable transfectant monoclonal NIH-3T3 cell line which overexpressed Jagged-1 protein, provides the conditions to further study the mechanism of the bone marrow stromal cell-mediated drug resistance and to discover the new drug targets.

Expression of Notch receptors, ligands and downstream target genes in epidermis of hypertrophic scar / 中华整形外科杂志

<p><b>OBJECTIVE</b>To study the expression of Notch receptors, ligands and downstream target genes in hypertrophic scar and normal skin, and to investigate its role in the development of hypertrophic scar.</p><p><b>METHODS</b>By immunohistochemistry, the expression of epidermal differentiation markers- beta1 integrin, keratin 14 (K14) and keratin 19 (K19), as well as Notch 1-4 and Jagged1 were examined in hypertrophic scars and normal skins. The expression of Notch downstream genes- P21 and P63 was analyzed with real-time quantitative PCR and immunohistochemistry staining.</p><p><b>RESULTS</b>Histological analysis revealed a significant epidermal thickening in the hypertrophic scars, with excessive cell layers above the basal layer. Compared to the normal epidermis, the expression of beta1 integrin, K19 and K14 decreased in hypertrophic scars (P <0.05). Positive expression rate of Notch1 and Jagged1 in keratinocytes was significantly higher in hypertrophic scar than in normal skin (P < 0.05), while there was no difference in Notch2 and 3 positive expression rate. Furthermore, the expression of P21 was significantly up-regulated, while the expression of P63 was down-regulated in keratinocytes of hypertrophic scar (P < 0.05).</p><p><b>CONCLUSIONS</b>Notch signal may play an important role in hypertrophic scar pathogenesis. Over-differentiation of Keratinocytes in hypertrophic scar may be related to the overexpression of Notch1 and Jagged1, up-regulation of P21 gene and down-regulation of P63 gene.</p>

Construction and expression of a fusion protein containing extracellular domain of human Jagged1 and Fc fragment of human IgG1 in Pichia Pastoris / 中国实验血液学杂志

In order to construct a pichia pastoris expression vector containing the extracellular domain of human Jagged1 and the Fc fragment of human IgG1 fusion gene, or containing only the Fc fragment of human IgG1 and to express them in pichia pastoris. The extracellular domain of human Jagged1 gene was cloned from normal human bone marrow cells. After DNA sequencing, the extracellular domain of Jagged1 gene was inserted into pIC-Fc vector constructed previously, which is Pichia pastoris expression vector containing only the Fc fragment of human IgG1. The constructed plasmid was transformed into yeast GS115 by means of electroporation. The recombinant transformants with a high copy number of the plasmid were selected on MD plate with G418. The expression of protein was induced by addition of methanol. Then, protein expression was analyzed by SDS-PAGE. The results indicated that the extracellular domain of human Jagged1 gene was effectively amplified. The DNA sequencing result showed that the constructed plasmid containing hJagged1(ext)-Fc fusion gene was the same as designed. The fusion protein was successfully expressed in Pichia pastoris. It is concluded that the hJagged1(ext) gene has been successfully cloned and expressed, which provides a new fusion protein for further experiments, the hJagged1(ext)-Fc fusion protein can be used as a new stimulator for proliferation of hematopoietic stem/progenitor cells in vitro.

Notch signaling in human breast cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the role of Notch signaling in human breast cancers, the expression of Notch1 and its ligand JAG1 in human breast cancers and their relationships with clinical stages of breast cancers were analyzed.</p><p><b>METHODS</b>RT-PCR was used to detect the expression of Notch1 and JAG1 in 62 breast cancer specimens and 22 normal breast tissues at the margin of tumor sections, and the statistical difference of expression rates and standardized coefficient between the two groups were analyzed. To compare the expression intensity of Notch1 and JAG1 at different development stages of the illness and at different stages with or without axillary node metastasis.</p><p><b>RESULTS</b>The expression rate and standardized coefficient of Notch1 in human breast cancers were significantly higher than those of normal breast tissues at the margin of tumor sections. The expression rate of JAG1 in human breast cancers was 15%, while JAG1 was not detected in normal breast tissues at the margin of tumor sections. The standardized coefficient of Notch1 in cases with axillary node metastasis was significantly higher than that in cases without axillary node metastasis. The standardized coefficient of Notch1 at stage I was significantly lower than that at stage II, and stage II was significantly higher than stage III. There was no statistically significant difference between stage I and stage III.</p><p><b>CONCLUSION</b>Notch1 and JAG1 are highly expressed in human breast cancers, indicating that the aberrant expression and activation of Notch1 may be related with tumorigenesis of human breast cancer. Notch1 may play different roles at different developmentl stages of human breast cancer.</p>

Expression changes of Notch-related genes during the differentiation of human mesenchymal stem cells into neurons / 生理学报

The Notch signaling pathway has been implicated in the regulation of cell-fate decisions such as differentiation of embryo stem cells and neural stem cells into neurons. We cultured human mesenchymal stem cells (hMSCs) in vitro and induced hMSCs to differentiate into neural cells by beta-mercaptoethanol (beta-ME), DMSO and 3-tert-butyl-4-hydroxyanisole (BHA). Immunocytochemistry was utilized to detect neuron-specific enolase (NSE) and Nissl body, and flow cytometry was used to determine cell growth phases. The expressions of signal molecules involved in the Notch pathway such as Notch1, Jagged 1 (JAG1), presenilin 1 (PS1) and hairy and enhancer of split 1(HES1) were observed by RT-PCR and immunofluorescent techniques. The results were as follows: (1) Before induction, the percentage of hMSCs at G(0)/G(1) was 58.5%, and the percentage at S+G(2)/M was 41.5%. After induction, the percentage of hMSCs at G(0)/G(1) increased to 73.1%, 76.2% and 78.1%, respectively on days 2, 4 and 6, and the percentage at S+G(2)/M decreased to 26.8%, 24.8% and 21.9%, respectively; The percentage of NSE-positive cells reached (77+/-0.35) %; Nisslos staining was positive in cytoplasm. (2) Notch1 and JAG1 were both expressed in hMSCs before and after induction, but the mRNA expressions of both Notch1 and JAG1, detected by RT-PCR, decreased obviously after induction(P<0.05). Notch1 mRNA/beta-actin was 1.157, 0.815, 0.756 and 0.570, and JAG1 mRNA/beta-actin was 0.437, 0.350, 0.314 and 0.362, respectively, on days 0, 2, 4 and 6 after induction. The Notch pathway activation participant PS1 mRNA and Notch pathway target gene HES1 mRNA also decreased apparently after induction (P<0.05), and their mRNA/beta-actin was 0.990, 0.449, 0.441, 0.454 and 0.370, 0.256, 0.266, 0.240 on days 0, 2, 4 and 6, respectively. These observations indicate that the expressions of Notch signal molecules were suppressed when hMSCs were induced to differentiate into neural cells. Based on these findings, we propose that low level of Notch signaling activation may contribute to neural cell differentiation.

Expression and significance of Notch1, Jagged1 and VEGF in human non-small cell lung cancer / 中南大学学报(医学版)

OBJECTIVE@#To determine the expressions of Notch1, Jagged1 and vascular endothelial growth factor (VEGF) in human non-small cell lung cancer (NSCLC) and to explore its clinical pathological significance.@*METHODS@#Immunohistochemical SP method was used to detect the expressions of Notch1, Jagged1 and VEGF in 65 patients with NSCLC and 15 normal epithelial tissues of the lung, and the relationship between them and clinic-pathological parameters were analyzed.@*RESULTS@#The positive rates of Notch1, Jagged1 and VEGF in NSCLC were 81.5%, 83.1% and 93.8%, respectively, higher than those in normal epithelial tissues of the lung (P<0.05). The positive expression levels of Notch1 and VEGF were closely associated with the tumor stage and the lymph node metastasis (P<0.05). The positive expression levels of Jagged1 was positively correlated with the pathological type and lymph node metastasis. There was a positive correlation between Notch1, Jagged1 and VEGF.@*CONCLUSION@#Notch1, Jagged1 and VEGF protein may play an important role in the pathway of carcinogenesis and progression of NSCLC. The up-regulation of Notch1, Jagged1 and VEGF protein expression probably predict NSCLC carrying relatively strong permeation and metastasis.

Function of Delta4 gene and its effects on 32D cell differentiation / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Notch activation leads to transcriptional suppression of lineage-specific genes, inhibiting differentiation in response to inductive signals. The Notch signal system contains three parts: Notch molecules, Notch ligands and effectors. Delta4 is a newly-discovered Notch ligand which has received the attention of few detailed studies. This study sought to explore the biological function of Delta4 and observe its effects on 32D cell differentiation.</p><p><b>METHODS</b>Delta4-expressing vector pTracer.CMV.Delta4.FLAG was constructed using molecular biological techniques. CHO cells stably transfected with pTracer.CMV.Delta4.FLAG were confirmed to have a Delta4 protein band via Western blotting. High-expression Delta4-CHO clones were selected for the following functional studies. Notch1-CHO and Notch2-CHO were used as host cells. After transiently transfecting with transition protein 1 (TP1), Delta4 activity was compared in both cell lines by means of luciferase analysis. CHO cells were incubated with Notch1-32D cells that had been transfected with Notch1 and were observed for granulocyte colony-stimulating factor (G-CSF)-induced differentiation. Jagged2-CHO and Delta4-CHO cells transfected with the Notch ligands Jagged2 and Delta4, respectively, were incubated with Notch1-32D cells to observed inhibition of Notch on G-CSF-induced differentiation.</p><p><b>RESULTS</b>The vector pTracer.CMV.Delta4.FLAG was constructed successfully. CHO cells were stably transfected with the vector pTracer.CMV.Delta4.FLAG. Two CHO cell lines expressing Delta4 at high levels were selected for use in the study. Delta4 was found to induce signal activity via both Notch1 and Notch2 and the induction of signaling activity was stronger in Notch2 cells than in Notch1 cells. Compared with other Notch ligands, Delta4 was slightly weaker than Jagged2, but stronger than Delta1 and Jagged1 in terms of Notch1 ligands. In terms of Notch2, Delta4 had a strong signaling activity, but was weaker than Delta1, Jagged1, and Jagged2. Jagged2 could inhibit Notch1-32D cell differentiation induced by G-CSF, but Delta4 could not.</p><p><b>CONCLUSIONS</b>Delta4 induces both Notch1 and Notch2 activity and is a ligand for both of them. The effect of Delta4 is stronger on Notch2 than that on Notch1. Jagged2 can inhibit Notch1-32D cell differentiation induced by G-CSF, but Delta4 cannot.</p>

Effects of Notch and its ligands on the differentiation of 32D cell / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the mechanism of Notch signaling transduction system and its effects on hematopoietic system.</p><p><b>METHODS</b>Notch ligands transfected CHO cells were added into Notch1 and Notch2 transfected CHO cells, which were transiently transfected with reporter gene TP1. PGL-100 was used as substrate to test the interaction between Notch and Notch ligands. CHO, Jagged2-CHO and Delta 4-CHO cells were seeded in the petri dish containing G-CSF, and then Notch 1-32D cells were added in it to observe the differentiation of Notch1-32D cell after incubation and staining.</p><p><b>RESULTS</b>All of the five Notch ligands binding to Notch1 could induce TP1 activity, it increased significantly the Jagged2-CHO, Delta 4-CHO1-4 and Delta 4-CHO1-5 cells. For Notch2, the TP1 activity induced by the five ligands in these cells was much higher than that of CHO. At the presence of G-CSF, Notch1-32D could differentiate to mature granulocyte. Jagged2 could inhibit G-CSF induced Notch1-32D cell differentiation, but Delta 4 could not.</p><p><b>CONCLUSION</b>Jagged2 and Delta 4 are the ligands of Notch1. Jagged2 can inhibit G-CSF induced Notch1-32D cell differentiation, but Delta 4 can not.</p>