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Objective To detecting the expression of nicastrin,N1ICD and hes1 proteins in normal liver tissues and liver cancer tissues of C57BL/6 mice.Methods Twelve 6-week-old male C57BL/6 mice were ran-domly equally divided into the control group and model group.In the model group,the in situ liver cancer model was established with injection of Hepa1-6 cells into the liver,and the control group was treated with injection of the same amount of normal saline.Liver cancer was verified by HE staining.The expression of nicastrin,N1ICD and hes1 proteins in the normal and cancerous liver tissues was detected by IHC and Western blot.Results IHC results showed that nicastrin,N1ICD and hes1 proteins were localized in the hepatic sinusoidal endothelial cells in the normal hepatocytes,but not expressed in the hepatocytes.The model group showed higher expression of nicas-trin protein but lower expression of N1ICD and hes1 protein in the cancer cells compared with the control group.Conclusion NCSTN gene may play a carcinogenic role in mouse hepatocellular carcinoma,while notch1 and hes1 may play a carcinostasis role in the carcinoma.
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@#[摘 要] 目的:探究牛蒡子苷元(ARC)通过调控Notch/Hes-1信号通路对口腔鳞状细胞癌(OSCC)HSC-3细胞增殖、凋亡和侵袭的影响及其机制。方法:使用不同质量浓度的ARC处理人HSC-3细胞,CCK-8法检测ARC对细胞增殖活力的影响,以选择适宜的药物浓度。将HSC-3细胞分为对照组、ARC-L组(10 mg/L ARC)、ARC-M组(20 mg/L ARC)、ARC-H组(40 mg/L ARC)和ARC-H+Jagged1/FC组(40 mg/L ARC+1.2 μg/mL Jagged1/FC)。采用EdU法检测细胞增殖能力,划痕愈合实验、Transwell实验和流式细胞术分别检测细胞的迁移、侵袭能力及细胞周期和细胞凋亡率,WB法检测增殖(c-Myc、cyclin D1)、凋亡(BAX、Bcl-2、survivin)、EMT(E-cadherin、vimentin、Snail)及Notch/Hes-1通路(Notch 1、Hes-1、NICD)相关蛋白的表达水平。结果:与0 mg/L相比,10~80 mg/L的ARC均能显著降低HSC-3细胞增殖活力(均P<0.05)。与对照组相比,ARC-L组、ARC-M组和ARC-H组HSC-3细胞EdU阳性率、划痕愈合率、侵袭细胞数、S期和G2/M期细胞占比及c-Myc、cyclin D1、Bcl-2、survivin、vimentin、Snail、Notch 1、Hes-1和NICD蛋白表达均显著降低(均P<0.05),细胞凋亡率、G0/G1期细胞占比及BAX、E-cadherin的蛋白表达均显著升高(均P<0.05),且呈浓度梯度依赖性。同时使用Notch激动剂Jagged1/FC,则可部分逆转ARC对HSC-3细胞增殖、迁移、侵袭、凋亡及相关蛋白表达的作用(均P<0.05)。结论:ARC可能通过抑制Notch/Hes-1信号通路抑制OSCC细胞HSC-3增殖和侵袭并促进细胞凋亡。
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OBJECTIVE@#To investigate the expressions of Notch1 and Hes1 in diffuse large B-cell lymphoma (DLBCL), and their correlations with clinical features.@*METHODS@#Immunohistochemistry (IHC) was performed on DLBCL samples (54 cases) and lymphadenitis tissues (20 cases) to evaluate the expressions of Notch1 and Hes1, and analyze their correlations with clinical characteristics of patients. Based on Oncomine database, the expressions of Notch1 and Hes1 mRNA and DNA were also explored.@*RESULTS@#IHC result showed that the positive expression rates of Notch1 and Hes1 in DLBCL patients were significantly higher than those in the control group (P <0.05). In DLBCL patients, the expression of Notch1 was closely associated with B symptoms, Ann Arbor stage, lymphocyte count and the level of lactate dehydrogenase (P <0.05), while the expression level of Hes1 was significantly higher in patients with B symptoms (P <0.05). Notch+/Hes1+ expression was found in 21 DLBCL tissues (38.9%), and there was a correlation between Notch1 and Hes1 expression (r =0.296, P <0.05). Bioinformatics analysis (Oncomine database) showed that the mRNA expressions of Notch1 and Hes1 in the Brune dataset were significantly higher than those in the control tissues (P <0.05).@*CONCLUSION@#The expressions of Notch1 and Hes1 in DLBCL are significantly higher than those in lymphadenitis, and correlated with B symptoms and Ann Arbor stage, suggesting that Notch1 and Hes1 play important roles in the occurrence and development of DLBCL.
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Humans , Cell Line , Clinical Relevance , Lymphadenitis , Lymphoma, Large B-Cell, Diffuse/pathology , Prognosis , RNA, MessengerABSTRACT
ObjectiveTo observe the effects of Bufei Yishen prescription on airway mucus hypersecretion and Notch signaling pathway related protein Notch3 and enhancer of split homologue 1 (HES1) in rats with chronic obstructive pulmonary disease (COPD) and to explore its action mechanism. MethodForty-eight SD rats were randomly divided into the control group, model group, Bufei Yishen prescription group, and aminophylline (APL) group,with 12 rats in each group. The stable COPD rat model was established via cigarette smoking exposure combined with Klebsiella bacterial infection for 12 weeks, and the corresponding drugs (3.7 g·kg-1·d-1 Bufei Yishen prescription and 54 mg·kg-1·d-1 APL) were administered by gavage during the next eight weeks. After the last administration at week 20, the lung tissue was sampled for observing the pathological changes and the rat lung function was detected. The tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and mucoprotein 5AC (MUC5AC) in bronchial alveolar lavage fluid and the mRNA and protein expression levels of Notch3, HES1, and MUC5AC in lung tissues were assayed. ResultCompared with the control group, the model group exhibited significantly weakened pulmonary function (P<0.05,P<0.01), reduced average number of alveoli (P<0.01), elevated mean linear intercept (P<0.01), and up-regulated TNF-α, IL-6, and MUC5AC in bronchial alveolar lavage fluid and Notch3, HES1, and MUC5AC mRNA and protein expression in lung tissue (P<0.05,P<0.01). Compared with the model group, Bufei Yishen prescription and APL remarkably enhanced pulmonary function, alleviated its pathological injury (P<0.05,P<0.01), and down-regulated TNF-α, IL-6, and MUC5AC in bronchial alveolar lavage fluid and the mRNA and protein expression levels of Notch3, HES1, and MUC5AC in lung tissues (P<0.05,P<0.01). ConclusionThe mechanism of Bufei Yishen prescription in inhibiting airway mucus hypersecretion of COPD rats was related to its regulation of Notch3 and HES1.
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Objective:To evaluate the role of Notch1/hairy and enhancer of split homolog1(Hes1) signaling pathway in high glucose and hypoxia-reoxygenation (H/R) injury to cardiomyocytes.Methods:H9c2 cardiomyocytes were cultured in low-glucose DMEM culture medium supplemented with 10% fetal bovine serum.The cells were divided into 6 groups ( n=12 each) using a random number table method: control group (group C), H/R group, H/R+ Jagged-1 group (group H/R+ J), high glucose group (group HG), high glucose+ H/R group (group HG+ H/R) and high glucose+ H/R+ Jagged-1 group (group HG+ H/R+ J). The cells were incubated in low-glucose culture medium for 72 h in group C. After incubated in low-glucose culture medium for 72 h, the cells were exposed to 24-h hypoxia in an incubator filled with 95% N 2-5% CO 2 at 37℃, immediately followed by 6-h reoxygenation in an incubator filled with 95% O 2-5% CO 2 at 37℃ in group H/R.In group H/R+ J, Jagged-1 (Notch1/Hes1 signaling pathway specific activator) 5μg/ml was added to low-glucose culture medium and the cells were incubated for 72h before H/R.In group HG, H9c2 cardiomyocytes were incubated in high-glucose culture medium containing 33 mmol/L glucose for 72 h. In group HG+ H/R, the cells were incubated in high-glucose medium for 72 h before H/R.In group HG+ H/R+ J, Jagged-1 5μg/ml was added to high-glucose culture medium, and the cells were incubated for 72 h before H/R.At 6 h of reoxygenation, the supernatant of the culture medium was collected for detection of the activities of superoxide dismutase (SOD) and lactic dehydrogenase (LDH), the cell viability (by CCK-8 assay) and the cell apoptosis rate (by flow cytometry) and for determination of expression of Notch1, Hes1 and c-caspase-3 (by Western blot). Results:Compared with group C, the cell survival rate and SOD activity were significantly decreased, and apoptosis rate and LDH activity were increased in H/R, H/R+ J and HG groups, expression of Notch1, Hes1 and c-caspase-3 was up-regulated in H/R and H/R+ J groups, and the expression of Notch1 and Hes1 was down-regulated and c-caspase-3 expression was up-regulated in group HG ( P<0.05). Compared with group H/R, the cell survival rate and SOD activity was significantly increased, apoptosis rate and LDH activity were decreased, expression of Notch1 and Hes1 was up-regulated, and c-caspase-3 expression was down-regulated in group H/R+ J, and the cell survival rate and SOD activity were significantly decreased, apoptosis rate and LDH activity were increased, expression of Notch1 and Hes1 was down-regulated, and c-caspase-3 expression was up-regulated in group HG+ H/R ( P<0.05). Compared with group HG, the cell survival rate and SOD activity were significantly decreased, and apoptosis rate and LDH activity were increased in HG+ H/R and HG+ H/R+ J groups ( P<0.05), and expression of Notch1 and Hes1 was down-regulated, and c-caspase-3 expression was up-regulated in group HG+ H/R ( P<0.05). Compared with group HG+ H/R, the cell survival rate and SOD activity were significantly increased, apoptosis rate and LDH activity were decreased, expression of Notch1 and Hes1 was up-regulated, and c-caspase-3 expression was down-regulated in group HG+ H/R+ J ( P<0.05). Compared with group H/R+ J, the cell survival rate and SOD activity were significantly decreased, apoptosis rate and LDH activity were increased, expression of Notch1 and Hes1 was down-regulated, and c-caspase-3 expression was up-regulated in group HG+ H/R+ J ( P<0.05). Conclusion:Activation of Notch1/Hes1 signaling pathway is the endogenous protective mechanism of high glucose and H/R injury to cardiomyocytes.
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NOTCH pathway proteins, including the transcriptional factor HES1, play crucial roles in the development of the inner ear by means of the lateral inhibition mechanism, in which supporting cells have their phenotype preserved while they are prevented from becoming hair cells. Genetic manipulation of this pathway has been demonstrated to increase hair cell number. The present study aimed to investigate gene expression effects in hair cells and supporting cells after Hes1-shRNA lentivirus transduction in organotypic cultures of the organ of Corti from postnatal-day-3 mice. Forty-eight hours after in vitro knockdown, Hes1 gene expression was reduced at both mRNA and protein levels. Myo7a (hair cell marker) and Sox2 (progenitor cell marker) mRNA levels also significantly increased. The modulation of gene expression in the organ of Corti upon Hes1 knockdown is consistent with cell phenotypes related to lateral inhibition mechanism interference in the inner ear. The lentivirus-based expression of Hes1-shRNA is a valuable strategy for genetic interference in the organ of Corti and for future evaluation of its efficacy in protocols aiming at the regeneration of hair cells in vivo.
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Animals , Rats , Cochlea , Basic Helix-Loop-Helix Transcription Factors/genetics , Organ of Corti , Cell Differentiation , Receptors, Notch , Transcription Factor HES-1/genetics , Hair Cells, AuditoryABSTRACT
OBJECTIVE@#To observe the effects of a traditional Chinese medicine (TCM) capsule for replenishing qi, nourishing yin and activating blood on Notch/Hes1 signaling pathway in the renal tissue and vascular endothelial CD34 and CD144 expressions in a rat model of diabetic nephropathy.@*METHODS@#Rat models of early-stage diabetic nephropathy were established by left nephrectomy and high- fat and high- sugar feeding combined with intraperitoneal injection of STZ. The rats were randomized into model group, benazepril group, and high-, moderate-, and low-dose TCM capsule groups for corresponding treatments, with 6 normal rats as the control group. After 8 weeks of drug treatment, blood glucose and 24-h urinary albumin of the rats were measured, and the renal histopathology was observed with HE staining; Hes1 expression in the renal tissue was detected with immunohistochemical staining, and the renal expressions of CD34 and CD144 were detected using Western blotting.@*RESULTS@#Compared with the normal control group, the rat models of diabetic nephropathy showed obvious abnormalities in 24- h urinary albumin and expressions of Hes1, CD34 and CD144d. The TCM capsule at both the high and moderate doses significantly reduced 24-h urinary albumin in the rats; the renal expressions of Hes1 and CD34 was significantly reduced in all the dose groups, and the expression of CD144 was significantly reduced in the high- dose group. Compared with benazepril group, the TCM capsule obviously reduced CD34 expression at all the 3 doses and lowered CD144 expression at the low dose. Histopathologically, the rats in the model group showed glomerular hypertrophy, increased mesenteric matrix, thickening and widening of the mesenteric membrane, and nodular hyperplasia. These pathologies were obviously alleviated by treatment with the TCM capsule at the high and moderate doses.@*CONCLUSIONS@#The Traditional Chinese medicine (TCM) capsule for replenishing qi, nourishing yin and activating blood can reduce Hes1, CD34 and CD144 in kidney tissue of model rats, play a protective role on kidney function and delay the development of DN.
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Animals , Rats , Diabetic Nephropathies , Drugs, Chinese Herbal , Medicine, Chinese Traditional , Qi , Signal Transduction , Transcription Factor HES-1ABSTRACT
Objective@#To evaluate the effect of Notch-Hes1 signaling blockade by a γ-secretase inhibitor on the expression of γδT17 cells in a mouse model of psoriasis-like skin inflammation.@*Methods@#Forty-five healthy specific pathogen-free (SPF) male BALB/c mice were randomly divided into control group, model group and intervention group by simple random sampling. The model group and intervention group were both topically treated with imiquimod 5% cream (62.5 mg once a day) on the shaved back, the intervention group were then intraperitoneally injected with the γ-secretase inhibitor DAPT (10 mg/kg once a day) immediately after topical application of imiquimod, and the control group were topically treated with equivalent amount of vaseline once a day. After 6-day treatment, psoriasis area and severity index (PASI) was used to evaluate changes of skin lesions. On day 7, blood samples were obtained from all the mice through heart puncture after anesthetization, and spleen and skin tissues were acquired to prepare single cell suspension. Spleen index was compared among the 3 groups. Skin tissues on the mouse back were resected and subjected to hematoxylin-eosin staining to observe histopathological changes. Flow cytometry was performed to determine the percentage of γδT17 cells in the spleen and skin tissues, real-time reverse transcription (RT) -PCR to measure the mRNA expression of Hes1 in single cell suspension of the spleen, and enzyme-linked immunosorbent assay (ELISA) to determine the serum level of interleukin (IL) -17A. Statistical analysis was carried out by using one-way analysis of variance and repeated measures analysis of variance for comparison of indices among groups, and Pearson correlation analysis for evaluating the correlation between different indices.@*Results@#Twenty-four hours after the final treatment, the intervention group showed milder psoriasis-like skin inflammation, lower PASI score, and milder degree of epidermal thickening and dermal inflammatory cell infiltration compared with the model group. The model group showed significantly increased spleen index (12.534 ± 1.636) , proportions of γδT17 cells in the spleen (24.659% ± 4.603%) and skin tissues (22.127% ± 5.670%) , mRNA expression of Hes1 in the spleen (4.867 ± 0.543) , and serum level of IL-17A ([22.478 ± 2.776] ng/L) compared with the control group (all P < 0.01) . However, the above indices were significantly lower in the intervention group (9.449 ± 1.040, 14.966% ± 5.770%, 13.631% ± 5.946%, 2.541 ± 0.347, [18.639 ± 1.816] ng/L) than in the model group (all P < 0.01) . In the model group and intervention group, there were positive correlations between the proportions of γδT17 cells in the spleen and serum levels of IL-17A (r = 0.56, 0.53 respectively, both P < 0.05) , between the proportions of γδT17 cells in skin lesions and PASI scores (r = 0.56, 0.52 respectively, both P < 0.05) , as well as between the mRNA expression of Hes1 in the spleen and the proportions of γδT17 cells (r = 0.61, 0.58 respectively, both P < 0.05) or serum levels of IL-17A (r = 0.60, 0.54 respectively, both P < 0.05) .@*Conclusion@#Notch-Hes1 signaling blockade by γ-secretase inhibitor can markedly inhibit the expression of γδT17 cells, and effectively alleviate the severity of psoriasis-like skin inflammation in mouse models.
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<p><b>OBJECTIVE</b>To observe the effect of acupuncture on the Notch signaling pathway in rats with traumatic brain injury and to explore the pathogenesis of acupuncture intervention on traumatic brain injury.</p><p><b>METHODS</b>Feeney's freefall epidural impact method was used to establish a traumatic brain injury model in rats; the rats were randomly divided into a normal group, sham operation group, model group and acupuncture group. Acupuncture was performed in the Baihui (DU 20), Shuigou (DU 26), Fengfu (DU 16), Yamen (DU 15) and Hegu (LI 4) acupoints in the rat, and Yamen was punctured via Fengfu. Then, the rats in each group were randomly divided into three subgroups, namely the day 3 subgroup, day 7 subgroup and day 14 subgroup according to treatment duration. The modified neurological severity scores (mNss) method was used to perform neurobehavioral scoring for evaluating the degree of injury in the rats. The hematoxylin-eosin (HE) staining method was used to observe the pathological change in the brain tissue of rats in each group. Real-time fluorescent quantitative polymerase chain reaction (Q-PCR) technology was used to detect changes in the Notch1, Hes1 and Hes5 gene expression levels in the cortex on the injured side. Western blot was used to detect the protein expression changes.</p><p><b>RESULTS</b>One day after modeling, the mNss scores in the model group and in the acupuncture group were significantly higher than those in the normal and sham operation groups (P<0.01) ; there was no statistically significant difference between the normal group and the sham operation group. The scores decreased with increased treatment time, and the scores in the acupuncture group decreased more significantly than those in the model group (P<0.01). The pathological examination by the HE staining method demonstrated that the brain tissue of the rats in the acupuncture and model groups relatively significantly changed. The Notch1 gene expression level in the acupuncture group was significantly higher than the level in all of the other groups (P<0.01) ; the Hes1 and Hes5 gene expression levels were also higher in the acupuncture group. The expression changes of the Notch1 and Hes1 protein were consistent with that of mRNA. In each experimental group, the mNss score and the pathological results by the HE staining method were consistent with the mRNA results.</p><p><b>CONCLUSION</b>Acupuncture could significantly promote high expression levels of Notch1, Hes1 and Hes5 in the brain tissue of traumatic brain injury rats. Therefore, acupuncture might be an important intervention for inducing endogenous stem cell proliferation and for promoting nerve repair.</p>
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Animals , Male , Rats , Acupuncture Points , Acupuncture Therapy , Brain Injuries , Genetics , Pathology , Therapeutics , Brain Ischemia , Pathology , Therapeutics , Nerve Regeneration , Genetics , Rats, Sprague-Dawley , Receptors, Notch , Genetics , Metabolism , Reperfusion Injury , Genetics , Therapeutics , Signal Transduction , GeneticsABSTRACT
Objective to construct the high titers rat Hes1 adenovirus expression vector (Ad-Hes1).Methods With the rat cDNA as a template,the Hes1 fragment was amplified by PCR,which constructed pShuttle-CMV-Hes1 shuttle plasmid by directly clone.Based on pShuttle-CMV-Hes1,pAdeno-Hes1 virus plasmid was constructed,pAdeno-Hes1 was transfected into 293 cells to package Ad-Hes1,virus titers were determined by modified TCID50.Hes1 was detected by Western blot after Ad-Hes1 infected with H9c2 myocardial cells.Results pShuttle-CMV-Hes1 shuttle plasmid and pAdeno-Hes1 plasmid were constructed successfully,with a general titer of 1.6 × 1011 PFU,Ad-Hes1 can be expressed in H9c2 myocardial cells,and its MOI value was 30.Conclusion Ad-Hes1 is successfully constructedand packaged,thus provide basis for further research on the protection effect of Hes1 on myocardium.
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@#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.
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AIM: To investigate the role of transcription factor hairy and enhancer of split 1 (Hes1) in the malignant transformation of human bronchial epithelial cell line BEP2D induced by tobacco.METHODS: The BEP2D cells were chronically exposed to cigarette smoke condensate (CSC) at 1 cigarette per L until the 70th generation.The phenotype of malignant transformation of the cells induced by CSC was detected by soft agar clony formation assay.RT-PCR and Western blot were used to determined the expression of Hes1 at mRNA and protein levels in each generation of the cells.The proliferation and apoptosis of the BEP2D cells exposed to CSC were analyzed with the methods of MTT assay, flow cytometry and cell colony formation assay after treatment with Notch pathway bloker DAPT or liposome transfection with Hes1-siRNA.The expression of Hes1 in the peripheral small airway tissues of the smoking rats was evaluated by immunohistochemical staining.The expression of Hes1 in non-small-cell lung cancer and normal airway tissues was also detected by the methods of immunohistochemistry and RT-PCR.RESULTS: The BEP2D cells in the 70th generation had a malignant transformation phenotype.The expression of Hes1 in the BEP2D cells exposed to CSC for different time showed an increa-sing trend.DAPT and liposome transfection with Hes1-siRNA down-regulated the expression of Hes1, inhibited the cell proliferation and induced cell apoptosis.The expression of Hes1 in the airway mucosa of the rats exposed to cigarette smoke for 1 month and 6 months was significantly higher than that in control group.Cigarette smoking induced the expression of Hes1 in lung cancer and normal airway tissues.CONCLUSION: Hes1 may be involved in smoking-induced lung cancer by promoting the imbalance between apoptosis and proliferation.
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Objective To investigate the expression of Notch1,Notch3 and Hes1 in gastrointestinal stromal tumors(GIST) and their clinical significance.Methods Quantitative real-time polymerase chain-reaction(Q-PCR) and Western blot were applied to detect the mRNA and the expression of Notch1,Notch3 and Hes1 in 135 matched GIST specimens and adjacent tissues.Meanwhile,the expression of Notch1,Notch3 and Hes1 was detected by immunohistochemistry,and the relationship between their expression and clinicopathological factors in GIST patients was analyzed.In addition,a total of 40 wild type mice(WT) and Notch1 knockout mice(KO) was divided into WT group,KO group,WT+ GIST group and KO+GIST group,and the expression of Notch1,Notch3 and Hes1 in each group was detected.Results Compared with adjacent tissues,the mRNA andthe expression of Notch1,Notch3 and Hes1 were up-regulated in GIST tissues(P<0.05).The positive rates of Notch1,Notch3 and Hes1 in the GIST specimens (59.26 %,65.19 % and 62.22 %) were higher than those in the adjacent tissues(17.780%,22.22 % and 17.78 %),and the difference was statistically significant(P<0.05).Statistical analysis showed that the expression of Notch1 was significantly correlated with the NIH grade of GIST(x2 =8.532,P=0.002);the expression of Notch3 was significantly related with tumor metastasis of GIST (x2 =7.532,P=0.003);the expression of Hes1 was significantly associated with the tumor size of GIST(x2 =6.781,P=0.012).The expression of Notch1,Notch3 and Hes1 was higher in WT+GIST group compared to the expression found in WT group(all P<0.05).There were no significant differences in the expression of Notch1,Notch3 and Hes1 between WT+ GIST group and KO+GIST group.The expression of Notch1,Notch3 and Hes1 was lower in KO+ GIST group compared to the expression found in WT+GIST group(all P<0.05).Conclusion The expression of Notch1,Notch3 and Hes1 related to Notch signaling pathway is elevated in GIST tissues,and the activation of Notch signaling pathway may play an important role in the occurrence and progression of GIST.
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Objective To study effects ofDanlong Xingnao Formula (DLXNF) on proliferation of neural stem cells (NSCs) and the expressions of Hes1 and Hes5 in sub ventricular zone (SVZ) in cerebral ischemia-reperfusion injury model rats; To explore the mechanism of promoting the proliferation of NSCsMethods Eighty male SD rats were randomly divided into sham-operation group, model group, edaravone group andDLXNF group. The focal cerebral ischemia reperfusion injury models were prepared by suture method, and 7 d after reperfusion, the SVZ brain tissue of ischemia side was taken. The proliferation of cells was detected by Brdu labeling fluorescence immunocytochemistry; Hes1, Hes5 mRNA and protein expressions were detected by fluorescence real-time quantitative PCR and Western blot method in each group.Results Compared with the sham-operation group, Brdu positive cell rate in other groups increased more obviously, and the expressions of Hes1, Hes5 mRNA and protein also increased significantly (P<0.01). Compared with the model group, Brdu positive cell rate increased significantly in edaravone group and DLXNF group, and the expressions of Hes1, Hes5 mRNA and protein increased significantly (P<0.01). The expression of Hes1 mRNA in DLXNF group was superior to that in edaravone group (P<0.01), and other indexes had no significant difference.Conclusion DLXNF can promote the proliferation of NSCs in SVZ in cerebral ischemia-reperfusion injury model rats, and up-regulate the expressions of Hes1 and Hes5, whose mechanism may be related to the activation of Notch signaling pathway.
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Objective To investigate the expression of Notch1,Delta-like ligand 4 (DLL4),hairy and enhancer of split-1 (HES-1),microvessel density (MVD),lymphatic vessel density (MLD) in gastric carcinoma,so as to discuss their roles in the development of gastric carcinoma.Methods Gastric carcinoma,paracancer tissues which was apart from the edge of cancer tissue > 60 mm obtained during operation and normal gastric mucosa obtained during gastroscopy were used as controls.All specimens were made tissue microarray.The expressions of Notchl,DLL4,HES-1 were detected by immunohistochemistry.Immunohistochenical double taining was used to detect MVD and MLD.The relationships between Notch1,DLL4,HES-1 expression and angiogenesis,lymphangiogenesis and their significances were analyzed.Results The positive rate of Notch1 in gastric carcinoma was 48.30%,significantly higher than that of paracancerous (25.00%,x2 =6.38,P < 0.05) and control group (16.67%,x2 =10.18,P <0.05).The differences of the positive rate of DLL4 in gastric carcinoma (55.94%),paracancerous (45.70%) and control group (56.67%) were not significant (x2 =1.18,P >0.05 ; x2 =0.005,P > 0.05).The differences of the positive rate of HES-1 in gastric carcinoma (36.64%),paracancerous (34.40%) and control group (33.33%) were not significant (x2 =0.05,P > 0.05 ;,x2 =0.11,P > 0.05).The mean of MVD in gastric carcinoma group was 28.84 ± 14.17,which was significantly higher than that in paracancerous group (17.02 ±8.54,t =4.03,P<0.05) and control group (16.69 ±7.21,t =5.01,P<0.05).The mean of MLD in gastric carcinoma group was 8.55 ±4.98,which was significantly higher than that in paracancerous group (4.05 ± 2.48,t =9.30,P < 0.05) and control group (3.99 ± 1.56,t =10.32,P < 0.05).The expression of DLL4 was correlated with MVD (t =2.77,P < 0.05),but wasn't correlated with MLD (t =1.89,P >0.05).There were no correlations between the expression of Notch1,HES-1 and tissues MVD,MLD (P >0.05).Conclusion Notch1 plays important roles in the development of gastric carcinoma.There are many angiogenesis and lymphangiogenesis in gastric carcinoma.The expression of DLL4 in gastric carcinoma has certain effect in the formation of microvessel.
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Objective To investigate the effect of Notch1 gene dowuregulated on migration and proliferation of human prostate cancer cell line PC-3.Methods The small interfering RNA (siRNA) targeted Notch1 gene or negative control sequences was transfected into PC-3 cells.The expression of Notch1 or Hes1 gene was detected by Real-Time PCR and Western Blot.Then ability of migration or proliferation was measured by Transwell assay or MTS Assay.Results Compared with negative control group (36.097±1.941) and untransfected group (38.762±1.897),Notch1 expression level in siRNA group (3.960±0.510) was significantly reduced (P < 0.01).Meanwhile,Hes1 level in siRNA group was decreased,expression in three groups as follows:siRNA group was 1.690±0.994,negative control group was 8.776±0.916,untransfected group was 9.803±1.001 (P < 0.01).In Transwell assay,the number of migration cells in siRNA group was 657.867±27.610,more than that in the negative control group (158.533±18.263) and untransfected group (146.933±15.733) (P < 0.01).In MTS assay,there was no significant difference among three groups at 0 h point,however,siRNA group was significantly raised at the time points of 24,48 and 72 h (P < 0.01).Conclusions Downregulation of Notch1 gene by transfection of the siRNA-Notch1 sequences significantly promoted ability of migration or proliferation in PC-3 cells,and the effect may be due to the down-regulation of Hes1 expression.
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Objective To observe the effects of exogenous basic fibroblast growth factor (bFGF) on radiation-induced apoptosis of C17.2 neural stem cells(NSCs) and explore the relationship between bFGF and Hes1,RBP-JK protein.Methods The cell viability was assessed using the MTT assay.After the cells attach to the flasks they were irradiated by the linear accelerator.And 5 min later,different concentrations of bFGF in accordance with the experimental design and cultured cells 48 h.Extracted total protein of each group and Western blot analysis showed the changes of the Hes1,RBP-JK protein.Results Compared with the control group,irradiation group cell growth was inhibited,the OD of 0 ng/ml bFGF group was 0.61±0.81,the OD of 80 ng/ml bFGF group was 1.21±1.01 and the control group was 1.51± 1.13.Compared with the control group,all groups showed statistically significant difference (P< 0.05).Western blot analysis showed that with the increasing concentrations of bFGF,RBP-JK expression gradually decreased,but Hes1 expression gradually increased.Conclusion Exogenous basic fibroblast growth factor (bFGF) can inhibit apoptosis of C17.2 NSCs.bFGF can regulate Notch signaling pathway downstream proteins Hes1,RBP-JK expression and inhibit neural stem cell apoptosis which were irradiated.
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Objective:To determine the effect of Hes1 on bone marrow CD34+cells in acute myeloid leukemia (AML). Meth-ods:Bone marrow mononuclear cells were isolated by using Ficoll. Then, the proportion and cell cycle of CD34+cells were analyzed by using fluorescence-activated cell sorting (FACS). CD34+cells were cultured in vitro for colony-forming cells (CFC). The expression of Hes1 in CD34+cells was evaluated by using real-time polymerase chain reaction. After upregulating the expression of Hes1 in CD34+cells, the cell cycle was analyzed through FACS, and the colony formation of CD34+Hes1+cells was analyzed by CFC. Results:The ra-tio of CD34+cells in the bone marrow was lower in the AML group than in the control group. In addition, more CD34+cells underwent quiescence in the AML group than in the control group. In vitro assay showed that the colony formation of CD34+cells was lower in the AML group than in the control group. The expression of Hes1 was higher in the CD34+cells from the AML patients than that in the CD34+ cells from normal donors. After Hes1 transduction, more CD34+ cells underwent quiescence and showed weak proliferation. Conclusion:The proportion of CD34+cells in the bone marrow was lower in AML patients than in normal donors. A large proportion of CD34+cells underwent quiescence, which was related to Hes1, in AML patients.
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tumours was found to be slightly faster than those of the controls.Conclusion:Hes1 is overexpressed in colon cancer than that in normal tissue, and Hes1 is upregulated in poorly differentiated cancer samples compared with well-differentiated tumour samples. Hes1 has a positive inlfuence on the ability of tumour-formation in SW620 cellsin vivo.
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Objective To obtain the expression pattern of imprint gene H19 in JEG-3 cell in order to explore the regulation mechanism of H19 on trophoblast cellular biological behavior .Methods After correct identification with sequencing for the recom-binant eukaryotic expression plasmid pRc/CMV which including the whole length of H19 cDNA ,the plasmid was transfected to the cell line JEG-3 .The expression of H19 mRNA was observed and the gene expression profile of three groups of JEG-3 cell were de-tected with Affymetri :U133 plus 2 .0 Array .Results After being transfected with target H 19 gene ,the expression of the mRNA level was significantly increased compared with control group .And the gene expression profile was changed significantly .19 genes were up-regulated ,77 genes were down-regulated .Expression levels of HES1 gene which being choosed as a different expression gene were detected by fluorescence quantitative PCR in severe preeclampsia placenta tissue and normal late pregnant placenta .The expression level of HES1 mRNA in severe preeclampsia placenta decreased significantly than normal late pregnant placenta tissues . Conclusion Many genes induced by H19 have been screened by high-throughput gene chip method .It provides the experimental ba-sis for advanced studying the regulation the cellular biological behavior with H 19 gene .