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Objective To explore the effects and mechanism of Baishile Capsules regulating SHH/Gli1 signaling pathway on hippocampal neurogenesis of depression model rats.Methods Totally 32 SD rats were randomly divided into control group,model group,fluoxetine(5.4 mg/kg)group and Baishile Capsules(2.88 g/kg)group,with 8 rats in each group.A depression rat model was established using chronic unpredictable mild stress and single cage feeding method.The model was established and administered simultaneously for 21 consecutive days.Depression-like behavior in rats were evaluated by sucrose preference experiment and open field experiment,ELISA was used to detect brain derived neurotrophic factor(BDNF)contents in rat serum and hippocampal tissue,the number of BrdU,BrdU/DCX,BrdU/NeuN positive cells in dentate gyrus of the hippocampus was observed by immunofluorescence,immunofluorescence and Western blot were used to detect the fluorescence intensity and protein expression of SHH,Gli1,Smo,Ptch in hippocampal tissue.Results Compared with the control group,the degree of sucrose preference significantly decreased in the model group(P<0.01),the number of horizontal and vertical movements significantly decreased(P<0.01),the contents of BDNF in serum and hippocampal tissue significantly decreased(P<0.05),the number of BrdU,BrdU/DCX,BrdU/NeuN positive cells in dentate gyrus of the hippocampus significantly decreased(P<0.01),and the fluorescence intensity and protein expression of SHH,Gli1,Smo,Ptch in hippocampal tissue significantly decreased(P<0.01,P<0.05).Compared with the model group,the degree of sucrose preference and the number of horizontal and vertical movements in fluoxetine group and Baishile Capsule group increased significantly(P<0.05,P<0.01),the contents of BDNF in serum and hippocampal tissue significantly increased(P<0.05,P<0.01),and the number of BrdU,BrdU/DCX,BrdU/NeuN positive cells in dentate gyrus of the hippocampus significantly increased(P<0.01,P<0.05),the fluorescence intensity and protein expressions of SHH,Gli1,Smo,Ptch in hippocampal tissue significantly increased(P<0.01,P<0.05).Conclusion Baishile Capsule can promote the hippocampus neurogenesis in depression model rats by regulating SHH/Gli1 signaling pathway,and play an antidepressant role.
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@#[摘 要] 目的:探讨扁蒴藤素(Pris)调节Shh/Gli1信号通路对宫颈癌HeLa细胞增殖、凋亡和血管生成拟态(VM)的影响及其机制。方法:采用MTT法检测不同浓度Pris对宫颈癌HeLa细胞增殖的抑制作用,以选取合适的干预浓度。将HeLa细胞分为对照组、环巴胺组、Pris组、Pris+pc-NC组和Pris+pc-Shh组。采用MTT法、EdU法检测各组细胞的增殖能力,Transwell小室法、流式细胞术检测各组细胞的迁移及侵袭能力和细胞凋亡率,体外血管生成实验观察VM形成情况,qPCR法检测各组细胞中Shh和Gli1 mRNA表达水平,WB法检测细胞中血管内皮生长因子A(VEGF-A)、血管内皮钙黏素(VE-cadherin)、Ki-67、caspase-3及与Shh/Gli1信号通路相关蛋白表达水平。结果:0.25~2.5 mmol/L的Pris对HeLa细胞增殖均有显著抑制作用,选择1.5 mmol/L的Pris进行后续实验。对照组细胞形成良好的管腔结构,与对照组相比,环巴胺组、Pris组和Pris+pc-NC组HeLa细胞管腔结构被明显破坏,细胞增殖活力和增殖率、迁移及侵袭细胞数目、Shh和Gli1 mRNA、VEGF-A、VE-cadherin、Ki-67、Shh、Gli1蛋白表达均显著降低(均P<0.05),细胞凋亡率和caspase-3表达均显著升高(均P<0.05);环巴胺组与Pris组HeLa细胞各项检测指标比较差异均无统计学意义(均P>0.05);与Pris+pc-NC组相比,Pris+pc-Shh组细胞管腔结构形成明显改善,细胞增殖活力和增殖率、迁移及侵袭细胞数、Shh和Gli1 mRNA、VEGF-A、VE-cadherin、Ki-67、Shh、Gli1蛋白表达均显著升高(均P<0.05),细胞凋亡率和caspase-3表达均显著降低(均P<0.05)。结论:Pris抑制宫颈癌HeLa细胞的增殖、迁移与侵袭和VM的形成并促进细胞凋亡,可能与阻断Shh/Gli1信号通路有关。
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BACKGROUND:Studies demonstrated that miR-135a-5p was highly expressed in mouse embryonic palatal mesenchymal cells with cleft palate induced by dexamethasone.The primary cilium and its mediated Shh signaling pathway were involved in the autophagy of mouse embryonic palatal mesenchymal cells.It is speculated that miR-135a-5p may regulate autophagy in mouse embryonic palatal mesenchymal cells through primary cilia and its mediated Shh signaling pathway. OBJECTIVE:To investigate the regulatory effect of miR-135a-5p on autophagy of mouse embryonic palatal mesenchymal cells. METHODS:In vitro,palatal mesenchymal cells from C57BL/6J mouse embryos were extracted and cultured.Cell transfections were set up as follows:(1)the cells were divided into control group,miR-135a-5p negative control group and miR-135a-5p mimic group;(2)NC+miR-NC group,KIF3B overexpression group,and miR-135a-5p+KIF3B group:qRT-PCR was performed to verify transfection efficiency of miR-135a-5p and KIF3B.A transmission electron microscope was used to observe the number of autophagosome/autophagolysosome in the cells of each group.The degree of fluorescence expression of autophagy marker LC3B was determined by the immunofluorescence technique.The protein expression of KIF3B,LC3 and P62 was determined by western blot assay.(3)The cells were divided into miR-135a-5p negative control group,and SAG treated group,and SAG+miR-135a-5p group.qRT-PCR was used to detect the mRNA expression levels of Gli3,a key transcription factor downstream of Shh signaling.The protein expressions of autophagy-related proteins LC3 and P62 were detected by western blot assay. RESULTS AND CONCLUSION:(1)After overexpression of miR-135a-5p,the number of autophagosome/autophagolysosome was significantly increased(P<0.01).The fluorescence density of LC3B increased significantly(P<0.01);the protein expression of KIF3B and P62 decreased(P<0.01),and the protein expression of LC3 increased.(2)After overexpression of KIF3B,the number of autophagosome/autophagolysosome was significantly decreased(P<0.01);the fluorescence density of LC3B was decreased(P<0.01);the protein expression of P62 was increased(P<0.01),and the protein expression of LC3 was decreased(P<0.01).Targeted expression of KIF3B was inhibited by miR-135a-5p(P<0.01);the number of autophagosome/autophagolysosome,the fluorescence intensity of LC3B as well as the protein expression of LC3 were reversed(P<0.01)and the protein expression of P62 was decreased(P<0.01).(3)SAG significantly increased the mRNA expression of Gli3(P<0.01),increased the protein expression of P62(P<0.01),and decreased the protein expression of LC3(P<0.01).When miR-135a-5p was added,Gli3 mRNA expression was significantly decreased(P<0.01);P62 protein expression was decreased(P<0.01),and LC3 protein expression was reversed(P<0.01).(4)These results indicate that miR-135a-5p targets the inhibition of KIF3B and promotes autophagy in mouse embryonic mesenchymal cells possibly by negatively regulating the Shh signaling pathway.
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BACKGROUND:The second heart field is crucial for the development of the embryonic heart.Abnormal development of the second heart field can result in multiple cardiac malformations.After Cx43 gene knockout,reduced formation and proliferation of cells of the second heart field can be observed,but the specific reason remains unclear. OBJECTIVE:(1)To determine whether β-catenin,Smo and Cx43 were co-expressed in the second heart field and the endoderm,we observed the expression patterns of these proteins.(2)To explore whether Cx43 interacts with the Wnt/β-catenin pathway or the Shh pathway in the development of the second heart field. METHODS:Serial paraffin sections of the mouse embryos at embryonic days 10-12 were selected for immunohistochemical staining,hematoxylin-eosin staining and immunofluorescence staining.The primitive gut of mouse embryos at embryonic day 11 was separated for western blot assay and co-immunoprecipitation. RESULTS AND CONCLUSION:(1)Cx43 and Isl1 were co-expressed in some mesenchymal cells on the ventral side of the foregut and dorsal wall of the pericardial cavity of mouse embryos at embryonic days 10-12;Isl1 positive cells increased while Cx43 positive cells increased.(2)Cx43 and β-catenin were co-expressed in the ventral part of the endoderm at embryonic days 10-12.(3)Cx43 and Smo were co-expressed in the endoderm at embryonic days 10-12.(4)The co-immunoprecipitation results confirmed that there was an interaction between Cx43 and β-catenin,which suggested that Cx43 interacted with β-catenin to participate in the development of the second heart field.
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The seat of human intelligence is the human cerebral cortex, which is responsible for our exceptional cognitive abilities. Identifying principles that lead to the development of the large-sized human cerebral cortex will shed light on what makes the human brain and species so special. The remarkable increase in the number of human cortical pyramidal neurons and the size of the human cerebral cortex is mainly because human cortical radial glial cells, primary neural stem cells in the cortex, generate cortical pyramidal neurons for more than 130 days, whereas the same process takes only about 7 days in mice. The molecular mechanisms underlying this difference are largely unknown. Here, we found that bone morphogenic protein 7 (BMP7) is expressed by increasing the number of cortical radial glial cells during mammalian evolution (mouse, ferret, monkey, and human). BMP7 expression in cortical radial glial cells promotes neurogenesis, inhibits gliogenesis, and thereby increases the length of the neurogenic period, whereas Sonic Hedgehog (SHH) signaling promotes cortical gliogenesis. We demonstrate that BMP7 signaling and SHH signaling mutually inhibit each other through regulation of GLI3 repressor formation. We propose that BMP7 drives the evolutionary expansion of the mammalian cortex by increasing the length of the neurogenic period.
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Animais , Camundongos , Humanos , Células Ependimogliais/metabolismo , Proteínas Hedgehog/metabolismo , Furões/metabolismo , Córtex Cerebral , Neurogênese , Mamíferos/metabolismo , Neuroglia/metabolismo , Proteína Morfogenética Óssea 7/metabolismoRESUMO
OBJECTIVE@#To investigate the effect of Zuogui Jiangtang Jieyu Decoction (ZJJ) on Shh signaling and self-renewal of neural stem cells in the hippocampal dentate gyrus of diabetic rats with depression.@*METHODS@#Diabetic rat models with depression were randomly divided into model group, positive drug (metformin + fluoxetine) group, and low-, medium-, and high-dose ZJJ groups (n=16), with normal SD rats as the control group. The positive drugs and ZJJ were administered by gavage, and the rats in the control and model groups were given distilled water. After the treatment, blood glucose level was detected using test strips, and behavioral changes of the rats were assessed by forced swimming test and water maze test. ELISA was used to examine the serum level of leptin; The expressions of nestin and Brdu proteins in the dentate gyrus of the rats were detected using immunofluorescence assay, and the expressions of self-renewal marker proteins and Shh signaling proteins were detected using Western blotting.@*RESULTS@#The diabetic rats with depression showed significantly increased levels of blood glucose and leptin (P < 0.01) and prolonged immobility time in forced swimming test (P < 0.01) and increased stage climbing time with reduced stage seeking time and stage crossings in water maze test (P < 0.01). The expressions of nestin and Brdu in the dentate gyrus, the expressions of cyclin D1, SOX2, Shh, Ptch1, Smo in the hippocampus and the nuclear expression of Gli-1 were decreased (P < 0.01) while hippocampal Gli-3 expression was increased significantly (P < 0.01) in the rat models. Treatment of rat models with high-dose ZJJ significantly reduced the blood glucose (P < 0.01) and leptin level (P < 0.05) and improved their performance in behavioral tests (P < 0.01). The treatment also obviously increased the expressions of nestin, Brdu, cyclin D1, SOX2, Shh, Ptch1, and Smo and the nuclear expression of Gli-1 in the dentate gyrus (P < 0.01) and reduced hippocampal expression of Gli-3 (P < 0.05) in the rat models.@*CONCLUSION@#ZJJ can significantly improve the self-renewal ability of neural stem cells and activate Shh signaling in dentate gyrus of diabetic rats with depression.
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Animais , Ratos , Glicemia , Bromodesoxiuridina , Autorrenovação Celular , Ciclina D1 , Giro Denteado , Depressão , Diabetes Mellitus Experimental , Hipocampo , Leptina , Nestina , Ratos Sprague-DawleyRESUMO
OBJECTIVE@#To investigate the effects of inhibiting Sonic Hedgehog (Shh) signaling on fibrous scar formation and functional outcome after ischemic brain injury.@*METHODS@#Adult SD rats were randomized into sham-operated group, middle cerebral artery occlusion (MCAO) and reperfusion (I/R) group, I/R with intraventricular empty adenoviral vector (rAd-NC) injection group, and I/R with adenovirus-mediated Shh knockdown (rAd-ShShh) group. After the treatments, the neurological deficits of the rats were assessed, and the protein and mRNA expressions of fibronectin (Fn), α-SMA, and Shh in the ischemic hemisphere were detected with immunofluorescence assay and qPCR; TUNEL staining was used for detecting neural cell apoptosis. In the cell experiment, primary meningeal fibroblasts isolated from neonatal SD rats were pretreated for 24 h with TGF-β1 or TGF-β1 plus cyclopamine (CYC) before oxygen-glucose deprivation for 150 min followed by reoxygenation for 72 h (OGD/R). CCK-8 assay and scratch test were performed to examine the changes in cell proliferation and migration, and immunofluorescence assay, qPCR and Western blotting were used for detecting cell transformation and the expressions of Shh, α-SMA, and Fn.@*RESULTS@#Cerebral I/R injury significantly increased the protein and mRNA expressions of Shh, α-SMA, and Fn in the ischemic hemisphere of the rats, but their expression levels were significantly lowered by intraventricular injection of rAd-Shshh (P < 0.05), which obviously increased cell apoptosis in the ischemic hemisphere (P < 0.05) and improved modified mNSS and modified Bederson scores of the rats (P < 0.05). In the cell experiment, pretreatment with TGF-β1 and TGF-β1+CYC both increased the viability of the primary meningeal fibroblasts after OGD/R. TGF-β1 significantly enhanced the migration ability and induced obvious transformation of the exposed cells (P < 0.05), but these effects were significantly attenuated by co-treatment with CYC (P < 0.05). The expressions of Shh, α-SMA and Fn in the TGF-β1 group were all significantly higher in TGF-β1-treated cells (P < 0.05) and were obviously lowered by co-treatment with CYC (P < 0.05).@*CONCLUSION@#Inhibition of Shh signaling may inhibit fibrous scar formation and functional recovery in rats after ischemic brain injury.
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Animais , Ratos , Lesões Encefálicas , Cicatriz , Proteínas Hedgehog , RNA Mensageiro , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta1RESUMO
OBJECTIVE@#To investigate the effect of TGF-β1 on Shh signaling pathway during the transformation of meningeal fibroblasts into myofibroblasts.@*METHODS@#Primary meningeal fibroblasts were isolated from neonatal (24 h) SD rats and purified using type Ⅳ collagenase. The isolated cells were treated with 10 ng/mL TGF-β1 alone or in combination with 20 μmol/L SB-431542 (a TGF-β1 receptor inhibitor) for 72 h, and the changes in proliferation and migration abilities of the fibroblasts were assessed with CCK-8 assay and cell scratch test. The expression of fibronectin (Fn) was detected with immunofluorescence assay, and Western blotting was performed to examine the expressions of Fn, α-SMA and Shh protein in the cells; the expression of Shh mRNA was detected with real-time fluorescence quantitative PCR.@*RESULTS@#TGF-β1 treatment obviously enhanced the proliferation and migration of primary meningeal fibroblasts (P < 0.05), and promoted the transformation of meningeal fibroblasts into myofibroblasts and the secretion of Fn (P < 0.05). TGF-β1 treatment also upregulated the expression of Shh at both protein and mRNA levels (P < 0.05). Treatment with SB-431542 partially blocked the effect of TGF-β1 on the transformation of meningeal fibroblasts (P < 0.05).@*CONCLUSION@#TGF-β1 can induce the transformation of meningeal fibroblasts into myofibroblasts by up-regulating Shh expression in Sonic Hedgehog signaling pathway.
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Animais , Ratos , Fibroblastos/metabolismo , Proteínas Hedgehog , Miofibroblastos/metabolismo , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta1/metabolismoRESUMO
OBJECTIVE: To observe the effect of penetrative needling from "Baihui" (GV20) to "Qubin" (GB7) on neural stem cell proliferation and sonic hedgehog (Shh) signaling in subventricular zone (SVZ) in intracerebral hemorrhage (ICH) rats so as to explore its mechanisms underlying improvement of ischemic injury of brain. METHODS: Male SD rats were randomly divided into blank control, model, acupuncture and agonist (Purmorphamine, an activator of Shh signaling pathway) groups (n=18 in each group, 6 for H.E. stain, 6 for examining neuronal cell proliferation, and 6 for immunohistochemistry). The ICH model was established by injecting autogenous blood (50 µL) into the right caudate nucleus. The neurological defect was scored with refe-rence to Bederson's method. Penetrative needling from GV20 to GB7 was performed by manipulating the needle for 6 min (repeated 3 times in 30 min), once daily for 7 days. Intraperitoneal injection of Purmorphamine (1 mg/mL, 1 mg/kg) was performed, once daily for 7 days. Histopathological changes of the hemorrhagic penumbra region were observed under microscope after H.E. stain, the newborn neural stem cell proliferation (BrdU+/Nestin+ double labeled cells) in the SVZ was observed by immunofluorescence after intraperitoneal injection of BrdU (50 mg/kg), and the expression of Shh and glioma-associated hemolog-1 (Gli1) detected by immunohistochemistry. RESULTS: After modeling, the neurological score and expression levels of Shh and Gil1 proteins were significantly increased in the model group relevant to the blank control group (P0.05). Outcomes of H.E. stain showed obvious edema, disordered arrangement of cells, infiltration of inflammatory cells and red blood cells with glial cell hyperplasia around the hematoma area in the model group, which was relatively milder in both acupuncture and agonist groups such as in basic disappearance of edema and inflammatory reaction. CONCLUSION: Penetrative needling from GV20 to GB7 can obviously improve neurological function in ICH rats, which is related to its effects in activating Shh/Gil1 signaling and in further promoting neural stem cell proliferation in the SVZ region.
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BACKGROUND: Studies have shown that Sonic Hedgehog (Shh) is expressed in mouse Notochordal cells, and India Hedgehog (Ihh) is mainly expressed in chondrocyte-like cells and end plates of mouse embryonic vertebral bodies. However, the expression of Shh and Ihh during the formation of intervertebral discs in mouse embryo is unclear. OBJECTIVE: To investigate the expression of Shh and Ihh during the formation of intervertebral discs in mouse embryo. METHODS: Male Shh-CreERT2, R26-mTmG/+ mice mated with female R26-mTmG/+ mice to obtain pregnant rats at different periods of pregnancy (E8.5, E11.5, E12.5, E14.5, E16.5, E18.5), followed by injection of 10 g/L tamoxifen at a dose of 10 μL/g. Intervertebral disc tissues were isolated from the mouse embryo at P0, followed by genotype identification. At the same time, male C57BL/6 mice mated with female C57BL/6 mice to obtain pregnant rats at different periods of pregnancy (E11.5, E12.5, E14.5, E16.5, E18.5), and the intervertebral disc tissues of mouse embryo were taken and analyzed. The expression of Shh and Ihh during the formation of intervertebral discs in mouse embryo was detected using immunofluorescence and immunohistochemistry, respectively. Implementation of animal experiments was approved by the Animal Ethics Committee of Soochow University. RESULTS AND CONCLUSION: Shh-CreERT2; R26-mTmG/+ mouse embryos were identified by PCR amplification. The results of immunofluorescent staining showed a gradual decrease in the expression of Shh in nucleus pulposus cells during the formation of intervertebral discs in mouse embryo. The results of immunohistochemical staining showed a gradual increase in expression of Ihh in nucleus pulposus cells during the formation of intervertebral discs in mouse embryo. In summary, Shh and Ihh are dynamically expressed during the formation of intervertebral discs in mouse embryo, and provide a basis for further research on the molecular mechanism of intervertebral disc degeneration.
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Sonic Hedgehog gene (SHH) plays a vital role in embryogenesis through its secreted protein sonic hedgehog protein (Shh). During embryogenesis, Shh acts as a morphogen controlling proximal and distant signaling in the specific development of tissue lineages, patterning, regulation of cell proliferation and suppression of tissue apoptosis. Shh also exerts its role in odontogenesis by determining the site of tooth bud formation, in tooth morphogenesis and root formation. The difference in the specific development of a region by Shh can be explained by its [a] 'Spatial gradient [b] the 'form' [c] Concentration gradient and [d] Temporal gradient. Shh signaling pathway has an extracellular and an intracellular component. A disruption of Shh pathway contributes to tumorigenesis of several cell types including those arising from odontogenic structures. This article reviews Shh from its formation in embryonic stages, its role in development and odontogenesis, to its reactivation in tumorigenesis and in specific to odontogenic pathologies.
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Objective To study the mechanism of the Hedgehog signaling transduction intervened by polypeptide extract from scorpion venom (PESV) on K562/BALB/c-nu leukemia mice. Trying to analyze the molecular mechanisms and targets of the inhibited effect of PESV on chronic myeloid leukemia (CML) in vivo. Methods After establishing the K562/BALB/c-nu leukemia mice successfully, the model mice were divided into six groups which were the blank group, the PESV high, medium, and low doses (0.3, 0.6, 1.2 mg/kg) group, the Imatinib (50 mg/kg) group, and the model group. After 14 d drug intervention, the levels of gene and protein expression of Hedgehog signaling pathway upstream factors Shh, Ptch, and Smo were detected by qRT-PCR and Western blotting, and the protein expression of downstream factor Gli1 was determined by ELISA test. Results Compared to the model group, the genetic and protein expression of Shh which was an upstream factor were increased in the PESV groups. The mRNA and protein expression of Ptch and Smo in PESV low-dose and medium-dose groups were decreased. There were no significant differences of upstream factors between Imatinib group and model group. The concentration of downstream Gli1 protein significantly decreased within low-dose and medium-dose PESV groups, while there was no significant difference between high-dose PESV group and Imatinib group. Conclusion PESV can inhibit the expression of Hedgehog signaling pathway upstream factor Ptch, Smo and downstream factor Gli1 on the mRNA and protein level, while Imatinib has no obvious inhibitory effect on the Hedgehog signaling pathway.
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@#Choroid neovascularization is the characteristic pathological change of many fundus diseases and is the most common cause for severe vision loss and metamorphopsia. Among the pathogenic factors, VEGF is considered to be the most important and treatment targeting VEGF showed promising results. However, anti-VEGF agents need to be administrated frequently and they are usually expensive. Also, some patients got no response to this treatment. These facts force us to find other pathway that involves in the formation of CNV. This article reviews the latest research on CNV-related signaling pathways so as to provide a deeper look into CNV and hopefully point out new directions for treating diseases that share similar pathogenesis.
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Objective: To investigate the effect of activation of SHH signaling pathway on the apoptosis and invasion of the trophoblast cells in the preeclampsia (PE) patients, and to clarify its mechanism. Methods: The HTR8/SVneo cells were divided into normal control group, hypoxia/reoxygenation (H/R) treatment group and purmorphamine+H/R treatment group. The expression levels of SHH signaling pathway related proteins SHH, Ptchl, Smo, Glil, and Gli3 in the placenta tissues of PE and late trimester of normal pregnancy were detected by Western blotting method; the apoptotic rates of HTR8/SVneo cells in various groups were detected by flow cytometry (FCM); Transwell assay was used to detect the number of transmembrane cells; the expression levels of matrix metalloproteinase-2 (MMP-2) and metalloproteinase-9 (MMP-9) proteins in the HTR8/SVneo cells in various groups were detected by Western blotting method. Results: The expression levels of SHH, Ptchl, Smo, Glil and Gli3 proteins in the placenta tissue of PE were significantly lower than those in normal pregnancy placenta tissue (P<0. 05); the expression levels of SHH, Ptchl, Smo, Glil, Gli3, MMP-2, and MMP-9 proteins and the number of transmembrane cells in H/R treatment group were significantly lower than those in normal control group (P
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AIM:To investigate the relationship between Sonic Hedgehog(Shh)signaling pathway and cell cycle and radioresistance of esophageal cancer by up-regulating Gli1,a key factor in Shh signaling pathway.METHODS:The human esophageal cancer cell line Eca 109 was transfected with plasmid to induce Gli 1 over-expression,which served as Eca109-ox-Gli1 group.In addition, Eca109 cells transfected with empty plasmid served as negative control group and the untreated Eca109 cells were used as normal control group.The expression of Gli1 was confirmed by real-time PCR and Western blot.The radiosensitivity of the cells in the 3 groups was determined by colony formation assay.The effect of irra-diation on the cell cycle was analyzed by flow cytometry.RESULTS:The expression of Gli1 in Eca109-ox-Gli1 group was higher than that in the other 2 groups(P<0.05).The survival fraction at dose of 2 Gy in Eca109-ox-Gli1 group was high-er than that in normal control group, indicating that the radioresistance of the Eca 109 cells transfected with Gli1 plasmid was increased.The cells in Eca109-ox-Gli1 group showed higher S phase proportion than that in normal control group and negative control group(P<0.01).After irradiation at dose of 6 Gy,all cells in the 3 groups found that the cell cycle was arrested at G2/M phase,while the cells in normal control group showed higher G 2/M phase proportion than that in Eca109-ox-Gli1 group(P<0.01).CONCLUSION: The up-regulation of Gli1 may enhance the radioresistance of esophageal cancer by regulating the cell cycle.
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Pituitary stalk interruption syndrome(PSIS) is a developmental defect,characterized by a thin or absent pituitary stalk. PSIS is a rare disease with combined pituitary hormone deficiencies, the pathogenesis of which is related to genetic mutations and environmental factors. It is also a genetically and clinically heterogeneous disease. Nine genes were found to be related to PSIS,including HESX1,LHX4,OTX2,SOX3,CDON,GPR161, PROKR2,TGIF and Wnt,Notch,SHH signalling pathways. In recent years,the intensive genetic studies show that four novel genes(CDON,GPR161,PROKR2,TGIF)and SHH pathway are related to PSIS,which provides a brand-new etiopathogenesis of PSIS.
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It has been demonstrated that hypoxia retards the growth of fish, reduces the survival of their larvae, deforms their vertebral column, but despite this teleost fish have the ability to completely regenerate many of their tissues, particularly the retina. As we do not have enough information about the effects of hypoxia on the eyeball, orbit and retina of Atlantic salmon (Salmo salar), we propose the following objectives: 1) Compare the morphological changes of the eyeball of fish subject to hypoxia and normoxia. 2) Determine changes in the orbit structure. 3) Describe the retina of salmon alevins. 4). Recognize hypoxic cells using the anti-Hif1a antibody in the retina of alevins as a sensor. 5) Determine the Shh morphogenic expression in alevins exposed to different times of hypoxia. Around 1,000 Salmo salar alevins were placed in a continuous water flow of 9 °C at 100 % SatO2 and alevins maintained at a hypoxia of 60 % SatO2. The latter were transferred to normoxia (at days two, four, and eight after hatching). A control group maintained at continuous normoxia and another at continuous hypoxia was also considered. All the alevins were euthanized at 950 UTAs (±2 months after hatching). Diaphonization (double-stain) according to the Hanken & Wassersug technique was undertaken to describe the morphology of the periocular cartilage and to measure the ocular diameter. The HIF-1a factor antibody 1:50, and the anti-Shh antibody dilution of 1:100 were used. The alevins after hatching had large eyeballs with the optic cup having an embryonic shape, even a choroidal fissure. The greatest thickness was observed in the nasal ventral zone which corresponds to a zone of pluripotent cells. The optic cup aspect with embryonic characteristics has only been reported in salmonids. The central retina of the alevins those were cultivated with a 60 % saturation of O2 for two, four or eight days had positive immunostaining when analyzed with the anti-HIF1a antibody hypoxia sensor. The inner ganglion and nuclear layers had immunopositive cells, with the highest in the alevins that were two days in hypoxia and the lowest when the hypoxia was chronic. Nevertheless, in the latter case the alevins had anatomical deformation of the eyeball and periocular cartilage. The anti-Shh antibody clearly shows a gradient that is expressed in the germinative zone and in the cells of the inner ganglion and nuclear layers. The eyeball and particularly the retina in salmon alevins are an example of neuronal plasticity and neurogenesis.
Se ha demostrado que la hipoxia retarda el crecimiento de los peces, reduce la supervivencia de sus larvas, deforma su columna vertebral, pero a pesar de esto, este pez teleósteo tiene la capacidad de regenerar completamente muchos de sus tejidos, en particular la retina. Como no existe suficiente información sobre los efectos de la hipoxia en el bulbo ocular, la órbita y retina del salmón del Atlántico (Salmo salar), los objetivos de este trabajo fueron: 1) Comparar los cambios morfológicos del bulbo ocular del pescado sujetos a hipoxia y normoxia; 2) Determinar los cambios en la estructura de la órbita; 3) Describir la retina de los alevines de salmón; 4) Reconocer las células hipóxicas utilizando el anticuerpo anti-Hif1a en la retina de alevines como un sensor; 5) Determinar la expresión morfogenética de Shh en alevines expuestos a diferentes momentos de hipoxia. Alrededor de 1.000 alevines Salmo salar se colocaron en un flujo continuo de agua a 9 °C, con 100 % de SatO2 y otros alevines se mantuvieron con una hipoxia de 60 % SatO2. Estos últimos fueron trasladados a normoxia (en los días dos, cuatro y ocho después de la eclosión). Un grupo control se mantuvo a normoxia continua y otro grupo a hipoxia continua. Todos los alevines se sacrificaron a 950 UTA (+ dos meses después de la eclosión). Se realizcón una diafonización (doble tinción), de acuerdo con la técnica de Hanken & Wassersug, para describir la morfología del cartílago periocular y para medir el diámetro ocular. Se utilizaron el anticuerpo anti-Hif1a a una dilución 1:50, y el anticuerpo anti-Shh a una dilución de 1:100. Los alevines después de la eclosión presentaron grandes bulbos oculares, con la copa óptica con forma embrionaria, incluso una fisura coroidea. El mayor espesor se observó en la zona ventral nasal que corresponde a una zona de células pluripotentes. El aspecto de la copa óptica con características embrionarias sólo se ha informado en los salmónidos. La retina central de los alevines fueron cultivadas con una saturación de 60 % de O2 para dos, cuatro y ocho días, y presentó inmunotinción positiva cuando se analizó con el sensor de hipoxia, el anticuerpo anti-HIF1a. El ganglio interior y las capas nucleares presentaron células immunopositivas, con los niveles más altos en los alevines con dos días de hipoxia y niveles más bajos en hipoxia crónica. Sin embargo, en éste último caso los alevines presentaron una deformación anatómica del bulbo ocular y el cartílago periocular. El anticuerpo anti-Shh mostró claramente un gradiente expresado en la zona germinativa y en las células del ganglio interior y las capas nucleares. El bulbo ocular y en particular la retina en alevines de salmón son un ejemplo de plasticidad neuronal y neurogénesis.
Assuntos
Animais , Olho/anatomia & histologia , Hipóxia , Órbita/anatomia & histologia , Retina/anatomia & histologia , Salmo salar/anatomia & histologiaRESUMO
@#Proliferation and differentiation of neural stem cells is regulated by autologous or external, adjacent or remote cell signaling pathways. This paper reviewed the studies about the Notch, BMP, Wnt, Shh signaling pathways related to brain neurogenesis.
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Proliferation and differentiation of neural stem cells is regulated by autologous or external, adjacent or remote cell signaling pathways. This paper reviewed the studies about the Notch, BMP, Wnt, Shh signaling pathways related to brain neurogenesis.
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To study the effect of primary cilia-mediated sonic hedgehog (Shh) signaling on differentiation of rat bone marrow stromal cells (MSCs) into neuron-like cells. Methods Rat MSCs were isolated and cultured. The study was divided into normal resveratrol-cultured group, resveratrol-induced group, SAG (Smoothened [Smo] agonist) group, and cyclopamine (Smo inhibitor) group. Cell morphology was observed under inverted microscope; the expressions of Ac-Tu, Ptc, Smo, and Glil were examined by immunofluorescence method. Western blotting analysis was used to detect the protein expressions of Smo and Gli, and RT-PCR was applied to detect mRNA expressions of Smo and Glil. Results No expression of primary cilia was found in the normally cultured MSCs. After pre-induction or 24 h starvation, MSCs expressed primary cilia, Ptc, Smo and Glil proteins, with Ptc protein in the primary cilia and Smo, Glil in the cytoplasm. In normal cultivation, resveratrol did not promote the translocation of Smo or induce differentiation of MSCs into neuronal-like cells. When MSCs expressed primary cilia, resveratrol and SAG led to translocation of Smo from the cytoplasm into primary cilia, accompanied by MSCs differentiation into neuronal-like cells and significantly increased expression of Smo and Glil mRNA and protein (P< 0. 05). When cyclopamine was added, Smo remained expressed in the cytoplasm, the expression of Smo and Glil mRNA and protein was significantly decreased (P < 0. 05), and MSCs differentiation into neuronal-like cells was inhibited. Conclusion MSCs express primary cilia and have Shh signaling. Primary cilia-mediated Shh signaling participates in MSCs differentiation into neuronal-like cells.