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Objective The aim of this study was to investigate the expression of cell cycle checkpoint kinase 1(Chk1)gene in glioblastoma cells( GBM) and its correlation with GBM cell proliferation,tumorigenic activity and prognosis. Methods The ex-pression of Chk1 in GBM cells was selected and analyzed by TCGA database and brain tumor molecular database( Rembrandt),and the level of Chk1 expression in GBM cells was detected by molecular biology techniques such as Western blot and Real-Time PCR. The expression of Chk1 was silenced by siRNA to investigate its effect on proliferation and colony-forming ability of GBM cells. The prognosis survival of GBM patients accompanying with Chk1 expression was analyzed by immunohistochemical staining and Rembrandt database. Results The results of TCGA database and Rembrandt showed that Chk1 gene was highly expressed in GBM tissues. West-ern blot and Real-Time PCR also showed that Chk1 gene was highly expressed in GBM cells. Lentiviral transfection siRNA-specific silencing of Chk1 significantly inhibited proliferation and colony-forming ability of U87 cells( P<0. 01 and P<0. 05). Prognostic survival analysis showed that GBM patients with low expression of Chk1 gene had a significantly better clinical outcome than those of GBM patients with high expression of Chk1 gene(P<0. 001). Conclusion Chk1 gene is overexpressed in GBM cells,up-regula-tion of Chk1 gene expression can promote the growth and proliferation of GBM cells,and Chk1 gene is associated with poor prognosis in GBM patients.
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Objective@#To discuss the etiology, pathogenesis, clinical manifestation, diagnosis and therapy of sphenoid wing dysplasia(SWD) associated with neurofibromatosis type Ⅰ(NF-Ⅰ).@*Methods@#We retrospectively reviewed its clinical manifestations, imaging, surgical treatment, complications and postoperative outcome of one NF-Ⅰ patient with SWD.@*Results@#A 14 years-old girl presented with pulsating exophthalmos, loss of vision and café au lait spots. Radiological studies showed right-side orbital enlargement and complete absence of the greater wing of the sphenoid. Titanium mesh was tailored intraoperatively to close the defect as a barrier between the orbital cavity and the cranium and then covered by periosteum.The patient developed postoperative infectious which was controlled by after antibiotic treatment and proper drainage. Proptosis improved significantly after surgery within a month. Ocular pulsation subsided and clinical symptoms improved at 28-month follow-up.@*Conclusions@#Sphenoid greater wing dysplasia associated with neurofibromatosis type Ⅰ is a rare inherited autosomal dominant disorders. The treatment should be customized to each patient. Titanium mesh reconstruction is patients with symptomatic sphenoid dysplasia. It can correct the proptosis and pulsating exophthalmos without the risk of bone resorption and recurrence.However, high risk of infection is associated with the procedure.
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Objective To investigate the clinical effect of cisplatin combined with cisplatin and radiotherapy in the treatment of glioblastomas patients receiving surgery.Methods 68 cases of glioblastomas patients with surgery were divided into control group (n=36) and treatment group (n=32).The control group was treated with temozolomide and radiotherapy;the treatment group was treated with cisplatin combined with temozolomide and radiotherapy.Short-term efficacy,adverse reactions,and survival time were documented during following up in both groups.Results The efficiency in the treatment group (87.5％) was significantly higher than that in the control group (66.67％),P＜0.05.There were no significant difference of adverse reaction including bone marrow inhibition,digestive tract symptoms,and psychiatric symptoms among the two groups,P＞0.05.The medium survival time of the treatment group (26.0 month) was significantly longer than that of the control group (17.5 month),P ＜0.05.There was no significant difference of 1-year survival rate between the treatment group (81.25％) with control group (63.89％),P＞0.05;however,2-and 3-year survival rate in treatment group (52.13％,31.25％) were significantly higher than that in the control group (27.78％,11.11％),P＜0.05.1-year progression free survival (PFS) rate of treatment group (68.75％) was significantly higher that of control group (41.67％),P＜0.05.Conclusion Cisplatin combined with temozolomide and radiotherapy can significantly improve the treatment efficiency without leading to higher rate of adverse reactions and significantly improve the 2-,3-year survival rate as well as 1-year progression free survival (PFS) rate in patients with glioblastomas,it is worth being promoted in clinical application.
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ABSTRACT:Objective To investigate the effects of poly ADP-ribose polymerase (PARP ) inhibitor 3-aminobenzene (3-AB)on the delayed development cerebral vasospasm (DCVS)after subarachnoid hemorrhage (SAH)and on the inflammatory factors,namely monocyte chemotactic protein 1 (MCP-1 )and hypersensitive c-reactive protein (hsCRP),and to explore the relationship between these and the signaling pathway of NF-kappa B (NF-κB).Methods Eighty male Sprague-Dawley rats were randomly divided into four groups:normal group (n =8),sham-operation group (n =8),SAH model group (n =32)and 3-AB group (n =32).We established 64 SAH model animals by double injection of blood into the cisterna magna.Half of the SAH model animals were treated with 3-AB by intraperitoneal injection (30 mg/kg).These rats were killed to obtain specimens respectively at days 3, 5,7 and 14 after the second blood injection.The morphological changes of basilar arteries were observed under the light microscope.The contents of PARP,MCP-1 and hsCRP in brain tissues were detected with enzyme-linked immunosorbent assay (ELISA).The expression of NF-κB in basilar arteries was determined by immunohistochemistry. Results Compared with those in the sham-operation group,the degree of basilar artery spasm reached the peak [(30.47±3.89)%]at day 5 after established SAH model;the thickness and diameter of basilar artery were (1 6.44 ±1.32)μm and (1 78.21 ± 1 1.13)μm,respectively.Cerebral blood flow was reduced by nearly 60% (P <0.01 ). The expression of NF-κB in the cytoplasm and nucleus and PARP content in brain tissue were both increased significantly (P < 0.01 ).MCP-1 [(365.29 ± 28.08 )pg/mL ] and hsCRP [(402.1 6 ± 48.99 )ng/mL ] were significantly enhanced (P <0.01).Compared with the SAH group,after 5 days’intervention with 3-AB,there was obvious alleviation in the spasm degree of basilar artery [(22.65±3.21)%],the thickness [(14.89±1.27)μm]and diameter [(1 98.56±10.91)μm],respectively (P <0.01).Cerebral blood flow was significantly enhanced,but the expression of NF-κB in the cytoplasm and nucleus was decreased and PARP in brain tissue was significantly decreased (P < 0.01 ).MCP-1 [(126.5 1 ± 18.67 )pg/mL]and hsCRP [(285.39 ± 39.07 )ng/mL]in brain tissue were significantly declined,respectively (P <0.01).Conclusion PARP inhibitor 3-AB can alleviate DCVS and inhibit the inflammatory response in brain tissue after SAH.The mechanism may be related to NF-κB signaling pathway.
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The previous pharmacokinetic methods can be only limited to drug analysis in vitro, which provide less information on the distribution and metabolismof drugs, and limit the interpretation and assessment of pharmacokinetics, the determination of metabolic principles, and evaluation of treatment effect. The objective of the study was to investigate the pharmacokinetic characteristics of gene recombination angiogenesis inhibitor Kringle 5 in vivo. The SPECT/CT and specific 131I-Kringle 5 marked by Iodogen method were both applied to explore the pharmacokinetic characteristics of 131I-Kringle 5 in vivo, and to investigate the dynamic distributions of 131I-Kringle 5 in target organs. Labeling recombinant angio-genesis inhibitor Kringle 5 using 131I with longer half-life and imaging in vivo using SPECT instead of PET, could overcome the limitations of previous methods. When the doses of 131I-Kringle 5 were 10.0, 7.5 and 5.0 g/kg, respectively, the two-compartment open models can be determined within all the metabolic process in vivo. There were no significant differences in t1/2α, t1/2β, apparent volume of distribution and CL between those three levels. The ratio of AUC(0 ? 1) among three different groups of 10.0, 7.5 and 5.0 g/kg was 2.56:1.44:1.0, which was close to the ratio (2:1.5:1.0). It could be clear that in the range of 5.0–10.0 g/kg, Kringle 5 was characterized by the first-order pharmacokinetics. Approximately 30 min after 131I-Kringle 5 was injected, 131I-Kringle 5 could be observed to concentrate in the heart, kidneys, liver and other organs by means of planar imaging and tomography. After 1 h of being injected, more radionuclide retained in the bladder, but not in intestinal. It could be concluded that 131I-Kringle 5 is mainly excreted through the kidneys. About 2 h after the injection of 131I-Kringle 5, the radionuclide in the heart, kidneys, liver and other organs was gradually reduced, while more radionuclide was concentrated in the bladder. The radionuclide was completely metabolized within 24 h, and the distribution of radioactivity in rats was similar to normal levels. In our study, the specific marker 131I-Kringle 5 and SPECT/CT were suc-cessfully used to explore pharmacokinetic characteristics of Kringle 5 in rats. The study could provide a new evaluation platform of the specific, in vivo and real-time functional imaging and pharmacokinetics for the clinical application of 131I-Kringle 5.
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Objective To investigate the effect of supernatant from carcinoembryonic antigen -related cell adhesion molecule 1(CEACAM1)gene RNAi glioma SHG44 cells on human umbilical vein endothelial cell proliferation and angiogenesis in vitro .Methods Three pairs of specific siRNA targeting CEACAM 1 were de-signed and synthesized , and then transiently transfected into SHG 44 cells via cathodolyte liposome transfection method.The supernatant from cultured glioma SHG 44 cells was collected as the conditional medium 48 h after transfection.RT-PCR was used to detect CEACAM1 and VEGF expression at mRNA level after CEACAM1 gene RNAi.The expression of VEGF in supernatant was measured by ELISA .The proliferation of HUVEC was assessed by MTT method after co-cultured with supernatant .The migration of HUVEC was examined by using a Transwell assay.The ability of angiogenesis was measured by capillary -like network formation assay .Results Forty -eight hours after the 3 pairs of specific CEACAM1 siRNA were transfected into SHG44 cells,RT-PCR results showed that the expression of CEACAM 1 mRNA was significantly inhibited compared with those in the normal control and the negative control groups (P<0.01).The most significant interference effect was CEACAM 1-siR-NA3.Expression of VEGF mRNA was remarkably reduced ,and expression of VEGF protein in the supernatant was also reduced after transfection of CEACAM 1-siRNA for 48 h.The proliferation of HUVEC was inhibited , HUVEC migration and tube capillary -like formation were decreased .Conclusion CEACAM1-siRNA can ef-fectively suppress the expression of CEACAM 1 mRNA in human glioma SHG44 cells,may inhibit HUVEC prolif-eration,migration and tube capillary -like formation via down -regulated the secretion of VEGF in vitro .
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<p><b>OBJECTIVE</b>To investigate the glycolytic phenotype of SHG44 human glioma cells under hypoxic conditions and the association between cell proliferation and apoptosis and the metabolic status.</p><p><b>METHODS</b>An in vitro hypoxic cell model was established in SHG44 cells using CoCl2. Real-time PCR and Western blotting were used to assess the expressions of hypoxia-inducible factor-1α (HIF-1α) and the enzymes involved in glycolysis including PDK1, PKM2, and LDHA. Intracellular ATP levels were measured by bioluminescence assay to assess the energy metabolic status of SHG44 cells. The viability and apoptosis of the cells were examined using MTT assay and flow cytometry, respectively.</p><p><b>RESULTS</b>The cells in hypoxic culture showed obviously increased expressions of HIF-1α, LDHA, PDK1, and PKM2 at both the mRNA and protein levels as compared to those in normal cell culture. Hypoxia of the cells also resulted in a lowered cell proliferative activity and an increased apoptosis rate with lowered intracellular ATP concentrations and elevated mitochondrial membrane potential.</p><p><b>CONCLUSION</b>Hypoxia can induce a glycolytic phenotype of tumor cells. The sensitivity of tumor cells to hypoxia-induced cell death is directly correlated with their metabolic status.</p>
Subject(s)
Humans , Adenosine Triphosphate , Metabolism , Apoptosis , Carrier Proteins , Metabolism , Cell Hypoxia , Cell Line, Tumor , Cell Proliferation , Central Nervous System Neoplasms , Metabolism , Pathology , Glioma , Metabolism , Pathology , Glycolysis , Hypoxia-Inducible Factor 1, alpha Subunit , Metabolism , Membrane Potential, Mitochondrial , Membrane Proteins , Metabolism , Protein Serine-Threonine Kinases , Metabolism , Thyroid Hormones , MetabolismABSTRACT
ObjectiveTo establish rat model of traumatic brain injury combined with hemorrhagic shock.Methods Rat models of traumatic brain injury (produced by free fall impact method) combined with hemorrhagic shock (produced by venous injury method) were established and the related physiological parameters were recorded.The neurological impairment score,cerebral edema degree and blood brain barrier (BBB) were determined by using neurofunction scales,dry-wet method and Evans blue (EB) respectively.HE staining and immunohistochemical staining were applied to evaluate the pathological changes in brain sections.ResultsBlood pressure dropped from 95 mm Hg to 25 mm Hg within three minutes after modeling and maintained around 60 mm Hg one hour later.Neurological impairment score was increased dramatically.The ratio of water content in the brain tissue was elevated nearly from 77％ to 81％.The concentration of EB residual in the brain tissue was increased more than one fold.Neuronal pathological abnormalities,including neuron shrinking,dark eosinophilic staining,perineuronal vacuole in HE staining,and positive staining of β-amyloid precursor protein (β-APP) in immunohistochemical staining were also observed. ConclusionsRat models of traumatic brain injury combined with hemorrhagic shock are successfully established.In addition,the main pathological changes,such as cerebral edema,disruption of BBB,neuron damage,and expression of β-APP are replicated.
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Objective Angiotensin Ⅱ (Ang Ⅱ ) contributes to modulating blood pressure by stimulation of Ang Ⅱ AT1 receptors. We devised a rat transient middle cerebral artery occlusion (MCAO) model to assess whether oxidative damage is decreased after pretreatment with Angiotensin Ⅱ AT1 receptor blocker (ARB). Methods After 2 weeks pretreatment with ARB 0. 5 and 1 mg/kg, the male Wister rats were subjected to 2 h middle cerebral artery occlusion (MCAO). At 24 h, the lumen diameter of middle cerebral artery, the plasma level of 8-hydroxy-2'-deoxyguanosine (8-OHdG), and HIF-1 a levels were recorded and compared. Results After pretrcatment with ARB 0.5 and 1 mg/kg, blood pressure did not significantly change compared with that of controls. In the group of candesartan at 1 mg/(kg· day), the lumen diameter was significantly increased compared to that in control group [(86.0±5.0) μm vs. (69.0± 2.1) μm; P<0. 01, n = 6- 8]. The plasma 8-OHdG levels of ARB pretreatment groups were decreased. In immunohistochemical findings, 8-OHdG- and HIF-1α-containing cells in ARB pretreatment groups were decreased. Conclusion Brain ischemia and oxidative damage can be reversed by AT1 receptor blockade in normotensive rats after transient cerebral artery occlusion.
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Objective To explore the role and significance of matrix metalloproteinase-9 (MMP-9) in angiogenesis through observing the relationship between the expression of MMP-9 and microvessel density (MVD) in glioma. Methods The expressions of MMP-9 and CD34 in 10 cases of normal brain tissues and 58 cases of glioma (14 cases of grade Ⅰ , 20 cases of grade Ⅱ , 15 cases of grade Ⅲ, and 9 cases of grade Ⅳ ) were detected by immunohistochemical streptavidin-peroxidase technique. The positive cells of MMP-9 and the positive microvessels were examined under binocular light microscope. Results The positive expression of MMP-9 in glioma was located in the tumor-cell cytoplast and endothelial cells. The positive rate of MMP-9 in glioma of grade Ⅰ, Ⅱ, Ⅲ and Ⅳ was 42.9%, 65.0%, 86.7% and 88. 9%, respectively. The expression of MMP-9 was obviously higher than that of normal brain tissues (P<0.01) and positively correlated with glioma malignancy (rz =0. 597, P<0.05). MVD was correlated with glioma malignancy (H=47. 865, P<0. 05). The expression of MMP-9 was significantly correlated with MVD (rz =0.897, P<0.01). Conclusion The expressions of MMP-9 and MVD are correlated with glioma malignancy, which may be helpful in judging the malignancy, invasion and prognosis. MMP-9 plays an important role in angiogenesis of glioma and accelerates glioma malignancy development by promoting angiogenesis.
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Objective To investigate the anti-tumor effect and mechanism of tamoxifen on rat C6 glioma cells. Methods C6 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 3% fetal calf serum (FCS), and treated with tamoxifen of different concentrations, i.e. group A (1.25μmol/L), group B (2.50 μmol/L), group C (5.00 μmol/L), group D (10.00 μmol/L), group E (20.00 μmol/L) and control group (0.00 μmol/L). Morphological changes, MTT assay and 5-bromo-2'-deoxyuriding labeling ratio were assessed. Apoptosis was observed by flow cytometry. Results C6 cells treated with different doses of tamoxifen for 24, 48, and 72 hours became irregular in shape, while cells treated with vehicle grew normally. MTT assay showed that tamoxifen did not suppress C6 cell growth until 72 hours after treatment. Seventy-two hours after treatment, there were significant differences in cell viable rate between group A versus groups C, D and E; so did group B versus group D as well as group E (P<0.05). BrdU incorporation assay indicated significant difference of BrdU labbled index (BrdU LI) among groups A, C, E and control group 48 hours after treatment (P<0.05). And the BrdU LI decreased with the increased concentration of tamoxifen. Flow cytometry (FCM) showed significant difference between treated group and control group at 24, 48, and 72 hours after treatment (P<0.05). Conclusion Tamoxifen significantly suppresses the growth of C6 glioma cells in a time- and dose-dependent manner. The mechanism of tamoxifen suppressing C6 glioma cells may be inhibiting proliferation and inducing apoptosis. Therefore, tamoxifen can be a candidate as a chemotherapy agent for glioma.
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Objective Angiotensin Ⅱ (Ang-Ⅱ) increases NADPH oxidase activity and stimulates the production of reactive oxygen species (ROS) including superoxide anion through Ang Ⅱ AT1-receptor (AT1-R) activation. ROS is involved in various pathological processes in brain ischemia. We investigated whether the AT1-R blocker (ARB) candesartan can protect normotensive rats against brain ischemia. Methods After 2-week pretreatment with candesartan, rats were subjected to 2 hours middle cerebral artery occlusion-reperfusion (MCAO-R) and 24 hours later, the infarct volume, iNOS, and eNOS mRNA in the internal carotid artery was recorded and compared. Results Candesartan pretreatment reduced cerebral ischemia and oxidative brain damage after MCAO-R in normotensive rats, resulting in a decreased cortical infarct volume [0.5 mg/kg candesartan, (46.8±13.2)mm3; 1.0 mg/kg candesartan, (19.3±15.3)mm3 vs. control, (111.7±14.3)mm3; P<0.05, P<0.01, respectively]. Candesartan pretreatment increased the eNOS mRNA level in the internal carotid artery. Conclusion In normotensive rats exposed to MCAO-R, candesartan protectes against brain ischemia. This effect may represent a significant therapeutic advantage and may induce end-organ protection even at normal blood pressure.
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Objective To explore the clinical significance of the differential expression of PTEN in glioma tissue.Methods The mRNA and protein expressions of PTEN were assayed by reverse transcription polymerase chain reaction(RT-PCR) and immunohistochemistry in 75 human brain glioma cases.Results The positive expression rate mRNA of PTEN differed significantly between brain glioma tissue and normal brain tissue(?2=22.66,P