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Objective:To investigate the role of microchromosome maintenance protein 5 (MCM5) in promoting malignant progression and its mechanism in glioblastoma.Methods:(1) Three freshly excised brain tissues from non-tumor patients and 3 grading IV glioblastoma tissues were collected in our hospital from September 2020 to September 2021; mass spectrometry and quantitative proteomic analysis were performed to screen differentially expressed proteins for functional enrichment analysis. (2) The target protein MCM5, which was highly expressed in glioblastoma, was screened on the basis of proteomics, and its expression in glioblastoma was verified using The Cancer Genome Atlas (TCGA) database and further validated at the mRNA level in the collected clinical specimens. (3) U251 cells were divided into negative control group, knockdown group-1 (si-1 group) and knockdown group-2 (si-2 group) by siNRA transfection. The regulation role of MCM5 in malignant phenotype of glioblastoma was detected by CCK-8 assay, clone formation assay, 5-ethynyl-2'-deoxyuridine (EdU)staining, Transwell invasion assay and flow cytometry. (4) The transcriptome data of glioma patients from TCGA database were used to explore the possible molecular mechanisms of MCM5 regulation in the malignant process of glioblastoma by gene set enrichment analysis (GSEA) algorithm.Results:(1) In clinical samples of glioblastoma, 322 up-regulated proteins and 94 down-regulated proteins were screened out; MCM5 was highly expressed in these 3 glioblastoma samples. (2) Based on TCGA database, results of 163 patients with glioblastoma and 207 patients with non-tumor brain tissues showed that MCM5 expression was statistically up-regulated in glioblastoma ( t=3.340, P<0.001). Real-time quantitative PCR results of 3 glioblastoma tissues and 3 non-tumor brain tissues clinically collected in our hospital also indicated that significantly increased MCM5 expression in glioblastoma was noted as compared with that in the non-tumor brain tissues ( t=3.876, P<0.001). (3) As compared with the negative control group, the si-1 and si-2 groups had significantly decreased MCM5 mRNA and protein expressions, significantly lower proliferation rate 5 d after inoculation, statistically decreased number of cell clones, significantly decreased proportion of EdU positive cells, and significantly increased proportion of cells at G1 phase, and significantly impaired migration ability ( P<0.05). (4) GSEA analysis showed that mRNA in MCM5 high expression group was enriched in DNA damage repair gene, E2F target gene, MYC target gene, epithelial-mesenchymal transformation (EMT), nterleukin 6-Janus kinase-signal transduction and transcription activation factor 3 (IL6-JAK-STAT3), interferon, KRAS, NOTCH, transforming growth factor-β (TGF-β), Wnt/β-Catenin and other characteristic genes. Conclusion:MCM5 is highly expressed in glioblastoma, and MCM5 regulates the malignant progression of glioblastoma through multiple mechanisms including E2F, MYC, EMT, and Wnt/β-Catenin.
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Objective:To observe the safety and efficacy of one-stage bilateral temporal lobe lesions resection in patients with severe bilateral radiation-induced temporal lobe injury after radiotherapy for nasopharyngeal carcinoma.Methods:Clinical data of 12 patients with severe bilateral radiation-induced temporal lobe injury after radiotherapy for nasopharyngeal carcinoma underwent one-stage bilateral temporal lobe lesions resection in our hospital from January 2013 to December 2017 were retrospectively analyzed. Karnofsky Performance Scale (KPS) scores were used as predictors of surgical outcomes before and two weeks after surgery. Imaging changes of radiation-induced brain injury lesions after surgery before and 3 months after surgery, surgical-related complications, and death were observed.Results:All 12 patients (100%) had improved postoperative KPS scores, which showed significant difference as compared with the preoperative KPS scores ( P<0.05). Two patients had pulmonary infection after operation, one patient had poor wound healing, and one patient had intracranial infection; all of them recovered after expectant treatment. None of the 12 patients had new neurological symptoms after surgery. During the follow-up period, the postoperative cranial MR imaging showed that the lesions were completely removed, and the leukoencephalopathy was alleviated or completely subsided. Conclusion:One-stage bilateral temporal lobe lesions resection is feasible for bilateral radiation-induced temporal lobe injury after radiotherapy for nasopharyngeal carcinoma.
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Objective To investigate the effect of micro RNA (miR)-125a-3p on proliferation and apoptosis of glioma cells and its role in MAPK signaling pathway. Methods (1) The miR-microarray data from the Cancer Genome Atlas (TCGA, https:// cancergenome.nih.gov/) database were downloaded, and the miR-125a-3p expressions in 565 gliomas tissues and 10 normal brain tissues were compared. (2) Clinical collection of 30 glioma specimens surgically resected in our hospital from April 2015 to April 2018, was performed, including 7 of low-grade glioma and 23 of high-grade glioma;8 normal brain tissues needed craniocerebral trauma excision were collected at the same time period;reverse transcription (RT)-real-time quantitative (q) PCR was used to detect the miR-125a-3p expressions in glioma tissues and normal brain tissues. (3) The normal brain glial cells HA1800 and glioma cells (U251, U138, U87, U373, and T98G) were routinely cultured in vitro; RT-qPCR was used to detect the miR-125a-3p expression in normal brain glial cells and glioma cell lines. (4) The cultured glioma cell lines U251 and U373 at logarithmic phase were divided into miR-125a-3p group and negative control group;and miR-125a-3p mimic or nonsense sequence were transfected using LipofectamineTM 2000;72 h after transfection, the miR-125a-3p expression was detected by RT-qPCR; the proliferation rate was detected by clone formation after transfection; the apoptosis rate was detected by flow cytometry 72 h after transfection; the cleaved-caspase-3, cleaved-caspase-9, cleaved-caspase-7, P38 and P-P38/MAPK protein expressions were detected by Western blotting. Results (1) In TCGA database, the miR-125a-3p expression in glioma brain tissues was statistically lower as compared with that in normal brain tissues (P<0.05). (2) The miR-125a-3p expressions in clinically collected normal brain tissues, low-grade glioma specimens and high-grade glioma specimens were decreased successively, enjoying statistically significant differences (P<0.05). (3) As compared with normal glial cells, the miR-125a-3p expressions in glioma cell lines were significantly lower (P<0.05), of which, U251 and U373 enjoyed the most obvious decrement. (4) As compared with the blank control group, the miR-125a-3p group had significantly increased miR-125a-3p expression, significantly decreased colony forming efficiency, significantly increased proliferation rate, significantly increased expressions of apoptosis-related proteins cleaved-caspase-3, cleaved-caspase-9, and cleaved-caspase-7, and statistically increased phosphorylated-P38/MAPK expressions (P<0.05). Conclusion The miR-125a-3p expression is low in glioma tissues and cells; miR-125a-3p over-expression can inhibit the proliferation of glioma cells and promote apoptosis through MAPK signaling pathway, which may provide a new potential target for treatment of glioma.
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Objective To investigate the expression and biological value of heme oxygenase I (HO-1) in gliomas. Methods Fifty-six patients with gliomas, admitted to and accepted surgery in our hospital from January 2010 to January 2015, were chosen in our study; WHO grade I was noted in 4 patients, grade Ⅱ in 16, grade Ⅲ in 10, and grade IV in 26 patients; patients with grade I and Ⅱ were as low-grade glioma group and patients with grade Ⅲ and IV were as high-grade glioma group. The HO-1 expression in the two groups was detected by immunohistochemistry. R-langrage survival tool was used to analyze the relation between HO-1 expression and prognosis of 1107 patients with gliomas selected from The Cancer Genome Atlas (TCGA) database. Results Significant differences of HO-1 expression were observed in different grades of gliomas (P<0.05), and HO-1 high-expression rate in the high-grade gliomas (75%) was significantly higher than that in the low-grade group (30%, P<0.05). HO-1 protein expression level in the high-grade gliomas was significantly higher than that in the low-grade group (P<0.05). Moreover, area under the curve (AUC) of the receiver operating characteristic curve was suggested that the HO-1 could be an ideal determine factor (AUC=0.747, P=0.002). Log rank analysis indicated that the accumulate survival rate in patients with low HO-1 expression was significantly higher than that in patients with high HO-1 expression (P<0.05). TCGA database analysis showed that simultaneous survival rate in patients with low HO-1 expression was significantly higher than that in patients with high HO-1 expression (P<0.05). Conclusion Expression of HO-1 is correlated with the malignant degrees and prognoses of gliomas, and it has potential to be a novel biological marker for the diagnosis and treatment of gliomas; furthermore, HO-1 could also be a target for the study and treatment of gliomas.
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Objective To investigate the sorting nexin 10 (SNX10) expression in glioma tissues and its relationship with prognosis of the patients.Methods Thirty glioma specimens,collected from surgery and conformed by pathology in our hospital from January 2007 to December 2012,were used in our study,and in them,9 were WHO grade Ⅰ and Ⅱ and 21 were WHO grade Ⅲ and Ⅳ;and 30 nonneoplastic tissue specimens collected during decompression were used as control group.Immunohistochemical staining using polyclonal SNX10 antibody was performed on paraffin embedded specimens.The staining intensity was stratified as absent (-),weak (+),moderate (++) and strong (4+++).The relationships of SNX10 expression with several clinic pathologic indicators and prognosis were analyzed.Results High SNX10 expression was noted in 12 specimens and low SNX10 expression in 18 specimens of the glioma group.High SNX10 expression was noted in 25 specimens and low SNX10 expression in 5 specimens of the control group;the high SNX10 expression rate in glioma tissues was significantly lower than that in non-neoplastic brain tissues (P＜0.05);the high SNX10 expression rate in high-grade glioma tissues was significantly lower than that in low-grade glioma tissues (P＜0.05).The median survival time ofglioma patients with high SNX10 expression was 22.50±8.27 months,and that of glioma patients with low SNX10 expression was 15.50±0.99 months.The survival rate of glioma patients with low SNX10 expression was significantly lower than that of glioma patients with high SNX10 expression (34％ vs.65％,P＜0.05).By Cox multi-factor risk scale model,the expression level of SNX10 and grading of tumors were identified as the independent risk factors of patient's post-operative death;following the decreased SNX10 expression,the risk of postoperative death increased 1.983 times (95％ confidence interval=1.602-2.314,P＜0.05).Conclusions Decreased SNX10 expression is associated with occurrence and development of gliomas,and has a significant effect on patients' post-operative survival time.Decreased SNX10 expression level may be an important index of poor prognosis in glioma patients.
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Objective To explore the changes of cell proliferation and differentiation of neural stem cells induced by fasudil treatment,and to primarily study the mechanism in C17.2 mice.Methods C17.2 cells were cultured in vitro; 5,25,50 and 100 μmol/L fasudil were given to the cells,respectively,for 24 h,and cells in the blank control group were given the same volume of culture medium.The changes of cell morphology were observed under a phase-contrast microscope; cell viability and cell necrosis rate were determined by MTT assay and lactate dehydrogenase (LDH) assay,respectively.Western blotting was applied to detect the expression levels of neural markers (nestin,glial fibrillary acidic protein [GFAP],double cortisol [DCX],microtubule-associated protein-2 [MAP-2]),and Notch Ⅰ and Hes 1 proteins in the notch signaling in cells from the 100 μmol/L fasudil treatment group and blank control group.Immunofluorescence staining was used to detect the nestin and GFAP expressions in the C 17.2 cells.Results As compared with that in the blank control group,the cell viability in the 50 and 100 μmol/L fasudil treatment groups was significantly decreased; that in the 100 μmol/L fasudil treatment group was significantly lower than that in 50 μmol/L fasudil treatment group (P＜0.05); LDH assay showed no significant difference of cell necrosis among the five groups (P＜0.05).Western blotting indicated that 100 μmol/L fasudil treatment group had significantly decreased nestin expression,significantly elevated DCX,MAP-2 and GFAP expressions,and statistically decreased expression levels of Notch 1 and Hes 1 as compared with blank control group (P＜0.05).Immunofluorescence staining indicated that the percentage of nestin positive cells was markedly decreased,the percentage of GFAP positive cells was significantly increased in the 100 μmol/L fasudil treatment group as compared with those in the blank control group (P＜0.05).Conclusion Fasudil treatment could inhibit the proliferation of C17.2 cells and promote them differentiate into neuronal and glial cells via decreasing the expression level of Notch signaling.
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AIM:TostudythechangeofradiosensitivityofU251cellsaftertreatedwithsodiumdichloroacetate ( DCA) and further to explore the possible mechanism .METHODS: The U251 cells were divided into 4 groups: control group, DCA group, X-ray irradiation without DCA pretreatment ( IR) group and X-ray irradiation with DCA pretreatment ( DIR) group.MTT assay was applied to determine the cell viability .The intracellular reactive oxygen species ( ROS) were detected by DHE fluorescence .The expression level of Bcl-2 was assessed by Western blot .The percentage of apoptosis of cells was determined by flow cytometry .RESULTS:No difference between control group and DCA group in cell viability (P>0.05) was observed.However, the cell viability of both IR group and DIR group was markedly reduced compared with control group ( P<0.05).Furthermore, the viability of DIR group was significantly decreased compared with IR group ( P<0.05 ) .The percentage of ROS positive cells was obviously increased in DIR group compared with IR group (P<0.05).The expression level of Bcl-2 was sharply decreased in DIR group (P<0.05) and the percentage of apoptosis of cells was significantly elevated ( P<0.05) in DIR group compared with IR group .CONCLUSION:The better antitu-mor effect was obtained by improving the radiosensitivity through pretreating the cells with DCA , and the possible mecha-nism was down-regulation of the Bcl-2 expression by developing the intracellular ROS .
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Objective To comparatively study the differences of 18F‐FDG imaging agent by three kinds of different intravenous injection method for conducting PET / CT examination in aspects of the puncture success rate ,residual amount of drug injection and staff ray exposure time and their significance .Methods 240 patients with PET /CT examination were randomly divided into the group A ,B and C ,80 cases in each group .The drug injection adopted the traditional direct injection ,indwelling catheter injection and scalp venous needle connecting syringe(indwelling bubbles) .The puncture success rate ,drug residues and staff contacting radio‐pharmaceuticals time were compared among 3 groups .The obtained relevant data were statistically analyzed .Results The puncture success rate in the scalp venous needle connecting syringe (indwelling bubbles) and the indwelling catheter injection was higher than that in the traditional direct injection and the staff contacting radiopharmaceuticals time was significantly decreased ,the differ‐ences among them were statistically significant(P< 0 .01) ;the radioactive drugs residue in the scalp venous needle connecting syr‐inge was significantly decreased than that in other two kinds of injection method ,the difference was statistically significant (P <0 .01) .Conclusion The injection method of scalp intravenous needle connecting syringe (indwelling bubbles) significantly increases the puncture success rate ,reduces the radioactive drug residue ,at the same time decreases the staff radiation exposure time ,this method has the advantage in the radionuclide injection .
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Cerebral vasospasm (CVS) is one of the serious complications of subarachnoid hemorrhage (SAH).Pathogenesis of CVS has not been fully clarified,and it may be associated with a variety of factors.With the development of molecular biology techniques,people have more understanding on SAH caused pathogenesis of CVS,and the research has also made considerable progress in the prevention of CVS.
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Chronic ulcer of the lower extremities amounts for a grave and serious problem for public health. Western medicine focuses on controlling infection, improving blood circulation, surgical debridement, skin grafting, etc, but there are bottlenecks in the treatment. Traditional Chinese medicine (TCM) has a long history and a legacy of sound clinical efficacy in this area. TCM has developed a unique, effective external theory, and a large number of topical prescriptions and external technology. Through this research, a safe and effective treatment protocol of TCM for chronic ulcer of the lower extremities can be formed. To this end, during China's "Eleventh Five-Year" Plan, special research committees and projects on TCM external treatments and external technologies were established. This study on ulcer of the lower extremities constitutes one of the major research topics.
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BACKGROUND: Recent researches indicate that ischemia and hypoxia can lead to abnormal brain metabolism and even energy failure, which is an important reason for brain damage and necrosis and identifies energy metabolism disorder as the key event in brain ischemia-reperfusion (IR)injury. Glucose transporter-3 plays the vital role in brain energy metabolism.OBJECTIVE: To observe the changes of cerebral infarct volume and glucose transporter-3 mRNA and protein expressions in cerebral cortical penumbra at different stages of focal cerebral ischemia and reperfusion in rats.DESIGN: Randomized controlled experiment.SETTING: Department of Neurosurgery, Second Hospital Affiliated to Sun Yat-sen University.MATERIALS: This experiment was conducted in the Animal Laboratory of Medical Research Center, Second Hospital Affiliated to Sun Yat-sen University between August and October 2002.Totally 56 SD rats were randomized into 3 groups which were subjected to ① ischemia for 1 hour followed by reperfusion (n=28), ② ischemia for 3 hours followed by reperfusion (n=24), and ③ sham operation (n=4). The rats in the first group were subdivided into 7 subgroups for examination at 1, 3, 6, 12, 24, and 72hours and 1 week after ischemia, with 7 rats in each subgroup; the rats in the second ischemia group were also subdivided in similar manner but without a 1 hour postischemic subgroup. The rats in the sham operation group only received the operation but without arterial occlusion.METHODS: Focal cerebral ischemia-reperfusion (IR) injury model was induced in the rats in the two ischemic groups by means of insertion of suture for arterial occlusion, and the ratio of central ischemic area to cerebral infarct volume in the ischemic penumbra was examined at the specified time points. Reverse transcription-PCR (RT-PCR) was used to detect the expression of glucose transporter-3 mRNA in the cerebral cortex in ischemic penumbra region, and semi-quantitative immunohistochemistry (IHC) employed to detect the level of glucose transporter-3 protein.MAIN OUTCOME MEASURES: Cerebral infarct volume after IR injury, changes of transporter-3 mRNA and protein expressions after IR injury.RESULTS: Totally 56 rats were used in this experiment and all entered result analysis. The post-IR cerebral infarct volume was obviously smaller in 1-hour ischemia group than in 3-hour ischemia group. Glucose transporter-3 mRNA expression began to increase 3 hours after ischemia in 1-hour ischemia group, reaching the peak level at 24 hours and still mainrained higher level than that of the sham operation 1 week; in 3-hour ischemia group, the mRNA expression was slightly decreased at 3 hours but began to increase afterwards till reaching the peak level at 24 hours, followed then by recovery of normal level at 1 week. The changes in glucose transporter-3 protein and mRNA expressions followed almost the same pattern.CONCLUSION: Glucose transporter-3 expression is up-regulated in the ischemic penumbra region, possibly as a protective response to cerebral IR injury.
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AIM: To investigate the volume percentage of infarct and expression of glucose transporter-1 (GLUT1) transcription and protein levels at different ischemic time point and different reperfusion time point in rat focal cerebral ischemic penumbra. METHODS: Focal ischemic models of middle cerebral artery occlusion (MCAO) in rats were made by inserting nylon thread. Brain samples were harvested from ischemic penumbra. Infarct volume were analyzed quantitively by Kontron IBAS 2.5 image auto-analyses system. The expressien of GLUT1 mRNA was assessed by RT-PCR, and the expression of GLUT1 protein was assessed by immunohistochemistry. RESULTS: The infarction volume in MCAO 1 h/reperfusion (R) group was obviously smaller than that in MCAO 3 h/R group. GLUT1 increased at (1 h) MCAO 1 h/R group, climbed to climax at 24 h and remained higher than normal at 1 week. In contrast, in the MCAO 3 h/R group, the corresponding index was at 3 h, 24 h and 1 week, but the increasing degree of GLUT1 was slighter than MCAO 1 h/R. GLUT1 protein began to ascend at 1 h, reached climax at 24 h and was higher than normal at 1 week in MCAO 1 h/R group, while in MCAO 3 h/R group, the corresponding index was at 3 h, 24 h and 1 week. CONCLUSION: GLUT1 expression is notably up-regulated in the penumbra region after focal cerebral ischemia, it may be a protective reaction against ischemic injury. [
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AIM: To investigate the volume percentage of infarct and expression level of glucose transporter-3 (GLUT3) transcription and protein at different ischemic time points and different reperfusion time points in rat focal cerebral ischemic penumbra. METHODS: Focal ischemic models of middle cerebral artery occlusion (MCAO) in rats were made by inserting nylon thread. Brain samples were harvested from ischemic penumbra. Infarct volume was analyzed quantitatively by Kontron IBAS 2.5 image auto-analyses system. The change of GLUT3 mRNA was assessed by RT-PCR, and the expression of GLUT3 protein was assessed by immunohistochemistry. RESULTS: The infarction volume in MCAO 1 h/R group was obviously smaller than that in MCAO 3 h/R group. GLUT3 began to ascend at 3 h in MCAO 1 h/R group, reached to climax at 24 h and remained higher than normal at 1 week. In contrast, in the MCAO 3 h/R group, GLUT3 had a descent at 3 h. Later on, it ascended rapidly, and reached climax at 24 h. At 1 week, it approached to normal. The expression level of GLUT3 protein corresponds with that of mRNA. CONCLUSION: GLUT3 expression is up-regulated in the penumbra region after focal cerebral ischemia, it may be a protective reaction against ischemia/reperfusion injury. [
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AIM:To study the effects of neurological improvement and remyelination after intracranial hemorrhage(ICH)in rats by a novel therapeutic strategy with hepatocyte growth factor(HGF)gene transfected human umbilical cord mesenchymal stem cells(hUCMSCs)by lentiviral vector.METHODS:ICH was induced in 60 adult male Sprague-Dawley rats by a stereotactically guided injection of bacterial type IV collagenase into the right internal capsule.Non-modified hUCMSCs,HGF-modified hUCMSCs with lentiviral vector or PBS were administered left intraventricularly 7 d after right internal capsule ICH.All rats underwent modified neurological severity scores for 35 d.Luxol fast blue staining,immunohistological staining and Western blotting assessments for myelin basic protein(MBP)were applied.RESULTS:The ICH rats receiving HGF-modified hUCMSCs demonstrated significant functional recovery,determined by modified neurological severity scores,compared to the other groups from 2 weeks after cell therapy.As indicated by Luxol fast blue staining,the percent area of demyelination was obviously reduced in the HGF-hUCMSC treatment group compared to the PBS control group and hUCMSC-only treatment group at 5 weeks after ICH.The expression of MBP detected by immunohistological staining and Western blotting was significantly higher in HGF-hUCMSCs treated brain than that in other groups(P
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Objective:After construction and identification of HBsAg gene recombinant backbone adenoviral vector,it is to prouduct HBsAg gene recombinant adenoviruses by packing PAd-Easy-1-HBs in 293 cells.Methods:The gene of interest was amplified from plasmid pEcob-6 PCR, the gene of interest which contained HBsAg gene was cloned into on adenoviral shuttle vector pAd-track-cmv. The pAd-track-cmv-HBs was linearized by digesting with restriction endonuclease Pme-1, and subsequently cotransformed into E.coli BJ-5183 cells with an adenoviral backbone vector pAd-Easy-1, Homologous recombinants were performed in bacterial cells. Finally, the linearized backbone adenoviral vector was transfected into adenoviruses packing cells lines,e.g. 293 cell by lipofectamine transfection. Transfections and viral productions can be minotored by green fluorescent protein(GFP). The expression of HBsAg in supermatant was investigated by ELISA. It was a certain HBsAg vaccine to amplify recombinant adenoviruses by repeating the infevtion cell to collect the viral supermatant.Results:GFP expression was visible by fluorescence microscopy after transfection. Adenoviral titer was monitored by GFP expression. GFP expression was visible after repeating the infection cell using the viral supernatant in more than 90 percent of the cells.The HBsAg also expressed in supermatant.Conclusion:HBsAg gene recombinant adenoviral backbone vector has been constructed successfully. HBsAg gene recombinant adenoviruses have been producted by packing in 293 cells. The study provides the possibility of further researches on the development of new anti-HBV vaccines.