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Objective To construct a eukaryotic expression vector for MEK2-siRNA to explore the expression of this endogenous MEK2 in glioma U87 cell line. Methods Four single-stranded template DNAs encoding siRNA against MEK2, each consisting of 60 bp, were synthesized chemically;based on these 4 single-stranded template DNAs, 2 double-stranded DNAs were formed by annealing,then identified by restriction analysis and inserted into vector pSUPER.basic by T4 ligase. Positive recombinants were indentified by EcoRI and HindⅢ double digestion and transfected into the U87 cells.The protein expression level of MEK2 was determined by using Western blotting. Results Both restriction analysis and sequencing proved that the eukaryotic expression vector for MEK2-siRNA was constructed correctly. MEK2-siRNA plasmid screened out by Western blotting. The MEK2 expression level in negative control group was (0.105±0.023) and that in transfected group was (0.030±0.006).Conclusion The eukaryotic expression vector for MEK2-siRNA is successfully constructed, which down-regulates the transcription of MEK2 protein in U87 cells. It provides a certain experimental basis for gene therapy of glioma by RNAi technique.
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Objective To explore the expression and function of correlative genes in the happening and developing of human pituitary adenoma-subtypes.Methods The whole genome oligonucleotide microarray (Affymetrix 133 plus 2.0) was used to examine the gene expressions of pituitary adenoma tissue in 8 patients with pituitary adenoma (2 with growth hormone adenomas,2 with prolactinomas,2 with gonadotroph adenomas and 2 with null cell adenomas) and normal pooled pituitary tissue.Differentially expressed genes were analyzed by Hierarchical method and bioinformatics.A candidate gene was selected to verify the microarray analyzed result by real-time quantitative PCR.Results Compared with associated genes with normal control,associated genes with pituitary adenoma mainly involved in the following biological processes analyzed from the view of function: binding,apoptosis-or-tumor correlation,metabolism,signal-transducer-activity,cell cycle,transcription-regulator-activity and transporter-activity.The specificity of expression in several differential genes was connected to the development of pituitary adenoma-subtypes.Conclusion The development of pituitary adenoma is a complex regulation process involving lots of genes,molecules and pathways.However,the molecular mechanism related to the individual pituitary adenoma-subtypes is different.
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Objective To detect the expression of leptin in pituitary adenomas and investigate the association of leptin expression with the invasiveness and proliferation of pituitary adenomas. Methods Sixty-one surgical specimens of pituitary adenomas and the clinical data of the cases were obtained from the Department of Nenrosurgery, Changzheng Hospital between February and October in 2007. Immunohistochemical staining was used to detect the expressions of leptin and Ki-67 in the pituitary adenomas to investigate the role of leptin in the occurrence and development of the tumor. Results The overall leptin positivity rate in these pituitary adenomas was 34.4%, significantly lower than that in normal pituitary tissues (P<0.05). The Ki-67 index and diameter of the leptin-positive pituitary adenomas were significantly lower than those of leptin-negative tumors (P<0.05). The leptin expression rate was significantly lower in invasive pituitary adenomas than in non-invasive adenomas (P<0.05). Conlusions Compared with normal pituitary tissues, pituitary adenomas have reduced leptin expression, which is significantly correlated to Ki-67 index, tumor invasiveness and diameter but not to the patients' age or gender.
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Objective To investigate the expression ofubiquitin conjugating enzyme UbcH10 gene at mRNA and protein levels in brain astrocytomas and its correlation to the pathological grades of astrocytomas. Methods Quantitative real-time RT-PCR analysis, immunohistochemistry and Western blot were performed to detect the expression of UbcH10 mRNA and protein in 32 astrocytomas of different pathological grades and 6 normal brain tissues. The correlation between UbcH10 and Ki-67 immunoreactivity was examined with the Spearman's correlation coefficient. Results Statistical analysis showed significantly increased expression levels of UbcH1O mRNA in high-grade astrocytornas (64.33±60.98) in comparison with those in low-grade astrocytomas (8.36±8.15) and normal brain tissues (1.00±1.57) (P<0.05). increased UbcH10 labeling index in immunohistochemistry was also noted in high-grade astrocytomas [(9.65±5.75)%] as compared with that in low-grade astrocytomas [(4.82± 3.30)%] (P<0.05) and normal brain tissues [0%, P<0.05). Western blot demonstrated significantly higher UbcH10 protein levels in high-grade astrocytomas (0.69±0.38) than in low-grade astrocytomas (0.10±0.08, P<0.05) and normal brain tissues (0.01±0.02, P<0.05). UbcH10 positivity rate was found to positively correlate to that of Ki-67 (r=0.67, P=0.000). Conclusion Overexpression of UbcH10 may play an important role in the development and progression of astrocytomas.
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Objective To identify the factors that influence the prognosis and particularly the survival of patients with corpus callosal gliomas. Methods A retrospective analysis of the clinical data was conducted involving 60 patients with corpus callosal gliomas treated between January, 1995 and December, 2007. All the patients underwent surgical tumor resection with postoperative radiotherapy and chemotherapy. Kaplan-Meier and Cox regression model were used to evaluate the possible prognostic factors including the patients' gender, age, preoperative Karnofsky performance status (KPS), tumor locations, preoperative epilepsy, histological grade, enhancement pattern on magnetic resonance imaging (MRI), extent of surgical resection, and tumor size. Results Kaplan-Meier analysis revealed that age, preoperative KPS score, and histological grade had significant influences on progression-free survival (PFS) and overall survival time of the patients. The tumor location had a significant impact on the overall survival time of the patients, but did not obviously affect the PFS. Multivariate analysis with the Cox regression model indicated that age, histological grade, and extent of surgical tumor resection significantly influenced the overall survival time of the patients, and age and histological grade of the tumor significantly affected the PFS. Conclusion A younger age, lower pathological grade and radical surgical resection of the tumor are the protective prognostic factors in patients with corpus callosal gliomas, while gender, tumor size, tumor location, and KPS score before operation have no prognostic significance.
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Objective To probe the technical processes of the suboccipital retrosigmoidal craniotomy with replacement of bone flap and deep muscles incision and suture and its clinical application. Methods The data of 40 patients undergoing suboccipital craniotomy to the cerehello-pontine angle lesions, deep muscles incision and suture in our hospital from May, 2003 to May, 2005 were analyzed retrospectively. Results All the procedures of craniotomy including removal of the bone flap, deep muscles incision and suture were accomplished safely with an average operation time of 40-60 min. Two patients appeared post-operative subcutaneous hydrops without CSF leakage and the majority can move their head freely sixth day after the operation. Conclusion Suboccipital retrosigmoidal craniotomy with replacement of bone flap, and deep muscles incision and suture, being a safe and feasible method, may decrease the happening of postoperative CSF leakage and encephalocele, and accelerate the recover of head movement.
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Objective To investigate the role of pituitary tumor transforming gene 1 (PTTG1) in the growth and invasion of human glioma cell line by introduction of exogenous microRNA to silence PTTG 1 gene expression. Methods Two double-stranded DNA pcDNA6.2-GW/EmGFP-miR vectors (MIR-1, MIR-2) targeting human PTTG1 mRNA and a negative control plasmid (Neg) were constructed, and were transfected into human U251 cells with high metastatic potentials. Real-time PCR and Western blotting were used to quantify the mRNA and protein levels of PTTG1, respectively. Proliferation and invasiveness of transfected U251 cells were analyzed by MTT assay and Matrigel invasion assay. Results After transfection, Expression of PTTG1 mR.NA was inbibited significantly with inhibitory rates of 87.6% in MIR-2 group, and the protein levels were significantly lower than those of the other groups. There was significant difference in cellular growth rate among the 3 groups. The growth inhibiting rates in the MIR-2 group are 10.7%-34.7%. The migrating number of U251 cells transfected with MIR-2 with relative percentage (12.3±1.0)% was also significantly decreased as compared the Neg group (24.7±1.4)% and Mock group (24.0±2.0)%. Conclusion Introduction of exogenous miRNA to U251 cell line by transfection of MIR-2 can effectively reduce the PTTG1 expression, which can significantly inhibit the proliferation and decrease the invasiveness of glioma cells.
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Objective To observe dynamically the response of the retinal ganglion cells (RGCs) following chronic optic nerve compression in cats. Methods Thirty adult cats were randomly divided into 6 groups (n=5): normal control group, sham operation group, 1-week compression group, 2-week compression group, 4-week compression group and 8-week compression group. The chronic optic nerve injury was produced by an inflatable balloon implanted under the optic chiasm. RGCs of all animals were labeled with Dil by retrograde tracing 2 weeks before operation. After each group animals were killed by perfusion, the retina were harvested to observe the pathological changes using the light microscope and electron microscope and the number of RGCs was counted under fluorescence microscope. Results There were three cell layers in normal HE stained retinas of cats with clear limits, named ganglion cell layer, bipolar cell layer and photoreceptor cell layer in sequence from vitreous body to selera. By 4 weeks after optic nerve compression, there were no obvious pathological changes in the retinas, however, at 8 weeks the nuclei of the RGCs became markedly thin, with the larger almost disappearing, and the total thickness of the retinas reduced with the glial cells proliferating. Under electron microscopy, the RGCs of the normal eats had large ovate nuclei with homogeneous karyoplasms. The cytoplasm occupied only small space of the cells, but contained a great of cellular organelle. At 4 and 8 weeks after compression, it was found in the retinal ganglion cells that the components of cytoplasm reduced, the endoplasmic reticulum expanded, the mitochondria was swollen, the vacuole occurred under the plasma membrane, the membrane of nuclei was shrunk and the chromatin was marginated and condensed. The density of the DiI labeled RGCs in the normal group animals ranged from 406 to 527 cells/mm2, with an average of (465±38) cells/mm2 and higher density in the central area than in the peripheral one. The number of the RC, Cs was unchanged by 4 weeks after optic nerve compression, but 8 weeks later, the number declined significantly to (293±32) cells/mm2 by about 37%. Conclusion The RGCs present delayed and secondary degeneration following chronic optic nerve compression, which gives an opportunity to protect the RGCs.
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Objective To evaluate the injuries in Jiangyou Field Hospital first week after Wenchuan earthquake,and accumulate experience for better medical services and better-built field hospitals to meet the demands of the earthquake-stricken areas.Methods Classification was performed in 1 905 patients with emergency treatment.The medical records of those with hospital stay were retrospectively analysed,including geographical distribution,trauma type,diagnosis,initial treatment and outcomes.Results Six hundred and twenty-nine(33%)patients were hospitalized,with an average of 2 to 100 years old(median,41.2).Three hundred and eighty-four patients(61%)were admitted within the first three days after earthquake.The patients were mainly from Jiangyou City(245,39%),Beichuan County(194,31%)and Pingwu County (158,25%).The most common types of injuries were superficial lacerations(326,51.8%),fractures(202,32.1%) and soft tissue contusions/sprains(87,13.9%),among whom 15 experienced severe squeeze.There were 318 injuries to pelvis and extremities(50.6%),117 to head(18.6%)and 43 to back(6.8%),and 73 cases had multiple injuries (11.6%).Of 376 patients(59.8%)with operation,326 received debridement,21 external fixation,19 internal fixation, 11 amputation,4 laparotomy,4 fasciotomy and 2 craniotomy.The incidence of postoperative infections was 11.8%.Death occurred in 39 patients due to cerebral trauma,pelvic fracture,abdominal hemorrhage and multiple injuries.Conclusion When the earthquake has destroyed the major medical facilities,rapid organization of medical rescue teams,making full use of remaining medical resources and recovering electricity play an important role in the rescue of victims and reduction of mortality.Orthopaedic surgeons must dominate the medical teams as most of the earthquake victims suffer from injuries to the extremities.