| Background:In recent years, the incidence of brain tumors is rising. According to the statistics, brain tumors have accounted for about5%of systemic cancer, and accounted for70%of children’s cancer, of which the most common brain tumors are glioma. And intracranial gliomas account for about40%-45%of tumors, which derived from various types of glial cells. In the broad sense, the entire nerve glioma should include all types of epithelial tissue derived glial cells and neurons of the tumors, including medullary epithelial tumors, medulloblastoma, glioblastoma, astrocytoma, small sticks of gum Stromal tumor, ependymoma, etc..Gliomas can be divided into (Ⅰ-Ⅳ) grade by according to malignancy.The clinical manifestations of glioma include intracranial hypertension clinical symptoms and the lesions localized symptoms. The mechanism of increased intracranial pressure of glioma induced:real space occupying; tumor edema; cystic tumor or hemorrhage. Whether the tumor intracranial hypertension occurs sooner or later depends on the speed of development and severity, the tumor growth rate, the cerebrospinal fluid pathway or the veins around the side or on situations such as posterior fossa. The clinical manifestations of gliomas is an important basis for the identification, in addition, neuroimaging basis, including lesion location, lesion signal lesion within the texture, peripheral shape, strengthening condition, the surrounding structure determines the nature and extent of the disease, which play an important part to the treatment programs.As for the treatment of glioma, the first is surgery, for the primary tumor, surgical resection may as far as possible remove tumor cells, avoid cell proliferation,and is still the most common mode of treatment. For benign tumors, with higher probability of complete surgical resection, the patient survival rate is higher. But for a higher degree of malignancy, especially of more than WHO grade Ⅲ tumors, given the invasive growth of glioma cells and abnormal biological characteristics, which is not sensitive to radiotherapy and chemotherapy, however the treatment is ineffective; and it often recur to the same level or high-level, which increased the patient’s suffering and economic burden. Therefore, it is of great importance to analyze the malignant phenotype of glioma by clinical and basic research.Cancer is caused by many factors. Experiments have proved that application of free radical scavenging agents and antioxidants in whole or in vitro experiments have inhibit some carcinogenic effect. This indicates that too much free radicals may cause tissue cell structure and function change through some mechanism, the growth level was out of control, thus cause and promote cancer occurrence, development and deterioration. Reactive Oxygen Species (ROS) refers to chemical properties active Oxygen or atoms, including containing superoxide radical, hydrogen peroxide (H2O2), plasmalemma peroxide free radicals, etc. ROS enzymes inside cells were mainly the Nicotinamide oxidase (NADPH vadenine dinucleotide phosphate oxidase, NOX), yellow rowl ridge oxidase etc, and the NOX is the main source of ROS. ROS is considered to be the toxic byproduct of cell metabolism, which can directly act with lipids and proteins, DNA molecular etc., causing histologic changes and dysfunction. Currently, more and more research shows that, ROS can be used as in signal transduction molecules at low concentration, participating in the various cell pathological physiology reaction process. Research shows that during the malignant transformation process of glioma cell, ROS in cells also increased significantly, ROS might have played an important role in the the pathological physiology. Research shows that ROS can activate protein kinase, transcription factors and gene expression, etc. NADPH oxidase is an important source of intracellular ROS. Normally, this enzyme in the resting state under appropriate stimulation, can quickly activation. NADPH oxidase can interact intracellular nucleic acid, protein, fats and sugars, causing cellular structure and function change and promoting the occurrence and development of cancer, especially glioma.On this basis, in consideration of some the problems of gliomas clinical diagnosis and treatment, we collect clinical cases and specimen of different grade of glioma to detect NAD(P) H oxidase and ROS level and investigate the correlation between the NAD(P) H oxidase and ROS level and the malignancy grade of glioma so as to give guidance to clinical diagnosis and treatment of gliomas.Purpose:To detect NAD(P)H oxidase and ROS level in gliomas of different malignancy degree, investigate the NAD(P) H oxidase and ROS level and investigate the correlation between the NAD(P) H oxidase and ROS level and the malignancy grade of glioma so as to give new idea to clinical diagnosis and treatment of gliomas.Methods:we collected30gliomas patients tissue samples of different levels during August in2007to August in2010in neurosurgery in Zhujiang hospital, referring to the malignant tumor classification and grading criteria of central nervous system of the declared by WHO, and10cases of normal patients’tissue samples as control group, the patients were3to68years old, with an average age of38.5years old, and divided into control group (10cases), low level-Ⅰ and Ⅱ grade gliomas (10cases), Ⅲgrade gliomas (10cases) and Ⅳgrade gliomas (10cases). Of all30patients18cases were males,12cases were women, there were no significant comparative differences for group in sex, age, duration, etc no, and the grouping followed the equilibrium and random principles. Methods:1. Use Real time PCR method to detect tissue (cells) in the expression of NOX gene of the gliomas tissue;2. Determining content of ROS in tissue with carboxy-H2DCFDA3. Western blot and immunofluorescence to detection expression of NOX;4. Do reasonable statistical analysis;Analysis and detection NAD(P)H oxidase and ROS in different grade gliomas, investigate the correlation between the NAD(P) H oxidase and ROS level and the malignancy grade of glioma so as to give guidance to clinical diagnosis and treatment of gliomas.Results:1. Detection of glioma cells NOX mRNA expression levels by Real time PCRIn this study,40patients were divided into normal control group patients, low-grade gliomas(Ⅰ and Ⅱ grade), Ⅲand IV grade glioma, used real time PCR method to analyzes NOX mRNA expression in each group and human GAPDH gene was as internal reference, results suggest that samples NOX1-5mRNA could be detected a certain degree expression in40the cases, and were significantly different to the control group, P<0.05, however, comparisons between the various groups, especially for NOX4expression in each experimental group had significantly difference comparing to the control group, for low-grade gliomas (Ⅰ and Ⅱ), Illand Ⅳgrade its level of expression was significant difference, P<0.05, however, NOX1,2,3,5didn’t expressed significantly differently in various grade tumor tissues, P>0.05.2. Measure the production of ROS with Carboxy-H2DCFDA in tissueGlioma issue were prepared to cells and added lOuM carboxy-H2DCFDA as a final concentration, using Flow cytometry detect the content of ROS, the result showed that, for the control group, low-level (Ⅰ and Ⅱ grade), Ⅲ and Ⅳ grade glioma, with the tumor grade increased, the fluorescence intensity of ROS in the tissue increased, and the production of ROS were significantly higher. Compared with the control group it was statistically significant difference, P<0.05, ROS levels in different group was statistically significant different, P<0.05, indicating that ROS level was related to glioma grade.3. Detect the expression of NOX4using Western blot methodWestern Blot indicated that NOX4protein in control group showed low or negative expression, while in low-grade gliomas (Ⅰ and Ⅱ grade) it increased and expression increased with the tumor malignant grade, it was significantly increased in grade Ⅲ and Ⅳglioma compared with the control group, P<0.05. This coincided with result trend by fluorescence quantitative PCR. Such result of different stages of N0X4protein level in different glioma progression suggested that the protein may participated malignant progression through certain signal transduction pathways.4. Detect NOX using immunofluorescence methodImmunofluorescence showed that NOX protein samples in the control group showed low expression or no expression, but low-grade gliomas (Ⅰ and Ⅱ grade), Ⅲ and Ⅳ grade glioma groups all showed fluorescence, suggesting that these organizations had NOX protein, and with the increase of the degree of malignancy, the expression of NOX increased correspondingly, the higher the degree of malignancy, the higher the NOX expression level.Conclusion:By studying expression of NOX and ROS levels of different degrees of malignant glioma tissue, we got the following conclusions:comparing the control group, the higher grade malignant glioma had higher NOX gene expression levels, which was significantly different; expression levels of NOX were significantly different between the groups; comparing with the control group, the higher grade malignant glioma generated more ROS, which was also significantly. NOX and ROS levels were positively correlated with the degree of malignancy. NOX4in gliomas may be the main enzyme body of ROS production, and oxidative enzyme NOX4and ROS production may played an important regulatory role in the malignant proliferation of glioma, which was closely related to malignant gliomas. The study results provided a new idea for clinical and basic research on diagnosis and treatment of glioma. |
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