The Expressions Of HIF-1α And NNOS In Human Cerebral Glioma

ObjectiveGlioma is one of the most common intracranial malignant tumors and it reaches to the 50-60% of the total intracranial tumors. Surgical excision is a chief treatment of glioma currently; however, it's hard to excise the glioma thoroughly, since between which and the normal tissues there is no border-line, due to it grows in a diffused and infiltrated way. Although combining with radiotherapy and chemotherapy after surgical treatment, most patients died of progression or recurrence of glioma, for which is insensitive to radio- or chemo-therapy. The pathological studies found that, there are anoxic zones, central necrosis and proliferation of abnormal vessels around the malignant glioma. For the purpose of investigating the molecular mechanism of glioma further, in this study, we examined the expression of hypoxia inducible factor 1 alpha (HIF-1α) and neurological nitric oxide synthase (nNOS) in human glioma; and then we discussed not only the roles of HIF-1 a and nNOS in heterogeneity of glioma but also the correlation between them. Our study would provide a basic theory to researches on the mechanisms of hypoxia angiogenesis in glioma, and establish new ideas for further treatment of the most common intracranial malignant tumor.MethodSubject: 30 cases of excised human glioma tissue samples (including grade I,II,III,IV), proved by post-operational pathological results, were obtained from the patients in charged in the Neurosurgery of the First Affiliated Hospital of China Medical University from June 2007 to December 2007. According to the pathological classification criteria of human glioma published by WHO in 1999, the samples were divided into two groups.Immunohistochemical stain was used to identify the expression of HIF-1 a and nNOS in different pathological grade of human glioma, respectively. Firstly, peroxidase inhibitors were used to inactivate the endogenesic peroxidate in frozen slices for incubating at room temperature for 30 minutes. After that the samples were washed with PBS and incubated with animal-serum for 30 minutes, then added with primary antibody and incubated at 4℃overnight. In the following day, the samples were added with second antibody and incubated for 60 minutes at room temperature. Then the samples were added with Streptavidin-horseradish peroxidase and incubated at room temperature for 60 minutes. Finally, the samples were stained by DAB method, and counterstained with haematoxylin. After that, the samples were fixed with resin.The semi-quantification criterion of the stain of HIF-1αand nNOS was identified, naked-eye observing method was used for cell counting, and automatic graphic analysis system was used to exam the optical density of a specified field. We processed the acquired data using the statistic software SPSS13.0 for Windows.ResultOur results of the staining suggest that, there are Hif-1αand nNOS expression in glioma tissues of all grades. Hif-1αis mainly stained in nuclear and nNOS is mainly stained in cytoplasm, respectively; and the expressions of both of them are correlated to the pathological grades. The expression level of higher grades glioma is obviously higher than the lower grades, and there is an obvious statistical distributional difference (P<0.01). Moreover, the result of average optical density determined by an automatic graphic analysis system shows that, there is an obvious distinction among the expression levels of Hif-1αand nNOS in different grades of glioma, and which are positively correlated with the pathological grades. And also we found that there is a relationship between the expression of Hif-1αand nNOS.DiscussionGlioma is one of the most common intracranial malignant tumors, and one of whose features is the invasion and infiltration of tumor cells, which is also the basic reason for recurrence and residual of tumor after surgical excision. The pathological studies found that, there are anoxic zones, central necrosis and proliferation of abnormal vessels around the malignant glioma. In recent studies, Hif-1αwas identified to be a most important regulator in the micro-circumstance of tumor cell, playing a central role in angiogenesis and invasion of glioma. The over expression of Hif-1αwill increase the expression of VEGF in transcriptional level, because Hif-1αcan combine with the consensus sequence in its downstream genes (i.e. hypoxia response elements in VEGF, HRE). Hif-1αcan increase the stability of VEGF mRNA obviously in hypoxia circumstances, make the expressing level of VEGF protein up-regulated, and therefore proliferate generation of vessels in hypoxia regions. This study demonstrated that Hif-1αis expressed in glioma cells and the expression level of Hif-1αis increased with the pathological grade of glioma accordingly. Furthermore, it was reported recently that nNOS also play an important role in proliferating angiogenesis in hypoxia circumstance in malignant glioma. nNOS can make the blood-brain barrier open through cGMP pathway, increase the level of VEGF, proliferate generation of new vessels; meanwhile, nitrogen monoxides can dilate the vessels and increase the blood flow to meet the need of tumor cells for oxygen. This study demonstrated that nNOS is expressed in glioma cells and the expression level of nNOS is increased with the pathological grade of glioma accordingly. Besides, this study also proved that the expression of Hif-1αand nNOS is correlated in malignant glioma, and presumed that both of them participate in promoting angiogenesis and increasing the ability of glioma in proliferation and invasion.ConclusionThe expression level of Hif-1αand nNOS grows with the increase of pathological grades. The expression of Hif-1αand nNOS are also positive correlation. Therefore, the quantitative evaluation to the expression of Hif-1αand nNOS would be an index for judging the malignance degree and the prognosis of the patients with brain glioma.