Change Of Tight Junctions Between Microvascular Endothelial Cells In C6 Glioma

BackgroundGlioblastoma multiforme(GBM) is an extremely invasive,well-vascularized tumor believed to be of astroglial origin.It is the most prevalent and lethal of all primary malignant brain tumors,with a median survival rate of months.Despite the standard treatment of surgical resection of all primary malignant brain tumors followed by radiation and/or chemotherapy,the survival rate has increased only slightly over the past three decades.Happen seen from the development process of disease,the reason that glioma decrease the quality of life and lead to death in patients with glioma mainly come from two aspects:①the malignant tumor cell proliferation itself;②the effects of glioma cells in patients and causing a variety of pathophysiological changes and symptoms and complications in central nervous system,researchers have made considerable progress in molecular mechanism on proliferation of malignant glioma,but how does glioma cell cause a series of pathophysiological changes in central nervous system? What is the molecular mechanism? These are weak links of current reaseach in glioma.As we all know,the blood-brain barrier is the basic structure to maintain a stable environment within the central nervous system.It is generally believed that the blood-brain barrier including:the brain capillary endothelial cells and their tight junctions and the membrane Picchi astrocytes formed end-feet glial membrane. Among them,the brain capillary endothelial cells and their tight junctions are the main basis of the blood-brain barrier function and structure.In recent years,with tight junctions research institute foundation's enormous progress,it is closely linked to the protein component,nano-structure and physiological function with a new understanding of the current view,tight junctions with nano-structure is molecular components of the complex by a group of proteins are posed.These molecular components including:①transmembrane protein,mainly by claudins,occludins, junction associated molecules(JAM) family composition,such as protein;②subsidiary cytoplasmic protein,mainly by the zonula occludens(ZO) protein family, such as composition.Membrane components of tight junctions molecules interact with each other and with their neighbors on tight junctions membrane protein polymerization,to promote local membrane "fusion" and then freeze-etch electron microscopy the formation of tight junctions by the performance of three-dimensional network of complex ultrastructure.The structure and function of blood-brain barrier to the maintenance of the integrity of the normal functioning of the nervous system is essential,at the same time,the changes of structure and function of tight junctions are key process of a variety of changes in the pathophysiology of diseases of the nervous system.With the development of neurooncology,it has been found that glioma cells can bring a variety of effects on blood-brain barrier during its occurrence and development and thus cause a variety of nervous system symptoms and complications.As to morphological changes of glioma in the blood-brain barrier,it is much differently described in literatures.Blood-brain barrier in gliomas of the impact of tight junctions,different scholars have also made an "open" and "integrity" of the results of different studies.To study the morphological changes of the blood-brain barrier in glioma,We established the animal model of glioma and explore the tumor cells to affect the ultrastructure of the blood-brain barrier;the same time,with advances of modern molecular biology,on the molecular level to clarify tight junctions of the blood-brain barrier in the glioma and its pathophysiological significance of the role has become the study of neural tumor priority,it has become the urgent work to clarify tight junctions of the blood-brain barrier in the glioma and its pathophysiological significance of the role on the molecular level.Studies have shown that tight junctions are compose of a variety of molecular components, claudins currently be considered the formation of tight junction ultrastructure,tight junctions barrier properties of the decision and ion permeability properties of the main components.In order to study the changes of the expression of molecular components of the blood-brain barrier tight junction in glioma,to clarify the molecular level from the glioma cells to the effects of blood-brain barrier tight junctions and their pathophysiological significance,we used to immunohisto chemistry and RT - PCR technology,to explore the glioma cells of the blood-brain barrier tight junction molecule components of claudin-5 and its possible role in molecular biology and pathophysiology mechanisms.Objective:1.Set up near the blood-brain barrier of human glioma in vivo model to study the blood-brain barrier changes in physiology and pathophysiology.2.To provide a theoretical foundation in the morphological,we studied on the blood-brain barrier tight junctions ultrastructure changes in glioma.3.To clarify pathophysiological significance in glioma cells affecting blood-brain barrier tight junctions.We studied in the expression of molecular components of blood-brain barrier tight junctions major changes. Methods:1.The models of C6 glioma were established by stereotaxical injection of C6 glioma cells into the caudate nuclei in male Wistar rats.2.In 21 day,we examined the brain tumor through the naked eye and the MRI examination.3.In 21 day,we selected interested spots included the tumor core,tissues on the edge of the tumor and ipsilateral hemisphere tissues out 2mm to the border of the tumor; The ultrastructures of BBB in the above-mentioned interested sports were examined by electron microscopy.4.In 21 day,immunohistochemistry were used to analyze their expression of claudin-5.5.In 21 day,reverse-transcriptase polymerase chain reaction was used to investigate their expression level of claudin-5.6.Statistical analysis:The use of SPSS 13.0 statistical analysis package.Data are expressed x~-±s,the normal groups and the different parts of claudin-5 mRNA bands OD values using a one-way analysis of variance and multiple comparisons,if met homogeneity of variance using Fisher analysis of variance and LSD multiple comparison method,if does not meet the homogeneity of variance using approximate F test analysis of variance as Welch and using Tambanes' s T2 to multiple comparison,meaning P≤0.05 for the difference was significant.Results:1.C6 tumor cell inoculation in the right side of Wister rat caudate nucleus,21 days later,the naked eye and brain MRI shows that the formation of glioma,it is obvious that glioma is oppression of surrounding tissue and there is local edema surrounding glioma.2.By transmission electron microscopy,in the control brain,the paracellular cleft between adjacent endothelial cells is sealed by continuous strands of tight junctions. In the tumor core of brain gliomas,intercellular tight junctions could be found in 22.23%of microvessels and a significant paracellular cleft could be found between adjacent endothelial cells in other micro vessel;In the tissues on the edge of the tumor, intercellular tight junctions could be found in 57.15%of microvessels and the continuous strands of tight junctions was not complete cleft in other microvessel.3.Immunohistochemical results show that there are strongly positive for claudin-5 in the control brain and negative for claudin-5 expression in the tumor core of brain gliomas.4.RT-PCR results showed:the tumor edge and tumor center claudin-5 expression was significantly reduced and compared with normal brain tissue,the difference was statistically significant(P=0.000,P=0.000),the ipsilateral hemisphere tissues out 2 mm to the border of the tumor expression of claudin-5 is also reduced,but the difference was not statistically significant(P=0.960)。Conclusion:1.C6 glioma animal mode can be successful setting up at the right side of Wistar rat caudate nucleus through stereotactic technology,and brain MRI shows that the formation of glioma,it is obvious that glioma is oppression of surrounding tissue and there is local edema surrounding glioma.2.Glioma in their occurrence and development can affect to blood-brain barrier endothelial cells and destroy blood-brain barrier tight junctions ultrastructure.3.At different locations of brain tissue in C6 glioma animal models,the blood-brain barrier damage closely linked with varying degrees of acceptance.4.Glioma cells can reduce the expression of claudin-5,this process may be involved in the destruction of the blood-brain barrier tight junctions.