Expression And Significance Of Chemokine Receptor CXCR4 In Glioma

Objective: Glioma is the most common malignant tumor in brain,which has high incidence, strongly invasive power and recurrence fast after surgery. The effects of a variety of traditional treatment methods are not ideal.At the present time, the specific pathogenesis of glioma is not clear.With the gradually deep study for the pathogenesis of glioma and the rapid development of molecular biology techniques, molecular target treatment of cancer is gradually becoming a promising new means of treatment.Studies have shown that chemokines SDF-1 and their receptors CXCR4 play important and complex roles in carcinogenesis and development of glioma, they can promote glioma cells prolifertion and migration,accelerate pervasion and canceration of glioma cells or angiogenesis in tumor tissue. Therefore,it is of great significance to study the roles of chemokines and their receptors in the process of carcinogenesis,invasion and angiogenesis of glioma for understanding the mechanism of carcinogenesis and development, anti-angiogenic and molecular target treatment of glioma.The objective of this study is to analyze the internal relations between the expression of chemokines receptors CXCR4 and invasion of glioma,and the relations with microvessel density (MVD) by studying the expression of CXCR4 in all grade gliomas.Methods: 38 glioma tissue specimens were collected from the glioma patients who were operated in the Neurosurgery of the Second Hospital of Hebei Medical University from 2009.10 to 2010.3(22 of them were male,16 of them were female),all of the patients are initial cases and not accepted the treatment of radiotherapy and chemotherapy etc. And 5 normal brain tissue specimens of intracranial decompression operation were gathered as control group.Two specimens were taken for each,one of which was fixed by 4% formaldehyde and paraffin-embedded;another was frozen saved in the liquid nitrogen immediately after operation. All of the specimens were confirned by histopathologic examination and clinical pathological staging was taken according to the WHO(2000) classifying and grading criteria of nervous system tumors.1 Expression of CXCR4 protein and anti-CD34 monoclonal antibody in these specimens were examined by immunohistochemistry.And MVD was counted.Judgment of immunohistochemistry result:By the methods of the percentage of positive cells and color intensity,(1) percentage of postive cells is divided into the following four grade:0 score:postive cell<10%;1 score:postive cell 10%～25%;2 scores:postive cell 25%～50%;3 scores:postive cell>50%. (2)dyeing intensity: 0 score: not stained;1 score:poor positive; 2 scores:moderate positive;3 scores:strong postive; The results can be gotten by adding (1)and(2):0～1score as negative,marked"-";2～4 scores as minute positive,marked"+";5 scores above as positive,marked"++". Method of counting MVD:Any endothelial cell or endothelial cell cluster which is stained brown by anti-CD34 monoclonal antibody in the tumor tissue is signed one positive blood vessel marker,with or without lumen,if only it is obvious different from other connective tissue components. We observe one slice of every specimen,avoid the tumor necrosis region or bleeding region, and select the most abundant area of micrangium under low power field(×40),which is"hot spot". we count the average of blood vessels under five high power fields(×200),and count in eyepiece micrometer grid range of 0.25mm~2,the arithmetic mean is signed as MVD measured value of this slice.2 The relative expression of chemokines receptors CXCR4 in glioma tissue specimen was determined by Quantitative real-time PCR.Use Trizol to extract total RNA,then make agarose gel electrophoresis detection, and RNA purity is detected by spectrophotometer.The mRNA is reversed transcription into cDNA ,and cDNA were detected,β-actin as internal reference.It is measured by strict re-hole. Statistical methods:According to the different methods of data acquisition and various requirements of statistical analysis,all of the measurement data is shown with Mean±SD. we choose x2-test,t-test,and t '-test. We use SPSS17.0 statistics software to processing data.When P<0.05,the data has significant statistics significance. When P<0.01,the data has very significant statistics significance.Results:1 Immunohistochemistry experiment showed that chemokines receptors CXCR4 was negatively expressed in normal brain tissues and positively expressed in 24 cases glioma tissue,the total positive expression rates in tumor group are 63.16%,and the positive expression rates were separately 44.44% and 80.00% in low-grade (gradeⅡ)glioma group and high-grade (gradeⅢandⅣ) glioma group.The positive expression rates of CXCR4 increased along with the pathological grades of glioma,and the positive expression rates of high-grade glioma group were significantly higher than low-grade glioma group, the data has significant statistics significance(P<0.05).2 The expression average of MVD labelled with anti-CD34 monoclonal antibody in normal brain tissues group and tumor group respectively were 3.76±0.48 and 35.25±9.75, the expression of MVD is higher in glioma tissues group than in normal brain tissues group, the data had very significant statistics significance(P<0.01).The expression of MVD in low-grade glioma group and high-grade glioma group respectively were 27.38±5.04and 42.34±7.12, the expression of MVD is higher in high-grade glioma group than in low-grade glioma group, the difference had very significant statistics significance(P<0.01).3 The expression average of MVD respectively were 26.37±5.11and 40.43±7.88 in 14 cases negative expression of CXCR4 and 24 cases positive expression of CXCR4 in glioma tissues, we can know that the data has very significant statistics significance through the statistical test (P<0.01). 4 Quantitative real-time PCR detection showed that the normalized chemokines receptors CXCR4 mRNA amount relative to normal brain tissues group was 2.22±0.80 in low-grade glioma group ,and 4.03±1.98 in high-grade glioma group,the data increased along with the pathological grades of glioma, the difference had significant statistics significance(P<0.05).Conclusion:1 Chemokines receptors CXCR4 expresses negatively in group of normal brain tissues,and expresses positively in group of glioma tissues,it suggests that CXCR4 is related to the invasiveness of glioma.2 In glioma tissue, chemokines receptors CXCR4 has higher expression at protein and molecular level,and the expression increased along with the pathological grades of glioma,,this suggests that CXCR4 is related to the malignancy of glioma.3 In glioma tissue, MVD which can reflect the increase of neovascularization, increased along with the pathological grades of glioma.It suggests that the relation between the angiogenesis of glioma and MVD is inseparable.The higher MVD,the more tumor angiogenesis,the higher malignancy of the tumors.It was proved that MVD was an important indicator of determining malignancy and prognosis of tumors.4 The experimental results show that the expression of MVD has significant statistics significance between the negative expression of CXCR4 and positive expression of CXCR4 in glioma tissue,and CXCR4 can also express in vascular endothelial cells of glioma,which suggests that CXCR4 might take part in the angiogenesis of glioma.