Investigation Of Correlation Between Expression Of Glioma Diphtheria Toxin Receptor And Pathological Grade Of Glioma

ObjectiveHuman glioma is a kind of high grade malignant disease in central neural system, which is easy to relapse after operation.Postoperative chemotherapy is currently one of the necessary means of treating glioma and extending the life of patients.However, due to the existence of blood tumor barrier founded by capillaries of glioma,how to selective transport chemotherapeutic drugs to brain tumors selectively is a difficult problem.In recent years,studies at home and abroad show that the brain drug delivery mediated by the endogenous receptor on the brain capillary endothelial cells may be the most effective way.The membrane-bound precursor of heparin-binding epidermal growth factor (proHB-EGF) is the transmembrane protein containing 206 amino acids.It can be combined with the diphtheria toxin receptor specifically through receptor-mediated endocytosis into cells,so that it is also known as the diphtheria toxin receptor (diphtheria toxin receptor,DTR).The expression of DTR is limited to in the smooth muscle cells,vascular endothelial cells,BBB and so on.In disease conditions(such as stroke,brain tumor),and its expression in the BBB was significantly upregulated;It further had been found that DTR also expresed in human glioma cells,and the expression is significantly higher than the one of vascular endothelial cells.Studies have shown that the diphtheria toxin non-toxic mutant-CRM197,coule be used as a safe and effective human vaccines.During the study of application to the diphtheria toxin-sensitive guinea pig,it was founded DTR expressed on vascular endothelial cells and the glial cell line in guinea pig brain,and it was detected the carrier protein of CRM197 could transfer the macromolecular horseradish peroxidase (horseradish peroxidase,HRP,40 kD) effectively through the blood-brain barrier, which proved that CRM-197 coule be used as a safe and effective vector to mediate the cross-cell transfer of macromolecular substances by DTR.These results led us to believe that,DTR-mediated transfer might become a safer and more effective channels of selective transshipment of macromolecular drug,so as to achieve the purpose of the treatment of tumor.In this study,the postoperative tissue samples of patients with glioma tumor was determined the expression levels of DTR in brain glioma.In addition,the relativity was analysised between the expression levels of DTR and its pathological levels.It provided the experimental basis for the DTR-mediated drug delivery method to be applies to clinical chemotherapy on brain tumors.Materials一,Experimental specimensFrom July 2007 to July 2008,32 cases fresh tissue samples were randomly obtainde from the pations with brain glioma who underwent surgery in the Department of Neurosurgery of the First Affiliated Hospital of China Medical University.At the same time,three cases of normal brain tissue samples was obtained after cerebral contusion surgery as a control group.32 cases of patients,18 cases of male(56%),14 cases of female(44%).At the age between 25 and 76,the average age is 47 years.The fresh tissue samples after surgical resection was frozen in liquid nitrogen immediately then was preserved at deep hypothermic refrigerator of -70℃.All the experiments about the human body were in line with the Declaration of Helsinki and obtained the patient's informed consent.二,Experimental regentsGoat anti-human DTR / proHB-EGF polyclonal antibody,mouse anti-β-actin monoclonal antibody and the Enhanced chemiluminescence method(enhanced chemiluminescence,ECL) kit were purchased in SANTA CRUZ BIOTECHNOLOGY company;goat anti-mouse IgG conjugated with horseradish peroxidase,rabbit anti-goat IgG conjugated with horseradish peroxidase and DAB stainning kit were obtained in Zhongshan Company,Beijing;Immunohistochemical strept avidin-peroxidase (SP) kit from Maixin biotechnology companies,Fuzhou.三,Experimental instruments:Homogenate machine,electrothermal constant-temperature dry box,Electronic Analytical Balance,Constant temperature water bath,low temperature refrigeration centrifuge,ultraviolet spectra photometer,automatic running gel imaging system, electrophoresis apparatus,transmark apparatus,-70℃deep freeze refrigerator, homeothermia freezing microtome,liquid nitrogen container.Methods一,H＆E staineHuman glioma diagnosis and classification of pathology were determined by reviewing all hematoxylin and eosin(H＆E) stained tissue sections.The tumor grade was determined according to the 2000 World Health Organization(WHO) Central Nervous System Tumor Classification.Each grade of pathology is a group.二,Western blot methodWestern Blot was used to detect the expression of DTR at the each level of pathology,moreover,to analyze the correlation between the expression level of DTR in human gliomas and its pathological grade.三,Immunohistochemical SP methodImmunohistochemical SP method was used to detect the expression level and distribution of DTR in different pathological grade of human glioma,moreover,to analysis the correlation between the expression level of DTR and its pathological grade.四,Image analysisThe datas obtained from western blot were scanned with Chemi Imager 5500 V2.03 software;Integrated Density Value(IDV) of the DTR was calculated with Fluor Chen 2.0 software.The data of Immunochemistry were obtained and analysed semiquantitatively with Motic Images Advanced 3.2 image analysis system.五,Statistics analysisData from Immunohistochemical and western blot analysis were expressed as mean±standard deviation(SD),and analyzed by using One-way ANOVA of SPSS 13.0 statistic software.The differences between groups were determined using the least significant difference(LSD) of One-way ANOVA.Correlative analysis and linear regression were used to compare the difference in the expression of DTR among three groups.A P-value of＜0.05 was considered significant. Results一,H＆E analysisDetermined according to the 2000 World Health Organization(WHO) Central Nervous System Tumor Classification,among 32 cases of glioma there were 10 cases of gradeⅠ,12 cases of gradeⅡ,10 cases ofⅢ.GradeⅣcases were not obtained because they were rare.Devide the specimens into 3 groups.Every grade was as one group.Three cases samples of normal brain tissue obtained in cerebral contusion surgery regard as a control group.二,Results of western blot analysisWestern blot analysis with anti-DTR polyclonal antibody of Santa Cruz Biotechnology revealed a 22 kDa protein.The results were determined by the IDV ratio of each group toβ-actin.DTR targeted at tumor cells,and in the control group did not express.There was significant difference of DTR expression level among different pathological grade of glioma(gradeⅠvs.gradeⅡ:0.1070±0.0265 vs.0.1592±0.0322, P=0.000;gradeⅠvs.gradeⅡ:0.2730±0.0425 vs.0.1070±0.0265,P=0.000;gradeⅡvs. gradeⅢ:0.1592±0.0322 vs.0.2730±0.0425,P=0.000).Comparison of the result showed differential expression and distribution of DTR of the three groups of glioma in order of gradeⅠ＜gradeⅡ＜gradeⅢ.There was a significant positive correlation between expression of DTR of glioma and pathological grade of glioma(r=0.877, P=0.000).Regression analysis,linear regression equation was Y(＾)=0.083X+0.0127.三,Results of Immunochemistry analysisSP immunohistochemical assay showed that there was no expression of DTR in control group while every grade glioma cells were showed positive staining by polyclonal anti-DTR antibody.The positive grown kernels were located in the cell membrane and cytoplasm while there was no colour staining in nuclear.There was significant difference of DTR expression level among different pathological grade of glioma(gradeⅠvs.gradeⅡ:0.1923±0.0203 vs.0.2084±0.0211,P=0.008;gradeⅠvs. gradeⅢ:0.1923±0.0203 vs.0.2455±0.0384,P=0.000;gradeⅡvs.gradeⅢ: 0.2084±0.0211 vs.0.2455±0.0384,P=0.000).Comparison of the result showed differential expression and distribution of DTR of the three groups of glioma in order of gradeⅠ＜gradeⅡ＜gradeⅢ.There was a significant positive correlation between expression of DTR of glioma and pathological grade of glioma(r=0.026,P=0.000). Regression analysis,linear regression equation was Y(＾)=0.026X+0.164.DiscussionWith human specimens this study firstly demonstrated that there was difference of DTR expression among human gliomas with different pathological grade.The difference was in order of gradeⅠ＜gradeⅡ＜gradeⅢ.There was a significant positive correlation between DTR expression of glioma and pathological grade of glioma.HB-EGF is a molecular weight of 20-22kDa glycoprotein.It belongs to the EGF superfamily which promote the differentiation and growth of protein^[8],.It was firstly synthesized as proHB-EGF.The proteolysis sHB-EGF contains a 76-87 split of the soluble amino acid residues.Immunohistochemistry showed that proHB-EGF is the main expression of HB-EGF in human glioma cells.The expression of HB-EGF in human glioma cells is bFGF,EGF or TGF-α-induced in vitro.Meanwhile human glioma cells produce TGF-A and bEGF in vivo.These improve the possibility of multiple tumor-derived growth factor mutual regulation of their signal-transduction pathway,as well as play a important role in stimulating the growth of glioma cells. At the same time,these cross-reaction of growth factors may also caused the HB-EGF overexpression in glioma.Accompanying with the geometric growth and the increase in pathological level of Human glioma cells,this role may have been enlarged, so that the expression level of HB-EGF,namely DTR increased with the pathological increasing.There is the expression of proHB-EGF in human vascular endothelial cells.As the DTR,proHB-EGF can mediate the exogenous drug which connected the receptor by the anti-DTR antibody transfer to the brain tissue by endocytosis.Also it was found that there was high expression of DTR in human glioma cells,which suggested that the drug could play specific role in brain glioma further.As the carrier,CRM197,the non-toxic diphtheria toxin mutant,can mediate the elements to enter the brain through the endothelial cells by the DTR-mediated drug transport,however the process do not damage the barrier properties.It suggest that DTR-mediated drug delivery may become a new way of the CNS targeted drug delivery,which can be used as a targeted treatment of human gliomaIn the study it was found that there was a significant positive correlation between the expression level of DTR of the brain glioma and its pathological level,moreover their linear regression equations have statistical significance.So that it can be speculated that the patients with high pathology level of brain glioma,because of its higher expression level of DTR,the effects of drug transporter mediated by DT and its analogues CRM197 may be more significant,so that it could achieve better therapeutic effect.Conclusion1,There was difference of DTR expression among human gliomas with different pathological grade.The difference was in order of gradeⅠ＜grade＜ⅡgradeⅢwith human specimens.2,There was significant positive correlation between DTR expression of glioma and pathological grade of glioma.