Expression Of Glut1 And HIF-1a In Human Cholangiocarcinoma And Its Clinical Significance

PrefaceIn malignant tumors, the energy of the cells proliferation surpass the energy supply, so cells to over-consumption of oxygen and nutrients can lead to tumor micro-environment as hypoxia, low-sugar and acid, this is more evident in the solid tumor. In the hypoxic state, tissue obtain energy through aerobic oxidation were markedly reduced, while the more important way is that it meets the needs of the energy for the tumour growth through the anaerobic glycolysis. This means that the cell should be intake more sugar to meet the rapid cell growth and proliferation of high-energy demand。The abnormal increase of Glucose transporter 1(Glutl) in cancer cell may be one of the mechanisms of glucose uptake. Hypoxia inducible factor-1a(HIF-la) is a key transcriptional regulatory gene, which can mediated cell to adapt the anoxic micro-environment, it can activate the expression of many hypoxia-responsive gene. So that the tissue can survive in the hypoxic state by series of adaptive response. Including HIF-1a can promote tumour cell glucose transporter proteins (Glut) gene expression in order to meet the energy needs of tumour growth. Recently the incidence of cholangiocarcinoma(CCA) has greatly increased, while the prognosis of cholangiocarcinoma is poor.It has not been reported about the expression of Glutl and HIF-la in cholangiocarcinoma by far, so the purpose of this research is to research the expression of Glutl and HIF-la in human cholangiocarcinoma and its clinical significance.Materials and MethodsⅠ,General Information Between 2005-2008,90 patients who received a diagnosis of cholangiocarcinoma were pathologically confirmed at the department of pathology of Shengjing Hospital which is affiliated to Chinese medical university including male 47 cases, female 43 cases,5 normal bile duct tissue, age from 34 to 82,the average age was 61 years, By cholangiocarcinoma pathology grade,well differentiated in 28 cases, moderately differentiated in 38 cases,24 cases of poorly differentiated, Lymph node metastasis in 34 cases. Without lymph node metastasis 56 cases. All samples were not recieived preoperative chemoradiotherapy. Phased according to UICC staging,Ⅰof 22 cases,Ⅱof 34 cases, III of 25 cases,Ⅳof 9 cases.Ⅱ,Experimental methods and reagents1,the main reagentsRabbit anti-human Glutl (bs-0472R) monoclonal antibodies were purchased from Beijing Boao Sen Biotechnology Co., Ltd., rabbit anti-human HIF-la (bs-0737R) monoclonal antibodies were purchased from Beijing Boao Sen Biotechnology Co., Ltd..2,experimental methodsExperiments were performed in strict accordance with the immunohistochemical S-P method steps of conventional staining.Glutl and HIF-1aⅠ-anti-concentration were 1:200. DAB colour, while using PBS instead of primary antibody as negative control, with known positive result for positive control.3,observeA doctor of pathology who did not know the clinical condition of the patient observed the staining slice.The positive staining of Glutl is that the membrane appeared brown yellow or brown granules. while the positive staining of HIF-la is that the cytoplasm or nucleus appeared brown yellow or brown granules. At high magnification (×400) randomly select five horizons, count 200 cells, a total of 1000, observe colour intensity and calculate the percentage of positive cells per slice. Glutl positive classification criteria:first by colour intensity rate:0 points colourless, light yellow 1 point,2 points brown, tan 3. And then rate the percentage of positive cells, reference Cooper (4) and other reports:no positive cells as 0, positive cells<10%1 point,10% to 50% for 2 points,> 50% 3 points.The product of the percentage of positive cells and color intensity:0 to 3 were divided into negative (-),4 to 6 divided into weak positive (+),7 to 9 are divided into strongly positive (++). HIF-la positive classification criteria:first by colour intensity rate:0 point colourless, light yellow 1 point,2 points brown, tan 3 points. And then rate the percentage of positive cells, reference Zhong and other reports:no positive cells as 0, positive cells<1% 1 point,1%-10% 2 points, 10% to 50% for 3 points,> 50%,4 ponits. The product of the percentage of positive cells and colour intensity:0 to 3 were divided into negative (-),4 to 6 divided into weak positive (+),7 to 9 were divided into moderately positive (++),10 to 12 were divided into strong positive (+++). Statistics dealed with the use of SPSS 13.0 Package, Correlation analysis used Spearman rank correlation analysis.Result1,Glutl expression resultsThe positive staining of Glutl is that the membrane appeared brown yellow or brown granules, and it expressed in the cholangiocarcinoma tissue(66.7%), while it did not express in the normal bile duct tissue. It has a correlation with tumour stage, lymph node metastasis and diffrerentiation, but not with tumour size.2,HIF-la expression resultsThe positive staining of HIF-la is that the nucleus appeared brown yellow or brown granules. And it expressed in the cholangiocarcinoma tissue(73.3%), while it did not express in the normal bile duct tissue. It has a correlation with tumour stage, differentiation and lymph node metastasis, but not with tumour size.3,Glut-1 expression and HIF-la expression was positively correlated.ConclusionThe expression of Glut1 and HIF-la in cholangiocarcinoma showed high expression, and there is a positive correlation between the expressions.