Expression And Clinical Significance Of P-gp And HSP70 In Cholangiocarcinoma Tissue

PrefaceCholangiocarcinoma is originated in bile duct epithelial cells,including intrahepatic cholangiocarcinoma(ICC) and extrahepatic cholangiocarcinoma(ECC). Because of its special anatomic location,the high degree of malignancy and the onset of misprision,the rate of early diagnosis are still low.When has been diagnosed,it has been too late to radical resection,the prognosis is pretty poor.Chemotherapy is ineffective for carcinoma which is well tolerated.The study showes that drug resistance of tumor is the main reason for the failure of chemotherapy.Since the year 1970 Biedler found the phenomenon of multidrug resistance,there has been a lot of research on it.P-glycoprotein has been confirmed to be one of the main resistance protein.The study has confirmed that heat shock protein 70 can protect the cells at stress state.The relationship of P-gp and HSP 70 has aroused the attention of scholars.This article tested the expression of P-gp and HSP70 in cholangiocarcinoma by immunohistochemistry to explore the relationship with tumor biological characteristics,as well as the interrelationship between them.Objective1.Research object98 pathological specimens of extrahepatic cholangiocarcinoma were from Pathology department of Shengjing Hospital of China Medical University (1996.01-2007.3),which were not for preoperative radiotherapy and chemotherapy, and confirmed by pathology.There were 51 cases of men and 47 cases of women aged from 40 to 70 years old with an average age of 63.High/medium/low differentiated cholangiocarcinoma were separately 47/35/16 cases.43 patients had lymph node metastasis,and the others 55 patients had not.Phased by UICC criteria,there are 46 cases in phaseⅠ-Ⅱ,52 cases in phaseⅢ-Ⅳ. 2.Main reagentMonoclonal mouse anti-HSP70 antibody(1:50),monoclonal mouse anti-human antibody P-gp(1:50) and SP kits were purchased from Wuhan Boshide Biological Engineering Limited.3.Immunohistochemical stainingSerial sections of wax block with 4um of thickness.Choose one for regular H-E staining to determine the histological diagnosis and the degree of differentiation. Operate strictly according to protocol to detect the expression of HSP70 and P-gp in the cholangiocarcinoma.Color DAB.SP method was used to all specimens for immunohistochemical staining,before which a microwave oven was used for antigen retrieval.Positive control was a known positive tissue slice,while PBS instead of first antigen as a negative control.4.Assessment of resultsObserved Comprehensively of each slice,selected randomly 10 high power field. Score of P-gp was according to staining intensity,scope.Staining intensity:colorless(0 points),light yellow(1 points),browm(2 points),yellow brown(3 points);dyeing range:＜5%(0 points),5%～20%(1 points),20%～50%(2 points),＞50%(3 points).The results divided into low-expression(0-1 point) and high expression(＞2 points).HSP70 staining were divided into three grade:positive cell count＜30%,staining particles show yellow,small piles as first grade;positive cells 30%-60%,staining particles show deep yellow,thick piles as second grade;positive cells＞60%,staining particles brown,form large pieces as third grade.5.Statistical analysis:SSPS 12.0 was applied for data processing.P＜0.05 has a statistical significance.Results1.P-gp was mainly located in membrane and cytoplasm,which HSP70 mainly in cytoplasm.98 in all patients,low expression of P-gp was 57 cases(58.2%),high expression 41 cases(41.8%).grade 1 staining of HSP70 protein was 27 cases(27.6%), grade 2 36 cases(36.7%),and grade 3 35 cases(35.7%).2.relationship between P-gp,HSP70 and clinicopathological parameters The expression of P-gp was positively correlated with differentiation of cholangiocarcinoma(r=0.254,P=0.028),expression of P-gp in well-differentiated cholangiocarcinoma was significantly higher than that of poorly differentiated cholangiocarcinoma.And its expression had no relationship with age,histology type, lymph node metastasis and TNM stage.The expression of HSP-70 was negative correlated with differentiation of cholangiocarcinoma(r=-0.236,P=0.031), expression of HSP 70 in poorly differentiated cholangiocarcinoma was significantly higher than that of highly differentiated cholangiocarcinoma.And its expression had no relationship with age,histology type,lymph node metastasis and TNM stage.3.relationship between HSP70 and P-gp The expression of P-gp was negetively correlated with HSP70(r=-0.352,P=0.001).Conculsions1.In cholangiocarcinoma tissue,there is a relationship betrwwn the expression of HSP70 and P-gp and the differentiation.The expression of P-gp was positively correlated with differentiation of cholangiocarcinoma.Expression of P-gp in well-differentiated cholangiocarcinoma was significantly higher than that of poorly differentiated cholangiocarcinoma.The expression of HSP-70 was negative correlated with differentiation of cholangiocarcinoma.expression of HSP 70 in poorly differentiated cholangiocarcinoma was significantly higher than that of highly differentiated cholangiocarcinoma.2.The expression of HSP70 and protein P-gp has no relationship with age, gender,lymph node metastasis and TNM stages in cholangiocarcinoma.3.HSP70 expression was nagatively correlated with P-gp.Anti-apoptotic effect of HSP70 may be due to the inhibition of P-gp activity to achieve.Howerer,the interaction mechaniam needa further experiments to prove.