Expression Of HSP70 And Caspase-3 In Cholangiocarcinoma Tissue And Their Significance

ObjectiveCholangiocarcinoma 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.Its etiology has not been clear yet. However,as the study for the incidence mechanism of cholangiocarcinoma continually deepening,people have begun to explore from the gene level.It was suggested that apoptosis and disorders of cell cycle regulation work in the pathogenesis of tumor.Heat shock proteins(HSP)are a group of protein family which widespread exist in organisms and are highly conserved in evolution,and are also known as molecular chaperones.HSP70 will increase significantly and protect cell in condition of stress. Tumor cells induced-HSP70,on the one hand may be related to a variety of oncogene interactions to regulate cell proliferation;on the other hand may also been involved in the inhibition of tumor cell apoptosis.HSP70 has a close relationship with many tumor development,biological behavior and prognosis.But it not was still explicit in the expression of cholangiocarcinoma and the clinical significance reported.Apoptotic cells degrade in the activation of Cysteinyl aspartate-specific protease(Caspase).Caspase family plays a important role in information transmission of tumor cell death process.Caspase-3 is an important element in the apoptotic signal transduction pathway,which once activated results in reversible cell apoptosis.Studies have shown that HSP70 as a "molecular chaperone",plays a key role in Caspase apoptosis pathway,possibly participates in regulate apoptosis of tumor cells.Immunohistochemical methods were used to test the expression of HSP70 and Caspase-3 in cholangiocarcinoma,and explore their relevance and the clinical significance.To test the expression of HSP70 and Caspase-3 in cholangiocarcinoma tissues,and explore the relevance and the clinical significance. To research the pathogenesis of cholangiocarcinoma and provide the basis for the fight against cholangiocarcinoma.Methods1.Research object68 pathological specimens of extrahepatic cholangiocarcinoma were from Pathology department of Shengjing Hospital of China Medical University (1996.01-2006.12),which were not for preoperative radiotherapy and chemotherapy, and confirmed by pathology.There were 35 cases of men and 33 cases of women aged from 45 to 70 years old with an average age of 62.High / medium / low differentiated cholangiocarcinoma were separately 32/24/12 cases.38 patients had lymph node metastasis,and the others had not.Phased by UICC criteria,there are 14 cases in phaseⅠ,18 cases in PhaseⅡ,28 cases in phaseⅢand 8 cases in phaseⅣ.2.Main reagentMonoclonal mouse anti-HSP70 antibody(1:50),monoclonal rabbit anti-human antibody caspase-3(1:50)and SP kits were purchased from Wuhan Boshide Biological Engineering Limited.3.Immunohistochemical stainingSerial sections of wax block with 4urn of thickness.Choose one for regular H-E staining to determine the histological diagnosis and the degree of differentiation.Ope-rate strictly according to protocol to detect the expression of HSP70 and Caspase-3 in the cholangiocarcinoma.Color DAB.SP method was used to all specimens for immu-nohistochemical staining,before which a microwave oven was used for antigen re- trieval.Positive control was a known positive tissue slice,while PBS instead of first antigen as a negative control.4.Assessment of resultsThe standard takes yellow or brown granules as positive.Random observed 100 cells,HSP70 staining were divided into three grade:positive cell count＜30%,stain-ing 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.Caspase-3 staining were divided into 5 grade:positive cell count＜1%as 0,1%-24%as first grade,25%-49%as second grade,50%-74%as third,75%-100%as forth,set the zero,first and second grade low expression and the forth the high expression.5.Statistical analysisSSPS 12.0 was applied for data processing.Rank sum test,Pearson correlation analysis.P＜0.05 has a statistical significance.Results1.Expression of HSP70 and Caspase-3 in cholangiocarcinomaHSP70 and Caspase3 were mainly expressed in the cytoplasm,a few positive in nucleus.The worse the cholangiocarcinoma differentiated,the stronger the expression of HSP70(P＜0.05),the weaker the expression of Caspase-3(P＜0.05).2.The relationship between the expression of HSP70 and Caspase-3 and cholangiocarcinoma clinicopathological parametersThe expression intensity of HSP70 was significantly positive correlated with the differentiation degree of cholangiocarcinoma(r=0.251,p=0.039).The expression intensity of Caspase-3 was significantly negatively correlated with the differentiation degree of cholangiocarcinoma(r=-0.310,p=0.01).The expression of HSP70 and Caspase-3 had no relationship with age,gender,lymph node metastasis and TNM stages.HSP70 expression was negatively correlated with Caspase-3 in cholangiocarcinoma(r=-0.412,p=0.000).Conculsions1.The expression of HSP70 and Caspase-3 protein in cholangiocarcinoma has certain relationship with differentiation degree of cholangiocarcinoma.The expression intensity of HSP70 is significantly positive correlated with the differentiation degree of cholangiocarcinoma.The expression,intensity of Caspase-3 protein is significantly negative correlated with the differentiation degree of cholangiocarcinoma.2.The expression of HSP70 and Caspase-3 protein has no relationship with age, gender,lymph node metastasis and TNM stages in cholangiocarcinoma.3.HSP70 expression was negatively correlated with Caspase-3 in cholangioc-arcinoma.