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Study On The Roles Of Hepatic Oval Cells, Local Microenvironment And Wnt5a Expression In The Formation And Metastasis Of Hepatocarcinoma

Posted on:2010-07-16Degree:DoctorType:Dissertation
Country:ChinaCandidate:J P ZhangFull Text:PDF
GTID:1114360302983777Subject:Surgery
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PartⅠStudy on the potentiality of hepatocellular carcinoma originating from hepatic oval cells by immunohistochemisty and its clinical significanceObjetive There are two theories on the origin of hepatocellular carcinoma and the two theories are still controversial.This experiment is to explore whether hepatocellular carcinoma(HCC)originates from hepatic oval cells and whether there is any relationship beween origin of HCC and clinicopathologic factors of HCC.Methods Specimens were obtained from 63 cases of hepatocellular carcinoma and its adjacent non-tumorous tissues,15 cases of liver cirrhotic tissues and 10 normal liver tissues,so they were divided into four groups.Cytokeratin(CK)8/18,CK7,CK19, C-KIT and AFP were used as surface marker of oval cells.The expressions of surface marker of oval cells were semiquantitiatively assessed by immunohistochemisty.Cell morphologic features and distributions labled by CK8/18,CK7 CK19,C-KIT and AFP were observed with optical microscope.Clinical and pathological data including gender,serum HBsAg status,HBcAb status,serum AFP,tumor size,number of intrahepatic lesions and so on were summuried.Results The positive rates labled by CK8/18 were 95.2%in HCC,96.8%in adjacent non-tumorous tissues,100%in cirrhotic tissues and 100%in normal tissues, respectively.There were no significant difference of the positive rates and intensity scores labled by CK8/18 among four groups(x~2=1.274,P>0.05;F=1.465,P>0.05). The positive rates labled by CK7 were 46.0%in HCC,58.7%in adjacent non-tumorous tissues of HCC,26.7%in cirrhotic tissues and negative in normal tissues,respectively.There were significant difference of the positive rates and intensity scores labled by CK7 among four groups(x~2=14.86,P<0.01;F=14.73, P<0.01).The positive rates and intensity scores labled by CK7 were significantly higher in HCC than those in cirrhotic and normal tissues(x~2=6.887,P<0.01; F=21.26,P<0.01),so were in adjacent non-tumorous tissues(x~2=13.133,P<0.01; F=21.26,P<0.01).The hepatic cells and tumor cells were no labled by CK19 in all specimens.There were significant difference of the positive rates and intensity scores labled by C-KIT among four groups(x~2=25.089,P<0.01).The positive rates and intensity scores labled by C-KIT were significantly higher in HCC,adjacent non-tumorous tissues and cirrhotic tissues than those in normal tissues.The intensity scores labled by C-KIT were significantly higher in adjacent non-tumorous tissues than those in HCC,cirrhotic and normal tissues(F=36.19,P<0.01).The positive rates and intensity scores labled by AFP were significantly higher in HCC than those in adjacent non-tumorous tissues,cirrhotic and normal tissues(x~2=5.567,P<0.05;F= 15.76,P<0.01).Hepatic oval cells labled by CK7,C-KIT and AFP were seen among normal or tumor cells,especially on the edge of tumor nodules.There is no correlation between CK7 positive expression in HCC and all involed clinicopathologic factors(P>0.05).C-KIT positive expression was associated with the following clinicopathologic factors,such as serum AFP,tumor metastasis,cancer cells ES grading and TNM staging(P<0.01),but not with the others(P>0.05).AFP positive expression in HCC was associated with the serum AFP,tumor metastasis,cancer cells ES grading,TNM staging and postoperative survival periods of 2 years(P<0.01),but not with the others(P>0.05).Conclusions The oval cells labled by CK7,C-KIT and AFP take part in the occurrence of HCC and may be one of the original cells for HCC.The oval cells labled by CK7,C-KIT and AFP represent various stem subpopulations and play different roles for repairment of chronic hepatitic injury and carcinogenesis.Expressions of stem cell phenotypic markers in HCC and paracancerous tissue represent a variety of clinical significance.Expressions of CK7 and C-KIT in adjacent non-tumorous tissues is related to the heterogeneity of hepatic oval cells,proliferation and tissue repairment.Expressions of C-KIT and AFP in HCC indicate malignant transformation of oval cells.PartⅡChanges of Tenascin and MVD-CD105 in hepatocellular cancer tissues and their correlations with metastasisObjective To study the expression of tenascin in hepatocellular carcinoma(HCC) and to investigate the roles of tenascin expression in microvessel angiogenesis,invasion, metastasis and prognosis of HCC.To evaluate the expression and distribution of CD105 in HCC and adjacent non-tumorous tissues,and then to discuss the specificity of CD 105 labling microvessel density(MVD) and its clinical significance.Methods Formalin fixed,paraffin wax embedded specimens from 63 patients with HCC were stained with anti-tenascin and anti-CD105 monoclonal antibody,So were done by adjacent non-tumorous specimens from the same patients.The correlation was analysed between expression of tenascin and clinicopathological features as well as microvessel angiogenesis.The correlation was analysed between the expression and distribution of MVD-CD 105 in HCC and clinicopathological features,so was also analysed in adjacent non-tumorous tissues.Results Tenascin immunoreactivity was seen in 65.1%specimens of HCC,69.8%of adjacent non-tumorous,20.0%of liver cirrhosis and riegative of normal liver specimens.Positivity and intensity of tenascin expressions in specimens of HCC and adjacent non-tumorous tissues were higher than those in specimens of cirrhotic and normal liver(x~2=27.67,P<0.01;F=12.82,P<0.01).Tenascin expression was associated with E-S histological grading,tumour numbers,capsule invasion,tumour metastasis and microvessel angiogenesis.CD105 immunoreactivity was seen in all specimens of HCC and adjacent non-tumorous tissues.Immunoreactivity intensity was higher in adjacent non-tumorous tissues than that in HCC(x~2=9.184,P<0.01). The mean scores of MVD-CD105 were higher in adjacent non-tumorous tissues than that in HCC.(81.62±19.86 vs 58.37±21.45;t=2.438,P<0.05).The levels of expression and distribution of MVD-CD105 in HCC were associated with tumor metastasis and TNM staging(P<0.01),but not with gendar,age,HBsAg status,AFP level,tumor size,tumor number,capsule infiltration,E-S histological grading,liver function,cirrhosis and survival periods of 2 years(P>0.05).The levels of expression and distribution of MVD-CD105 in adjacent non-tumorous tissues were associated with TNM staging and survival periods of 2 years(P<0.01),but not with gendar,age, HBsAg status,AFP level,tumor size,tumor number,capsule infiltration,E-S histological grading,liver function,cirrhosis and tumor metastasis(P>0.05).Conclusions The up-regulation of tenascin expression may play an important role in invasion and metastasis of HCC and lead to poor prognosis.The superiority of MVD labled with CD 105 is not reflected compared with MVD labled with CD34,The latter is pan-endothelial cell maker that reacts not only with prolifering vessles but also with established vessles in the tumor.The higher level of expression and distribution of MVD-CD105 in HCC means that the local tumor metastasis and progress.CD105 can not be serve as an appropriate targeting for antiangenesis therapy in HCC,but the study on MVD-CD105 in HCC may guide the comprehensive treatment postoperatively.PartⅢThe roles of Wnt5a in hepatocellular carcinoma development, invasion and metastasisObjective To detect the expression of Wnt5a at the levels of transcription and translation and to evaluate the roles of Wnt5a expression in microvessel angiogenesis, invasion,metastasis and prognosis of HCC.Methods The level of Wnt5a mRNA expression in HCC and adjacent non-tumorous tissues of 28 cases was detected by real-time fluorescence quantitative RT-PCR.The level of Wnt5a protein expression was detected with the same specimens by immunohistochemistry,so was done the level of expression and distribution of MVD-CD105.The correlation was analysed between the expression level of Wnt5a mRNA,Wnt5a protein and MVD-CD105 in HCC and clinicopathological features.Results Wnt5a mRNA transcription was seen in all specimens harvested from cancerous and adjacent non-tumorous tissues of 28 cases with HCC and normal liver tissues of 10 cases.The mean expression level is 0.78±0.15 in HCC,0.81±0.17 in adjacent non-tumorous tissues and 0.41±0.11 in normal liver tissues.The expression levels were significantly higher in HCC and adjacent non-tumorous tissues than that in normal liver tissues(F=6.958,P<0.001).There was no significant difference of Wnt5a mRNA transcription between cancerous and adjacent non-tumorous tissues (t=1.16,P=0.227).Wnt5a immunoreactivity was 32.1%in specimens of HCC, 89.3%in the adjacent non-tumor and 30.0%in normal liver.Wnt5a immunoreactivity in adjacent non-tumorous tissues were significantly higher than that in HCC and normal liver tissues(x~2=21.808,P<0.001).There were no significant differences of the mean scores of MVD-CD105 between Wnt5a protein expression higher and lower specimens.(46.51±15.38 vs 38.24±8.32;t=1.132,P>0.05).The level of Wnt5a mRNA transcription in HCC was associated with serum AFP level,E-S histological grading and proliferation of hepatic oval cells(P<0.05),but not with gendar,age,HBsAg status,tumor size,tumor numbers,capsule infiltration,tumor metastasis,liver function,cirrhosis and TNM staging(P>0.05).The level of Wnt5a protein in HCC was associated with serum AFP level,E-S histological grading and TNM staging(P<0.05),but not with gendar,age,HBsAg status,tumor size,tumor numbers,capsule infiltration,tumor metastasis,liver function,cirrhosis and immunoreactivity of C-KIT(P>0.05).Conclusions It is not incompatible that Wnt5a mRNA is increased transcription expression and Wnt5a protein is reduced expression in HCC.Increased transcription expression of Wnt5a mRNA can regulate cell growth,migration,differentiation and transformation bidirectionally and play a tumor suppressor role,which embodys one kinds of repairing mechanisms of preserving cells normal morphology and function. The reduced or lost expression of Wnt5a protein in HCC should be a late event during development of HCC,which implicates tumor invasion and metastasis.The expression of Wnt5a protein and the distribution of MVD-CD105 in HCC are two independent cliniclpathological factors associated with TNM staging.
Keywords/Search Tags:HCC, hepatic stem/oval cells, cytokeratin, immunophenotype, immunohistochemisty, hepatocellular carcinoma, tenascin, microvessle density, CD105, Wnt5a, RT-PCR immunohistochemisty
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