| IntroductionTumor is a pathological progression consisting of many genes, steps and stages. As a ser/thy protein kinase,ILK is a upstream cross point of some tumor — associated factors. ILK can interact with GSK -3,PKB, E - cadherin et al,and has effect on formation, proliferation, invasion and metastasis of tumors. Now it has been a hot spot in cancer research field.The level of ILK in tumor tissues is noticeably higher than that in the normal tissues, and it keeps correlation to tumor malignancy, clinical stage, lymph metastasis and survival time. Now there have been little reports about the expression of ILK in lung cancer tissue and the relationship with E - cadherin, so we investigated the expression of ILK and E - cadherin in lung squamous carcinoma and adenocarcinoma cases with the neighboring noncancerous tissues,to analyse weather they were related to clinical pathological factors and survival time. Thus we can evaluate their role and significance in lung cancer metastasis and prognosis estimation.Materials and Methods1. Samples:44 lung squamous carcinomas and 32 lung adenocarcinomas with neighboring noncancerous tissue samples were obtained from patients who had surgery in the LiaoNing Province Tumor Hospital between 1980 and 2001, fixed in 4% paraformaldehyde,embedded in paraffin. 30 freshly(12 lung squamous carcino-mas12 lung adenocarcinomas and 6 non - cancerous tissues) were obtained from the First Affiliated hospital of China Medical University between September to November ,2003.2. ReagentThe antibody anti - human ILK mouse monoclonal antibody was provided by Upstate Biotechnology Lake Placid company( USA). The anti - human E - cad-herin mouse monoclonal antibody was provided by Santa Cruze company (USA) . The Ultro — sensitive S — P kit and DAB agent kit were bought from Fujian Maxin Biological Company. The goat - anti - mouse second antibody and NBT/ BCIP agent kit were bought from Huamei Biological Company. Protein marker was bought from Invitrogen company.3. Method3. 1 Immunohistochemistry: Detect the ILK expression by immunohisto — chemistry S - P method. Reasult determination: ( 1) ILK was a plasm staining protein. A: The staining intensity was scored as negative (0) , weak staining ( V ) , morderate or strong staining ( 3/ ) ; B: The number of cell stained was graded as follows ;0', less than 5% of tumor cells showed immunoreactivity; 1' ,6 -50% of tumor cells showed immunoreactivity ;2',50 - 80% of tumor cells showed immunoreactivity; over 80% of tumor cells showed immunoreactivity. Ax B , Grades 0-1 were regarded negative, grades 2—3 were regarded weak positive and grades 4-6 were regarded positive. In order to numerate simply,we defined 0 - 3 as negative and 4 - 6 as positive. ( 2) E - cadherin was a membrane staining protein, less than 60% of tumor cells showed negative, and over 60% of tumor cells showed positive.3.2 Western blot; Detect the expression of ILK and E - cadherin in lung cancer tissues and neighboring noncancerous tissues. Rapidly homogenize tissues (1 - 2g) in 5 volumes of lysis buffer centrifuge the homogenate (10, OOOrpm, 4X. ) for 60 minutes to pellet insoluble material. (DElectrophoresis; ?transfer proteins from gel to PVDF membrane;(D blocking;? incubation with primary antibody;?enzyme conjugate incubation;?substrate incubation.4. Statistical analysisAll the data were analyzed with SPSS for Windows 10.0 software. Postoper-ative survival periods were computed by the Kaplan - Meier method and compared by the Log - rank test. Hie Cox stepwise regression analysis was used to e-valuate the statistical strength of independent association between selected cova-riates and patient survival, P <0.1. Spearman test was to compare the clinico-pathological characteristics with the expressions of ELK and E - cadherin.Result1. Expression of ILK in lung squamous carcinomas N lung adenocarcinomas and normal tissuesILK was localized in cytoplasm. It showed a strong expression in many stro-mal cells including smooth muscle cells, fibroblasts and vascular endothelial cells,and it was predominantly positive in tumor cells. However,ILK was not detected in neighboring noncancerous tissues, except that weak expression in bronchus and alveolar epithelial cells occasionally. Western Blot Method showed the level of ILK in the tumor tissues is noticeably higher than that in the normal tissues.2. Expression of E -cadherin in lung squamous carcinomasNadenocarcinomas and normal tissuesE - cadherin expression was confined to the cell membrane in epithelial cells of bronchus and alveolus, while the tumor cells showed weak or negative staining. Western Blot Method showed the level of E - cadherin in the tumor tissues is noticeably lower than that in the normal tissues.3. Clinical Significance of ILK and E - cadherinIn lung squamous carcinoma , the expression of ILK was related to differentiation ( P <0.01) , clinical stage( P <0.01) and lymph metastasis( P <0.01) ; in adenocarcinoma ,its expression was related to clinical stage (P <0.01) Nmetastasis ( P < 0.01) , but was not related to the differentiation ( P = 0.352 ). The expression of E - cadherin was related to differentiation ( F^ < 0. 05, PAd < 0. 05 ) N clinical stage ( PgQ < 0.05; PAd < 0.01) and lymph metastasis( Pgg < 0.01; PAd <0.01). There was a significant inverse correlation between ILK and E -cadherin expression ( P <0.01). However neither of two proteins was related to...
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