The Preliminary Study Of Stromal Fibroblasts In Different Stages Of Canceration In Cardiac Glandular Epithelia

Objective:The most important ingredient in tumor stromalmicroenvironment is activated fibroblasts,which called cancer-associatedfibroblasts(CAFs).They can secrete several growth factors, Chemokines,andregulate tumor development through multiple signaling pathways.Byobservating the expression of Alpha-SMA and CD34of stromal fibroblasts inthe cardiac normal, precancerosis, and cancer and culturing them,thisexperimental aimed to explore the features and the changes of the fibroblastsin cardiac different carcinogenesis stages and to provide a good platform forfurther research about the regulation of cardiac cancer-associated fiberblasts(CAFs) in cardiac epithelial carcinogenesis process.Methods:1Used the S-P methods of immuunohistochemistry to measure theexpression of alpha-SMA and CD34in100cases of cardiac tissue,including5groups: normal group, low level intraepithelial neoplasia,high levelintraepithelial neoplasia,early cancer and advanced cancer. SPSS13.0statisticssoftware was applied to analyze the results of experiment.2Used the organization block adherent method and collagenasedigestion method to culture cardiac normal fibroblasts,precancerousfibroblasts and cancer-associated fibroblasts.When cells crawled the entireculture bottle,used trypsin digestion and repeated attachment method to purifythe cells.3Used the inverted microscope to observe the cell morphology.4Used the MTT method to test the cell growth curves.5Producted cell crawling, then used Reye-Giemsa staining methodto observe cell coloring and used the cell Immunohistochemistry techniques tomeasure the expression of keratin,vimentin, desmin, α-SMA and CD34. 6Used the transmission electron microscope to observe the internalstructure of the cells.Results:1The positive expression rate of α-SMA in normal group, low levelintraepithelial neoplasia,high level intraepithelial neoplasia,early cancer andadvanced cancer respectively was (0/20),10%(2/20),45%(9/20),75%(15/20),90%(18/20). Positive cells were spindle-shaped, and Ribbon-likearound the lesion, nests of carcinoma were wrapped with rope-like positivecells in advanced cancer. Results showed a ascendant trend in α-SMAexpression of fibroblasts along with the disease development.The expressionwas not obviously correlated with patient’s gender and age.2The positive expression rate of CD34in normal group, low levelintraepithelial neoplasia,high level intraepithelial neoplasia,early cancer andadvanced cancer respectively was95.0%(19/20),85%(17/20),55%(11/20),30%(6/20),(0/20);in advanced cancer group stromal fibroblasts did not expressCD34, positive expression only in vascular endothelial cell.Results showed adescendant trend in CD34expression of fibroblasts along with the diseasedevelopment.The expression was not obviously correlated with patient’sgender and age.3There were three kinds of growth curves in different cells, whichwere "S" shape growth curves. As activated fibroblasts,the proliferation ofCAFs was faster than the other two, the proliferation ability of precancerousstromal fibroblasts was between NFs and CAFs.4Morphological results:①Cardiac normal fibroblasts: flat longfusiform, basically the same size, arranged in a certain direction, single-layercell growth and had density inhibition and contact inhibition. Cytoplasmicstaining evenly, single core, oval, located in cytoplasmic Central. Cytoplasmicorganelles within the more developed, rich in rough endoplasmic reticulum,Ribosome, Gower, and a small amount of microfilament.②Cardiaprecancerous stromal fibroblasts: morphological indeterminate, growth ofpartial overlap. Cytoplasmic staining evenly, increasing cell nuclear, some cell nuclear of irregular shape. Cytoplasmic organelles developed, you can seelarge areas of microfilaments and a small amount of attachment plaque.③Cardia carcinoma associated fibroblasts: shape irregular, arrangement disorder,non-directional, overlapping, density and contact inhibition were disappear.Cytoplasmic staining light, shape of cell nucleus were irregular, nucleolusvisible, with1-3kernel. Cytoplasmic extension, form a more ridged,cytoplasmic organelles developed, rough endoplasmic reticulum expansion,you can clear a lot of microfilaments and attachment plaque, and massivedistribution of microfilament and attachment plaque away from the nucleus.5Cell Immunohistochemistry results: The expression of three speciescells of keratin and desmin were negative, vimentin were positive, theexpression situation of α-SMA in normal fibroblasts was negative,precancerous fibroblasts was weak positive and carcinoma-associatedfibroblasts cardiac cancer was positive.The expression situation of CD34innormal fibroblasts was positive,precancerous stromal fibroblasts was weakpositive and carcinoma-associated fibroblasts was negative.Conclusions:1Immunohistochemical and cell culture results confirmed that thephenotypes of cardia fibroblasts changed, precancerous stromal fibroblastshad CAFs characteristics.2Through the organization block adherent method and collagenasedigestion method, successfully established a in vitro model of cardiacinterstitial fibroblasts about different stages of carcinogenesis,to provide aplatform for further research into fiber cell phenotype transformationmechanism and internal structure and biological characteristics.