Study On The Expression And Function Of FAP In The Eyelid Basal Cell Carcinoma And Benign Tumor Associated Fibroblasts

Objective: In order to separate and obtain purified eyelid basal cell carcinoma related fibroblasts(CAFs),eyelid benign tumors(intradermal nevus)associated fibroblasts(BAFs)and fibroblasts from normal eyelid skin(NFs)by primary cells culture.And to detect the difference in expression of fibroblast activating protein(FAP)in three different fibroblasts,and further explored the role and significance of FAP in eyelid basal cell carcinoma.Methods:(1)The primary CAFs,BAFs and NFs were obtained by tissue block wall culture,and there were separated and purified by trypsin differential digestion and repeated passage.(2)And the expression of vimentin(VIM),alpha smooth muscle actin(alpha-SMA),cytokeratin(CK)and FAP in these three fibroblasts was tested and identified by Cell Immunohistochemical staining technique.(3)Subsequently,MTT colorimetric assay was used to ascertained the growth and proliferation of the three fibroblasts and the growth curve was plotted.Finally,the difference in the expression of FAP mRNA and FAP protein in CAFs,BAFs and NFs cells were detected by real-time fluorescence quantitative PCR and Western blotting testing respectively.Results:(1)Successfully cultured and obtained the purified eyelids CAFs,BAFs and NFs.(2)CAFs like long spindle or spindle shape,proliferate faster,with fewer cytoplasmic processes and more granules in the cytoplasm.CAFs with different sizes and the arrangement are disorder,overlapping growth phenomenon is obvious,and the contact inhibition phenomenon is absent.As for BAFs,the growth rate between CAFs and NFs,the majority of them were fusiform,radiate arrangement,with overlapping growth phenomenon,to some extent,the contact inhibition phenomenon is lost.As far as NFs is concerned,they are long spindle shaped and radially arranged,there were few particles in the cytoplasm and the proliferation rate is slower than that of CAFs and BAFs.It has non-existent overlapping growth phenomenon,however,the contact inhibition phenomenon is significant.(3)The Cell Immunohistochemical staining results showed as follows: In CAFs group,the expression of VIM,a-SMA and FAP are positive,and the expression of CK is negative.BAFs with the positive expression of VIM and negative expression of CK,nevertheless,the expression of a-SMA and FAP is w+.In NFs group,merely with expression of VIM is positive,however,the others all negative.(4)The consequences of RT-PCR of FAP mRNA amplification in CAFs,BAFs and NFs respectively were 3.6715±0.2212,1.4810±0.0904,1.0340±0.0236,and the statistical difference is significant in the three groups in ONE-WAY ANOVA SNK test(F=103.676,P< 0.005).(5)The outcome of Western blot indicated that high expression of FAP protein in CAFs,low expression or no expression of BAFs,and no expression of NFs group.It is apparent that the expression level of FAP protein in the 3 groups is various.Conclusion: It has transformed in cell morphology,structure and secretion function of fibroblasts in the tumor microenvironment,and which play a crucial role in tumor occurrence and progression.There are significant differences in biological characteristics of CAFs,BAFs and NFs of eyelids in morphology,growth and proliferation,growth factor expression and etc.It is suggested that the microenvironment of the eyelid basal cell carcinoma has changed which could induce the changes of the biological characteristics and functions of NFs,and eventually switches to CAFs and BAFs.