The Studies On Separation And Culture And Biologic Characteristics Of The Eyelid Basal Cell Carcinoma-associated Fibroblasts

Objective: To separate, culture, purify and identify the eyelid basal cell carcinoma associated fibroblasts(CAFs) and eyelid normal fibroblasts(NFs), and also to research their biological character differences in cellular morphology and growth and proliferation and expression of periostin(PN or POSTN).Methods: 1.Primary eyelid basal cell carcinoma associated fibroblasts(CAFs) and eyelid normal fibroblasts(NFs) were cultivated by tissue block adhering wall method and purified by subculturing cells with pancreatic enzyme differential digestion. 2.The morphology of CAFs and NFs was observed by inverted phase contrast microscope. CAFs and NFs were identified by immunocytochemical staining to detect cell specific protein expression.3.The cells were continuous cultured for 7 days, and the cells number was determined by MTT colorimetric method every day, from which the growth curve was drawed mean while. It was detected by enzyme-linked immunosorbent assay(ELISA) method that the content of PN from the CAFs and NFs cell culture supernatant. The expression of PN in CAFs and NFs was detected by immunofluorescence cytochemistry staining method.Results: 1.It was gotten that the purified eyelid basal cell carcinoma associated fibroblasts(CAFs) and eyelid normal fibroblasts(NFs). 2.Eyelid CAFs are long spindle, cytoplasmic processes are reduced, much particle can be seen in the cytoplasm, cell sizes are different, cells grow fast with disordered arrangement and overlap growth phenomenon. CAFs had high expression of vimentin, α-smooth muscle actin and fibroblast activation protein but no expression of cytokeratin, however NFs only had high expression of vimentin and no expression of cytokeratin, α-smooth muscle actin and fibroblast activation protein. 3.The growth and proliferation of CAFs are fast than NFs, and the doubling time of CAFs is shorter. The contents of PN which were from the CAFs and NFs cell culture supernatant detected by ELISA were(9.26±2.35)ng/ml and(2.57±0.41)ng/ml; The expression of PN in CAFs which was detected by immunofluorescence cytochemistry staining method was more stranger than NFs, and the difference of which was statistically significant(P<0.05).Conclusion: Compared with the eyelid normal fibroblasts, the eyelid basal cell carcinoma associated fibroblasts had many differences in biologic characteristics, such as morphology, expression of periostin and growth way so on, which showed that the growth micro-environment of the eyelid basal cell carcinoma cell had been changed, so we speculated that it may play an important role in the genesis and development of eyelid basal cell carcinoma.