Analysis Of Secreted Proteins In Oral CAFs And The Regulation Role Of CAFs In Hyperthermia-induced Apoptosis In Tca8113 Cells

[Objective] Analysis of secreted proteins in oral CAFs and the role of CAFs in hyperthermia-induced apoptosis in Tca8113 cells.[Methods] (1) Using primary culture method cultivate fibroblast of operating oral tongue squamous carcinoma tissue and oral mucousmembrane tissue. Immunocytochemistry was used to identify oral CAFs and fibroblast after purification. Celluar was observed by the microscope. (2)The third generation of oral CAFs and fibroblast was cultured conventionally for 72 hours, clear away the supernate, continuely cultured in serum-free media, harvested the cell supernate after 24 hours, proceed cytokines antibody microarray testing. The third generation of oral CAFs and fibroblast supernate was harvested, prepare conditioned medium on volume ration of 1/6,2/6,3/6,4/6, co-cultivate Tca8113 indirectly in conditioned medium. Livability of TCA8113 was detected after 24 hours. Immunocytochemistry was used to bal-2 and bax of Tca8113. The cell were treated at temperature of 43 degree Celsius in a heating waterbath for 80mins, continue cultured for 48 hours, harvested cells to analysis apoptosis rate by FCM with propidium iodide (PI) stain.[Result] (1) Oral CAFs and fibroblasts were obtained. Compared with the normal fibroblasts, oral CAFs grows faster and observe more cell overlap, squat spindle-shaped, nuclear single dye or multinuclear celluar gibbosity clear and irregular; a-SMA positive. (2) Protein-factor have significant differences which secreted by oral CAFs and fibroblast. CD40/TNFRSF (TNF 4.39017408 times) IL-17C (IL 6.55024497 times),MFRP (16.70024954 times),MIG (CF 19.22187517 times),Smad 4 (5.954984175 times) were significantly higher fibroblasts, CCL28/VIC(CF 0.0889778 times,GFR alpha-1 (growth factor 0.0754521 times) Neuregul(growth factor 0.0365995 times) were significantly down oral CAFs. (3) Fibroblast supernate of 3/6 volume indirectly co-cultivate Tca8113 influence on survival rate of cell. Expressions of bax protein in Tca8113 has no obviously influence to fibroblast supernate, expressions of bcl-2 protein in Tca8113 has no obviously influence to normal fibroblast supernate, but oral CAFs supernate obviously increased expressions of bcl-2 protein in Tca8113. (4) Normal fibroblast supernate has no obviously influence to hyperthermia-induced apoptosis in Tca8113 cells (P＞0.05); but oral CAFs supernate in volume ration of 2/6 obviously inhibited cell apoptosis of Tca8113 (P＜0.05)[Conclusion] (1) Oral CAFs and, normal fibroblast are different in growth characteristics and expressions of protein-factor, some protein-factor have obvious differences. (2) Oral CAFs decreased hyperthermia-induced apoptosis in Tca8113 cells by increasing expressions of bcl-2 protein in Tca8113.