The Effects Of Down-regulated ITGB5 Expression On The Function Of Keloid Fibroblasts

Object:Through knocking down the expression of ITGB5, observing the effect of ITGB5 on the function of keloid fibroblasts, such as proliferation, invasion and migration ability, analyzing the function of ITGB5 on the formation and development of keloid, to provide theoretical basis for the study of the pathogenesis of keloid and do some preliminary work for the clinical application of therapeutic target to treat keloid.Methods:1. Constructing lentiviral shRNA-Expression Vector targeting ITGB5.2. Fibroblasts were isolated from tissue specimens of Keloid. Cells from passages 3-5 were randomly divided into 3 groups(LV-KFb group, LV-NC group and KFb group). The cells of LV-KFb group and LV-NC group were infected by the lentivirus, stable cell lines were selected by flow cytometry.3. The expression of ITGB5 were detected by PCR and Western Blotting.4. The proliferation ability was identified by MTT, the invasion and migration ability were tested by transwell plates and the changes of cell cycle and apoptosis were tested by flow cytometry.5. The interactions between TGF-β and ITGB5 were detected with immune co-precipitation.Results:1. Lenti viral shRNA-Expression Vector targeting ITGB5 was constructed successfully and the effecting rate can reach to 80% when the value of MOI is 50.2. The expression quantity of ITGB5 mRNA and protein in LV-KFb group, LV-NC group and KFb group are 0.34±0.01,1.08±0.05,1.00±0.00 and 0.91±0.03, 0.93±0.02.0.28±0.07. Compared with LV-NC group and KFb group, the expression quantity of ITGB5 mRNA and protein in LV-KFb group decreased significantly(p<0.01).3. Compared with LV-NC group and KFb group, the proliferation rate, the number of cells at S-Phase and the migration and invasion rate decreased significantly in LV-KFb group(P<0.01).4. The positive bands of ITGB5 and TGF-β can be detected both in immune co-precipitation.Conclusion:1. Lentiviral shRNA-Expression Vector targeting ITGB5 can be constructed successfully;2. Stable cell strain of KFb effected by Lentiviral Vector can be acquired;3. Lentiviral shRNA-Expression Vector targeting ITGB5 infected KFb successfully and inhibited the expression of ITGB5 significantly;4. ITGB5 can promote the ability of cell division, proliferation, invasion and migration and inhibit apoptosis of KFb significantly;5. The function of ITGB5 can be effected by TGF-β.6. ITGB5 may be one of the therapeutic targets to treat keloid.