Effect Of Smad7 Protein On Keratinocytes In Keloids

Objective(s):Previous studies on the pathogenesis of keloids have focused on fibroblasts,extracellular matrices,etc.There is increasing evidence that keratinocytes are also actively involved in the development of keloids,and their pathogenesis in keloids The role needs to be further explored.This study constructed Keloid keratinocyte(KK)with high expression of Smad7,detected the functional changes of KK,and explored the relationship between Smad7 protein and epithelial-mesenchymal transition and TGF-β signaling pathway between KK and keloid.Relationships provide a scientific basis for the development of new strategies and methods for the treatment of keloids.Methods:A male patient’s shoulder keloid was taken(with the consent of the patient),and the surgically removed tissue was used as a source of experimental specimens.KK was cultured in vitro under sterile conditions using cell culture.A lentivirus with high expression of Smad7 was constructed in vitro,and a lentivirus was produced using the corresponding empty vector.Cell lines were constructed using the infection enhancer Polybrene and grouped according to the cell line:KK group(normally isolated keloid keratinocytes),GFP group(empty vector lentivirus infection group)and mSmad7 group(high expression of Smad7 lentivirus infection)Group),the detection of Smad7 mRNA expression level by QPCR and the detection of Smad7 protein expression level by Western Blotting were used to evaluate the infection efficiency.Based on efficient infection and expression of Smad7:flow cytometry to detect cyclic changes,CCK-8 kit to detect cell proliferation,Transwell chamber to detect cell migration rate,Western Blotting and immunofluorescence double labeling to detect the role of Smad7 in related pathways.Results:The expression of Smad7 protein was detected by QPCR when the Smad7 lentivirus was highly expressed in KK to 72h.The mSmad7 group was significantly higher than the GFP group,P<0.01.The difference was statistically significant.The results of Western Blotting were the same as above.When the cell line was successfully established for 72 hours,the mSmad7 group was compared with the GFP group,and the cell cycle G0/G1 phase was significantly increased(P<0.01),and the G2/M phase was significantly decreased(P<0.01).The proliferation of the three cells was continuously detected at 1 day,2 days,3 days,4 days,5 days,6 days,and 7 days.The statistics of the GFP group were slowed down and statistically compared with the mSmad7 group on the 3rd day.significance.From the beginning to 24h,20,000 cells were added to each chamber,and the number of migrated cells in the GFP group was significantly decreased in the mSmad7 group by the cell count of 0.5%crystal violet migration membrane(P<0.01,statistically significant).Total protein was extracted by Western Blotting to detect EMT down-regulated protein Occludin,E-cadherin and up-regulated proteins Vimentin and N-cadherin,and TGF-P and Smad3 proteins associated with TGF-β pathway.The mSmad7 group compared GFP group Occludin and E-cadherin significantly.Rising;TGF-β,Smad3,Vimentin,and N-cadherin decreased significantly;P<0.01,the difference was statistically significant.The expression of N-cadherin and Occludin protein was detected by immunofluorescence double labeling,and the results were consistent with those of Western Blotting.Conclusion(s):I.Smad7 plays an important role in the formation of keloids by affecting the cycle,proliferation and migration of KK;2.Smad7 participates in epithelial-mesenchymal transition in keloid formation by affecting EMT-related proteins Occludin,E-cadherin,Vimentin and N-cadherin;3.Smad7 participates in the signal transduction of the keloid TGF-P pathway by affecting TGF-β and Smad3 proteins.