Researches On The Function Of Severe Acne Susceptible Gene Ddb2 In Han Nationality

Objective: To investigate the effect of DDB2 gene on the proliferation and apoptosis of human HaCaT cells and the related target genes and signal pathways.Methods:1 Using ELISA to detect serum DDB2 concentration of severe acne pateints and healty control.2 Construct the overexpression lentiviral vector of DDB2 gene and three interference vectors, including PGMLV-SC5-DDB2-Shl, PGMLV-SC5-DDB2-Sh2,PGMLV-SC5-DDB2-Sh3 and PGMLV-PA6-DDB2. Using the 293T cell packaging to produce overexpression lentivirus vector and interference vectors. Then human HaCaT cells were infected by those lentivirus, and using puromycin to screen cells,which led to the establishment of stable infectious cell lines.3 Cell proliferation was detected by Cell Counting Kit-8 (CCK-8) and EdU, and the cell apoptosis was detected by flow cytometry before and after infection.4 The possible target gene NF- κ B of DDB2 was confirmed by immunofluorescence and Western blot at the cellular level.Results:1 Compared with healty control, the serum DDB2 concentration of severe acne patients was decreased (P<0.05).2 Western blot showed that DDB2 gene could be overexpressed in HaCaT cells which were infected by overexpression lentiviral vector. And the expression of DDB2 gene was down-regulated in HaCaT cells which were infected by interference vectors.3 The experiment was divided into three groups: NC group, DDB2 overexpression group and DDB2 interference group.3.1 CCK8 method was used to detect the proliferation of HaCaT cells in 12h, 24h,36h, 48h, 72h. The results showed that the proliferative activity of HaCaT cells infected with overexpression lentivirus vectors was decreased (P<0.05),and the proliferative activity of HaCaT cells infected with interference lentivirus vectors was significantly increased compared with the NC group (P <0.05).3.2 EdU test showed that the proliferative activity of HaCaT cells infected with overexpression lentivirus vectors was decreased (P<0.01), and the proliferative activity of HaCaT cells infected with interference lentivirus vectors was significantly increased compared with the NC group (P <0.01).3.3 Flow cytometry was used to detect the apoptosis of HaCaT cells after 24 hours of inoculation. The apoptotic rate of DDB2 overexpression group was significantly increased compared with the NC group.4 Compared with NC group, the expression of NF-κB / p65 protein in HaCaT cells infected with overexpression lentivirus vectors was decreased, and the expression of NF-κB / p65 protein in HaCaT cells infected with interference lentivirus vectors was significantly increased. Compared with NC group, the expression of IκBα in HaCaT cells infected with overexpression lentivirus vectors was increased, and the expression of IκBα in in HaCaT cells infected with interference lentivirus vectors was decreased.5 The expression of NF-κB / p65 in HaCaT cells infected with overexpression lentivirus vectors was decreased compared with the NC group, and the expression of NF-κB / p65 in HaCaT cells was significantly decreased in DDB2 overexpression group.Conclusion:1 Susceptible Gene DDB2 was down-regulated in the serum of severe acne patient.2 HaCaT cell lines that overexpress of DDB2 gene and HaCaT cell line of which DDB2 gene was down-regulated were successfully established.3 DDB2 may be involved in the pathogenesis of acne by regulating the proliferation and apoptosis of HaCaT cells. In addition, DDB2 may target NF-κB signaling pathway,which participates in the inflammatory reaction of acne and promotes the occurrence and development of acne. Overall, it can provide new insights into the pathogenesis diagnosis and treatment of severe acne.