Expression And Significance Of Negative Co-stimulatory Molecule PD-L1 In Keloid

Objective Taking the negative costimulatory molecule PD-L1 as the target molecule and anti-epidemic targeted therapy as the breakthrough point,the immune escape effect and significance of keloid mediated by negative costimulatory molecule were discussed by means of histology,cell and molecular biology,zoology and so on.Method 1.Immunohistochemical(IHC)method was used to detect the expression of PD-L1,PD-1 and CD8 in normal skin,hypertrophic scar and keloid,and to anal yze the expression trend,characteristics and correlation in normal skin,hypertrophic scar and keloid.2.Small molecular interference(Small interfering RNA,si RNA)technique was adopted to silence the expression of PD-L1 in fibroblasts,and reverse transcriptase pol ymerase chain reaction(RT-PCR)was conducted to detect the effect of interfering with the expression of PD-L1 m RNA and COL-1 m RNA.The expression of PD-L1 and COL-1 protein in transfected fibroblasts was detected by Western Blot,the growth of keloid fibroblasts was detected by scratch test and CCK-8 method,and the apoptosis was detected by flo w cytometry.3.The keloid model of nude mice was established.16 days later,the animal model was verified.Local injection of three different doses of PD-1 blocking antibody was used for immunotherapy,and a blank control group was set up.The effect of PD-L1 and COL-1 m RNA expression after blocking was detected by reverse transcriptase pol ymerase chain reaction(RT-PCR).Sirius red staining was used to detect the changes of total collagen and COL-1 in the blocked specimens,and the blood of nude mice was taken to detect the toxicit y and side effects of different doses of antibodies on animal models.Results 1.The expression intensity of PD-L1,PD-1 and CD8 in normal skin,hypertrophic scar and keloid was keloid > hypertrophic scar > normal skin,the expression in keloid was statisticall y significant with P<0.05,and the expression of PD-L1 was positivel y correlated with CD8 in keloid.2.After silencing the expression of PD-L1 molecule in fibroblasts by si RNA,the proliferation of fibroblasts was inhibited and the invasive abilit y of fibroblasts was significantl y decreased.It was screened that the expression levels of PD-L1 and PD-L1 genes in human keloid fibroblasts were significantl y inhibited after si RNA-PD-L1-C1 transfection,and the inhibition rates we re 61.5% and55.5%,respectivel y.After transfection,the expression levels of PD-L1 and COL-1 proteins were significantl y suppressed.The inhibition rates were 58.9% and 58.7.6%,respectivel y.Scratch test showed that the migration ability of fibroblasts c omposed of si RNA-PD-L1-C1 decreased after transfection.CCK-8 results showed that the growth rate of scar fibroblasts in group C1 was significantl y lower than that in the control group(P < 0.05).The apoptosis rate of keloid fibroblasts in each group was detected by flow cytometry with26.63%?8.45%?7.72%?5.92%,respectivel y,and the apoptosis rate in C1 group was significantl y higher than that in the control group(P < 0.05).3.16 days after the establishment of the nude mouse model,there was no significant change in the volume and composition of keloid specimens,and different doses of PD-1 blocking antibody could block the local immunit y of the nude mice model,the total collagen and COL-1,PD-L1,COL-1 m RNA and protein expression decreased(P<0.05),but the effect of low concentration treatment dose was not good,the animal had obvious residual injury under high concentration treatment dose.Conclusion Overexpression of PD-L1,negative costimulatory molecule PD-L1 in keloid tissue can promote fibrosis,ef fectivel y block PD-L1/PD-1binding,reduce the synthesis of total collagen and COL-1,and effectivel y inhibit keloid proliferation.It provides a new target for inhibiting the proliferation of keloid and lays the groundwork for further research on immunotherapy of keloid.