Objective To compare the induction effects of direct treatment with low-temperature plasma(LTP)and treatment with plasma-activated medium(PAM)on immunogenic cell death(ICD)of melanoma cells.Methods After direct treatment of melanoma cell line B16F10 with LTP and treatment of it with PAM for 24 hours,cell viability was detected by MTT assay.Flow cytometry was used to detect cell apoptosis and the expression of calreticulin(CRT)on the cell surface.The adenosine triphosphate(ATP)content in the culture medium was detected by an ATP detection kit.The content of high-mobility group box 1(HMGB1)in the cell culture medium was detected by ELISA.B16F10 cells treated with LTP were co-cultured with immature dendritic cells(DC)DC2.4 cell line,and flow cytometry was used to detect DC surface molecules CD80 and CD86.Results Compared with the control group,both direct treatment and indirect treatment could lead to a decrease in the viability of B16F10 cells,an increase in the apoptosis rate,an increase in intracellular ROS,an increase in CRT expression,and an increase in the secretion of ATP and HMGB1(P＜0.05).At the same treatment time,the expression of CRT and the release of ATP in B16F10 cells directly treated with LTP were higher than those indirectly treated with PAM(P＜0.05).Compared with the DC2.4 group,the expression proportion of the DC cell maturation marker molecule CD80 was significantly increased in LTP-120s group,LTP-180s group,PAM-120s group,and PAM-180s group.The expression proportion of the DC cell maturation marker molecule CD86 was significantly increased in LTP-120s group,LTP-180s group,and PAM-180s group,and the difference was statistically significant(P＜0.05).Conclusion Both direct treatment with LTP and indirect treatment with PAM can induce ICD in melanoma cells.The direct treatment with LTP has a better induction effect.

Objective To compare the induction effects of direct treatment with low-temperature plasma(LTP)and treatment with plasma-activated medium(PAM)on immunogenic cell death(ICD)of melanoma cells.Methods After direct treatment of melanoma cell line B16F10 with LTP and treatment of it with PAM for 24 hours,cell viability was detected by MTT assay.Flow cytometry was used to detect cell apoptosis and the expression of calreticulin(CRT)on the cell surface.The adenosine triphosphate(ATP)content in the culture medium was detected by an ATP detection kit.The content of high-mobility group box 1(HMGB1)in the cell culture medium was detected by ELISA.B16F10 cells treated with LTP were co-cultured with immature dendritic cells(DC)DC2.4 cell line,and flow cytometry was used to detect DC surface molecules CD80 and CD86.Results Compared with the control group,both direct treatment and indirect treatment could lead to a decrease in the viability of B16F10 cells,an increase in the apoptosis rate,an increase in intracellular ROS,an increase in CRT expression,and an increase in the secretion of ATP and HMGB1(P＜0.05).At the same treatment time,the expression of CRT and the release of ATP in B16F10 cells directly treated with LTP were higher than those indirectly treated with PAM(P＜0.05).Compared with the DC2.4 group,the expression proportion of the DC cell maturation marker molecule CD80 was significantly increased in LTP-120s group,LTP-180s group,PAM-120s group,and PAM-180s group.The expression proportion of the DC cell maturation marker molecule CD86 was significantly increased in LTP-120s group,LTP-180s group,and PAM-180s group,and the difference was statistically significant(P＜0.05).Conclusion Both direct treatment with LTP and indirect treatment with PAM can induce ICD in melanoma cells.The direct treatment with LTP has a better induction effect.

Objective To explore the plasma-activated medium(PAM)produced by low temperature plasma(LTP)on the proliferation and angiogenesis of human umbilical vein endothelial cells(HUVECs)so as to provide theoretical basis for the future use of PAM to promote wound healing and inhibit tumor angiogenesis.Methods HUVECs were selected as the in vitro research model.The PAM-containing medium after LTP treatment for different time points(0 s,15 s,30 s,45 s,60 s,and 75 s)was used for intervention.The influence of PAM on HUVECs viability was assessed using the MTT assay and cell cycle analysis.The effects of PAM on angiogenesis were examined through angiogenesis experiments.Intracellular levels of reactive oxygen species(ROS)were measured using fluorescence probes.A melanoma mouse model was established,and CD31 expression was detected by immunohistochemistry.Results As the treatment time increased,the intracellular levels of ROS also elevated.PAM derived from LTP exhibited a bidirectional effect on angiogenesis in HUVECs.Compared to the control group(0 s),low-dose treatments(15 s and 30 s)enhanced HUVECs viability,while high-dose treatments(45 s,60 s,and 75 s)significantly decreased cell viability(P＜0.05).The proportion of HUVECs in the S phase was significantly increased in the PAM-15 s and PAM-30 s groups,but markedly decreased in the PAM-45 s,PAM-60 s,and PAM-75 s groups,with statistically significant differences(P＜0.05).The HUVECs tube formation ability was enhanced in the 15 s and 30 s PAM groups,but diminished in the PAM-45 s,PAM-60 s,and PAM-75 s groups,characterized by the decreased numbers of vascular nodes,intersections,meshes,and branching points(P＜0.05).After PAM treatment in the melanoma mouse model,the control group exhibited widespread distribution of CD31 in tumor tissue,while the PAM-5 min and PAM-10 min groups displayed reduced distribution of CD31.Conclusion Short-term exposure to PAM enhances HUVECs proliferation and angiogenesis,whereas prolonged exposure suppresses cell viability and inhibits angiogenesis.