Artocarpus Lingnanensis Lectin Stimulates Lymphocyte Proliferation And The Anti-Hepatocarcinoma Effect After Stimulation

OBJECTIVE:To study the effect of Artocarpus lingnanensis lectin（ALL）on the proliferation of lymphocytes and its subsets,and the anti-tumor effect of lymphocytes stimulated by ALL on hepatocellular carcinoma cells.It provides a new theoretical and experimental basis for clinical immunotherapy.METHODS:（1）Peripheral blood lymphocytes from healthy people were isolated by density gradient centrifugation.The concentrations of ALL,IL-2（interleukin,IL-2）and ALL combined with IL-2,which were most suitable for lymphocyte proliferation,were screened by CCK8 method and morphological observation,respectively.Apoptosis test were used to detect the effect of ALL combined with IL-2 on the activity of lymphocytes.Finally,flow cytometry was used to detect the changes of ALL on T cells and stem cell like memory Tcells(T_SCM).（2）Study on the killing effect of ALL-stimulated lymphocytes on SK-Hep1and MHCC97L by CCK8 method and morphological observation,and to find out the most suitable ratio of effector to target.The apoptosis of SK-Hep1 and MHCC97L hepatocellular carcinoma cells by ALL-stimulated lymphocytes by Hochest33258 staining,thereby further confirming the effective target ratio.Lactate dehydrogenase（LDH）assay and ELISA assay were used to detect the release of LDH and the secretion of perforin（PRF1）when ALL-stimulated lymphocytes were co-cultured with SK-Hep1 and MHCC97L hepatoma cells.Scratch method and Transwell method were used to detect the effect of ALL-stimulated lymphocytes and SK-Hep1 co-culture supernatant on SK-Hep1migration and invasion,so as to detect the indirect effect of ALL-stimulated lymphocytes on hepatocellular carcinoma cells.Finally,morphological method,CCK8 method,apoptosis method,scratch method and Transwell method were used to verify the effect of low concentration ALL on hepatocellular carcinoma cells.RESULTS:（1）The morphological observation showed that after stimulated by ALL,lymphocytes grew well and a large number of proliferating colonies appeared,and the effect of ALL at the concentration of 7.5μg/m L and 10μg/m L was better.The maximum proliferation rate of ALL with 7.5μg/m L,10μg/m L was（50.75±0.87）%and（50.31±4.46）%,respectively,the maximum proliferation rate was higher than（9.25±1.58）%in 2.5μg/m L group and（21.46±2.06）%in5μg/m L group（P<0.05）.IL-2 could also promote the proliferation of lymphocytes,but not colony growth.When the concentration of IL-2 was12.5ng/m L,the maximum proliferation rate was（21.88±1.05）%,which was significantly higher than that in the group below the concentration of 12.5ng/m L（P<0.05）.When stimulated by ALL combined with IL-2,the maximum proliferation rate was（68.56±4.05）%in 7.5μg/m L ALL+12.5ng/m L IL-2 group and（64.65±7.41）%in 10μg/m L ALL+12.5ng/m L IL-2 group.The proliferation effect of both groups was better than that of stimulation alone（P<0.05）,and the effects of the two groups were similar（P>0.05）.Therefore,the concentration of 7.5μg/m LALL+12.5ng/m L IL-2 can be selected to stimulate the proliferation of lymphocytes.The apoptotic activity test showed that the apoptosis rate in7.5μg/m L ALL+12.5ng/m L IL-2 group was the lowest（23.40±1.32）%,followed by 7.5μg/m L ALL group（27.53±2.44）%.The apoptosis rate of both groups was significantly（P<0.05）higher than that of non-stimulation group（39.73±4.98）%,so ALL can increase the activity of lymphocytes.By further detecting the effect of ALL on cell subsets,The positive rate of CD3 in the non-stimulation group was（55.8±0.45）%,while the positive rate of CD3 in the 7.5μg/m L ALL+12.5ng/m L IL-2 group was（61.35±1.35）%,which was higher than that in the non-stimulation group（P<0.05）.The positive rate of T_SCMin non-stimulation group was（1.42±0.0.31）%,and the positive rate of T_SCMstimulated by7.5μg/m L ALL+12.5 ng/m L IL-2 for 72h was（6.65±0.08）%,which was significantly higher than that in non-stimulation group（P<0.0001）.Therefore,ALL can promote the proliferation of T and T_SCMcells.（2）CCK8 experiment and morphological observation showed that the effect-target ratio of co-culture of ALL-stimulated lymphocytes with SK-Hep1and MHCC97L was E:T=10:1 and E:T=20:1,respectively,and could inhibit the proliferation of Hepatocellular carcinoma cells.There was significant difference between the control group and the control group（P<0.05）.Hochest33258staining showed that ALL-stimulated lymphocytes could also promote the apoptosis of SK-Hep1 and MHCC97L,and the apoptosis effect was better when the effector-target ratio was E:T=10:1 and E:T=20:1,respectively.LDH test showed that the co-culture of ALL-stimulated lymphocytes and SK-Hep1hepatocellular carcinoma cells could increase the activity of LDH in the supernatant of co-culture.The highest activity was 111.38±13.87 U/L,which was significantly higher than that in the control group（P<0.0001）.When the ALL-stimulated lymphocytes were co-cultured with MHCC97L hepatocellular carcinoma cells,the highest activity of LDH was 146.20±10.08 U/L,which was significantly higher than that in the control group（P<0.0001）.ELISA assay showed that ALL-stimulated lymphocytes were co-cultured with SK-Hep1 and MHCC97L liver cancer cells.It could make lymphocytes secrete a large amount of perforin（PRF1）.When co-cultured with SK-Hep1,the maximum secretion of PRF1 was 497.55±2.18 pg/m L,which was significantly higher than that in the control group（P<0.0001）.When co-cultured with MHCC97L,the maximum secretion of PRF1 was 344.79±12.85 pg/m L,which was significantly higher than that in the control group（P<0.001）.In addition,through scratch test,it was found that the migration of SK-Hep1 cells was significantly inhibited by the supernatant of ALL-stimulated lymphocytes co-cultured with SK-Hep1,and the migration area at 12h and 36h in the control group was lower than that in the control group（P<0.0001）.Transwell test showed that the number of transmembrane cells in the control group was 71.33±2.52,while the number of cells in the ALL-stimulated lymphocytes and SK-Hep1 co-culture group was27.00±2.64.the invasion of SK-Hep1 was significantly inhibited（P<0.0001）.However,the ALL of 5μg/m L,15μg/m L and 25μg/m L could not directly affect the proliferation,apoptosis,invasion and migration of SK-Hep1.CONCLUSION:（1）Low concentration of ALL combined with IL-2 can stimulate the rapid proliferation of lymphocytes and promote the proliferation of T and T_SCMcells.（2）When the effector-target ratio of co-culture of ALL-stimulated lymphocytes and hepatocellular carcinoma cells was E:T=10:1 and E:T=20:1,respectively,it could significantly inhibit the proliferation of hepatocellular carcinoma cells and promote their apoptosis.After co-culture of ALL-stimulated lymphocytes and hepatocellular carcinoma cells,lymphocytes secrete a large number of PRF1,to kill liver cancer cells and cause liver cancer cells to release LDH.The supernatant of ALL-stimulated lymphocytes co-cultured with Hepatocellular carcinoma cells can inhibit the invasion and migration of Hepatocellular carcinoma cells,while low concentration of ALL had no effect on the proliferation,apoptosis,invasion and migration of Hepatocellular carcinoma cells.