| Immune escape mechanisms are important during the occurrence and development course of tumor, including complex contents related to tumor and host. The mechanisms of immune escape, related to tumor, are involved in two aspects. The first, some ways such as mutation or downregulation of tumor antigens and MHC genes, lack of costimulatory molecules result in unavailable immune recognization. Thus, the tumor can avoid anti-tumor immune reaction.The second, more importantly, the tumor can positively affect the proliferation, activation or the function of anti-tumor immune cells by the mechanisms of"Fas counterattack", secretion of immunosuppression molecules and so on.Ginsenoside and berberine are the composition of Panax ginseng (tonic) and Coptis chinensis (heat-clearing and detoxicating drug) respectively. Both have the obvious anti-tumor effects. The former anti-tumor researchs about Ginsenoside and berberine focused on inhibiting proliferation, inducing apoptosis, suppressing invasion and metastasis or inducing cell differentiation. But the researchs as to immune escape were less reported.Our research observed the Jurkat T cells'proliferation and apoptosis phenomenons by cocultured with hmuan lung carcinoma PG cells,the expressions of FasL and Fas molecules and the secretion of transforming growth factorβ1 (TGF-β1) and prostaglandin E2(PGE2) about PG cells. Further, we studied the intervention effects on the above-mentioned phenomenons and indexs by applying Ginsenoside and berberine. The aim of our research was to investigate the positive tumor immune escape mechanisms and the effects of Ginsenoside and berberine.Our research would be showed with details respectively.1. Firstly, we established the coculture system with PG cells and Jurkat T cells,and observed the growth and proliferation of Jurkat T cells.(1)By inverted microscope, we could observe the growth status of Jurkat T cells directly. After cocultured 24 hours, the Jurkat T cells cultured solely grew fascicularly, but the Jurkat T cells cocultured with PG cells (the cocultured group) grew dispersedly. The Gs group and Ber group'Jurkat T cells in the cocultured group, which respectively cocultured with PG cells treated with Ginsenoside or berberine, displayed less cell numbers. However, there was a more obvious difference between Gs group and Control group than between Ber group and Control group (2) The Jurkat T cells'proliferation was also evaluated by cells counting after typan blue staining. The results displayed that the cocultured group had the smaller cell numbers and had an obvious difference in statistics (p<0.05) after cocultured 6 hours and 24 hours. More ,the Gs group'proliferation was inhibited remarkably compared with the Control group after cocultured 6h or 24h with the very significant difference(p<0.01). Similar result also occurred in the Ber group after cocultured 24h. (3) The Jurkat T cells'proliferation was estimated after cultured with PG cells'culture supernatant by MTT reduction assay. According to the results, all of the groups, which grouped by the different volume ratio (supernatant/cell suspension) such as 2:1, 1:1 and 0.5:1, discovered a suppressed proliferation. A majority of the groups had a statistics difference compared with the Jurkat T cell group, and had a volume dependence. Nevertheless, there were no a statistics difference in different volume ratio groups.All results showed that PG cells could suppress the growth of Jurkat T cells, and the effect might be strengthened by treating PG cells with Ginsenoside or berberine. We could conclude that the fortified effect of Ginsenoside or berberine might be attributed to the influence on the PG cells surface molecules'expression.2. Apoptosis of the Jurkat T cells cocultured with PG cells was evaluated by morphology observation and flow cytometry (FCM) detection.(1) We could observe that the Jurakt T cell non-cocultured with PG cells had the following features by Giemsa staining, such as uniform globular appearance, bigger cell nucleus which almost occupied whole endochylema and showed violet or black. But the cocultured group showed inhomogenous appearance and cell nucleus'crazing and pycnosis.(2) Under the fluorescence microscope, the Jurkat T cells sololy cultured had the uniform round and dominant volume cell mucleus with green or flavo-green flourescence, had the jacinth endochylema or no flourescence. However, the cocultured groups presented inhomogenous nucleus (demi-moon, pyknosis etc.) with green flourescence and dominant endochylema with red flourescence, additionly appeared the weak flourescence nucleus.(3) To evaluate the apoptosis difference in all groups, we applied the FCM to apoptosis detection. The Jurkat T cells, which not cultured with PG cells, showed a lower natural apoptosis ratio(early apoptosis ratio was 5.57%, middle-terminal apoptosis ratio was 4.67%). Inversely, the cocultured groups displayed the higher apoptosis ratio. Their early apoptosis ratio reached over 10% and middle-terminal apoptosis ratio went up about 50%. Among the cocultured groups, the Gs group's early apoptosis ratio was 15.79% which had the statistica difference compared with the Control group's 11.40% and the Ber group's had not the difference in early apoptosis ratio. Both of the Gs and Ber group hardly showed the difference in middle-terminal apoptosis ratio. More attentively, the whole apoptosis ratio, including the early and the middle-terminal gave us a obvious statistica difference vs. the Control group.The part revealed that PG cells could induce the apoptosis of Jurkat T cells, further Ginsenoside and berberine might enhance PG cells'abilitities.3. To answer the question"why PG cells could induce the apoptosis of Jurkat T cells?",we detected the FasL and Fas expression of PG cells with immunocytochemical method. The results illustrated that (1) PG cells expressed the FasL molecules, mainly distributing in endochylema. According the photos, we could know that Ginsenoside and berberine could up-regulate the FasL expression.(2)Fas molecules were expressed on PG cells, which mainly distributed in endochylema or also on cell membrane. Ginsenoside and berberine might have no obvious effect on Fas expression by obvserving photos. (3)The following image analysis about FasL and Fas expression showed us the semi- quantitation results. Ginsenoside and berberine could up-regulate the FasL expression, which showed the same conclusion with naked eye observation, and had the difference in statisticis vs. the Control group. More, the Ber group could up-regulate FasL expression very obviously (p<0.01 vs. the control group). Ginsenoside also up-regulate Fas expression but berberine failed to affect it.The part results could tell us that PG cells expressed FasL and Fas molecules, Ginsenoside and berberine could influence their expression and these influences might increase the apoptosis induction of PG cells.4. Additionally, we also detected the concentration of transforming growth factorβ1 (TGF-β1) and the prostaglandin E2 (PGE2) . The results were as follows. Firstly, PG cells could produce TGF-β1 and PGE2. And 100μg/ml Ginsenoside could improve the secretion of TGF-β1 as well as 5μg/ml and 2.5μg/ml berberine. But Ginsenoside and berberine had no effect on the secretion of PGE2.This part results hinted that PG cells could secrete TGF-β1 and PGE2 and Ginsenoside and berberine could intervene in the former's secretion.To sum up, the experiments in vitro approved that lung carcinoma PG cells could supress Jurkat T cells growth effectively. The results might be related to Jurkat T cells growth supression and apoptosis induction. These effects were immediately prominent after PG cells'treatment with Ginsenoside and berberine which might result from up-regulate FasL and Fas expression and increase the secretion of TGF-β1. So we could also conclude that Ginsenoside and berberine either resisted tumor by up-regulating FasL and Fas etc.expression which were a major mechamism of anti-tumor as many drugs or developed a pathway of tumor immune escape. |
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