| Objective: 1. To investigate the effects of novel synthetic immunoregulator CH1b、CH2b on the proliferation and function of i NKT(invariant nature killer T)cells in active RA patients and healthy control in vitro,and to compare the different effects between active RA patients and healthy control.2.To investigate the mechanism of immunoregulator CH1b、CH2b in activation and molecule recognition to i NKT cells,to explore the recognition manner and signal pathway of i NKT cells stimulated by CH1b、CH2b,that is to explore the effects of immunostimulants on key proteins and signal transduction pathway in RA relying on i NKT cells.It will be significant to provide the relationship between i NKT and RA,then supply an new strategy for treatment of RA.Method: ① In this study, i NKT cells in healthy adults and active RA patients were regarded as subjects.First of all,blood samples were collected from active RA patients and healthy control,and peripheral blood mononuclear cells(PBMCs)were isolated.Then,PBMCs were cultured with stimulation of α-Galcer and IL-2 in vitro,i NKT cells expanded were then separated by using magnetic-activated cell sorting(MACS)method with i NKT isolation kit.At last,i NKT cells were incubated with CH1b、CH2b,for proliferational and functional analysis respectively.The effects on the proliferation of i NKT cells were analyzed by using MTT assay;MILLIPLEX MAP Human Cytokine/Chemokine kit was used to evaluate the secretion of IFN-γ and IL-4 in culture media; The expressions of IFN-γ m RNA and IL-4 m RNA in i NKT cells were analyzed by RT-PCR;The effects on the cytotoxicity to K562 cells of i NKT cells were analyzed also by using MTT assay.②In this study, i NKT cells in healthy adults were regarded as subjects.First of all,blood samples were collected from healthy control,and peripheral blood mononuclear cells(PBMCs)were isolated.PBMCs were cultured with stimulation of α-Galcer and IL-2 in vitro,i NKT cells were separated by using magnetic-activated cell sorting(MACS)method with i NKT isolation kit.Then,i NKT cells were incubated with CH1b、CH2b and anti-CD1 d groups to CH1b、CH2b,for proliferational and functional analysis respectively.The effects on the proliferation of i NKT cells were analyzed by using MTT assay; MILLIPLEX MAP Human Cytokine/Chemokine kit was used to evaluate the secretion of IFN-γ and IL-4 in culture media; The expressions of IFN-γ m RNA and IL-4 m RNA in i NKT cells were analyzed by RT-PCR;The effects on the cytotoxicity to K562 cells of i NKT cells were analyzed also by using MTT assay.At last,the expression of T-bet and GATA3 in i NKT cells were analyzed by Western-Blot,to ensure the recognition manner and signal pathway of i NKT stimulated by CH1b、CH2b.Result: 1. The proliferation of i NKT cells in two groups of IL-2、CH1b、CH2b and control were not obvious(P>0.05).Compare with control respectively,the proliferation of i NKT cells of IL-2+α-Galcer were much higher(P<0.05).Compare with IL-2+α-Galcer respectively,the proliferation of i NKT cells in healthy control and active RA patients of IL-2+CH1b 、IL-2+CH2b were much higher(P<0.05). However, the proliferation of i NKT cells in active RA patients were much lower than healthy control respectively(P<0.05);Compare with control respectively,α-Galcer could significantly promote the secretion of IFN-γ in i NKT cultured(P<0.05), CH1 b 、CH2b markedly promote the secretion of IL-4 and decrease the ratio of IFN-γ/IL-4(P<0.05),but there were no obvious difference on secretion of IFN-γbetween control and CH1b、CH2b(P>0.05).The secretion of IFN-γ and IL-4 in active RA patients were much lower than healthy adults,but were much higher than control in patients(P<0.05);Compare with control,expressions of IFN-γ m RNA were much higher inα-Galcer,expressions of IL-4 m RNA were much higher in CH1b、CH2b(P<0.05),but there were no obvious difference on expressions of IFN-γ m RNA between control and CH1b、CH2b(P>0.05).More importantly,CH1b、CH2b could also markedly enhance the cytotoxicity of i NKT cells,but the cytotoxicity of i NKT in active RA patients were much lower than healthy adults(P<0.05).2.The i NKT cell proliferation,the secretion of IL-4,the expressions of IL-4 m RNA and cytotoxicity of anti-CD1 d groups were much lower than CH groups correspondingly,there were no obvious difference between anti-CD1 d and control.Anti-CD1 d decrease the function of CH1b、CH2b to stimulate the i NKT proliferation,to promote the secretion of IL-4,to decrease the ratio of IFN-γ/IL-4,to elevate expressions of IL-4 m RNA and to enhance the cytotoxicity of i NKT cells;What’s more,there were statistical significance on expressions of GATA3 not T-bet between CH1b、CH2b and control.Conclusion: 1.There were not obvious proliferation of i NKT cells in two groups of IL-2、CH1b、CH2b and control alone, CH1b、CH2b alone could not stimulate the proliferation of i NKT cells in healthy adults and active RA patients.CH1b、CH2b were found to significantly stimulate the proliferation of i NKT cells in healthy adults and active RA patients induced by IL-2 in vitro,there were profound deficiency on i NKT cells proliferation and activity in active RA patients. Immunostimulator CH1b、CH2b could promote the production of Th2 1ike cytokines by i NKT cells in active RA patients and healthy control, and induce differentiation of Th0 to Th2 cells. CH1 b 、 CH2 b could possibly regulate immunity disorder and the excessive inflammatory(CH2b was better).Immunoregulator CH1b、CH2b could promote the function of i NKT cells by elevate the expression of IL-4 m RNA, regulate Th1/Th2 imbalance and suppress excessive inflammatory in active RA patients(CH2b was better).Moreover,CH1b、CH2b could significantly enhance the cytotoxieity of i NKT cells,but the cytotoxicity of i NKT in active RA patients were much lower than healthy adults.There were profound deficiency on i NKT cells function in active RA patients.2.The i NKT cell proliferation,the secretion of IL-4,the expressions of IL-4 m RNA and cytotoxicity of anti-CD1 d groups were much lower than the CH groups correspondingly,there were no obvious difference between anti-CD1 d and control.So that,CH1 b and CH2 b were likely recognized by relying on TCR-CD1 d dependent manner; There were statistical significance on expressions of GATA3 not T-bet between CH1b、CH2b and control. In healthy adults,CH1 b and CH2 b were likely recognized by activating and differentiating i NKT cells by GATA-3 pathway,modulating the immune function by promoting production of IL-4(Th2 trend).Base on these capabilities, the agonist of i NKT cells- CH1 b and CH2 b will be proposed as the potential agent with immunotherapeutic capabilities in RA targeting to i NKT cells. |
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