Toll样受体信号通路在调控树突状细胞成熟中的作用研究

Study on the effect of Toll like receptor signaling pathway on the regulation of dendritic cell maturation

目的探讨TLR信号通路在调控树突状细胞(DC)熟过程中的作用。方法用密度梯度离心法从人外周富集血中分离单个核细胞(PBMC),以GM-CSF和IL-4体外诱导成未成熟DC(imDC),用电穿孔法把A549的总RNA转染入imDC,分别用传统细胞因子鸡尾酒(IL-1β、IL-6、TNF-α、PGE2)和改良细胞因子鸡尾酒(IL-1β、IL-6、TNF-α、PGE2、polyⅠ∶C、CpG ODN)刺激DC的成熟。实验分为4组,对照组:GM-CSF、IL-4;传统组:传统细胞因子鸡尾酒;改良1组:IL-1β、IL-6、TNF-α、polyⅠ∶C、CpG ODN);改良2组:IL-1β、IL-6、TNF-α、PGE2、polyⅠ∶C、CpG ODN。用RTPCR法检测RNA的转染效率,流式细胞术检测DC和DC致敏T细胞的表面标志,ELISA法检测IL-12的分泌水平,MTT法检测T细胞的增殖。结果 1)RT-PCR检测:电穿孔法能使A549的总RNA成功转入DC,并使DC表达组织多肽特异性抗原(TPS)、癌胚抗原(CEA)和细胞角蛋白19片段(Cyfra21-1)等肿瘤抗原。2)流式检测:CD83各组表达率分别为(15.17±1.61)%、(64.33±7.58)%、(60.26±4.18)%和(82.89±3.06)%,CD1a表达率分别为(25.74±6.06)%、(69.37±8.16)%、(61.66±5.82)%和(86.07±5.63)%(P<0.05),各组CD8+T细胞比例分别为(36.43±3.15)%、(61.91±2.33)%、(54.81±2.55)%和(66.78±2.74)%(P<0.05)。3)各组IL-12的分泌水平分别为(55.86±14.07)、(1 243.49±57.57)、(744±49.51)和(2 385±128.98)pg/mL(P<0.05)。4)各组刺激指数分别为11.85±2.61、54.14±3.81、47.57±3.14和64.43±5.88,阳性对照组为135.29±5.06(P<0.05)。结论TLR配体polyⅠ∶C和CpG ODN可以促进DC的分化成熟。

Objective To explore the effect of TLR signaling pathway in the process of DC maturation. Methods Peripheral blood mononuclear cells （PBMC） were isolated using density gradient centrifugation. Immature dendritic cells （imDC ） were induced by GM-CSF and IL-4 in vitro and were transfected with the total RNA extracted from A549 cells into imDC using electroporation. And then the imDC with A549-RNA transfacted were stimulated with traditional cocktail of cytokines （ IL- 1β, IL-6, TNF-α, PGE2 ） and modified cocktail of cytokines （ IL-1β, IL-6, TNF-α, PGE2, poly I ： C, CpG ODN ）, respectively. The experiments were divided into four groups, control group：GM-CSF, IL-4;traditional group：traditional cocktail of cytokines ; modified group 1 ： IL-1β, IL-6, TNF-α, poly I ： C, CpG ODN ; modified group 2 ： IL-1β, IL-6, TNF-α, PGE2, poly I ： C, CpG ODN. RNA transfection efficiency was detected by RT-PCR, surface markers of the DC and DC sensitized T cell were detected by flow cytometry,T cell proliferation was judged by MTY, the secretion of IL-12 were detected by ELISA. Results 1 ）RT-PCR showed that the A549-RNA were transfected into the DC successfully by the method of electroporation, and these DC could express three tumor antigens, including tissue polypeptide specific antigen （TPS）, carcino-embryonic antigen （CEA） and cytokeratin 19 fragment （Cyfra21-1）. 2） Flow showed that CD83 expression rates in the above groups were （15.17 ±1.61） % ,（64. 33 ±7.58）% ,（60.26 ±4.18）% and （82.89±3.06）% ,CDla expression rates were （25.74± 6.06）% ,（69.37 ±8.16）% ,（61.66 ±5.82）% and （86.07 ±5.63）% ,the proportion of CD8^＋T cells were （36.43 ±3. 15）% ,（61.91 ±2.33）% ,（54.81 ±2.55）% and （66.78 ±2.74）% ,all these differenees were statistically significant （P 〈 0.05 ）. 3 ） The secretion of IL- 12 in the above groups were （55.86 ±14.07 ） pg/mL, （ 1 243.49 ±57.57 ） pg/mL, （744±49.51 ） pg/ml and （ 2 385 ±128.98 ） pg/mL（P 〈 0. 05 ）. 4 ） Stimulation index in the above four groups and positive control group were 11.85 ± 2.61,54.14 ± 3.81,47.57 ± 3.14,64.43 ±5.88 and 135.29 ±5.06 （P 〈 0. 05 ）. Conclusion TLR ligand poly I ： C and CpG ODN can optimize the maturation of DC.