尿酸辅助树突状细胞诱导抗乙肝病毒免疫效应的实验研究

发布时间：2018-05-19 00:32

本文选题：尿酸 + 树突状细胞　；参考：《华中科技大学》2007年博士论文

【摘要】： 目的: 研究尿酸(uric acid, UA)联合HBsAg负载的树突状细胞疫苗接种小鼠后产生的抗乙肝病毒(HBV)免疫效应,包括HBsAg特异性细胞毒性T细胞(CTL)杀伤效应,T淋巴细胞增殖反应,Th1/Th2型细胞因子的分泌等。并对尿酸促进DC成熟与功能提高的机理进行探讨。方法: 1.体外分离小鼠骨髓细胞,使用rmGM-CSF、rmIL-4诱导分化为DC,以UA或LPS刺激其成熟。分不同剂量尿酸组(70μg/ml、100μg/ml、200μg/ml、400μg/ml),LPS组、RMPI-1640培养基对照组。用流式细胞仪技术检测DC细胞表面分子CD11c、CD83、CD86、IA/IE的表达,用MTT法检测DC刺激同基因小鼠T淋巴细胞的增殖反应;ELISA法测定DC上清IL-12p70的分泌水平。 2.尿酸体外刺激未成熟DC 48小时,提取DC总RNA。RT-PCR方法检测TLR2 mRNA、TLR3 mRNA、TLR4 mRNA、IL-12p70 mRNA等的表达。 3.使用尿酸或联合抑制剂( p38 MAPK抑制剂SB203580、ERK1/2抑制剂PD98059、JNK抑制剂SP600125、NF-κB抑制剂PDTC )体外刺激未成熟DC。在0min、15 min、30 min、45 min时,分别提取DC细胞总蛋白与核蛋白。免疫印迹方法检测DC p- p38、p-ERK1/2、p-JNK、NF-κB p65表达量。分别使用SB203580、PD98059、SP600125、PDTC与尿酸联合刺激DC 48h后,收集细胞,用流式细胞仪技术检测DC细胞表面分子CD83、CD86、IA/IE的表达;收集DC培养上清,ELISA法测定IL-12p70的水平。 4.DC体外负载HBsAg,联合尿酸接种正常BALB/c小鼠。尾静脉注射方式接种,DC接种数量为1×106/每只小鼠,每周接种一次,共两次。分高、中、低剂量(分别为400μg、200μg、100μg)尿酸联合HBsAg-DC组、负载或未负载HBsAg的DC单独接种组、尿酸单独接种组(200μg)及PBS对照组。同时以负载HBS28-39的DC单独或联合尿酸免疫小鼠,作为平行对照观察组。一周和两周时,以体内荧光测定CTL活性方法,用流式细胞仪检测特异性CTL活性;免疫两周时,取小鼠脾T淋巴细胞,CFSE染色,体外以HBsAg或HBS28-39刺激培养72h后,流式细胞仪测定其增殖反应;取小鼠脾淋巴细胞体外HBsAg或HBS28-39刺激培养72h后,以ELISA法测定细胞上清IL-4和IFN-γ的分泌水平。结果: 1.通过鉴定体外成功诱导培养出DC。尿酸浓度为400μg/ml、200μg/ml、100μg/ml时能增强DC细胞表面CD83、CD86、IA/IE分子表达率;提高IL-12p70分泌水平(与阴性对照组相比,P0.05);尿酸能增强DC刺激T细胞增殖能力(与阴性对照组相比,P值均小于0.05);尿酸浓度为70μg/ml时,细胞表面分子表达、IL-12p70分泌水平、刺激T细胞增殖作用与阴性对照组比较均无明显差别(P0.05)。UA促进DC成熟的能力呈尿酸剂量依赖性增强。 2. RTPCR结果示尿酸组与培养基对照组相比,TLRs mRNA表达出现明显的变化。TLR2 mRNA、TLR3 mRNA、TLR4 mRNA表达下降(P0.05),以TLR4 mRNA下降最明显;IL-12p70表达显著增高(P0.05);与LPS组相比,TLRs mRNA与IL-12p70 mRNA表达水平相似,无统计学差异,P0.05。 3.(1)免疫印迹示,尿酸刺激后,15min时,p- p38、p-ERK1/2、p-JNK、NF-κB表达量明显增加,30min时达到最大值,45min时则开始下降,与DMSO对照组相比,P值均小于0.05。使用相应抑制剂后,相关蛋白表达表不能测出。LPS组上述分子在45min时仍大量表达。 (2)与未用相应抑制剂相比,使用SB203580、SP600125、PDTC等抑制剂预处理后,CD83、CD86、IA/IE表达及IL-12p70分泌水平均出现下降(P0.05或0.01),其中,以SB203580的作用最明显;使用PD98059后,CD83、CD86、IA/IE表达及IL-12p70分泌水平则出现上升(P0.05)。 4. (1)免疫后一周或两周,HBsAg特异性CTL,以负载HBsAg的DC联合尿酸(400μg、200μg、100μg)接种组杀伤效应最强,400μg尿酸联合组一周时为32.45%±11.63%,两周时为74.56%±12.38%;与DC对照组相比,均具有显著性差异,P0.05。DC对照组杀伤效应,一周时为14.33%±2.04%,两周时为28.18%±2.38%,与其它对照组相比,均具有显著性差异,P0.05;单独尿酸免疫组、unpulsed-DC组的HBsAg特异性CTL杀伤效应与PBS对照组无显著性差异,P0.05。HBsAg负载的DC免疫各组与HBS28-39负载各组,特异性CTL杀伤效应,无明显差异,P0.05。荷肽或抗原DC免疫产生的特异性CTL杀伤效应呈尿酸剂量依赖性升高,与尿酸的接种次数亦有关。 (2)免疫后两周,脾脏T细胞增殖反应,以负载HBsAg-DC联合尿酸(400μg、200μg、100μg)接种组最强,P-I值为1.764±0.114;与DC对照组相比,均具有显著性差异,P0.05。DC对照组脾脏T细胞增殖反应,PI值为1.342±0.093,与其它对照组相比,均具有显著性差异,P0.05;单独尿酸免疫组、unpulsed-DC组,脾脏T细胞增殖反应与正常对照组无显著性差异,P0.05。HBsAg负载的DC免疫各组与HBS28-39负载DC免疫各组,脾脏T细胞增殖反应,无明显差异,P0.05。荷肽或抗原DC免疫后脾脏T细胞增殖反应呈尿酸剂量依赖性升高。 (3)免疫后两周,负载HBsAg的DC联合尿酸(400μg、200μg、100μg)接种组IFN-γ水平最高,IL-4水平最低,与DC对照组相比,均具有显著性差异,P0.05;400μg尿酸联合HBsAg-DC免疫组IFN-γ、IL-4水平分别为552.361±24.034 pg/ml, 14.265±1.333 pg/ml。DC对照组脾脏细胞IFN-γ水平和IL-4水平,高于其它对照组,分别为266.575±51.324 pg/ml,22.385±2.252 pg/m(lP0.05);单独尿酸免疫组、unpulsed-DC组,脾脏细胞IFN-γ水平和IL-4水平与正常对照组相比无明显变化(P0.05)。HBsAg负载的DC免疫各组和HBS28-39负载各组,IFN-γ和IL-4水平,无明显差异,P0.05。荷肽或抗原DC免疫后脾脏细胞分泌的IFN-γ和IL-4水平呈尿酸剂量依赖性。结论: 1.对体外培养诱导扩增的DC,尿酸可促进其成熟,提高表面共刺激分子表达;能增强其刺激T细胞增殖能力和分泌IL-12p 70的水平。UA的这些活性呈剂量依赖性。 2.尿酸能调节未成熟树突细胞TLR2 mRNA、TLR3 mRNA、TLR4 mRNA等受体mRNA及IL-12 p70 mRNA表达。此可能为其诱导DC成熟及免疫功能提高的机理之一。 3.未成熟树突状细胞p38、ERK1/2、PI3K、NF-κB等信号分子可由尿酸刺激磷酸化,相应信号分子抑制剂可抑制尿酸这种刺激作用。阻断相应上述信号分子通路,可对尿酸诱导的树突状细胞活性产生影响。阻断p38、JNK、NF-κB等信号通路能抑制尿酸对树突状细胞CD83、CD86、IA/IE等分子表达及IL-12p 70分泌的上调作用;阻断ERK1/2信号通路能增强尿酸对树突状细胞CD83、CD86、IA/IE等分子表达及IL-12p 70分泌的上调作用。尿酸可以调节P38、ERK1/2、JNK、NF-κB等信号分子的活化,从而促进DC表面分子表达及IL-12p 70分泌。此可能为尿酸能诱导DC成熟及免疫功能提高的机理之一。 4.联合尿酸免疫接种,可增强负载HBsAg或S28-39的树突状细胞疫苗接种所诱导抗HBV的T细胞免疫应答,包括HBsAg特异性CTL、IFN-γ细胞因子的分泌、对T淋巴细胞的增殖能力。这些免疫效应的增强呈尿酸剂量依赖性。
[Abstract]:Purpose :

To study the immune effects of hepatitis B virus ( HBV ) induced by dendritic cell vaccine combined with HBsAg in uric acid ( UA ) , including cytotoxic T cell ( CTL ) killing effect of HBsAg specific cytotoxic T cell ( CTL ) , proliferation of T lymphocyte , secretion of Th1 / Th2 cytokines , etc . The mechanism of uric acid to promote DC maturation and function improvement was discussed .

Method :

1 . Bone marrow cells were isolated from mouse bone marrow cells in vitro . rmGM - CSF and rmIL - 4 were used to induce differentiation into DC .

2 . The expression of TLR2 mRNA , TLR3 mRNA , TLR3 mRNA , IL - 12p70 mRNA and so on was detected by RT - PCR .

3 . DCs were stimulated in vitro by using Uric acid or a combination inhibitor ( p38 MAPK inhibitor SB203580 , ERK 1 / 2 inhibitor PD98059 , inhibitor SP600125 , NF - 魏B inhibitor PDTC ) . The total protein and nucleoprotein were isolated from DC cells at 0 min , 15 min , 30 min , 45 min .

4 . In vitro loading of HBsAg and Uric acid into normal BALB / c mice .

Results :

1 . When Uric acid concentration was 400 渭g / ml , 200 渭g / ml , 100 渭g / ml , the expression rate of CD83 , CD86 , IA / IE in DC cells was enhanced . The level of IL - 12p70 secretion was increased ( P < 0.05 ) . The level of IL - 12p70 secretion was not significantly different from that of negative control group ( P0.05 ) .

2 . Compared with the control group , the expression of TLRs mRNA and TLR3 mRNA in TLRs mRNA and TLRs mRNA decreased ( P0.05 ) , and the expression of IL - 12p70 increased significantly ( P0.05 ) .

3 . ( 1 ) Western blot showed that the expression of p - p38 , p - 1 / 2 , p - p38 , NF - 魏B was significantly increased at 15 min after uric acid stimulation , and the maximal value was reached at 30 min . The P - value was less than 0.05 when compared with DMSO control group . After the corresponding inhibitor was used , the expression of related protein was not detected .

( 2 ) After pretreatment with inhibitors such as SB203580 , SP600125 , PDTC and so on , the levels of CD83 , CD86 , IA / IE and IL - 12p70 secretion decreased ( P0.05 or 0.01 ) .

4 . ( 1 ) After one week or two weeks after immunization , the anti - tumor effect of HBsAg - specific CTL and HBsAg - loaded DC combined uric acid ( 400 渭g , 200 渭g , 100 渭g ) was the strongest , compared with the control group , there was a significant difference ( P < 0.05 ) .

Compared with the control group , the proliferative response of spleen T cells was significantly higher than that in the control group ( P0.05 ) . Compared with the control group , there was no significant difference in the proliferative response of spleen T cells and the proliferation of spleen T cells .

The levels of IFN - 纬 , IL - 4 and IFN - 纬 and IL - 4 in spleen cells were significantly higher than those in the control group ( P0.05 ) . The levels of IFN - 纬 and IL - 4 in spleen cells of the control group were 262.361 卤 24.34 pg / ml , 14.265 卤 1.333 pg / ml respectively .

Conclusion :

1 . The DC and uric acid induced by culture in vitro can promote their maturation and enhance the expression of co - stimulatory molecules on the surface . It can enhance their ability to stimulate T cell proliferation and secrete IL - 12p 70 . These activities of UA are dose - dependent .

2 . Uric acid can regulate the expression of TLR2 mRNA , TLR3 mRNA , TLR3 mRNA and IL - 12 p70 mRNA in immature dendritic cells , which may be one of the mechanisms to induce DC maturation and immune function .

3 . It is possible to inhibit the activation of Uric acid on DC surface molecule expression and IL - 12p - 70 secretion by blocking the signaling pathway of p38 MAPK and NF - 魏B in immature dendritic cells . It is possible to block the activation of the signal molecules such as p38 , MAPK and NF - 魏B to inhibit the expression of CD83 , CD8, IA / IE , and the up - regulation of IL - 12p 70 secretion in the dendritic cells .

4 . Combined Uric acid vaccination can enhance the T cell immune response against HBV induced by dendritic cell vaccines loaded with HBsAg or S28 - 39 , including the secretion of HBsAg specific CTL , IFN - 纬 cytokines , and the proliferative ability of T lymphocytes . These immune responses enhance the dose dependence of uric acid .
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

【参考文献】