本文選題：尿酸 + 樹(shù)突狀細(xì)胞�。� 參考：《華中科技大學(xué)》2007年博士論文

【摘要】： 目的: 研究尿酸(uric acid, UA)聯(lián)合HBsAg負(fù)載的樹(shù)突狀細(xì)胞疫苗接種小鼠后產(chǎn)生的抗乙肝病毒(HBV)免疫效應(yīng),包括HBsAg特異性細(xì)胞毒性T細(xì)胞(CTL)殺傷效應(yīng),T淋巴細(xì)胞增殖反應(yīng),Th1/Th2型細(xì)胞因子的分泌等。并對(duì)尿酸促進(jìn)DC成熟與功能提高的機(jī)理進(jìn)行探討。 方法: 1.體外分離小鼠骨髓細(xì)胞,使用rmGM-CSF、rmIL-4誘導(dǎo)分化為DC,以UA或LPS刺激其成熟。分不同劑量尿酸組(70μg/ml、100μg/ml、200μg/ml、400μg/ml),LPS組、RMPI-1640培養(yǎng)基對(duì)照組。用流式細(xì)胞儀技術(shù)檢測(cè)DC細(xì)胞表面分子CD11c、CD83、CD86、IA/IE的表達(dá),用MTT法檢測(cè)DC刺激同基因小鼠T淋巴細(xì)胞的增殖反應(yīng);ELISA法測(cè)定DC上清IL-12p70的分泌水平。 2.尿酸體外刺激未成熟DC 48小時(shí),提取DC總RNA。RT-PCR方法檢測(cè)TLR2 mRNA、TLR3 mRNA、TLR4 mRNA、IL-12p70 mRNA等的表達(dá)。 3.使用尿酸或聯(lián)合抑制劑( p38 MAPK抑制劑SB203580、ERK1/2抑制劑PD98059、JNK抑制劑SP600125、NF-κB抑制劑PDTC )體外刺激未成熟DC。在0min、15 min、30 min、45 min時(shí),分別提取DC細(xì)胞總蛋白與核蛋白。免疫印跡方法檢測(cè)DC p- p38、p-ERK1/2、p-JNK、NF-κB p65表達(dá)量。分別使用SB203580、PD98059、SP600125、PDTC與尿酸聯(lián)合刺激DC 48h后,收集細(xì)胞,用流式細(xì)胞儀技術(shù)檢測(cè)DC細(xì)胞表面分子CD83、CD86、IA/IE的表達(dá);收集DC培養(yǎng)上清,ELISA法測(cè)定IL-12p70的水平。 4.DC體外負(fù)載HBsAg,聯(lián)合尿酸接種正常BALB/c小鼠。尾靜脈注射方式接種,DC接種數(shù)量為1×106/每只小鼠,每周接種一次,共兩次。分高、中、低劑量(分別為400μg、200μg、100μg)尿酸聯(lián)合HBsAg-DC組、負(fù)載或未負(fù)載HBsAg的DC單獨(dú)接種組、尿酸單獨(dú)接種組(200μg)及PBS對(duì)照組。同時(shí)以負(fù)載HBS28-39的DC單獨(dú)或聯(lián)合尿酸免疫小鼠,作為平行對(duì)照觀察組。一周和兩周時(shí),以體內(nèi)熒光測(cè)定CTL活性方法,用流式細(xì)胞儀檢測(cè)特異性CTL活性;免疫兩周時(shí),取小鼠脾T淋巴細(xì)胞,CFSE染色,體外以HBsAg或HBS28-39刺激培養(yǎng)72h后,流式細(xì)胞儀測(cè)定其增殖反應(yīng);取小鼠脾淋巴細(xì)胞體外HBsAg或HBS28-39刺激培養(yǎng)72h后,以ELISA法測(cè)定細(xì)胞上清IL-4和IFN-γ的分泌水平。 結(jié)果: 1.通過(guò)鑒定體外成功誘導(dǎo)培養(yǎng)出DC。尿酸濃度為400μg/ml、200μg/ml、100μg/ml時(shí)能增強(qiáng)DC細(xì)胞表面CD83、CD86、IA/IE分子表達(dá)率;提高IL-12p70分泌水平(與陰性對(duì)照組相比,P0.05);尿酸能增強(qiáng)DC刺激T細(xì)胞增殖能力(與陰性對(duì)照組相比,P值均小于0.05);尿酸濃度為70μg/ml時(shí),細(xì)胞表面分子表達(dá)、IL-12p70分泌水平、刺激T細(xì)胞增殖作用與陰性對(duì)照組比較均無(wú)明顯差別(P0.05)。UA促進(jìn)DC成熟的能力呈尿酸劑量依賴(lài)性增強(qiáng)。 2. RTPCR結(jié)果示尿酸組與培養(yǎng)基對(duì)照組相比,TLRs mRNA表達(dá)出現(xiàn)明顯的變化。TLR2 mRNA、TLR3 mRNA、TLR4 mRNA表達(dá)下降(P0.05),以TLR4 mRNA下降最明顯;IL-12p70表達(dá)顯著增高(P0.05);與LPS組相比,TLRs mRNA與IL-12p70 mRNA表達(dá)水平相似,無(wú)統(tǒng)計(jì)學(xué)差異,P0.05。 3.(1)免疫印跡示,尿酸刺激后,15min時(shí),p- p38、p-ERK1/2、p-JNK、NF-κB表達(dá)量明顯增加,30min時(shí)達(dá)到最大值,45min時(shí)則開(kāi)始下降,與DMSO對(duì)照組相比,P值均小于0.05。使用相應(yīng)抑制劑后,相關(guān)蛋白表達(dá)表不能測(cè)出。LPS組上述分子在45min時(shí)仍大量表達(dá)。 (2)與未用相應(yīng)抑制劑相比,使用SB203580、SP600125、PDTC等抑制劑預(yù)處理后,CD83、CD86、IA/IE表達(dá)及IL-12p70分泌水平均出現(xiàn)下降(P0.05或0.01),其中,以SB203580的作用最明顯;使用PD98059后,CD83、CD86、IA/IE表達(dá)及IL-12p70分泌水平則出現(xiàn)上升(P0.05)。 4. (1)免疫后一周或兩周,HBsAg特異性CTL,以負(fù)載HBsAg的DC聯(lián)合尿酸(400μg、200μg、100μg)接種組殺傷效應(yīng)最強(qiáng),400μg尿酸聯(lián)合組一周時(shí)為32.45%±11.63%,兩周時(shí)為74.56%±12.38%;與DC對(duì)照組相比,均具有顯著性差異,P0.05。DC對(duì)照組殺傷效應(yīng),一周時(shí)為14.33%±2.04%,兩周時(shí)為28.18%±2.38%,與其它對(duì)照組相比,均具有顯著性差異,P0.05;單獨(dú)尿酸免疫組、unpulsed-DC組的HBsAg特異性CTL殺傷效應(yīng)與PBS對(duì)照組無(wú)顯著性差異,P0.05。HBsAg負(fù)載的DC免疫各組與HBS28-39負(fù)載各組,特異性CTL殺傷效應(yīng),無(wú)明顯差異,P0.05。荷肽或抗原DC免疫產(chǎn)生的特異性CTL殺傷效應(yīng)呈尿酸劑量依賴(lài)性升高,與尿酸的接種次數(shù)亦有關(guān)。 (2)免疫后兩周,脾臟T細(xì)胞增殖反應(yīng),以負(fù)載HBsAg-DC聯(lián)合尿酸(400μg、200μg、100μg)接種組最強(qiáng),P-I值為1.764±0.114;與DC對(duì)照組相比,均具有顯著性差異,P0.05。DC對(duì)照組脾臟T細(xì)胞增殖反應(yīng),PI值為1.342±0.093,與其它對(duì)照組相比,均具有顯著性差異,P0.05;單獨(dú)尿酸免疫組、unpulsed-DC組,脾臟T細(xì)胞增殖反應(yīng)與正常對(duì)照組無(wú)顯著性差異,P0.05。HBsAg負(fù)載的DC免疫各組與HBS28-39負(fù)載DC免疫各組,脾臟T細(xì)胞增殖反應(yīng),無(wú)明顯差異,P0.05。荷肽或抗原DC免疫后脾臟T細(xì)胞增殖反應(yīng)呈尿酸劑量依賴(lài)性升高。 (3)免疫后兩周,負(fù)載HBsAg的DC聯(lián)合尿酸(400μg、200μg、100μg)接種組IFN-γ水平最高,IL-4水平最低,與DC對(duì)照組相比,均具有顯著性差異,P0.05;400μg尿酸聯(lián)合HBsAg-DC免疫組IFN-γ、IL-4水平分別為552.361±24.034 pg/ml, 14.265±1.333 pg/ml。DC對(duì)照組脾臟細(xì)胞IFN-γ水平和IL-4水平,高于其它對(duì)照組,分別為266.575±51.324 pg/ml,22.385±2.252 pg/m(lP0.05);單獨(dú)尿酸免疫組、unpulsed-DC組,脾臟細(xì)胞IFN-γ水平和IL-4水平與正常對(duì)照組相比無(wú)明顯變化(P0.05)。HBsAg負(fù)載的DC免疫各組和HBS28-39負(fù)載各組,IFN-γ和IL-4水平,無(wú)明顯差異,P0.05。荷肽或抗原DC免疫后脾臟細(xì)胞分泌的IFN-γ和IL-4水平呈尿酸劑量依賴(lài)性。 結(jié)論: 1.對(duì)體外培養(yǎng)誘導(dǎo)擴(kuò)增的DC,尿酸可促進(jìn)其成熟,提高表面共刺激分子表達(dá);能增強(qiáng)其刺激T細(xì)胞增殖能力和分泌IL-12p 70的水平。UA的這些活性呈劑量依賴(lài)性。 2.尿酸能調(diào)節(jié)未成熟樹(shù)突細(xì)胞TLR2 mRNA、TLR3 mRNA、TLR4 mRNA等受體mRNA及IL-12 p70 mRNA表達(dá)。此可能為其誘導(dǎo)DC成熟及免疫功能提高的機(jī)理之一。 3.未成熟樹(shù)突狀細(xì)胞p38、ERK1/2、PI3K、NF-κB等信號(hào)分子可由尿酸刺激磷酸化,相應(yīng)信號(hào)分子抑制劑可抑制尿酸這種刺激作用。阻斷相應(yīng)上述信號(hào)分子通路,可對(duì)尿酸誘導(dǎo)的樹(shù)突狀細(xì)胞活性產(chǎn)生影響。阻斷p38、JNK、NF-κB等信號(hào)通路能抑制尿酸對(duì)樹(shù)突狀細(xì)胞CD83、CD86、IA/IE等分子表達(dá)及IL-12p 70分泌的上調(diào)作用;阻斷ERK1/2信號(hào)通路能增強(qiáng)尿酸對(duì)樹(shù)突狀細(xì)胞CD83、CD86、IA/IE等分子表達(dá)及IL-12p 70分泌的上調(diào)作用。尿酸可以調(diào)節(jié)P38、ERK1/2、JNK、NF-κB等信號(hào)分子的活化,從而促進(jìn)DC表面分子表達(dá)及IL-12p 70分泌。此可能為尿酸能誘導(dǎo)DC成熟及免疫功能提高的機(jī)理之一。 4.聯(lián)合尿酸免疫接種,可增強(qiáng)負(fù)載HBsAg或S28-39的樹(shù)突狀細(xì)胞疫苗接種所誘導(dǎo)抗HBV的T細(xì)胞免疫應(yīng)答,包括HBsAg特異性CTL、IFN-γ細(xì)胞因子的分泌、對(duì)T淋巴細(xì)胞的增殖能力。這些免疫效應(yīng)的增強(qiáng)呈尿酸劑量依賴(lài)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 - 緯 , IL - 4 and IFN - 緯 and IL - 4 in spleen cells were significantly higher than those in the control group ( P0.05 ) . The levels of IFN - 緯 and IL - 4 in spleen cells of the control group were 262.361 鹵 24.34 pg / ml , 14.265 鹵 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 - 緯 cytokines , and the proliferative ability of T lymphocytes . These immune responses enhance the dose dependence of uric acid .
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 陳文凱,陸春海,許嬌,楊迎春,李俊{，

本文編號(hào)：1907925