摘要
目的为研究人PBMC TLR3在人HBV感染和HBV宫内感染中的作用,本课题采用人群研究和体外实验相结合的方法,从基因和蛋白水平初步了解正常孕妇人群与HBsAg阳性孕妇人群PBMC TLR3的表达情况以及与HBV感染和HBV宫内感染的关系,根据人群研究的线索进一步进行体外实验,探索TLR3在抗HBV感染中的作用机制。
1.了解HBsAg阳性孕妇及其新生儿外周血单个核细胞(PBMC)HBV感染的状况;
2.了解TLR3在PBMC的表达情况;
3.探讨TLR3与HBV感染和HBV宫内感染的关系;
4.探讨TLR3抗病毒生物学效应的机制。
方法1.人群研究
①以2004年7月至2006年1月由太原市传染病院妇产科进行产前检查并分娩的HBsAg阳性孕妇为研究队列,从产前检查追踪观察至分娩,共收集HBsAg阳性孕妇及其新生儿228对。采集孕妇分娩前肘静脉血和新生儿出生24小时内注射乙肝疫苗前的股静脉血,同时收集其流行病学资料,用免疫荧光双标记的方法检测HBsAg阳性孕妇及其新生儿PBMC涂片上HBsAg的表达及TLR3的分布;用巢式病例对照研究的方法进行TLR3与PBMC HBV感染,TLR3与PBMC HBV宫内感染的研究。以PBMC HBV感染者为病例组,非感染者为对照组,分析HBsAg阳性孕妇及新生儿PBMC TLR3表达与PBMC HBV感染的关系;
②于2006年11月至2007年4月,从太原市传染病院妇产科选取外周血HBsAg阳性的足月分娩孕产妇及其新生儿共28对,同期从山西省妇幼保健院妇产科收集正常足月分娩孕产妇26例作为正常孕妇对照,采集孕妇肘静脉血和新生儿出生后24小时内注射乙肝疫苗前的股静脉血,用荧光定量PCR定量检测HBsAg阳性孕妇及其新生儿血清HBVDNA,用酶联免疫吸附试验(ELISA)检测乙肝标志物,用RT-PCR方法检测正常孕妇和HBsAg阳性孕妇PBMC TLR3 mRNA,分析PBMC TLR3 mRNA与HBV感染的关系;用real-time RT-PCR方法检测HBsAg阳性孕妇及其新生儿PBMC TLR3 mRNA,分析PBMCTLR3 mRNA与HBV感染及HBV宫内感染的关系;ELISA方法分别检测HBsAg阳性孕妇和正常孕妇血清中IL-2、6的浓度,分析HBV感染与细胞因子的关系及TLR3与细胞因子分泌的关系。
2.体外实验
①于2007年4月至2007年10月从山西医科大学第一医院妇产科收集正常足月分娩孕产妇26例,收集其肘静脉及流行病学资料。分离外周血PBMC,用含10%胎牛血清的1640培养液进行原代细胞培养,设HBV刺激组和正常对照组,在培养2、4、6天后分别收集PBMC细胞和培养上清,real-time RT-PCR方法检测PBMC的TLR3 mRNA,FACs方法检测PBMC的TLR3蛋白,ELISA方法检测培养上清液IL-2、6、10、12、IFN-γ和TNF-α的浓度;
②用RNAi技术沉默正常献血者PBMC TLR3 mRNA,用TLR3的配体PolyI:C刺激活化TLR3,并在相应处理后与一定量的HBV共孵育6天,在2,4,6天分别收集PBMC细胞和培养上清,real-time RT-PCR检测TLR3 mRNA,FACs检测TLR3蛋白,荧光定量PCR定量检测培养上清和PBMC中HBV DNA含量。
3.统计分析资料查实后输入数据库,采用SPSS 13.0 for windows软件进行统计分析,计数资料采用χ~2分析,计算各指标阳性率、χ~2值、OR值及其95%可信区间;计量资料采用t检验、配对t检验、方差分析及相关性分析。
结果1.HBsAg阳性孕妇PBMC TLR3蛋白和PBMC HBV感染的关系
①HBsAg分布在PBMC胞膜和胞浆部位;TLR3分布在胞膜上,TLR3在不同PBMC涂片中表达强度不同,同一视野下不同的细胞表达强度也不同,且在TLR3表达强的细胞上HBsAg表达弱。
②228例HBsAg阳性孕妇有64例(28.07%) PBMC HBsAg阳性;161例(70.61%)TLR3强表达;TLR3在PBMC HBV感染组和非感染组的表达差异有统计学意义(χ~2=30.944,P＜0.0001);新生儿有35例(15.35%) PBMC HBsAg阳性;HBsAg阳性孕妇PBMC TLR3的表达与新生儿PBMC HBV宫内感染有关(χ~2=12.354,P＜0.0001)。TLR3可能是HBsAg阳性孕妇PBMC HBV感染的保护因素(OR=0.181,95%CI=0.091-0.340),也是PBMC HBV宫内感染的保护因素(OR=0.279,95%CI=0.133-0.585)。
2.人群PBMC TLR3 mRNA与HBV感染的关系
①用RT-PCR检测HBsAg阳性孕妇组和正常孕妇组PBMC TLR3 mRNA,HBsAg阳性孕妇组TLR3 mRNA的相对表达量(0.8164±0.1608)高于正常孕妇组(0.3892±0.1879),其差异有统计学意义(t=-8.997,P＜0.001)。
②用real-time RT-PCR检测正常孕妇、HBsAg孕妇及其新生儿PBMC TLR3 mRNA,HBsAg阳性孕妇组TLR3 mRNA的相对表达量高于正常孕妇组(t=9.14,P＜0.0001)。在HBsAg阳性孕妇组中又分为DNA阴性组,≤10~4copies/ml组和＞10~4copies/ml组,TLR3 mRNA在这三组中差别无统计学意义(F=0.468,P＞0.05);且在HBsAg阳性孕妇组中的大三阳组和非大三阳组TLR3 mRNA差别无统计学意义(t=0.355,P=0.725);在HBsAg阳性孕妇组中的HBV宫内感染组和非HBV宫内感染组,TLR3 mRNA差别无统计学意义(t=-1.525,P=0.139)。
③根据新生儿血清中乙肝标志物的检测结果将新生儿分为HBV宫内感染组和非HBV宫内感染组,新生儿HBV宫内感染组TLR3 mRNA高于非HBV宫内感染组,差别有统计学意义(t=2.613,P=0.015)。
3.人群TLR3 mRNA与IL-2、6分泌的关系
①HBsAg阳性孕妇血清中IL-2的浓度(32.95±9.93pg/ml)比正常孕妇(27.65±2.86pg/ml)高,而IL-6浓度(52.35±22.09pg/ml)较正常孕妇(544.6±615.4pg/ml)低,差别均有统计学意义(t=-2.291,P=0.028;t=3.575,P=0.002),并且以IL-2代表Th1型细胞因子,IL-6代表Th2型细胞因子计算Th1/Th2比值,HBsAg阳性孕妇组Th1/Th2(0.22±0.25)比正常孕妇组(0.71±0.27)小,差别具有统计学意义(t=-5.86,P＜0.05)。
②HBsAg阳性孕妇的HBV宫内感染组血清中IL-6浓度(81.003±21.64pg/ml)高于非HBV宫内感染组(42.80±11.7 pg/ml),差别具有统计学意义(t=-3.77,P=0.014)。Th1/Th2比值(0.43±0.13)低于非HBV宫内未感染组(0.80±0.24),差别具有统计学意义(t=3.334,P=0.004)。
③以TLR3/β-actin Ct值与相应标本的IL-2,6的浓度作相关分析,得出TLR3/β-actin Ct值与IL-2的浓度呈负相关关系,r=-0.56,P＜0.05,随着TLR3的增加,IL-2分泌增加。而未发现与IL-6有相关性,r=0.096,P＞0.05。
4.HBV体外刺激正常孕妇PBMC TLR3的表达及与细胞因子分泌的关系
①正常孕妇PBMC TLR3的表达在HBV刺激组与正常对照组差异有统计学意义(t=-2.36,P=0.029),但对培养2、4、6天PBMC TLR3 mRNA的表达量进行比较,差别无统计学意义(F=0.045,P=0.965)。
②正常孕妇PBMC培养上清中IL-2、TNF-α在HBV刺激组浓度增加,与正常对照组相比差别有统计学意义(t=2.415,P=0.023;t=2.277,P=0.031)。IL-6、10、12和IFN-γ浓度,Th1/Th2(IL-2/IL-6)比值在HBV刺激组有所增加,但差别无统计学意义。TLR3/β-actinCt值与IL-10,IL-6,IFN-γ呈负相关关系,相关系数r分别为-0.331(P=0.043),-0.46(P=0.014),-0.557(P=0.025),随着TLR3的活化增多,其浓度增加。各个细胞因子之间的关系IL-12与IFN-γ,IL-2呈正相关关系,r分别为0.588(P=0.027),0.475(P=0.014),与IL-10呈负相关关系,r=-0.752(P=0.001)。IL-10与IFN-γ呈负相关关系,r=-0.633,(P=0.006),TNF-α和IL-6呈负相关关系,r=-0.467(P=0.019),IFN-γ和IL-6呈负相关关系,r=-0.507(P=0.045)。
5.RNAi沉默TLR3和PolyI:C活化TLR3后对HBV感染的影响
①RNAi技术对PBMC TLR3 mRNA的抑制效率为67.83%,正常人PBMC在HBV刺激,RNAi,PolyI:C刺激后和正常对照组的TLR3 mRNA,蛋白表达四组相比有统计学意义(F=5.33,4.89;P=0.018,0.031)。PolyI:C刺激24h后TLR3显著增高,RNAi48h后TLR3显著降低。
②RNAi 48h可以下调TLR3 mRNA和TLR3蛋白,且在干扰后加HBV刺激继续培养6天TLR3 mRNA随刺激时间的延长总体有回升的趋势,在第四天TLR3 mRNA增加到最大,之后又有所下降。TLR3蛋白也随时间的延长表达增加,开始时增加缓慢,在第五天达到最高。
③PolyI:C刺激24小时后TLR3 mRNA和TLR3蛋白显著增加,且在加HBV继续培养5天,TLR3 mRNA随刺激时间的延长继续增加,在第二天TLR3 mRNA增加到最大,第三天又开始下降。但TLR3蛋白在加入HBV刺激1天时,表达减少,后又逐渐增加。
④正常人的PBMC与HBV共培养,在第4,6天PBMC中检出了HBV DNA,且培养上清和PBMC中HBV DNA的拷贝数在第6天有所下降,但在培养2、4、6天后培养上清中、PBMC中HBV DNA的拷贝数差异无统计学意义(F=1.24,1.83,P=0.489,0.397)。
⑤PolyI:C刺激24h后加HBV共培养,在第4天PBMC中检出了HBV DNA。在培养上清和PBMC中HBV DNA含量随时间的延长明显下降,差异有统计学意义(F=6.34,7.01,P=0.036,0.027)。
⑥RNAi 48h后加HBV共培养在第2天,第6天PBMC中检出了HBV DNA。在培养上清中HBV DNA含量随时间的延长有增高的趋势,但在统计学上差异无显著性(F=2.34,P＞0.05)。在PBMC中HBV DNA含量随时间的延长有显著增高,差异有统计学意义(F=4.67,P=0.042)。
结论1.TLR3在PBMC有表达,在PBMC不同细胞上表达强度不同。在TLR3表达强的细胞上HBsAg表达弱或无表达。
2.TLR3与HBV感染有关,HBV阳性的PBMC TLR3低表达,TLR3是PBMC HBV感染的保护因素;以新生儿PBMC HBV感染判为新生儿PBMC HBV宫内感染,母亲TLR3蛋白是新生儿PBMC HBV宫内感染的保护因素。以新生儿血清HBV标志物HBsAg,HBV DNA任一阳性定义新生儿HBV宫内感染,母亲PBMC TLR3表达与新生儿HBV宫内感染无统计学关联,但新生儿PBMC TLR3表达是新生儿HBV宫内感染的保护因素。
3.TLR3与Th1细胞因子的分泌呈正相关关系,TLR3可能通过增加细胞因子的分泌参与HBV的清除过程。
4.以HBV刺激正常孕妇PBMC可使PBMC TLR3 mRNA和TLR3蛋白表达上调,IL-10,IL-6,IFN-γ随着TLR3的上调而分泌增加,而TNF-α,IL-2分泌增加显著,说明HBV感染PBMC以启动Th1型免疫反应为主。
5.RNAi后TLR3在蛋白水平和基因水平上有所降低,一定程度上增加了PBMC对HBV的易感染性。PolyI:C刺激后TLR3在蛋白水平和基因水平上有所升高,并一定程度上降低了PBMC对HBV的易感染性。TLR3可一定程度上影响HBV的复制增殖。
6.可以考虑将TLR3作为乙型肝炎治疗性疫苗或佐剂及辅助性治疗药物的作用靶点来开发应用。
OBJECTIVE:The aim is to study the role of toll like receptor 3 playing in HBV infection and HBV intrauterine infection,based in population and in vitro.The clues of the relationship between TLR3 and HBV infection and HBV intrauterine infection from normal and HBsAg-positive pregnant women at gene and protein level were made,and then a series of experiments in vitro were demonstrated.
1.To understand the HBV infection state of Peripheral Blood Mononuclear Cells(PBMC) in HBsAg positive pregnant women and their newborns.
2.To understand the distribution of Toll Like Receptor 3(TLR3) on PBMC.
3.To explore the role of TLR3 in human HBV infection and HBV intrauterine infection.
4.To study the anti-virus mechanism of TLR3.
METHODS:1 population-based studies:
①228 HBsAg positive pregnant women were selected from July,2004 to January,2006 in Taiyuan infectious hospital.Followed studies were performed from the time of their beginning antenatal examination to the time of their giving birth to newborns and their newborns were also selected.Newborns' femoral vein blood within 24 hours before injecting HBV vaccine and mother's elbow vein blood before delivery were collected.At the same time,the epidemiological base line data involving gestation and postpartum were also collected.HBsAg and TLR3 in PBMC were detected by double labeling immunofluorescent histochemistry assay.
②28 HBsAg positive pregnant women and their newborns were selected from November, 2006 to April,2007 in Taiyuan infectious hospital,at the same period,26 normal pregnant women were selected in Maternal and Child Health hospital of Shanxi province as the control, Newborns' femoral vein blood within 24 hours before injecting HBV vaccine and mother's elbow vein blood were collected,TLR3 mRNA in PBMC of HBsAg positive pregnant women and normal pregnant women was detected by RT-PCR;TLR3 mRNA in HBsAg positive pregnant women and their newborns PBMC was detected by real-time RT-PCR.HBsAg and HBeAg in gravidas' and newborns' serum were tested by ELISA.HBV DNA in both serums was detected by Fluorescence Quantitative PCR(FQ-PCR).The concentration of IL-2,6 in both serums of HBsAg positive and normal pregnant women were analyzed by ELISA.
2.Studies in vitro:
①26 normal pregnant women in the first affiliated hospital of Shanxi Medical University were selected from April,2007 to October,elbow vein blood and the epidemiological base line data were collected.PBMC were isolated to culture with 1640 which contains 10%FBS.2 groups were defined:HBV stimulus and non- stimulus in each objects,PBMC and culture supernatant were selected respectively at 2nd,4th,6th culture day,TLR3 mRNA in PBMC was detected with real-time RT-PCR,the protein of TLR3 was detected by FACs,IL-2,6,10,12, IFN-γand TNF-αin supernatant were analyzed by ELISA.
②TLR3 were silenced by RNAi and activated using PolyI:C that is a ligand of TLR3,then PBMC were cultured with HBV for 6 days,PBMC and culture supematant were selected respectively at 2nd,4th,6th culture day,TLR3 mRNA in PBMC was detected with real-time RT-PCR,the protein of TLR3 was detected by FACs,HBV DNA in both supernatant and PBMC were detected by FQ-PCR.
3.Statistical analysis:Check and input the data,all of the data were analyzed by SPSS 13.0 for windows.PBMC HBV infection was analyzed by nested case-control study,χ~2-test,t-test, ANOVAL and correlation analysis.
RESULTS:1.The relationship between TLR3 protein of HBsAg positive pregnant women'PBMC and PBMC HBV infection.
①HBsAg was mostly located in the cytoplasm,a part on the cell membrane.TLR3 was mostly located on the cell membrane,while the distribution density of TLR3 was different at different PBMC smears,even at the same smears;the distribution was also different in each scope.The thicker TLR3 was,the thinner HBsAg was at one cell.
②64 HBsAg positive were detected on PBMC smears of 228 HBsAg positive pregnant women and positive rate was 28.07%(64/228);161 PBMC smears were detected TLR3 positive and the positive rate was 70.61%(161/228),the difference of TLR3's expression between PBMC HBV infection group and non-infection group was statisticaly significant(χ~2=30.944,P＜0.0001); 35 PBMC smears were detected HBsAg positive from 228 HBsAg positive(in serum) pregnant women's newborn infants and the positive rate was 15.35%(35/228);The expression of TLR3 on pregnant women's PBMC was relative with PBMC HBV intrauterine infection of newborn infants(χ~2=12.354,P＜0.0001).TLR3 was probably a protective factor of PBMC HBV infection of HBsAg positive(in serum) pregnant women(OR=0.181,OR95%CI=0.091-0.340) and was also a protective factor of PBMC HBV intrauterine infection of newborn infants (OR=0.279,OR95%CI=0.133-0.585).
2.The relationship between TLR3 mRNA of pregnant women'PBMC and PBMC HBV infection.
①The performed tests of RT-PCR showed that TLR3 mRNA were higher in HBsAg positive pregnant women group than that in the normal.(t=-8.997,P＜0.001).
②The performed tests of real time RT-PCR showed that TLR3 mRNA were significantly higher in HBsAg positive pregnant women than that in the normal(t=9.14,P＜0.0001 ).The rate of TLR3Ct/β-actin Ct was not significant difference among DNA negative group,HBV DNA≤104copies/ml group and HBV DNA＞104copies/ml group(F= 0.468,P＞0.05 ),and there were not significant difference between HBsAg,HBeAg and HBcAb positive group and the group that no same time positive group that in HBsAg positive pregnant women(t=0.355,P=0.725). HBsAg positive pregnant women were divided by their newborns having HBV intrauterine infection or not,the rate of TLR3Ct/β-actin Ct was not significant difference between the two groups(t=-1.525,P=0.139).
③Newborns were divided by their having HBV intrauterine infection or not,the rate of TLR3Ct/β-actin Ct increased in HBV intrauterine infection group(t=2.613,P=0.015).
3.The relationship between TLR3 mRNA of pregnant women'PBMC and the concentration of IL-2,6.
①The concentration of IL-2 in HBsAg positive pregnant women serum(32.95±9.93 pg/ml) was higher than the normal pregnant women's(27.65±2.86 pg/ml ),while IL-6 was opposite to IL-2,(52.35±22.09 pg/ml ) vs.(544.6±615.4 pg/ml )(t=-2.291,P =0.028;t=3.575,P=0.002),the value of Th1/Th2 in HBsAg positive pregnant women(0.22±0.25) decreased compared with the normal(0.71±0.27)(P＜0.05).
②The concentration of IL-6(81.00±21.64 pg/ml) in serum of HBsAg positive pregnant women with their newborns having HBV intrauterine infection highly increased compared with not having HBV intrauterine infection women's(42.80±11.7 pg/ml),(P=0.014).The value of Th1/Th2((0.43±0.13) vs.(0.80±0.24)) significantly decreased in these two groups(P=0.004).
③Correlation analysis showed that the rate of TLR3Ct/β-actin Ct was negatively correlated with the concentration of IL-2,(r=-0.56,P＜0.05),the concentration of IL-2 increased with the TLR3 mRNA increasing.
4.The relationship between TLR3 of normal pregnant women'PBMC which were stimulated by HBV DNA and the concentration of cytokines
①PBMC from normal pregnant women were cultured,TLR3 increased in the HBV stimulus group compared to the HBV non-stimulus group(t=-2.36,P=0.029),but TLR3 mRNA at 2nd,4th,6th day was not significant difference(F=0.045,P=0.965).
②The concentration of IL-2,TNF-αin supematant of normal pregnant women PBMC cultured with HBV stimulus increased compared to the HBV non-stimulus group(t=2.415, P=0.023;t=2.277,P=0.031).IL-6,10,12,IFN-γand Th1/Th2(IL-2/IL-6) lightly increased but no significance.Correlation analysis showed that the value of TLR3Ct/β-actin Ct was significantly correlated with the concentration of IL-10,IL-6,IFN-γ,r was -0.331(P=0.043), -0.46(P=0.014),-0.557(P=0.025),respectively,that was to say,IL-10,IL-6 and IFN-γwill rise with TLR3 mRNA increasing.IL-12 was significantly correlated with the concentration of IFN-γ,IL-2,IL-10,rwas 0.588(P=0.027),0.475(P=0.014),-0.752,(P=0.001).IL-10 was significantly correlated with the concentration of IFN-γ,r was-0.633(P=0.006).IL-6 was significantly correlated with the concentration of TNF-α,IFN-γ,r was -0.467(P=0.019),-0.507 (P=0.045).
5.TLR3 was silenced by RNAi or activated by PolyI:C which influence the replication of HBV
①The depressed rate of PBMC TLR3 mRNA by RNAi was 67.83%,there was a significant difference at TLR3 mRNA level,as well as TLR3 protein,among healthy blood donor group, with HBV stimulus group,RNAi group and PolyI:C stimulus group(F=5.33,4.89; P=0.018,0.031).
②TLR3 was depressed by RNAi for 48 hours both in mRNA and protein levels,contrast to no treatments,TLR3 mRNA gradually raised after culturing with HBV for 6 days and to maximum at the 4th day.The rising trend of TLR3 protein was the same as TLR3 mRNA,but it was to maximum at the 5th day.
③TLR3 was augmented with PolyI:C provoking for 24 hours contrast to no treatments, TLR3 mRNA was continuously raised after culturing with HBV for 6 days and to maximum at the 2nd day.Then it was down at the 3rd day.TLR3 protein decreased at the 1st day,then rose gradually.
④PBMC from healthy blood donors were cultured with HBV for 6 days,HBV DNA were detected at the 4th day and 6th day,the HBV DNA copies in supernatant and PBMC were no difference at the 2nd,4th,and 6th day(F=1.24,1.83,P=0.489,0.397).
⑤PBMC activated by PolyI:C were cultured with HBV for 6 days,HBV DNA were detected at the 4th day,the content of HBV DNA in supernatant and PBMC decreased with the extension of time(F=6.34,7.01,P=0.036,0.027).
⑥PBMC with RNAi were cultured with HBV for 6 days and at the 2nd day and the 6th day, HBV DNA were detected.Moreover,the content of HBV DNA in PBMC were significantly increased with the time(F=4.67,P=0.042 ),but there was no statistical significance in supernatant (F=2.34,P=0.249).
CONCLUSIONS:1.TLR3 was expressed in PBMC and the density distribution of TLR3 was different at different kinds of cells.The thicker the TLR3 was,the thinner the HBsAg was at one cell.
2.TLR3 was relative with the HBV infection,TLR3 mRNA of PBMC increased from HBsAg positive pregnant women,but TLR3 protein of PBMC that were infected by HBV decreased.TLR3 was a protective factor of PBMC HBV infection.Newborns' PBMC HBV intrauterine infection was definded by Newborns' PBMC HBV infection.The higher expression of TLR3 on pregnant women's PBMC was a protective factor of PBMC HBV intrauterine infection of their newborns.Newborns' HBV intrauterine infection was definded by HBsAg or HBV DNA positive in Newborns' serum,TLR3 on pregnant women's PBMC had no relations to HBV intrauterine infection of their newborns,TLR3 on newborns was a protective factor of newborns HBV intrauterine infection.
3.TLR3 was positively correlated with Th1 cytokines,TLR3 may participate in anti-virus by increasing the production of cytokines.
4.The concentration of IL-2,TNF-αin supernatant of normal pregnant women PBMC cultured with HBV stimulus were more than no HBV stimulus group.IL-6,10,12,IFN-γand Th1/Th2(IL-2/IL-6) were lightly increased,that showed PBMC can predominantly promote Th1 immunological reaction against HBV infection.
5.PBMC were cultured with HBV stimulus,TLR3 increased both in mRNA and protein levels.When TLR3 was depressed by RNAi both in mRNA and protein levels,the susceptibility of PBMC to HBV was up-regulation.Moreover,if TLR3 were augmented with PolyI:C provoking both in mRNA and protein levels,the susceptibility of PBMC to HBV was down-regulation,TLR3 may influence HBV replication.
6.TLR3 can be exploited as effect target of adjuvanticity,HBV vaccine or HBV treatment medicine.
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