【作者】 阮杨；

【导师】 孙铁英；

【作者基本信息】 北京协和医学院 ， 内科学（专业学位）， 2013， 博士

【摘要】 目的：抗菌肽在机体防御中起重要作用。目前已经发现包括cathelicidin多种抗菌肽。Cathelicidin是由保守的N末端及可变的C末端组成,蛋白酶切割前体形成。LL-37是人体内唯一的cathelicidin。LL-37由37个氨基酸组成,呈螺旋状,有亲水和疏水部分,具有广谱抗菌活性。LL-37的防御作用包括调节炎症反应,趋化免疫细胞至损伤或感染部位,结合并中和LPS,促进再上皮化及损伤修复。本研究通过建立LTA诱导的巨噬细胞炎症模型,分析炎症时LL-37的变化,探讨LL-37在LTA诱导巨噬细胞炎症中的作用及机制。方法：LTA(2ug/ml)刺激巨噬细胞系RAW264.7和C57BL/6小鼠原代腹腔巨噬细胞,建立炎症的细胞模型。以ELISA及实时定量PCR方法测定炎症因子的表达,以免疫荧光检测LL-37的细胞定位,联合Western blot方法检测LL-37在炎症时的变化,以Western blot检测LL-37对LTA诱导的炎症的作用机制。结果：1.LL-37细胞定位以免疫荧光证实LL-37是否存在于巨噬细胞中及在巨噬细胞中的分布。结果显示LL-37主要分布在细胞质中。2.LTA诱导巨噬细胞炎症反应以不同浓度来源于金黄色葡萄球菌的LTA刺激RAW264.7及原代小鼠腹腔巨噬细胞,通过ELISA检测培养基中TNF-a的水平,确定LTA是否可以诱导巨噬细胞炎症反应及LTA的最佳浓度。在RAW264.7细胞系中,当LTA浓度从0至2ug/mL时,TNF-α浓度由4.56±0.89升高到7.49±1.80ng/L,在小鼠原代腹腔巨噬细胞中,则由5.18±0.91升至11.22±0.88ng/L。但当LTA浓度为5-20ug/mL时能够引起急性细胞坏死,从而导致TNF-α表达降低。结果提示0至2ug/mL的LTA可以诱导巨噬细胞炎症反应,但过高浓度的LTA导致细胞损伤和死亡3.LL-37在LTA诱导的RAW264.7炎症中的表达以免疫荧光及Western blot方法检测在LTA诱导的RAW264.7细胞炎症中LL-37的表达情况。将化学合成的LL-37加入到RAW264.7细胞培养基中以免疫荧光及Western blot检测检测LL-37的摄取效率。本实验分4组：对照组,LL-37干预组,LTA干预组及LTA与LL-37同时干预组。LTA干预组LL-37表达比对照组增加1.33±0.13倍,有统计学意义(p<0.05)。LTA与LL-37同时干预组LL-37表达比对照组增加1.93±0.18倍,有统计学意义(p<0.05)。LTA与LL-37同时干预组中,LL-37的表达较LTA干预组明显升高,有统计学意义(p<0.05),但与LL-37干预组无明显差异(p>0.05)。由此提示LTA可以诱导LL-37的表达,LL-37可以透膜进入RAW264.7细胞。4.LL-37可以减弱由LTA诱导的巨噬细胞炎症反应通过ELISA方法检测是否LL-37可以减弱由LTA诱导的RAW264.7细胞及原代小鼠腹腔巨噬细胞的炎症反应。首先以2ug/mL的LTA诱导RAW264.7细胞及小鼠原代腹腔巨噬细胞产生炎症。随后以0-40ug/mL的LL-37干预。结果显示：在RAW264.7细胞中,1-15ug/mL的LL-37以浓度依赖的方式降低TNF-α的表达,TNF-α浓度由基础值6.46±0.36ng/L降低至4.63±0.81ng/L, LL-37的最适浓度为15ug/mL;而在小鼠原代腹腔巨噬细胞中,1-10ug/mL的LL-37以浓度依赖的方式降低TNF-α的表达,TNF-α浓度由基础值10.52±0.92ng/L降低至6.69±0.64ng/L, LL-37的最适浓度为10ug/mL。结果所示,若LL-37浓度超过最适浓度,则可导致细胞损伤,增加TNF-α释放。因此适当浓度的LL-37可以减弱LTA诱导的巨噬细胞的炎症反应,但是过高浓度则可以导致细胞损伤。本实验以15ug/mL的LL-37干预RAW264.7细胞,以10ug/mLELISA的LL-37干预小鼠原代腹腔巨噬细胞。5.LL-37可以通过降低炎症因子的表达减弱LTA诱导的巨噬细胞炎症反应TNF-α及IL-6是炎症中的主要炎症因子。我们通过实时定量PCR检测RAW264.7细胞中及ELISA检测小鼠原代巨噬细胞培养基中的TNF-α及IL-6表达来证明LL-37是否可以通过降低炎症因子的表达减弱LTA诱导的巨噬细胞炎症反应。结果显示：在RAW264.7细胞中与对照组比较,LTA干预组中,TNF-α及IL-6的mRNA分别增加8.75±0.47倍及34.12±9.68倍；与LTA干预组比较,LTA和LL-37同时干预组TNF-α及IL-6的mRNA明显降低。提示LL-37可以明显降低TNF-α及IL-6的mRNA表达水平。在小鼠原代巨噬细胞中,与对照组比较,LTA干预组中,TNF-α及IL-6的浓度分别由基础值的4.98±1.30ng/L,16.75±9.06ng/L增加至7.53±2.21ng/L,180.38±29.46ng/L;与LTA干预组比较,LTA和LL-37同时干预组TNF-a及IL-6的浓度明显降低。因此LL-37可以通过降低炎症因子TNF-a及IL-6的表达减弱LTA诱导的炎症反应。6.LL-37通过抑制p38MAPK及Akt的磷酸化抑制LTA诱导的巨噬细胞炎症反应通过验证RAW264.7细胞及小鼠原代巨噬细胞中p38MAPK及Akt的磷酸化水平检测LL-37的信号通路。结果显示：LTA通过增加巨噬细胞p38MAPK及Akt的磷酸化水平促进炎症产生；LL-37通过抑制巨噬细胞p38MAPK及Akt的磷酸化水平抑制LTA诱导的巨噬细胞炎症产生。结论：1.在LTA诱导的金黄色葡萄球菌感染的巨噬细胞炎症模型中,TNF-α、IL-6及LL-37表达水平上升,提示LTA可以引起巨噬细胞产生炎症反应,LL-37参与LTA引起的炎症反应。2.LL-37降低炎症因子表达,提示LL-37可以缓解炎症反应。3.LTA可以促进p38及Akt磷酸化,LL-37可以抑制p38及Akt的磷酸化,提示LL-37可以通过抑制p38及Akt磷酸化参与LTA诱导的炎症反应。更多还原

【Abstract】 Objectives:Antimicrobial peptides play an important role in host defense. Various families of antimicrobial peptides have been identified, including the cathelicidin. Cathelicidin is characterized by a conserved N-terminal cathelin domain and a variable C-terminal part of the only human cathelicidin identified to date called human cationic antimicrobial protein (hCAP-18). LL-37is the only human cathelicidin. LL-37is a37-residue, amphipathic, helical peptide found throughout the body and has been shown to exhibit a broad spectrum of antimicrobial activity. It has been found to have defensive roles such as regulating the inflammatory response and chemoattracting cells of the immune system to wound or infection sites, binding and neutralizing LPS, and promoting re-epthelialization and wound repair. In this study, we established the LTA-induced inflammation models in vitro and analyzed the expression, function and mechanism of LL-37in macrophages.Methods:We established cellular inflammation model by treatment of RAW264.7cells and primary C57BL/6mouse peritoneal macrophage with2ug/mL LTA for24h. The expression of inflammatory cytokines were determined by ELISA and real-time quantitative PCR method, LL-37was measured by immunofluorescence and Western blot method. The mechanism of LL-37in LTA induced inflammation was deteced by Western blot.Results:1. Distribution of LL-37in macrophagesTo determine whether LL-37was expressed in macrophages and to determine its expression pattern, we did LL-37immunofluorescence staining in RAW264.7cells. The immunofluorescence staining showed that LL-37was mainly expressed in the cytoplasma but not in the nucleus. The merged figure also showed that LL-37was mainly expressed in the cytoplasma with uneven distribution.2. LTA could induce inflammatory reaction in macrophagesLTA is an exotoxin of S. aureus. In order to determine whether LTA could cause an inflammatory effect in macrophages, we used different concentrations of LTA to treat the RAW264.7cells and primary mouse peritoneal macrophages and analyzed TNF-a expression by ELISA. When the concentration of LTA is from0to2μg/mL, the con-centration of TNF-a increased from4.56±0.89to7.49±1.80ng/L in RAW264.7cells and from5.18±0.91to11.22±0.88ng/L in primary mouse peritoneal macrophages, in a concentration dependent manner while high doses of LTA from5to20μg/mL could cause acute cell death and attenuate the expression of TNF-a. This result indicates that0-2μg/mL of LTA can induce inflammation reaction in macrophages, but a high concentration of LTA could induce acute cell injury and death.3. LL-37expression in LTA treated RAW264.7cellsTo investigate the change of LL-37in LTA induced inflammation in RAW264.7cells, we checked LL-37expression in RAW264.7cells after LTA treatment. We also put chemically synthesized LL-37in the culture medium of RAW264.7cells to check its uptake efficiency by immunofluorescence staining and Western blot. There are four groups in the experiment:control group, LL-37treated positive control group, LTA treated group, and LTA and LL-37double treated group. After LTA treatment, the expression of LL-37is1.33±0.13fold compared with the control group (p<0.05). In the chemically synthesized LL-37and LTA double treated group, the expression of LL-37increases1.93±0.18fold compared with the control group (p<0.05). The LL-37expression in the double treated group is higher than that in the LTA treated group (p<0.05), but no significant difference with the LL-37treated group (p>0.05). These results indicated that LTA could effectively increase LL-37expression and chemically synthesized LL-37could be transported into RAW264.7cells.4. LL-37could attenuate LTA induced inflammation in macrophagesTo investigate whether LL-37could attenuate LTA induced inflammation reaction in macrophages, we check the TNF-a expression in different concentrations of LL-37in RAW264.7cells and primary mouse peritoneal macrophages by ELISA. We used2μg/mL of LTA treated RAW264.7cells and primary mouse peritoneal macrophages to induce inflammation. Then we used0-40μg/mL of LL-37to treat the RAW264.7 cells and primary mouse peritoneal macrophages.1-15μg/mL of LL37could effectively attenuate TNF-a expression in a concentration dependent manner, and decrease TNF-a to4.63±0.81ng/L compared with the basal level of6.46±0.36ng/L after LTA treatment in RAW264.7cells. And in primary mouse peritoneal macrophages,1-10μg/mL of LL37could effectively attenuate TNF-a expression in a concentration dependent manner, and decrease TNF-a to6.69±0.64ng/L compared with the basal level of10.52±0.92ng/L. The medium TNF-a concentration decreased to the lowest at the LL-37concentration of15μg/mL in RAW264.7cells and10μg/mL in primary mouse macrophages. On the other hand, if LL-37concentration is increased at over15μg/mL in RAW264.7cells and10μg/mL in primary mouse peritoneal macrophages, it will cause cell damage and increase TNF-a expression. Thus a moderate concentration of LL-37could attenuate LTA induced inflammation in macrophages. A high concentration of LL-37will cause cell damage. According to this, we used the optimal LL-37concentration of15μg/mL in RAW264.7cells and10μg/mL in primary mouse peritoneal macrophages for the rest of the experiments.5. LL-37attenuates LTA induced inflammatory reaction by decreasinginflammatory cytokine expression TNF-a and IL-6are two important inflammatory cytokines during inflammation. We checked TNF-a and IL-6expression by real time RT-PCR in RAW264.7cells and ELISA in primary mouse peritoneal macrophages. In the LTA treated group, the inflammatory cytokines TNF-a and IL-6mRNA increased8.75±0.47and34.12±9.68fold compared with the control group. The mRNA of TNF-a and IL-6in the LL-37and LTA double treated group decreased obviously compared with the LTA treated group.LL-37treatment alone could decrease cell TNF-a and IL-6mRNA expression. Similarly, in the LTA treated primary mouse peritoneal macrophages, the inflammatory cytokine TNF-a and IL-6expression increased to7.53±2.21ng/L and180.38±29.46ng/L compared with the basal level of4.98±1.30ng/L and16.75±9.06ng/L, respectively. The concentration of TNF-a and IL-6in the LL-37and LTA double treated group decreased obviously compared with that in the LTA treated group, and LL-37treatment alone could also decrease cell TNF-a and IL-6expression. So the exogenous chemically synthesized LL-37could attenuate LTA induced inflammatory reaction by the down regulation of TNF-a and IL-6expression in macrophages.6. LL-37attenuates LTA induced inflammation by the inhibition of p38MAPK andAkt activation in macrophages To investigate the signal transduction pathway of LL-37, we tested the activation of p38MAPK and Akt in the RAW264.7cells and primary mouse peritoneal macrophages. LTA could increase p38MAPK and Akt phosphorylation and cause the inflammatory reaction in the RAW264.7cells and primary mouse peritoneal macrophages. LL-37treatment could inhibit p38MAPK and Akt activation compared with the LTA treated group, which could attenuate inflammation reaction and modulate immune response. It suggested that LL-37could regulate inflammation by inhibition of p38MAPK and Akt signal transduction pathways in macrophages.Conclusions:1. In the cell model of Staphylococcus aureus infection induced by LTA, the expression of TNF-a, IL-6and LL-37increases, suggesting that LTA can induce macrophages to produce inflammatory reaction, LL-37is involved in the inflammatory response induced by LTA.2. LL-37reduced the expression of inflammatory factors, suggesting that LL-37can attenuate the inflammatory reaction.3. LTA could promote the phosphorylation of p38and Akt, however LL-37could inhibit the phosphorylation of p38and Akt, suggesting that LL-37takes part in the inflammatory reaction induced by LTA by inhibiting phosphorylation of p38and Akt.更多还原