【作者】 张军；

【导师】 李元海；

【作者基本信息】 安徽医科大学 ， 麻醉学， 2017， 博士

【摘要】 研究背景曲马多(Tramadol),是一种中枢神经镇痛药,在临床上广泛用于治疗多种形式的急慢性疼痛,包括肿瘤/癌症疼痛、急性严重疼痛、神经性疼痛、骨关节炎疼痛、分娩疼痛以及剖宫产疼痛等。目前研究认为,曲马多的镇痛效果主要是通过曲马多以及其代谢产物M1激活μ-阿片样受体,以及通过两对对映体抑制去甲肾上腺素(NE)和5-羟色胺受体(5-HT)的再摄取。曲马多较其他止痛药物有很多临床优势,但其潜在机制还有待进一步研究。越来越多的证据表明,曲马多可以刺激免疫细胞的免疫反应过程,例如增加白细胞介素-2(IL–2)的生成,增强自然杀伤细胞(NK细胞)的活性,脾淋巴细胞的增殖,T淋巴细胞的增殖以及激活腹膜吞噬细胞。然而,之前的研究多是检测全身注射曲马多后身体的免疫反应。在这些情况下,并不能区分出曲马多对免疫细胞的直接作用与通过NE和5-HT介导的间接作用。巨噬细胞是主要的免疫细胞之一,但是曲马多是如何调节巨噬细胞的目前还不清楚,本研究选择人脐带血,从中分离出巨噬细胞进行离体培养,通过流式细胞仪、酶联免疫标记试验(ELISA)、染色质免疫沉淀反应(Ch IP)检测等方法来研究曲马多对人巨噬细胞的直接作用,包括细胞的数目、形态、细胞因子产物的变化以及潜在的分子信号通路等。研究目的(1)研究曲马多对离体培养的人脐带血中巨噬细胞的直接影响,包括存活数量、形态以及抗原呈递功能等。(2)研究曲马多对人脐带血中M1型和M2型巨噬细胞参与免疫反应的调节方式以及相关的分子机制。研究方法(1)采集脐带血:胎儿分娩出后立即抽取5 ml脐带静脉血,储存于-80℃冰箱中备用。每次实验开始前血液样本在37℃环境下解冻。(2)巨噬细胞的分离、培养和诱导:分离的单核细胞悬液加入双抗PE-结合的anti-CD68和APC-结合的anti-CD11b进行孵育,通过对CD11b-PE抗体和CD68-PE抗体标记的细胞进行流式分析,确定巨噬细胞的数量及所占比率;分离的巨噬细胞在24孔板中悬浮培养并观察;5天后,细胞饥饿过夜并进行感染,然后暴露于LPS(100 ng/m L)和IFN-γ(10 ng/ml),或者IL-4(10 ng/ml),加入曲马多的含量为80或400μg/ml,倒置显微镜下观察细胞的形态学特征并照相。(3)采用Giemsa染色方法检测纯培养系统中巨噬细胞的存活情况。(4)q PCR检测分别由LPS和IFN-γ或者IL-4刺激下的巨噬细胞相关细胞因子(IL-6、TNF-a和IL-12)m RNA含量。(5)ELISA检测分别由LPS和IFN-γ或者IL-4刺激下的巨噬细胞相关细胞因子(IL-6、TNF-a和IL-12)蛋白质含量。(6)染色质免疫沉淀反应(CHIP)检测巨噬细胞中Arg1 DNA与STAT6结合的情况。(7)Western blotting实验分析分别由LPS和IFN-γ或者IL-4刺激下的巨噬细胞中的信号转导通路相关分子的变化,包括:NF-κB级联中的IKBa、在ERK1/2级联中的ERK1/2和磷酸化的ERK1/2(P-ERK1/2),P38级联中的P38和磷酸化P38,STAT6级联中的STAT6和磷酸化的STAT6(P-STAT6)。研究结果在人脐带血的血液样本中加入曲马多对巨噬细胞细胞百分比和数量没有明显差异。对照组和实验组在各时间点,脂多糖(LPS)诱导导致的巨噬细胞表面的主要组织相容性复合体II(MHCII)、两个表面共刺激物(CD80和CD86)三种细胞因子水平之间没有显著差异。曲马多显著降低了巨噬细胞中IL-6 m RNA、IL-12a m RNA和IL-12b m RNA的表达(P<0.05),而TNF-α得下降并没有明显的统计学意义(P>0.05)。80 ng/ml和400 ng/ml的曲马多刺激都显著增加了IL-4诱导的M2型巨噬细胞中细胞因子Arg1、Mrc1、Ym1以及Fizz1的表达,400 ng/ml组的增强效果要明显高于80 ng/ml组(P<0.01)。研究结论(1)曲马多并没有改变离体培养的来自人脐带血中巨噬细胞的存活数量和形态,也没有改变静息态巨噬细胞对LPS诱导的炎症反应的抗原呈递功能。(2)曲马多通过不同的方式调控了人脐带血中M1和M2型巨噬细胞分泌细胞因子:曲马多一方面抑制了M1型巨噬细胞的细胞因子的IL-6、IL-12a和IL-12b的生成和分泌,另一方面促进了M2型巨噬细胞合成炎症反应相关分子Arg1、Mrc1、Ym1、Fizz1。(3)曲马多对脐带血中M1型巨噬细胞参与炎症反应的调节并不依赖于NF-κB和MAPK通路的级联反应;曲马多对M2型巨噬细胞的调节,可能是STAT6级联反应所介导。因此,本研究揭示了曲马多主要是通过抑制脐带血中M1型巨噬细胞的功能(杀灭过程),同时促进M2型巨噬细胞的功能(愈合过程),这两种不同的调节方式和参与机制来调节炎症反应。曲马多对脐带血中巨噬细胞的影响可能是一种对缓解神经性疼痛的一种潜在的机制。更多还原

【Abstract】 Background Tramadol is an analgesic drug,widely used to treat multiple forms of pain,including acute severe pain,neuropathic pain,tumor/cancer pain,osteoarthritis,pain during labor and cesarean section,etc.Tramadol is putatively considered to exert its analgesic effects through activating μ-opioid receptors and inhibiting serotonin and noradrenaline reuptake.Emerging advantages of tramadol over other analgesic drugs have been discovered,but the underlying mechanisms await further investigations.Accumulating evidence shows that tramadol stimulates immune responses,such as an increase of IL-2 production,enhancement of NK cell activity,splenocyte proliferation,T-lymphocyte proliferation,and activation of peritoneal phagocytes.These effects could be mediated by μ-opioid receptors expressed in the immune cells.However,the previous studies tested immune responses after systemic administration of tramadol.In these situations,direct effects of tramadol on immune cells cannot be dissociated from its indirect effects via noradrenaline and serotonin receptors.Considering macrophages are a type of immune cell but are not known to be modulated by tramadol,we isolated macrophages from human umbilical cord blood and examined the direct effects of tramadol on macrophages,including number,morphology,cytokine products,and underlying signal transduction pathways,with the methods of flow cytometry,enzyme-linked immunoabsorbent assay(ELISA),chromatin immunoprecipitation(Ch IP)assays,luciferase assay.Objective(1)To study the direct effects of tramadol on macrophages cultured in vitro from human umbilical cord blood,including the number of surviving cells,morphology and antigen presenting function.(2)To study the regulation of tramadol on the immune responses of M1 and M2 macrophages in human umbilical cord blood and related molecular mechanisms.Metholds(1)Blood collection and storage: 5 ml blood was collected from umbilical cord from each subject,and was immediately stored at-80 ℃.Before experiments,the samples were thawed at 37 ℃.(2)Macophage isolation,culture and induction: The white blood cells were incubated with PE-conjugated anti-CD68 and APC-conjugated anti-CD11 b.Flow Cytometry was employed to sort cells positive for both CD11 b and CD68,representing macrophages.Isolated macrophages were cultured in suspension in 24-well plates and observed.After 5 days,the cells were starved overnight and infected,then exposed to LPS(100 ng / m L)and IFN-γ(10 ng / ml),or IL-4(10 ng / ml).The content of tramadol was added to 80 or 400 μg / ml.Morphological characteristics of the cells were observed under an inverted microscope.(3)Giemsa staining was used to test the survival of macrophages in pure culture system.(4)q PCR was used to test the m RNA levels of macrophage-associated cytokines(IL-6,TNF-a and IL-12)stimulated by LPS and IFN-γ or IL-4,respectively.(5)ELISA was used to detect the protein content of macrophage-related cytokines(IL-6,TNF-a and IL-12)stimulated by LPS and IFN-γ or IL-4 respectively.(6)Chromatin immunoprecipitation(CHIP)was used to detect the binding of Arg1 DNA to STAT6 in macrophages.Results The results of this study showed that the percentage and quantity of macrophage cells in the blood samples of human cord blood were unchanged.Control group and experimental group at various time points,lipopolysaccharide(LPS)induced macrophage surface caused by the major histocompatibility complex II(MHCII)and two surface stimuli(CD80 and CD86)there were no significant differences between three kinds of cytokine levels.The expression of IL-6 m RNA,IL-12 a m RNA and IL-12 b m RNA in macrophages was significantly reduced by tramadol(P < 0.05),while the decline of TNF-alpha was not statistically significant(P > 0.05).80 ng/ml and 400 ng/ml tramadol stimulation were significantly increased the IL-4 cytokines induced type M2 macrophages Arg1,Mrc1,Ym1 and Fizz1 expression,enhancement effect to 400 ng/ml group was obviously higher than 80 ng/ml group(P < 0.01).(1)Tramadol treatment did not alter the percentage and number of macrophage cells in umbilical cord blood after treatment.Meanwhile,tramadol did not affect the levels of CD80,CD86,and MHC,suggesting that it does not change antigen-presenting responses of macrophages to LPS.Conclusion(1)Tramadol did not change not only the apoptosis,survival,and morphology of resting macrophages,but also the antigen-presenting function of macrophages.(2)Tramadol differentially regulated the secretion of cytokines by M1 and M2 macrophages in human umbilical cord blood.We found that tramadol inhibited the cytokines IL-6,IL-12 a and IL-12 b in M1 macrophages,except TNF-a,and promoted M2 macrophages synthesis inflammation-related molecules Arg1,Mrc1,Ym1,Fizz1.(3)Tramadol did not affect Ik B,and phosphorylation of ERK1/2 and p38,suggesting that tramadol may exert its effects on inflammation responses to LPS through the signaling pathways other than NF-k B,ERK1/2 and p38 cascades.Meanwhile,STAT6 pathway may underlie the effects of tramadol.Therefore,this study reveals that tramadol regulates inflammation by controlling the function of M1 macrophages(killing process),but promoting the function of M2 macrophages(healing process).These effects may contribute to its therapeutic benefit for chronic neuropathic pain.更多还原