电针抑制脊髓内趋化因子CX3CL1表达减轻炎性痛的实验研究

目的:通过观察电针对完全弗氏佐剂(CFA)所致大鼠炎性痛急性期脊髓组织内趋化因子CX3CL1(Fractalkine,FKN)及其受体CX3CR1表达的影响,探讨FKN是否可能参与电针镇痛的相关机制。方法:清洁级雄性SD大鼠82只,分两批进行实验。第1批大鼠40只,随机分为4组(n=10):空白对照组(Control组),CFA模型组(CFA组),电针治疗组(EA组)和非穴位电针组(Sham组),其中CFA、EA和Sham组使用CFA足底注射造成炎性痛模型;造模后EA组给予电针刺激双侧"足三里"(30 min,2 Hz连续波,1~2 m A),而Sham组接受非穴位针刺处理;分别于造模前及造模后1~7 d观察并测量大鼠机械缩足反应阈值(paw withdrawal mechanical threshold,PWMT)及热缩足反应阈值(paw withdrawal thermal latency,PWTL),观察电针对CFA所致慢性痛的治疗作用。第2批大鼠42只,仍按上述分组方法随机分入7组(其中CFA、EA、Sham组取造模后1、3 d两个时间点,Control组取一个时间点),在相应时间点处死实验动物取腰段脊髓组织进行Western Blot检验,观察电针干预对FKN及CX3CR1表达的影响。结果:CFA造模致实验动物PWMT及PWTL显著下降(P0.01),提示电针处理部分拮抗了CFA的致痛作用(P0.05);Western Blot结果显示,电针处理能够降低CFA所致动物脊髓组织升高的FKN表达,在造模后3 d最为显著(P0.01),但其受体CX3CR1表达无显著性差异。结论:电针刺激大鼠"足三里"能够改善CFA所致炎性痛症状,其中抑制脊髓的FKN表达可能是电针镇痛的机制之一。     

福辛普利对糖尿病大鼠肾组织中Fractalkine及CD68阳性细胞表达的影响

目的探讨福辛普利对糖尿病大鼠肾组织中CX3C类趋化因子Fractalkine和CD68(巨噬细胞标记物)阳性细胞表达的影响及其肾脏保护作用的机制。方法采用免疫组织化学法及RT-PCR检测肾组织中Fractalkine表达,同时采用免疫组织化学法检测肾组织CD68阳性细胞的分布,并对结果进行半定量分析。结果治疗8周后,免疫组织化学法及RT-PCR检测DM组大鼠肾组织中Fractalkine表达增强(P<0.05),肾小球内CD68阳性细胞显著增多,DF组Fractalkine表达明显减弱(P<0.05),同时肾小球内CD68阳性细胞亦显著减少,相关分析显示肾小球CD68阳性细胞数与Fractalkine mRNA表达呈正相关。结论Fractalkine在糖尿病肾组织中表达明显增强,且与肾小球内CD68阳性细胞表达正相关,说明该趋化因子在糖尿病性肾病炎症反应中起重要作用;治疗组大鼠Fractalkine较模型组明显降低,表明福辛普利对肾脏的保护作用可能与下调fractalkine,抑制糖尿病炎症状态有关。     

Detection of fractalkine in human seminal plasma and its role in infertile patients

BACKGROUND: Fractalkine is a relatively newly discovered CX(3)C chemokine, which is a chemoattractant for T cells, monocytes and natural killer cells. Several reports have demonstrated the association between chemokine levels in seminal plasma and semen quality. The fractalkine levels in ejaculates from normal donors and infertile male patients with or without asthenozoospermia, were examined and correlated with sperm motility and morphology. METHODS AND RESULTS: Western blot analysis showed fractalkine protein to be present in the seminal plasma. Fractalkine titres in the seminal plasma of infertile men with asthenozoospermia (0.64 +/- 0.04 microg/ml; n = 58) were lower than those in patients without asthenozoospermia (0.94 +/- 0.10 microg/ml; n = 22, P < 0.01) and fertile donors (1.04 +/- 0.07 microg/ml; n = 10, P < 0.001). There was no significant difference between fractalkine levels in patients with and without leukospermia. No significant correlation was found between fractalkine and interleukin-8 levels in seminal plasma. Sperm motility was positively correlated (R(2) = 0.14, P < 0.001) with fractalkine concentration. The existence of CX(3)CR-positive leukocytes in semen was confirmed using specific primers for CX(3)CR. CONCLUSIONS: These results suggest that fractalkine is a chemokine associated with sperm motility and the migration of CX(3)CR-positive leukocytes into semen.     

CX3CR1 receptor is up-regulated in monocytes of coronary artery diseased patients: impact of pre-inflammatory stimuli and renin-angiotensin system modulators

Fractalkine/CX3CR1 pathway is considered a major modulator of atherosclerosis. In the present study, expression of CX3CR1 on PBMCs/monocytes of healthy individuals and coronary artery diseased patients was initially assessed by flow cytometry. Effects of pre-inflammatory cytokines interferon (INF)-gamma and tumor necrosis factor (TNF)-alpha on expression of CX3CR1 and a single representative of each major chemokine family (CCR5 and CXCR4) were further assessed in three cell models: THP-1 monocytes, Jurkat T lymphocytes and primary monocytes isolated from healthy donors. Finally, effects of angiotensin-converting enzyme (ACE) inhibitors captopril, lisinopril and angiotensin receptor blocker (ARB) losartan on chemokine receptor expression were evaluated in the same cell models either in a naive or stimulated state. INF-gamma significantly affected the chemokine receptor phenotype of THP-1 cells by increasing the rate of CX3CR1-positive cells. Pre-treatment with the ACE inhibitors, captopril and lisinopril, and the ARB, losartan, did not influence these effects. Captopril and lisinopril similarly had no effect on either stimulated or naive primary monocytes. Yet, a small but repeatable increase in CX3CR1 expression after treatment with losartan was noted. Nevertheless, the latter observation did not retain statistical significance after applying the Bonferroni correction. In conclusion, our data did not indicate any significant effect of the ACE inhibitors on the chemokine receptor phenotype of monocytes.     

Fractalkine transgene induces T-cell-dependent antitumor immunity through chemoattraction and activation of dendritic cells

Fractalkine (FK, also called neurotactin or CX3CL1) is a CX3C chemokine that can chemoattract T lymphocytes, monocytes and NK cells. In our study, we investigated the induction of antitumor response by FK gene transfer. FK gene-modified 3LL lung carcinoma cells (3LL-FK) could both secrete soluble form and express membrane-bound form of FK. The tumor growth of 3LL-FK was decreased. Vaccination with 3LL-FK was effective in the induction of protective immunity and CTL. In vivo depletion analysis demonstrated that CD8(+) T cells are the main participating cells of the antitumor response. Obvious infiltrations of CD8(+) T cells, CD4(+) T cells and dendritic cells (DC) were observed in the tumor sites, suggesting that 3LL-FK might induce antitumor immunity through chemoattraction and activation of T cells and DC. Then we investigated the chemoattraction and activation of DC by 3LL-FK. Chemotaxis assay showed that the supernatants of 3LL-FK could chemoattract immature DC, which were found to express FK receptor CX3CR1, and the immature DC could obviously adhere to 3LL-FK. Adherence of DC to 3LL-FK resulted in phenotypic maturation and upregulated IL-12 secretion of DC, and more strong stimulation of allogeneic T-cell proliferation by DC. The increased production of IL-2 and IFNgamma in 3LL-FK tumor tissue was also observed. Our data suggested that FK gene transfer to tumor cells could induce T-cell-dependent antitumor immunity through chemoattraction and activation of DC.     

分泌型白细胞蛋白酶抑制剂对急性肺损伤大鼠趋化因子fractalkine表达的影响

目的: 探讨分泌型白细胞蛋白酶抑制剂(SLPI)对急性肺损伤新生大鼠肺组织中趋化因子fractalkine表达的影响.方法: 利用内毒素(LPS)气管内滴入制备新生大鼠急性肺损伤的模型;设立SLPI治疗组、LPS组、生理盐水(NS)对照组.SLPI治疗组在气管内滴入LPS前预先滴入400 μg SLPI.分别在6,12,24 h后取肺组织匀浆,用ELISA法分别测定各组趋化因子fractalkine蛋白水平.RT-PCR测定fractalkine的mRNA水平.结果: 在各时间点,SLPI治疗组肺组织中Fractalkine蛋白水平及mRNA水平均显著低于LPS组.24 h时SLPI治疗组肺组织中fractalkine蛋白为(3 124.9±155.6)pg/ml,显著低于LPS组的(4 231.4±177.9)pg/ml和NS对照组的(4 412.4±201.5)pg/ml,F=42.8, P＜0.05,差异有统计学意义.24 h时SLPI治疗组肺组织中fractalkine的mRNA相对水平为47.0±5.1,显著低于LPS组的87.3±8.2和NS对照组的81.1±9.9,F=33.2, P＜0.05,差异有统计学意义.结论: SLPI能有效抑制新生大鼠急性肺损伤时肺组织中趋化因子fractalkine的表达.     

Effect of breviscapine on fractalkine expression in chronic hypoxic rats

Fractalkine （FKN） is the only known chemokine that fulfils the dual functions of an adhesive molecule and a soluble chemoattractant.1 FKN expression was reported increase in lungs of patients with severe pulmonary arterial hypertension,2 suggesting that FKN may participate in pathogenesis of pulmonary hypertension. Breviscapine is a flavonoid extracted from Erigeron breviscapus （Vant.） Hand. Mazz. Breviscapine can prevent the development of hypoxic pulmonary hypertension^3 but the mechanism is unknown. This study evaluated the role of FKN in the pathogenesis of hypoxic pulmonary hypertension and the effect of breviscapine on FKN in hypoxic pulmonary hypertension.     

Enhanced recruitment of CX3CR1+ T cells by mucosal endothelial cell-derived fractalkine in inflammatory bowel disease

BACKGROUND & AIMS: Fractalkine (FKN/CX3CL1) is a unique chemokine combining adhesive and chemotactic properties. We investigated FKN production by the mucosal microvasculature in inflammatory bowel disease (IBD), its capacity for leukocyte recruitment into the gut, and the number of CX3CR1+ cells in the circulation and mucosa of IBD patients. METHODS: The expression of FKN by human intestinal microvascular endothelial cells (HIMECs) and CX3CR1 by circulating cells was evaluated by flow cytometry, and mucosal CX3CR1+ cells were enumerated by immunohistochemistry. The capacity of FKN to mediate leukocyte binding to HIMECs was assessed by immunoblockade, and to induce HIMEC transmigration by a Transwell system. RESULTS: The spontaneously low HIMEC FKN expression was enhanced markedly by tumor necrosis factor-alpha plus interferon-gamma stimulation, or direct leukocyte contact. This effect was significantly stronger in IBD than control HIMECs. Up-regulation of HIMEC FKN expression was dependent on p38 and extracellular signal-regulated kinase phosphorylation, as was abrogated by selective mitogen-activated protein kinase inhibitors. Circulating T cells contained significantly higher numbers of CX3CR1+ cells in active IBD than inactive IBD or healthy subjects, and IBD mucosa contained significantly more CX3CR1+ cells than control mucosa. Antibody-blocking experiments showed that FKN was a major contributor to T- and monocytic-cell adhesion to HIMECs. Finally, FKN enhanced the expression of active beta1 integrin on leukocytes and mediated leukocyte HIMEC transmigration. CONCLUSIONS: In view of the capacity of FKN to mediate leukocyte adhesion, chemoattraction, and transmigration, its increased production by mucosal microvascular cells and increased numbers of circulating and mucosal CX3CR1+ cells in IBD point to a significant role of FKN in disease pathogenesis.     

Mito-KATP对缺氧复氧大鼠心肌微血管内皮细胞凋亡的影响及作用机制

目的探讨线粒体ATP敏感性钾通道(mito-KATP)开放影响缺氧复氧大鼠心肌微血管内皮细胞(MMECs)凋亡的作用机制。方法培养大鼠心肌微血管内皮细胞,随机分为四组:对照组(N组)、模型组(H/R组)、开放剂组(DZ组)、阻断剂组(5-HD组)。DZ组加入100μmol/L二氮嗪预处理2 h,5-HD组在加入100μmol/L二氮嗪前,先用100μmol/L 5-羟葵酸预处理2 h,然后上述两组和H/R组同样进行缺氧2 h复氧2 h。Hoechst染色方法观察凋亡细胞形态,Annexin V-FITC/PI双标记法测定各组细胞凋亡率、RT-PCR法检测各组NF-κB、FKN和p53 mRNA转录水平。结果与N组比较,H/R组可见大量细胞坏死、脱落,细胞凋亡率升高(P<0.01),NF-κB、FKN和p53 mRNA表达上调(P<0.01);与H/R组比较,DZ组可见部分细胞坏死脱落,细胞凋亡率降低(P<0.01),NF-κB、FKN和p53 mRNA表达下调(P<0.01);5-HD组与H/R组比较无显著差异。结论 mito-KATP开放可抑制缺氧复氧所致MMECs凋亡,其机制可能与抑制NF-κB、FKN及p53 mRNA表达有关。     

冠心病患者血清趋化因子和白细胞介素-18水平分析

目的:检测冠心病(CHD)患者血清趋化因子(FKN)和白细胞介素-18( IL-18)水平.方法:经冠脉造影确诊的CHD患者62例,其中稳定性心绞痛32例(SAP组),急性冠脉综合征30例(ACS组);另选经冠脉造影排除CHD者21例为对照.用酶联免疫吸附法测定各组患者血清FKN和IL-18的水平;记录Gensini积...