特应性皮炎患者外周血T淋巴细胞Rho激酶活性变化及其意义

摘要/Abstract

摘要：

目的观察特应性皮炎（AD）患者外周血T淋巴细胞中Rho激酶活化情况，分析其临床意义。方法收集60例AD患者和60例健康儿童外周肝素抗凝血8 ml，分离提取T细胞和血清。分别用AD血清和健康对照血清培养AD患者T细胞或健康对照T细胞，分为患者T细胞 + 自身血清组、患者T细胞 + 健康对照血清组、健康对照T细胞 + 自身血清组、健康对照T细胞 + AD血清组。此外，分别用Rho激酶特异性抑制剂Y27632（Y27632组）、CD3/CD28单抗（CD3/CD28单抗组）、Y27632 + CD3/CD28单抗（Y27632 + CD3/CD28单抗组）处理AD患者T细胞，自身血清培养AD患者T细胞为患者T细胞组。采用Western印迹法检测各组Rho激酶活性，四甲基偶氮唑蓝比色法（MTT）检测T细胞增殖活性，ELISA检测白细胞介素（IL）6、IL-10水平。结果新鲜分离的AD患者外周血T细胞Rho激酶活性（2.47% ± 0.89%）显著高于健康对照组（0.65% ± 0.35%，t = 2.729，P < 0.05）。AD患者T细胞在体外经10%胎牛血清培养24 h后Rho激酶活性（0.70% ± 0.38%）较培养前显著降低（t = 2.658，P < 0.05），但与培养24 h后健康对照T细胞（0.63% ± 0.32%）相比，差异无统计学意义（t = 1.010，P > 0.05）。与健康对照血清培养T细胞相比，AD血清培养T细胞会增加Rho激酶活性（F = 8.22，P < 0.001），患者T细胞 + 自身血清培养24 h后Rho激酶活性（2.41% ± 0.87%）明显高于患者T细胞 + 健康对照血清组（0.76% ± 0.41%），健康对照T细胞 + AD血清组（2.17% ± 0.85%）显著高于健康对照T细胞 + 自身血清组（0.64% ± 0.33%），差异均有统计学意义（P < 0.05）。Y27632可显著抑制AD患者T细胞的增殖和IL-6的分泌（F = 18.68、22.95，P < 0.001），Y27632组T细胞增殖率、IL-6的表达均显著低于患者T细胞组（均P < 0.05），且Y27632 + CD3/CD28单抗组也显著低于CD3/CD28单抗组（均P < 0.05），而Y27632对AD患者T细胞IL-10分泌无显著影响。结论 AD患者T淋巴细胞存在Rho激酶信号活化异常，提示Rho激酶信号异常在AD发病过程中可能发挥着一定的作用。

Abstract:

Liang Yinghong, Wei Ming, Tu Ling, Liu Jia, Gong Yanjie, Zhang Yihua Clinical Laboratory, Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China Corresponding author: Wei Ming, Email: gushiweiming@126.com 【Abstract】 Objective To evaluate changes of Rho kinase （ROK） activity in peripheral blood T lymphocytes from patients with atopic dermatitis （AD）, and to analyze their clinical significance. Methods Eight milliliters of heparin-anticoagulated blood samples were collected from 60 patients with AD and 60 healthy human controls followed by separation of T lymphocytes and sera from these blood samples as well as culture of isolated T lymphocytes with 10% fetal bovine serum for 24 hours. Both patient- and control-derived T lymphocytes were classified into two groups to be cultured with patient- or control-derived sera. In addition, some patient-derived T lymphocytes were classified into 4 groups: Y27632 group treated with the Rho kinase-specific inhibitor Y2763, CD3/CD28 group treated with anti-CD3/anti-CD28 monoclonal antibodies, Y27632 + CD3/CD28 group treated with Y27632 and anti-CD3/anti-CD28 monoclonal antibodies, and control group treated with patient-derived sera. Subsequently, Western-blot analysis was performed to evaluate ROK activity in cells, methyl thiazolyl tetrazolium （MTT） assay to evaluate proliferative activity of T lymphocytes, and ELISA to measure interleukin 6 （IL-6） and IL-10 levels in supernatants of T lymphocytes. Results ROK activity was significantly lower in fresh T lymphocytes from patients than in those from healthy controls （2.47% ± 0.89% vs. 0.65% ± 0.35%, t = 2.729, P < 0.05）. After 24-hour culture with 10% fetal bovine serum in vitro, ROK activity was significantly decreased in patient-derived T lymphocytes compared with those before culture （0.70% ± 0.38% vs. 2.47% ± 0.89%, t = 2.658, P < 0.05）, but no significant difference was observed between patient- and control-derived T lymphocytes （0.70% ± 0.38% vs. 0.63% ± 0.32%, t = 1.010, P > 0.05）. Compared with T lymphocytes cultured with control-derived sera, those cultured with patient-derived sera showed significantly increased ROK activity （F = 8.22, P < 0.001）. Concretely speaking, ROK activity was significantly higher in patient-derived T lymphocytes cultured with patient-derived sera than in those cultured with control-derived sera （2.41% ± 0.87% vs. 0.76% ± 0.41%, P < 0.05）, and higher in control-derived T lymphocytes cultured with patient-derived sera than in those cultured with control-derived sera （2.17% ± 0.85% vs. 0.64% ± 0.33%, P < 0.05） at 24 hours. Y27632 could significantly inhibit the proliferation of as well as secretion of IL-6 （F = 18.68, 22.95, respectively, both P < 0.001） by patient-derived T lymphocytes, but had insignificant effects on secretion of IL-10. The cellular proliferative activity and IL-6 supernatant level were significantly lower in the Y27632 group than in the control group, and lower in the Y27632 + CD3/CD28 group than in the CD3/CD28 group （all P < 0.05）. Conclusion Aberrant activation of ROK exists in T lymphocytes from patients with AD, which may play a certain role in the pathogenesis of AD.

梁颖红魏明涂玲刘佳龚艳杰张宜花. 特应性皮炎患者外周血T淋巴细胞Rho激酶活性变化及其意义[J]. 中华皮肤科杂志, 2016, 49(4): 256-260.

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