A Study of Activation and Apoptosis of Peripheral Blood Mononuclear Cells and Lamina Propria Mononuclear Cells in Inflammatory Bowel Diseases

分别从外周静脉血和手术标本中分离外周血单核细胞(PBMCs)和固有层单核细胞(LPMCs)，分别用6%葡聚糖沉淀和Ficoll-Hypaque密度梯度离心法分离。从PBMCs和LPMCs中，用磁性细胞分选法分离出CD4阳性T细胞。流式细胞仪分析证实分离的CD4+T细胞纯度很高(PBMCs：99.0±0.3%，LPMCs：98.5±0.4%)。在包被抗CD3和抗CD28抗体的微孔板中培养CD4阳性T细胞。IL-2受体(IL-2R)α链和膜型肿瘤坏死因子-α(TNF-α)在PBMC和LPMCs中的表达均在96h达到高峰。与PBMC中的CD4阳性T细胞相比，LPMCs中的CD4阳性T细胞中IL-2R和TNF-αmRNA的表达更为常见。一种嵌合的抗肿瘤坏死因子-α抗体(英夫利昔单抗)诱导了CD_4阳性T细胞的凋亡，表明…更多的证据表明，在体外治疗之前，预测药物的疗效是可能的。13例克罗恩病患者应用英夫利昔单抗治疗。用克罗恩病活动指数(CDAI)评定临床疗效，用双抗体夹心法检测治疗前后血清可溶性白介素2受体(sIL-2R)、白介素6(IL-6)、肿瘤坏死因子-α(TNF-α)水平。用流式细胞仪检测英夫利昔单抗培养后外周血和固有层T淋巴细胞的凋亡率。英夫利昔单抗治疗后CDAI和血清sIL-2R、IL-6、TNF-α水平多数下降。CDAI下降70以上的6例患者治疗前血清肿瘤坏死因子-α水平均低于5 ng/ml，英夫利昔单抗可诱导固有层T淋巴细胞的凋亡，但不能诱导外周T淋巴细胞的凋亡。低水平的血清肿瘤坏死因子-α可能是英夫利昔单抗治疗的一个指标。抗细胞因子药物诱导固有层T淋巴细胞凋亡可能是其抗炎症性肠病的重要机制之一。较少

Peripheral blood mononuclear cells (PBMCs) and lamina propria mononuclear cells (LPMCs) were isolated from the peripheral venous blood and the surgical specimens, respectively, by 6% dextran sedimentation followed by density gradient centrifugation on Ficoll-Hypaque. From PBMCs and LPMCs, CD4-positive T cells were isolated using magnetic cell sorting. Flow cytometric analysis confirmed that isolated CD4-positive T cells were very pure (PBMCs: 99.0±0.3%, LPMCs: 98.5±0.4%). CD4-positive T cells were cultured in microwell plates coated with anti-CD3 and anti-CD28 antibodies. The expression of IL-2 receptor (IL-2R) alpha chain and membranous type TNF-alpha mRNA were peaked after 96h in CD4-positive T cells from either PBMCs or LPMCs. Both IL-2R and TNF-alpha mRNA expressions were detected more commonly in CD4-positive T cells from LPMCs compared with CD4-positive T cells from PBMCs. A chimeric anti-TNF-alpha antibody (infliximab) has induced apoptosis on CD4-positive T cells, which indicat … More es that it might be possible to predict the efficacy of drugs before treatment in vitro. Thirteen patients with Crohn's Disease were treated with infliximab. Clinical response was evaluated using the Crohn's Disease Activity Index (CDAI), and serum soluble IL-2 receptor (sIL-2 R), IL-6, and TNF-alpha levels were analyzed by ELISA before and after treatment. Apoptosis of peripheral and lamina propria T-lymphocytes after culture with infliximab was detected by flow cytometry. CDAI and serum sIL-2 R, IL-6 and TNF-alpha levels decreased in most patients after treatment with infliximab. Serum TNF-alpha levels before treatment in the six patients in whom CDAI decreased by more than 70 were less than 5 ng/ml. Infliximab induced the apoptosis of lamina propria but not peripheral T-lymphocytes. A low level of serum TNF-alpha might be an indicator for infliximab treatment. The induction of apoptosis of lamina propria T-lymphocytes by anti-cytokine drugs may be one of the important mechanisms of their anti-inflammatory effect in patients with inflammatory bowel disease. Less