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目的:建立猪外周血来源的未成熟树突状细胞(im-DC)体外诱导、扩增方法。探讨负载猪源性pCTLA4-Ig重组腺病毒的猪imDCs对大鼠脾细胞增殖的影响。方法:家猪外周血单个核细胞(PBMC),以rpGM-CSF(30μg/L)和rpIL-4(20μg/L)体外诱导培养,收集悬浮细胞经电镜、流式细胞仪鉴定。利用Adv-pCTLA4-Ig转染猪imDCs,RT-PCR鉴定。对比转染组与未转染组imDCs刺激SD大鼠脾细胞增殖的能力。结果:获得第4～5天悬浮细胞具有典型的imDCs形态特征,表达猪源性DC的特异性标志SLA-DR、CD172a,而CD80/CD86表达较低。Adv-pCTLA4-Ig转染猪imDCs行RT-PCR出现pCTLA4-Ig、吲哚胺2,3-二氧化酶(IDO)基因的表达。混合淋巴细胞培养转染组与未转染组imDCs刺激指数有显著性差异(P<0.05)。结论:联合应用rpGM-CSF和rpIL-4可诱导猪PBMC衍生成大量的imDCs。负载pCTLA4-Ig基因的猪imDCs,能够表达IDO,降低对异种抗原的免疫应答,显著抑制异种脾细胞增殖。
Objective: To establish a method for in vitro induction and expansion of immature dendritic cells (im-DC) derived from porcine peripheral blood. To investigate the effect of porcine imDCs carrying pig-derived pCTLA4-Ig recombinant adenovirus on the proliferation of rat spleen cells. Methods: Porcine peripheral blood mononuclear cells (PBMCs) were cultured in vitro with rpGM-CSF (30μg / L) and rpIL-4 (20μg / L). The suspension cells were collected and identified by electron microscopy and flow cytometry. The imDCs were transfected with Adv-pCTLA4-Ig and identified by RT-PCR. The transfected group and untransfected group imDCs stimulated the proliferation of splenic cells in SD rats. RESULTS: Suspension cells obtained typical morphological features of imDCs on day 4 to 5, expressing SLA-DR and CD172a, but not CD80 / CD86. The expression of pCTLA4-Ig and indoleamine 2,3-dioxygenase (IDO) gene was detected by RT-PCR in pig imDCs transfected with Adv-pCTLA4-Ig. The stimulation index of imDCs in mixed lymphocyte culture transfected group and untransfected group was significantly different (P <0.05). CONCLUSION: Combined use of rpGM-CSF and rpIL-4 can induce porcine PBMC to derive a large number of imDCs. Pig imDCs harboring pCTLA4-Ig gene can express IDO, reduce the immune response to xenoantigen, and significantly inhibit the proliferation of xenogeneic splenocytes.