Chinese Journal of Tissue Engineering Research ›› 2014, Vol. 18 ›› Issue (32): 5120-5125.doi: 10.3969/j.issn.2095-4344.2014.32.007
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Yiliyaer Yilihamu1, Wang Yun-hai1, Wang Li1, Xu Xin-cai1, Aimulaguli Abula2, Zhang Wen-bin1
Received:
2014-07-09
Online:
2014-08-06
Published:
2014-09-18
Contact:
Zhang Wen-bin, M.D., Associate chief physician, Department of Gastrointestinal Surgery, First Affiliated Hospital of Xinjiang Medical University, Urumqi 830000, Xinjiang Uygur Autonomous Region, China
About author:
Yiliyaer Yilihamu, Studying for master’s degree, Physician, Department of Gastrointestinal Surgery, First Affiliated Hospital of Xinjiang Medical University, Urumqi 830000, Xinjiang Uygur Autonomous Region, China
Supported by:
the Tissue Engineering Special Fund of the First Affiliated Hospital of Xinjiang Medical University, No. 201022GC02; the Natural Science Foundation of Xinjiang Uygur Autonomous Region, No. 2014211C036; the National Key Clinical Specialty: the Project of General Surgery
CLC Number:
Yiliyaer Yilihamu, Wang Yun-hai, Wang Li, Xu Xin-cai, Aimulaguli Abula, Zhang Wen-bin. Interferon-gamma enhances immunosuppression mediated by adipose-derived stem cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(32): 5120-5125.
2.1 脂肪源性干细胞表面白细胞抗原Ⅱ的表达 流式细胞仪检测结果显示白细胞抗原Ⅱ在脂肪源性干细胞表面阳性表达较弱,但在γ型干扰素刺激48 h后,白细胞抗原Ⅱ类分子表达明显上调(图1),说明脂肪干细胞免疫原性低,经γ型干扰素诱导后,白细胞抗原Ⅱ表达增强,免疫抑制能力可能增强。 2.2 经γ型干扰素诱导后脂肪源性干细胞中吲哚胺-2,3-双加氧酶表达 研究表明人间充质干细胞的免疫抑制作用是通过吲哚胺-2,3-双加氧酶介导的[4],因此,为观察脂肪源性干细胞是否也通过吲哚胺-2,3-双加氧酶介导免疫抑制作用,将脂肪源性干细胞用γ型干扰素处理后,实时PCR及Western blot法分别检测其吲哚胺-2,3-双加氧酶基因和蛋白的表达。 实时PCR结果显示,脂肪源性干细胞低表达吲哚 胺-2,3-双加氧酶基因,但在用γ型干扰素处理后,吲哚 胺-2,3-双加氧酶基因表达明显上调,是未处理时的(2.86±0.32)倍(图2 A)。 Western blot结果显示脂肪源性干细胞低表达吲哚 胺-2,3-双加氧酶蛋白,同时在γ型干扰素诱导后吲哚 胺-2,3-双加氧酶蛋白表达增加(图2 B)。 2.3 脂肪源性干细胞单向刺激淋巴细胞增殖反应 根据测量的CPM值,可见未经处理的脂肪源性干细胞并不能刺激淋巴细胞增殖(1 652±189) CPM;同样,当脂肪源性干细胞经γ型干扰素处理后,淋巴细胞也未见明显增殖(1 735±201) CPM,两者差异无显著性意义(P > 0.05),说明无论脂肪源性干细胞是否高表达白细胞抗原Ⅱ,脂肪源性干细胞均不会刺激异体淋巴细胞增殖,间接证明脂肪源性干细胞具有低免疫原性。 但在对照组中,加入异体淋巴细胞,淋巴细胞有明显增殖(52 886±3 862)CPM,与经干扰素处理的细胞的差异有显著性意义(P < 0.05)。 2.4 脂肪源性干细胞对非特异性淋巴细胞反应的作用 当淋巴细胞用植物血凝素刺激后,会出现明显增殖,加入脂肪源性干细胞后,除1×102组脂肪源性干细胞外,其余浓度的细胞均可以明显抑制淋巴细胞的增殖(P < 0.05);但当脂肪干细胞用多聚甲醛固定失活后,所有浓度的抑制作用均消失(图3A)。 2.5 脂肪源性干细胞对混合淋巴细胞反应的作用 为了观察脂肪源性干细胞能否抑制异体淋巴细胞刺激导致的淋巴细胞增殖,在双向混合淋巴细胞培养中加入脂肪源性干细胞,作为第三者细胞。 共培养7 d后可见,除1×102组外,1×103-1×105数量级的脂肪源性干细胞均使淋巴细胞的增殖得到明显抑制。脂肪源性干细胞以多聚甲醛作用失活后,所有浓度的抑制作用均消失(图3B)。 流式细胞术检测到具有活性脂肪源性干细胞组的上清液中有大量γ型干扰素(21.354±1.78) μg/L,而失活脂肪源性干细胞组的上清液中γ型干扰素含量较少,几乎检测不到,证明脂肪源性干细胞在抑制淋巴细胞增殖过程中,会释放大量的γ型干扰素,从而促使吲哚胺-2,3-双加氧酶表达,发挥免疫抑制作用。"
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