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目的探讨不同发育阶段树突状细胞(DC)的微观流变学特性及其差异。方法应用密度梯度离心法从正常人外周血分离获得单个核细胞,经贴壁培养和免疫磁珠阴性选择获取纯化的单核细胞,以此作为DC前体(pDC)在体外培养诱导获得未成熟DC(imDC)和成熟DC(mDC)。并用免疫细胞化学、免疫荧光、酶细胞化学、流式细胞仪等技术对不同发育阶段的DC进行鉴定;应用微观流变学研究方法对以上细胞的电泳率、渗透脆性、膜流动性和傅立叶红外光谱(FT-IR)进行测量和分析。结果改进的分选法所获得的CDl4+pDC>97%。体外加入rhGM-CSF和IL-4培养6d后,S-100+imDC占细胞总数的96.9%;再加入rhGM-GSFI、L-4和TNF-α培养48 h后,CD86+mDC占细胞总数的87%。其微观流变学特性为:mDC电泳率明显高于pDC和imDC(P<0.05);3种细胞均具有较强的抗低渗能力,但imDC最强(细胞未破碎率为27%);mDC的膜流动性最大,其荧光偏整度P值为0.062±0.001(P<0.01);FT-IR显示mDC中的蛋白质二级结构发生了较大变化。结论DC的微观流变学特性随其不同的发育阶段而发生了明显变化。
Objective To investigate the micro-rheological properties and differences of dendritic cells (DCs) at different developmental stages. Methods Mononuclear cells were isolated from normal human peripheral blood by density gradient centrifugation. Purified monocytes were obtained by adherence culture and negative selection with immunomagnetic beads. These cells were used as DC precursors (pDC) to induce immature DC (imDC) and mature DC (mDC). Immunocytochemistry, immunofluorescence, enzyme cytochemistry, flow cytometry and other techniques were used to identify DCs at different developmental stages. The micro-rheological methods were used to evaluate the electrophoresis rate, osmotic fragility, membrane fluidity and Fourier transform infrared spectroscopy Spectra (FT-IR) were measured and analyzed. Results Improved sorting yielded CD14 + pDC> 97%. After cultured with rhGM-CSF and IL-4 for 6 days, S-100 + imDC accounted for 96.9% of the total number of cells. After 48 hours of addition of rhGM-GSFI, L-4 and TNF- 87%. The micro-rheological properties were: mDC electrophoresis rate was significantly higher than pDC and imDC (P <0.05); 3 kinds of cells have strong anti-permeability, but imDC the strongest (cell unbroken rate of 27%); The membrane fluidity of mDC was the largest with a P value of 0.062 ± 0.001 (P <0.01). FT-IR showed that the secondary structure of protein in mDC changed greatly. Conclusions The micro-rheological properties of DC have changed obviously with different developmental stages.