Flt-3L、SCF在食管癌患者外周血细胞的诱导及树突细胞的培养

Experimental study on proliferation of DC from peripheral blood of patients with esophageal carcinoma

目的探讨从食管癌患者外周血分离、诱导与扩增树突细胞(DC)的有效方法。方法采用密度梯度离心法从食管癌患者外周血分离单个核细胞,分别加入GM-CSF(100 ng/ml)+IL-4(50 ng/ml)(作为对照组);GM-CSF(100 ng/ml)+IL-4(50 ng/ml)+Flt-3L(40 ng/ml)(作为实验1组);加入GM-CSF(100 ng/ml)+IL-4(50 ng/ml)+Flt-3L(40 ng/ml)+SCF(100 ng/ml)(作为实验2组)。于37℃、5%CO2环境下进行培养,动态观察细胞生长情况,流式细胞仪检测培养第6天时DC表面分子CD1a、CD83、CD80、CD86的表达水平。结果上述不同刺激物均能从外周血单个核细胞成功诱导出DC,与对照组相比,实验组诱导出更多数量的DC,其中以实验2组最多;培养至第6天时,两实验组DC细胞表型CD1a表达率与对照组无明显差别(P>0.05),CD83、CD80、CD86表达水平明显高于对照组(P<0.01),其中以实验2组表达水平最高(P<0.01)。结论联合应用GM-CSF、IL-4、flt-3L和SCF能有效地从食管癌患者外周血诱导培养出大量具有活性的DC,为进一步的临床研究奠定基础。

Objective To study the efficient method to induce and culture DC from peripheral blood of esophageal carcinoma patients. Methods The peripheral blood mononuclear ceils were isolated by Ficoll-Paque density gradient centrifugation from peripheral blood monoeytes of esophageal carcinoma patients and cultured to differentiate into DC. The DC were divided into three groups to culture in 37℃, 5% CO2 environment： cultured with GM-CSF （100 ng/ml） ＋IL-4 （50 ng/ml）, as control group; cultured with GM-CSF （100 ng/ml） ＋ IL- 4 （50 ng/ml ） ＋ flt3-L （ 40 ng/ml ）, as experimental one group ; cultured with GM-CSF （ 100 ng/ml ） ＋ IL-4 （ 50 ng/ml ） ＋ flt3-L （40 ng/ml） ＋ SCF （ 100 ng/ml） , as experimental two group. The number of DC was counted and the morphology of DC was observed under inverted microscope and election microscope. The expression of cell surface molecule like CDla, CD80, CD83 and CD86 on DC were exam- ined On the 6 th day culture by flow cytometry. Results Three groups all succeeded to induce DC from peripheral blood mononuclear cells. A larger number of DC was found in experimental group than that of control group. The DC number in experimental two groups was the largest in three groups. On the 6 th day after cell culture, the cell phenotype CD1 a expression on peripheral blood DC had no significant difference in experimental group compared with that of control group （ P 〉 0. 05 ） , while CD83, CD80 and CD86 showed significantly higher expressions as compared with those in control group （P 〈 0. 01 ）, and DC cell phenotype expressions in experimental two group were the highest （P 〈 0. 01 ）. Conclusions It is an effective way to cultivate large amount of active DC in peripheral blood from esophageal carcinoma patients by using GM-CSF, IL-4, flt-3L and SCF, which will lay a foundation for further clinical trial.