目的：建立一种准确且对细胞活性干扰少的分离人肠道组织中CD3＋CD4＋ T淋巴细胞的分选方法。方法首先用消化酶分解人肠道组织，40μm细胞滤器过滤获得细胞悬液。Percoll连续密度梯度离心法分离细胞悬液收集单个核细胞，然后通过流式细胞仪分选法（FACS ）分选单个核细胞中的CD3＋CD4＋ T淋巴细胞。分选后的细胞采用细胞存活率、细胞纯度和形态观察对分选方法进行评价。结果消化酶能够很好地分解人肠道组织获得细胞悬液，Percoll连续密度梯度离心法收集的单个核细胞中CD3＋CD4＋ T淋巴细胞纯度为（43．9±7．3）％，FACS分选后CD3＋ CD4＋ T 淋巴细胞纯度为（96．9±1．2）％，差异有统计学意义（P＜0．01）。分选后的细胞存活率为（97．8±1．6）％，细胞的形态保持完整。结论 Percoll连续密度梯度离心法联合FACS收集人肠道组织的CD3＋CD4＋ T淋巴细胞纯度高，且对细胞形态和存活率影响小并可在无菌条件下进行，分选的细胞可继续用于其他功能的研究。%Objective To establish an accurate sorting method with less interference to the cell activity for isolating CD 3+CD4+T lymphocytes in human intestinal tissue .Methods Firstly the digestive enzymes were adopted to decompose the human intestinal tissue ,and then the cell suspension was obtained by the filtration of 40 μm cell strainer .Mononuclear cells were collected by using continuous density Percoll gradient centrifugation technique ,then the flow cytometry was adopted to sort CD3+ CD4+ T lympho-cytes in mononuclear cells .The survival rate ,cell purity and morphological observation of the sorted cells were adopted to conduct the evaluation on the sorting method .Results The digestive enzymes well decomposed human intestinal tissue into cell suspension , the purity of CD3+CD4+ T lymphocytes by using continuous density Percoll gradient centrifugation was (43 .9 ± 7 .3)% ,while the purity of CD3+CD4+ T lymphocytes was(96 .9 ± 1 .2)% after fluorescence-activated cell sorting ,the difference between them was statistically significant(P<0 .01) .The survival rate of sorted CD3+CD4+ T lymphocytes was(97 .8 ± 1 .6)% with intact cell shape . Conclusion The Percoll gradient centrifugation technique combined with the fluorescence-activated cell sorting not only collects highly pure human intestinal CD3+CD4+ T lymphocytes with little influence on the cell activity and shape ,but aslo may be carried out under sterile conditions ,the sorted cells can continue to be used in the other functional researches .
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