人结直肠癌肝转移瘤中CD11b~+髓系细胞分选方法的建立与鉴定

目的:从结直肠癌肝转移瘤组织中分选出高纯度的CD11b~+髓系细胞,从而建立从一种组织中获得特异单细胞的方法。方法:采用机械联合酶消化法将实体瘤组织制备成单细胞悬液;利用流式细胞术分选出CD11b~+髓系细胞,并回测细胞纯度;采用免疫细胞化学技术对分选出细胞的特异性进行鉴定;采用吉姆萨和台盼蓝染色对获得细胞进行评价。结果:流式细胞术能从机械联合酶消化法制备的单细胞悬液中分选出高纯度、足够数量的CD11b~+细胞;分选前细胞阳性率与分选后纯度的差异有统计学意义(P0.01),分选后纯度达到实验研究的要求(P0.05);免疫细胞化学技术验证了分选细胞的特异性;分选后的细胞形态完整,活性良好。结论:机械联合酶消化法制备的单细胞悬液,经过流式细胞术分选,能够分选出高纯度、活性良好且形态完整的特异性细胞,操作方法稳定可重复,满足进一步研究的要求。

Identification and establishment of sorting method for isolating CD11b+ myeloid cells in human hepatic metastases from colorectal cancer

AIM: To establish a method for obtaining specific cells in solid tumor tissue by sorting of CD11b⁺ myeloid cells in hepatic metastases from colorectal cancer.METHODS: Tumor tissues were prepared into single cell suspension by mechanical method combined with enzyme digestion, and then the CD11b⁺ myeloid cells were isolated by flow cytometry. The sorted cells were identified by immunocytochemistry. The viability and morphologiy of the sorted cells were evaluated by Giemsa and Typan blue staining. The cell purity was evaluated by flow cytometry.RESULTS: Sufficient numbers of CD11b⁺ cells with high purity were isolated by sorting with flow cytometry from the single cell suspension prepared by mechanical and enzyme digestion. The purity of the cells was confirmed by statistical analysis (P<0.05). The positive rates of the cells before and after sorting were significantly different (P<0.01). The positive cells were verified by immunocytochemical method. Meanwhile, the sorted cells had complete morphology and good activity.CONCLUSION: The CD11b⁺ myeloid cells in solid tumor tissue can be isolated by flow cytometry from the machine-enzyme digestion suspension with high purity, good activity and complete morphology.