目的评估免疫磁珠分选技术分选肝癌干细胞的效率。方法 (1)CD90的表达检测:采用免疫组织化学染色SP法检测8例正常肝组织、58例肝癌及其癌旁组织中CD90的表达。(2)细胞系筛选:将Huh-7、MHCC97-H、SMMC-7721及Bel7402细胞系分为空白对照组和实验组(细胞数量均为5.5×10~5个,均为1孔),实验组细胞中加入5μL CD90~–PE抗体,空白对照组加入5μL CD90~–无荧光抗体。采用流式细胞术检测4种细胞系空白对照组和实验组的CD90~+细胞比例,以筛选细胞。(3)免疫磁珠分选:将Huh-7和MHCC97-H细胞系分别进行磁珠标记后进行磁珠分选。收集流经磁珠分选柱(MS)后的细胞悬液1 m L,作为CD90~–组;将MS移出磁场区域,加入1 m L PBS缓冲液快速冲洗至离心管中,标记为CD90~+组;以未分选细胞作为空白对照组(加入CD90~–无荧光抗体)和未分选实验组(加入CD90~–PE抗体)。对Huh-7细胞系进行二次分选。采用流式细胞仪检测4组细胞中的CD90~+细胞比例。(4)无血清培养和含血清培养:将Huh-7细胞接种到无血清培养基专用6孔板中进行培养(无血清和含血清悬浮培养均为1孔),1周后采用流式细胞仪检测无血清培养和含血清培养后细胞中的CD90~+细胞比例。结果 (1)正常肝组织中CD90的表达阳性率为0(0/8),肝癌组织为65.5%(38/58),癌旁组织为20.7%(12/58)。肝癌组织中CD90的表达阳性率较正常肝组织(χ2=6.78,P<0.05)和癌旁组织高(χ~2=20.83,P<0.05)。(2)Huh-7、MHCC97-H、SMMC-7721及Bel7402细胞系中实验组的CD90~+细胞比例分别为0.851%、1.090%、2.710%及4.050%,空白对照组分别为0.241%、0.688%、1.890%及2.080%,故选择Huh-7和MHCC97-H细胞系进行下一步的免疫磁珠分选。(3)Huh-7细胞经一次分选后:未分选空白对照组的CD90~+细胞比例为0.241%,未分选实验组为0.851%,CD90~–组为0.574%,CD90~+组为1.100%;二次分选后:未分选空白对照组的CD90~+细胞比例为0.032%,未分选实验组为0.961%,CD90~–组为0.426%,CD90~+组为9.700%。结论正常肝组织实质细胞不表达CD90,肝癌组织高表达CD90。肝癌细胞系Huh-7和MHCC97-H中存在少量的CD90~+细胞,免疫磁珠分选法能明显提高CD90~+细胞比例,但作用十分有限。无血清悬浮培养法对富集CD90~+细胞无明显作用。 Objective To evaluate the efficiency of magnetic bead sorting in the sorting of hepatoma stem cells. Methods (1) The expression of CD90: Immunohistochemical SP method was used to detect the expression of CD90 in 8 cases of normal liver tissue, 58 cases of hepatocellular carcinoma and its adjacent tissues. (2) Cell line screening: The Huh-7, MHCC97-H, SMMC-7721 and Bel7402 cell lines were divided into blank control group and experimental group (the number of cells were 5.5 × 10 ~ 5, 5μL of CD90 ~ -PE antibody was added into the group of cells, and 5μL of CD90 ~ - non-fluorescent antibody was added into the blank control group. Flow cytometry was used to detect the percentage of CD90 ~ + cells in the blank control group and the experimental group in four cell lines to screen the cells. (3) Immunomagnetic bead sorting: Magnetic beads sorting was performed on Huh-7 and MHCC97-H cell lines respectively. Collect 1 ml of cell suspension after passing through magnetic separation column (MS) as CD90 ~ - group; remove MS from the magnetic field and quickly wash with 1 ml PBS buffer to centrifuge tube and mark as CD90 ~ + Group; unsorted cells as blank control group (add CD90 ~ - non-fluorescent antibody) and unsorted experimental group (add CD90 ~ -PE antibody). Huh-7 cell lines were sorted twice. Flow cytometry was used to detect the percentage of CD90 ~ + cells in the four groups of cells. (4) Serum-free culture and serum-containing culture: Huh-7 cells were inoculated into a special 6-well plate in serum-free medium (1 well in serum-free and serum-containing suspension culture) The concentration of CD90 ~ + cells in serum-free medium and serum-containing medium was measured. Results (1) The positive rate of CD90 expression in normal liver tissue was 0 (0/8), that of liver cancer was 65.5% (38/58) and that of paracancerous tissues was 20.7% (12/58). The positive expression of CD90 in hepatocellular carcinoma was significantly higher than that in normal liver tissue (χ2 = 6.78, P <0.05) and paracancerous tissues (χ ~ 2 = 20.83, P <0.05). (2) The percentage of CD90 ~ + cells of the experimental group in Huh-7, MHCC97-H, SMMC-7721 and Bel7402 cell lines were 0.851%, 1.090%, 2.710% and 4.050% respectively, while the blank control group was 0.241% and 0.688 %, 1.890% and 2.080%, Huh-7 and MHCC97-H cell lines were selected for further immunomagnetic bead sorting. (3) After a single sorting of Huh-7 cells, the percentage of CD90 ~ + cells in the untreated blank control group was 0.241%, 0.851% in the untreated control group, 0.574% in the CD90 ~ - group, Was 1.100%. After secondary sorting, the percentage of CD90 ~ + cells in the untreated blank control group was 0.032%, that in the unselected experimental group was 0.961%, that in the CD90 ~ - group was 0.426%, and that in the CD90 ~ + group was 9.700% . Conclusion The normal liver cells do not express CD90, and the high expression of CD90 in HCC tissues. A small amount of CD90 ~ + cells were found in Huh-7 and MHCC97-H cell lines. Immunomagnetic beads method could significantly increase the proportion of CD90 ~ + cells, but the effect was very limited. Serum-free suspension culture has no obvious effect on the enrichment of CD90 ~ + cells.