免疫磁珠及无血清悬浮培养法对肝癌干细胞的分离与富集

Isolation and enrichment of liver cancer stem cells by magnetic cell sorting and serumfree suspension culture

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 The aim is to sort CD90＋ subpopulation cells in human liver cancer cell lines and investigate efficiency of magnetic cell sorting （MACS） on sorting the liver cancer stem cells. Methods ①Expressions of CD90. Immunohistochemical method was used to determine the expressions of CD90 in normal liver tissues in 8 cases, liver cancer and adjacent liver cancer tissues in 58 cases. ②Screened the cell lines. Huh-7, MHCC97-H, Bel-7402, and SMMC-7721 cell lines were divided into blank control group and experimental group （5.5×105 cells per hole, 1 hole）, cells of the experimental group were added with 5 μL CD90–PE while cells of the blank control group were treated with 5 μL CD90–PE non fluorescent antibody. Determined the proportion of CD90＋ cells in the 2 groups by flow cytometry （FCM）. ③MACS. Huh-7 and MHCC97-H cell lines were labeled with magnetic beads respectively and sorted by MACS, 1 mL cell suspensionsorted by magnetic sorting （MS） was collected as CD90– group, and 1 mL PBS after MS wash was collected as CD90＋ group, as well as blank control group and experimental group. Determined the proportion of CD90＋ cells in 4 groups by FCM. Two times of MACS were performed in Huh-7 cells. ④Serum free culture and serum culture. Huh-7 cells were divided into serum-free culture group and serum culture group （1 hole）, and proportions of CD90＋ cells were determined by FCM at 1 week after culture. Results ①The positive rate of CD90 was 0 （0/8）, 65.5% （38/58）, and 20.7% （12/58） in normal liver tissues, liver cancer tissues, and adjacent liver cancer tissues respectively, and the positive rate of CD90 was higher in liver cancer tissues than those of normal liver tissues （χ2=6.78, P〈0.05） and adjacent liver cancer tissues （χ2=20.83, P〈0.05）. ②For Huh-7, MHCC97-H, SMMC-7721, and Bel7402 cell lines, the proportions of CD90＋ cells in the experimental group was 0.851%, 1.090%, 2.710%, and 4.050% respectively, the proportions of CD90＋ cells in the blank control group was 0.241%, 0.688%, 1.890%, and 2.080% respectively, so we chose Huh-7 and MHCC97-H cell lines to perform MACS. ③Results of MACS for Huh-7 cell line. For the first MACS, the proportions of CD90＋ cells in the blank control group, experimental group, CD90– group, and CD90＋ group was 0.241%, 0.851%, 0.574%, and 1.100% respectively. For the second MACS, the proportions of CD90＋ cells in the blank control group, experimental group, CD90– group, and CD90＋ group was 0.032%, 0.961%, 0.426%, and 9.700% respectively. Conclusions The normal liver tissues do not express the CD90, but the liver cancer tissues express CD90 highly. There is a few CD90＋ cells in Huh-7 and MHCC97-H liver cancer cell lines. The MACS has a certain effect on improving the proportion of CD90＋ cells in the cell lines. The serum-free suspension culture has no effect on enriching CD90＋ cells.