Combined use of positive and negative immunomagnetic isolation followed by real-time RT-PCR for detection of the circulating tumor cells in patients with colorectal cancers
Combined use of positive and negative immunomagnetic isolation followed by real-time RT-PCR for detection of the circulating tumor cells in patients with colorectal cancers
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DOI：
10.1007/s00109-004-0590-8
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发表时间：
2004-11-01
影响因子：
4.7
通讯作者：
Liu, Z
中科院分区：
文献类型：
--
作者：
Guo, JM;Xiao, BX;Liu, Z
为建立一种新的检测循环肿瘤细胞（CTC）的分子诊断方法，将结肠癌细胞LS 174 T用正常人血液连续稀释。从25例结直肠癌患者中收集额外的外周血样本。收集单核细胞（MNCs），等分为4份，用4种方法富集癌细胞：方法A，非免疫珠法;方法B，阴性免疫珠法：用CD 45免疫磁珠去除白细胞;方法C，阳性免疫头法：用Ber-EP 4免疫磁珠富集癌细胞;方法D，阴性和阳性免疫珠法：首先用CD 45免疫磁珠去除MNC中的白细胞，然后用Ber-EP 4免疫磁珠富集癌细胞。实时荧光定量RT-PCR检测β 2-微球蛋白（β 2 M）和癌胚抗原（CEA）mRNA的表达。根据用cDNA的10倍系列稀释液（1-10（4）个IS 174 T细胞）制备的CEA mRNA外标物和用cDNA的10倍系列稀释液（10（2）-10（7）个白细胞）制备的β 2 M mRNA外标物，将相对CEA mRNA值参考β 2 M mRNA校正为CEA mRNA/β 2 M mRNA比值。在恢复实验中，发现当单独使用CD 45或Ber-EP 4免疫磁珠时，癌细胞数量与CEA mRNA表达之间存在显著相关性。当连续使用CD 45和Ber-EP 4免疫磁珠时，发现高度显著相关。方法D的灵敏度为每毫升血液中有一个癌细胞。在25例结直肠癌患者中，19例检测到循环癌细胞。D法测得的CEA mRNA相对值最小。Dukes' B、C、D期患者循环癌细胞阳性检出率分别为25.0%（1/4）、83.3%（10/12）、88.9%（8/9）。免疫磁性分离结合实时RTPCR是一种检测结直肠癌患者循环肿瘤细胞的有用技术。先后应用阴性和阳性免疫磁珠与肿瘤细胞数量的相关性最高。
To establish a novel molecular diagnostic method of detecting circulating tumor cells (CTCs) LS174T colon cancer cells were serially diluted with normal blood. Additional peripheral blood samples were collected from 25 patients with colorectal carcinoma. Mononuclear cells (MNCs) were collected, equally divided into four parts, and then cancer cells were enriched by four methods: method A, nonimmunobead method; method B, negative immunobead method: CD45 immunomagnetic beads were used to deplete the leukocytes; method C, positive immunohead method: Ber-EP4 immunomagnetic beads were used to enrich cancer cells; method D, negative-and-positive immunobead method: CD45 immunomagnetic beads were first used to deplete the leukocytes from MNC and then Ber-EP4 immunomagnetic beads were used to enrich cancer cells. Finally, real-time quantitative RT-PCR was used to monitor mRNA expression Of beta(2)-mircoglobulin (beta2M) and carcinoembryonic antigen (CEA). The relative CEA mRNA values were corrected with reference to beta2M mRNA, to CEA mRNA/beta2M mRNA ratios according to a CEA mRNA external standards prepared with tenfold serial dilutions (1-10(4) IS174T cells) of cDNA and beta2M mRNA external standards prepared with tenfold serial dilutions (10(2)-10(7) leukocytes) of cDNA. In recovery experiments a significant correlation between the number of cancer cells and CEA mRNA expression was found when CD45 or Ber-EP4 immunomagnetic beads were used alone. A highly significant correlation was found when CD45 and Ber-EP4 immunomagnetic beads were used successively. The sensitivity of method D was one cancer cell per milliliter of blood. Circulating cancer cells were detected in 19 of 25 patients with colorectal cancers. The relative CEA mRNA value obtained by method D was the smallest. The positive detection rate of circulating cancer cells in patients at Dukes' B, C, and D stages were 25.0% (1/4), 83.3% (10/12), and 88.9% (8/9). Combinative use of immunomagnetic isolation followed by real-time RTPCR is a useful technique to detect circulating tumor cells in patients with colorectal carcinomas. Applying negative and positive immunomagnetic beads successively yields the highest correlation with amount of tumor cells.