Waguri Nobuo, Suda Takeshi, Nomoto Minoru, Kawai Hirokazu, Mita Yusaku, Kuroiwa Takashi, Igarashi Masato, Kobayashi Makoto, Fukuhara Yasuo, Aoyagi Yutaka
Division of Gastroenterology and Hepatology, Department of Cellular Function, Graduate School of Medical and Dental Science, Niigata University, Niigata 951-8122, Japan.
Clin Cancer Res. 2003 Aug 1;9(8):3004-11.
We evaluated whether detection of human telomerase reverse transcriptase (hTERT) mRNA after immunomagnetic separation is useful to detect circulating cancer (CC) cells.
Two ml of peripheral blood were collected from 55 cases with hepatocellular carcinoma (HCC), 20 cases with chronic liver diseases devoid of cancer, and 20 healthy volunteers. Then 1500 and 500 micro l were subjected to immunomagnetic separations using Ber-EP4 and anti-CD45 antibodies, harvested and supernatant cells were collected as epithelial and nonleukocyte fractions, respectively. Samples of each fraction were subjected to reverse transcription-PCR detecting beta-actin, interleukin-2 receptor (IL-2r), alpha-fetoprotein, and hTERT mRNAs. The cases were judged to be positive, equivocal, or negative for CC cells when hTERT positivity with IL-2r negativity, hTERT positivity with IL-2r positivity, or hTERT negativity was seen in epithelial and/or nonleukocyte fractions, respectively.
The dilution experiments revealed that our system could detect 10(0-1) HeLa cells involved in 2 ml of blood. The Ber-EP4-harvested cells from cases with distant metastasis were positive for immunostaining using Hep Par 1 monoclonal antibody. CC cells were judged to be positive in 29 of 55 (53%) HCC cases. On the contrary, no cases without HCC were determined to be positive. The frequency of positivity was significantly correlated with disease extent of HCC.
These results strongly suggest that detection of hTERT mRNA after immunomagnetic separation is a specific and sensitive tool to detect CC cells and that it would provide useful source for further investigation of cancer metastasis.
我们评估了免疫磁珠分离后检测人端粒酶逆转录酶（hTERT）mRNA是否有助于检测循环肿瘤（CC）细胞。
采集55例肝细胞癌（HCC）患者、20例无癌慢性肝病患者及20名健康志愿者的2ml外周血。然后分别取1500μl和500μl使用Ber-EP4和抗CD45抗体进行免疫磁珠分离，收获细胞，上清液中的细胞分别作为上皮细胞和非白细胞组分收集起来。各组分样本进行逆转录聚合酶链反应检测β-肌动蛋白、白细胞介素-2受体（IL-2r）、甲胎蛋白和hTERT mRNA。当上皮细胞和/或非白细胞组分中分别出现hTERT阳性且IL-2r阴性、hTERT阳性且IL-2r阳性或hTERT阴性时，这些病例被判定为CC细胞阳性、可疑或阴性。
稀释实验表明，我们的系统能够检测出2ml血液中混入的10（0 - 1）个HeLa细胞。远处转移患者经Ber-EP4收获的细胞用Hep Par 1单克隆抗体免疫染色呈阳性。55例HCC病例中有29例（53%）的CC细胞被判定为阳性。相反，无HCC的病例均未判定为阳性。阳性频率与HCC的疾病范围显著相关。
这些结果强烈表明，免疫磁珠分离后检测hTERT mRNA是检测CC细胞的一种特异且灵敏的工具，并且它将为癌症转移的进一步研究提供有用的资源。
