乳腺癌MCF-7细胞系转移亚克隆的筛选及其生物学特性研究

目的将乳腺癌MCF-7细胞接种SC ID鼠,筛选具有高转移潜能的转移亚克隆,阐明其生物学特性。方法接种培养的乳腺癌MCF-7细胞,取接种的SC ID鼠肺组织做原代细胞培养,将获得的细胞命名为LM-MCF-7。对该细胞进行生物学鉴定,研究其形态学、生长、细胞周期、染色体和乳腺癌特异性标志物等特征,与其亲本MCF-7细胞进行比较,检测二者肿瘤转移相关蛋白表达的差异。将LM-MCF-7细胞回接SC ID鼠,观察其成瘤和转移的能力。结果在接种MCF-7细胞第68天时处死SC ID鼠,经肺组织原代细胞培养,成功获得了MCF-7细胞的转移亚克隆LM-MCF-7。在形态学上LM-MCF-7细胞为典型的上皮样多角形;应用流式细胞仪检测分析显示,G0/G1期细胞为53.40%,S期细胞为17.10%,G2+M期细胞为23.20%;细胞群体平均倍增时间为20 h±14 h;染色体分析显示为人源性异倍体,其数量为16~123条;免疫细胞化学检测显示,乳腺癌特异性标志物CA15-3在LM-MCF-7细胞中呈阳性反应;W estern印迹检测结果显示,与MCF-7细胞相比,LM-MCF-7细胞中肿瘤转移抑制基因nm23和细胞周期相关蛋白p27的表达水平明显下调,而与细胞运动有关的肌球蛋白轻链激酶(MLCK)及抗细胞凋亡蛋白bc l-2和survivin的表达水平明显上调。将该细胞株接种SC ID小鼠,与亲本MCF-7细胞相比,成瘤时间由7.4 d±1.3 d缩短至5.0 d±0.0 d,在肺脏、肾脏、脾脏、骨髓、淋巴结和心脏等脏器有广泛转移。结论LM-MCF-7细胞株是乳腺癌MCF-7细胞的转移亚克隆,具有高转移潜能。

Screening of a sub-clone of human breast cancer cells with high metastasis potential

OBJECTIVE: To screen a sub-clone of human breast cancer cell of the MCF-7 line with high metastasis potential. METHODS: Human breast cancer cells of the MCF-7 line were injected subcutaneously into 10 severe combined immunodeficiency (SCID) mice. Sixty-eight days after the mice were killed and their lungs were taken out. Primary cell culture was conducted. When the cells were passed on to the third generation a sub-clone was screened from the lung tissue and termed LM-MCF-7. Microscopy was performed on the lung tissues. The growth curve was drawn. Flow cytometry was used to examine the cell cycle. Chromosome analysis was done. Immunohistochemistry was used to detect the expression of breast cancer specific antigen CAI5-3. Western blotting was used to detect the protein expression of the protein associated with tumor metastasis: nm23 (a metastasis-suppressing gene), myosin light chain kinase (MLCK, a kinase related to cell movement), survivin, bcl-2 and p27 (a gene related to cell cycle). LM-MCF-7 cells were injected into other SCID mice and these mice were killed 30 days later to observe the metastasis of cancer so as to detect the tumorigenic ability of the LM-MCF-7 cells. RESULTS: When the cells from the mouse lung tissues were passed on to the third generation a sub-clone with high metastasis potential was screened and termed LM-MCF-7. The morphology of the new cell line was typically epithelioid. Flow cytometry showed that the DNA relatively contents were 53.40% of the LM-MCF-7 cells were in the G(0)/G(1) phase, a lower percentage than that of the MCF-7 cells, and 17.10% in the S phase and 23.20% in the G(2+)M phase, both percentages higher than those of the MCF-7 cells. The proliferating time of the LM-MCF-7 cell population was about 20 +/- 14 hours, much shorter than that of the parent strain cells. The chromosomes of the LM-MCF-7 cells, numbering 16-123, showed the morphology characteristic c of human chromosomes. The marker of human breast cancer CA15-3 was detected in both MCF-7 and LM-MCF-7 cells. The protein expression of nm23 and p27 was down-regulated, but the protein expression of MLCK, bcl-2 and survivin was up-regulated in LM-MCF-7 cells in comparison with those in MCF-7 cells. The tumorigenesis rate of LM-MCF-7 cells was 100% (5/5), with a latent period of 5.0 +/- 0.0 d, and the tumor metastasized to lung, kidney, spleen, bone marrow, lymph node and heart. CONCLUSION: A human breast cancer line, LM-MCF-7 cell line, with high metastasis potential has been derived from the human breast cancer cells of MCF-7 line.