人大肠癌多药耐药细胞LoVo/5-FU的建立及其生物学特性的初步研究

Establishment of multidrug-resistant human colorectal cancer cell line LoVo/5-FU: a preliminary study of biological characterization

目的建立人大肠癌ＬｏＶｏ细胞多药耐药细胞株ＬｏＶｏ／５－ＦＵ，并探讨其生物学特性及耐药机制。方法人大肠癌细 胞系 ＬｏＶｏ在体外经 ２．５μｇ／ｍｌ５－氟尿嘧啶（５－ＦＵ）作用，成功诱导 ＬｏＶｏ／５－ＦＵ耐药细胞株。体外细胞毒性实验观察它 们对５－ＦＵ、丝裂酶素（ＭＭＣ）、阿霉素（ＡＤＭ）、顺铂（ＤＤＰ）、氨甲喋呤（ＭＴＸ）和阿糖胞苷（ＡｒａＣ）等６种药物的敏感性。 用噻唑蓝（ＭＴＴ）法、光镜及扫描电镜观察两种细胞形态及结构并绘制出细胞体外生长曲线。免疫组化ＬＳＡＢ法检测细 胞中Ｐ２６－Ｂｃｌ－２的表达。应用原位ＤＮＡ末端转移酶标记法检测５－ＦＵ在两种细胞中诱导的细胞凋亡。结果ＬｏＶｏ／５－ＦＵ 细胞株对５－ＦＵ、ＭＭＣ和ＡＤＭ均有耐药性，且对５－ＦＵ的耐药程度较亲本细胞提高。与亲本细胞相比，耐药细胞株生长 慢，倍增期延长，汇合密度低，异型性明显。免疫组化ＬＳＡＢ法提示，ＬｏＶｏ／５－ＦＵ细胞的凋亡与Ｐ２６－Ｂｃｌ－２过度表达有 关。ＬｏＶｏ细胞原位ＤＮＡ末端转移酶标记阳性率高于ＬｏＶｏ／５－ＦＵ。结论ＬｏＶｏ／５－ＦＵ多药耐药细胞株耐药性稳定，在相 同条件下与敏感细胞株ＬｏＶｏ相比，细胞凋亡受到抑制，提示ＬｏＶｏ／

Objective To investigate the changes in biological properties of the multidrug-resistant (MDR) variant of human colorectal cancer LoVo cell lines and to explore the mechanism for MDR generation in human colorectal cancer cells. Method A MDR valiant of human colorectal cancer to 5-FU treatment LoVO/5-FU, was established in vitro by exposing parent LoVo cells to pulse treatment with 2.5 μg/ml 5-FU over a period of 6 months. Its sensitivity to 6 antitumor agents was observed by MTT method, and morphological Observation Under light microscope and electron microscope of both LoVo and LoVo/5-FU cells were performed. Bcl-2 gene expressions in two cells were assayed immunohischemically. Results The LoVo/5-FU cells possessed tolerance for 5-FU 16.48 times higher than that of parent LoVo cells,and exhibited cross-resistance to mitomycin,cisplatin and adriamycin, but not to methobexate or Ara-C. Compared with the parent cells, the LoVo/5-FU cells showed a lower growth rate and confluent density with positive findings of P-GP expression in approximately 85% of them. Bcl-2 gene expression was detected in the cytoplasm of both LoVo and LoVo/5-FU cells,but the latter had higher expression levels. Conclusion The changed biological properties of LoVo/5-FU might be related to its MDR phenotype, and the generation of MDR, which may be due to multiple mechanisms, reduces the effectiveness of chemotherapy for colorectal cancer.