两种人鼻咽癌活检组织上皮细胞原代培养方法比较

Comparison of two methods for primary culture of epithelial cells from human bioptic tissue of nasopharyngeal carcinoma

目的比较两种人鼻咽癌活检组织上皮细胞原代培养方法,并初步探讨鼻咽癌原代培养细胞的生长特性。方法收集未经放、化疗的鼻咽癌初诊病人鼻咽部肿瘤活检组织33例,其中17例采用组织块培养法,16例采用组织块预消化培养法,用含2%胎牛血清的Keratinocyte-SFM培养基培养,比较两种方法干贴壁时间、成功率及细胞长出所需平均天数,并通过倒置显微镜、抗人角蛋白单克隆抗体免疫细胞化学染色、生长曲线、生存分析来观察原代培养细胞的特性。结果组织块培养法成功率为23.5%(4/17),干贴壁时间为4.47±0.48h,细胞长出所需时间为13.75±1.5d;组织块预消化培养法成功率为62.5%(10/16),干贴壁时间为7.88±1.01h,细胞长出所需时间为8.3±4.55d,差异有统计学意义(P<0.05)。组织块培养法,细胞多层重叠排列,结构不清,始终没有继续繁殖生长;组织块预消化培养法,细胞呈梭形,核大居中,多个核仁,抗人角蛋白单克隆抗体免疫细胞化学染色阳性,生存时间均数为62.72d。结论组织块预消化培养法较组织块法成功率高,细胞长出所需平均天数短,干贴壁时间长;低浓度血清的Keratinocyte-SFM培养基适合鼻咽癌原代上皮细胞体外培养。

Objective To compare two methods for primary culture of the epithelial cells from human bioptic specimens of nasopharyngeal carcinoma （NPC）,and preliminarily study the biological characteristics of the primarily cultured cells. Methods Thirty-three bioptic specimens were collected from patients with pathologically confirmed NPC who received no previous radiotherapy or chemotherapy. Seventeen samples were cultured using tissue culture method,and tissue predigestion culture was used for the other 16 samples. The cells were cultured in Keratinocyte-SFM medium containing 2% fetal bovine serum. The two culture methods were compared for successful rate,attachment time and average time of cell growth. The biological features of the cultured cells were observed by reverse microscope,cytokeratin immunocytochemistry,growth curve and survival analysis. Results The successful rate,attachment time and days for cell growth of the tissue culture method and tissue predigestion culture method were 23.5% （4/17） vs 62.5% （10/16）,4.47±0.48 h vs 7.88±1.01 h,and 13.75±1.5 days vs 8.3±4.55 days,respectively,showing significant differences between the two methods （P0.05）. The cells resulting from tissue predigestion method,with a average survival of 62.72 days,were characterized by a fusiform morphology with large nuclei,multiple nucleoli,and cytokeratins positivity. In contrast,the cells obtained by tissue culture method showed multilayer alignment without distinct cell structures and failed to grow consistently. Conclusion Compared with tissue culture method,tissue predigestion cell culture results in high successful rate and requires shorter cell growth time with longer attachment time. Keratinocyte-SFM medium supplemented with low concentrations of FBS is suitable for the growth of primarily cultured epithelial cells from NPC biopsy samples in vitro.