正常宫颈上皮细胞原代培养体系的改良

An improved culture system for the primary culture of normal cervical epithelial cells

目的:探索一种高效、可重复的正常宫颈上皮细胞原代培养方法。方法:取60例正常宫颈组织,分成2组,分别采用Ⅱ型中性蛋白酶联合0.25 g/mL胰蛋白酶-EDTA(中性蛋白酶联合法)及Ⅰ型胶原酶消化分离后进行原代培养,比较两种分离效果;观察采用中性蛋白酶联合法消化不同时间对分离效果的影响。观察添加体积分数5%FBS dK-SFM培养基与单纯dK-SFM培养基培养原代宫颈上皮细胞的生长曲线差异,并用形态学、广谱角蛋白免疫细胞化学染色鉴定细胞来源。结果:正常宫颈上皮细胞原代培养总成功率为40%。中性蛋白酶联合法成功率、分离所得细胞密度、原代细胞融合时间及细胞纯度均高于Ⅰ型胶原酶消化法(P<0.05)。中性蛋白酶消化20 h的分离效果优于消化16和24 h(P<0.05)。体积分数5%FBS dK-SFM培养基原代培养的宫颈上皮细胞增殖活性高于单纯dK-SFM培养基(P<0.05)。细胞生长状况良好,可在体外传代至5~6代,并可冻存与复苏,免疫细胞化学染色显示广谱角蛋白表达阳性。结论:Ⅱ型中性蛋白酶联合0.25 g/mL胰蛋白酶-EDTA分离法和体积分数5%FBS dK-SFM培养基培养原代正常宫颈上皮细胞,高效、可重复性好。

Aim： To explore an effective and repeatable method for the primary culture of normal cervical epithelial cells. Methods： A total of 60 normal human ectocervical tissue were collected and divided into two groups,primarily cultured using combined type Ⅱ dispase and 0. 25 g/mL trypsin-EDTA digestion method and type Ⅰ collagenase digestion method separately,and to compare the effectiveness of isolation,furthermore,to observe the detached effectiveness of combined dispase digestion method after digestion for different time. Cervical epithelial cells were cultured in 5% FBS dK-SFM and dK-SFM alone,respectively,to observe the growth of the primary cervical cells. The cultured cells were identified by the morphological analysis and pancytokeratin was identified by immunocytochemistry. Results： The total success rate of the primary culture in this study was 40%. The success rate,density,primary cell fusion time and purity of cells of the dispase digestion group were all higher than those of collagenase digestion group（ P 〈 0. 05）. The dispase digestion for 20 h was more effective than digestion for 16 and 24 h（ P 〈 0. 05）. The cell growth curve showed that the cells cultured in 5%FBS dK-SFM grew better and had a higher viability than cells cultured in dK-SFM alone（ P 〈 0. 05）. The primary cultured cells grew well and could be subcultured for five to six generations,and they also could be cryopreserved and resuscitated.Pancytokeratin positive staining was determined by immunocytochemistry. Conclusion： It is an effective and repeatable method for primary culture of normal cervical epithelial cells by using combined type Ⅱ dispase and 0. 25 g/mL trypsinEDTA digestion and 5% FBS dK-SFM medium.