洗涤方法和冻存温度对外周血单个核细胞内HCV RNA含量的影响

Effects of washing method and preserving condition on detecting HCV RNA in peripheral blood mononuclear cells

目的分析洗涤方法和冻存温度对外周血单个核细胞(PBMC)内HCVRNA含量的影响。方法收集同一天内17例血清HCVRNA阳性慢性丙型肝炎患者PBMC,随机分成A组(6例)和B组(11例),A组把细胞分成2份后分别采用常规法及改良法洗涤细胞5次,并留取每次洗涤液及细胞;B组采用改良法洗涤细胞后把细胞分成2份,分别作为新鲜组和-20℃冻存组,然后采用离心柱型RNA提取法和实时荧光定量聚合酶链反应(FQPCR)检测洗涤液和细胞中HCVRNA含量。结果(1)洗涤完3次时,常规法有1份洗涤液HCV为阳性,改良法均为阴性;与第1次洗涤液相比,改良法再洗涤1次的病毒含量下降幅度明显高于常规法;(2)新鲜组与冻存组HCVRNA含量不存在明显差异。结论洗涤PBMC以采用改良法洗涤3次为宜;-20℃简易冻存6周的PBMC标本仍可用于FQPCR检测。

Objective To investigate the effects of washing method and preserving condition on detecting HCV RNA in peripheral blood mononuclear cells（MC）. Methods The MCs of 17 chronic hepatitis C patients with serum HCV RNA positive were isolated and divided randomly into group A （6 cases ） and B （11 cases ） within the same day. In group A, two methods, a conventional method and a modified method, were used to wash the cells five times, respectively, for collecting each washing liquid and all cells. In group B, the cells were washed using the modified method and divided into two parts, the fresh group and the other as a frozen group. After RNA was extracted from each washing liquid and all MCs, HCV RNA was detected by fluotescence quantitative-PCR（FQ-PCR）. Results （1） After the MCs were washed for three times, only one with conventional method was HCV positive, whereas all washing liquids with modified method were HCV negative. Compared with the first washing liquid, the droping degree of viral loads using modified method was significantly higher than that of using conventional method. （2） HCV RNA loads of MCs were not significantly different between fresh group and frozen group. Conclusion Washing cells 3 times using modified method is better for disposal of MCs. MC samples plainly frozen at --20 ℃ for 6 weeks can be used for detecting HCV RNA by FQ-PCR.