摘要
为证明Lin28B/let-7通路与肝癌紫杉醇耐药密切相关和采用浓度梯度间歇刺激法建立Hep3B/PTX耐药细胞系。通过四甲基偶氮唑盐比色(methyl thiazolyl tetrazolium,MTT)分析Guava微流式分析对Hep3B/PTX耐药细胞系进行鉴定,realtime PCR检测耐药细胞中Lin28B及let-7表达水平。经过6个月的诱导,Hep3B/PTX耐药细胞系对紫杉醇的耐药指数为13.55;与亲本细胞相比,Hep3B/PTX处于G0/G1期的细胞比例较高,S期的细胞比例较低;0.2μmol/L紫杉醇处理亲本细胞24h后,有(41.73±1.80)%细胞发生凋亡,而Hep3B/PTX细胞凋亡率只有(17.21±3.60)%;real-time PCR发现,Hep3B/PTX与亲本细胞相比,Lin28B基因表达水平升高了49倍,而Lin28B调控的let-7家族成员表达水平均有不同程度的降低。Hep3B/PTX细胞中Lin28B基因和let-7家族成员表达量的变化揭示了肝癌细胞对紫杉醇的耐药与Lin28B/let-7通路相关,Lin28B基因可能通过影响let-7家族miRNA生物发生过程而改变肝癌细胞对紫杉醇的敏感性。
To determine whether the Lin28B/let-7 pathway is tightly related to paclitaxel resistance in hepatocellular carcinomacells, a paclitaxel-resistant cell line Hep3B/PTX was established by intermissive and stepwise exposure to paclitaxel. TheHep3B/PTX cell line was identified by methyl thiazolyl tetrazolium （MTT） assay and GuavaThe expressions of Lic28B and let-7 were detected by rell-time PCR. After 6 months induction, the Hep3B/PTX cell line was established, and the drug resistance mdex was 13. 55. Compared with parental cells, the Hep3B/PTX cells had a higher percent-age of G0 /G 1 phase and a lower percentage of S phase. After treatment by 0. 2 pmol/L paclitaxel for 24 h, the apoptosis rate of parental cells was （41. 73士 1. 80） % but that of Hep3B/PTX was only （17. 21 士3. 60） %. The real-time PCR analysis showed that the expression of Lic28B in Hep3B/PTX cells was 49 times more than parentll cells, but the expressions of let-7 miRNAs which were regulated by Lin28B were all decreased in different degree. Aberrant expression of Lic28B and let-7 in Hep3B/PTX cells suggested that paclitaxel resistance m hepatocellular carcinoma cells was related to the Lin28B/let-7change the chemosensitivity of human hepatocellular carcinoma by affecting let-7 processing.
出处
《中国科技论文》
北大核心
2017年第18期2118-2123,共6页
China Sciencepaper
基金
高等学校博士学科点专项科研基金资助项目(20133322120002)
国家自然科学基金资助项目(81602624)
作者简介
第一作者：田男（1983-）,女,副教授,主要研究方向为肿瘤分子靶向治疗,tiannanlux@126.com
