肝癌干细胞的分离及对索拉菲尼耐药的机制初探

Isolation of Hepatocellular Carcinoma Stem Cells and Preliminary Investigation into Their Mechanisms of Sorafenib Resistance

摘要: 目的：探讨肝癌干细胞对索拉菲尼耐药的作用机制，为肝癌的靶向治疗提供理论依据。方法：采用无血清培养法分离培养肝癌干细胞，通过Qrt-pcr检测肝癌干细胞表面标志物的表达情况；将肝癌干细胞和亲本肝癌细胞分别置于含有不同浓度索拉菲尼的培养基中培养，采用CCK8法检测细胞的存活率；利用Western Blot检测肝癌干细胞中OCT4A、STST3和p-STAT3的表达情况。结果：肝癌干细胞在无血清培养基中能够形成典型的干细胞球，且干细胞CD133、EpCAM及CD44等标志物的表达水平较亲本肝癌细胞高；在索拉菲尼作用下，肝癌干细胞的存活率高于亲本肝癌细胞。Western Blot检测发现，肝癌干细胞中OCT4A、STAT3和p-STAT3的表达水平显著高于亲本肝癌细胞。结论：采用无血清培养法能够从肝癌细胞中分离出具有肿瘤干细胞特性的肝癌干细胞。肝癌干细胞可能是导致肝癌对索拉菲尼耐药的重要原因，其机制可能涉及OCT4A通过激活STAT3信号通路来调控肝癌干细胞耐药性。

Abstract: Objective: To investigate the mechanism underlying sorafenib resistance in hepatocellular carcinoma stem cells (HCSCs) and to provide a theoretical basis for targeted therapy of hepatocellular carcinoma (HCC). Methods: HCSCs were isolated and cultured using serum-free medium. Expression of HCSC surface markers was determined by quantitative real-time PCR (qRT-PCR). HCSCs and parental HCC cells were exposed to graded concentrations of sorafenib, and cell viability was assessed by CCK-8 assay. Protein levels of OCT4A, STAT3, and phosphorylated STAT3 (p-STAT3) in HCSCs were evaluated by Western blot. Results: Under serum-free conditions, HCSCs formed typical spheroids and exhibited significantly higher expression of the stemness markers CD133, EpCAM, and CD44 compared with parental HCC cells. Upon sorafenib treatment, HCSCs displayed higher viability than their parental counterparts. Western blot analysis revealed markedly elevated expression of OCT4A, STAT3, and p-STAT3 in HCSCs. Conclusion: Serum-free culture successfully enriches HCSCs with tumor-initiating properties. HCSCs may be a critical driver of sorafenib resistance in HCC, and the mechanism may involve OCT4A to regulate HCC stem cell resistance by activating the STAT3 signaling pathway.