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目的建立人肝癌组织中肿瘤相关肝星状细胞(tHSCs)分离、培养方法,探讨tHSCs对肝癌细胞生物学行为的影响。方法采用密度梯度离心法从新鲜切除的人肝癌组织样本中分离tHSCs锥虫蓝染色检测细胞活力,细胞免疫荧光鉴定活化tHSCs表达α-平滑肌肌动蛋白的特征,光镜观察培养的tHSCs形态学变化;tHSCs无血清培养后,收集条件培养液,分别与肝癌细胞系QGY-7701、BEL-7402、SMMC-7721共培养,并以肝星状细胞系LX-2做对照,采用MTT法、细胞划痕实验、蛋白质印迹等方法检测肝癌细胞增殖、迁移和上皮间质转化(EMT)相关指标的表达。结果从肝癌组织中分离的tHSCs活率在95%以上,纯度接近100%,在体外可进行传代培养与冻存。tHSCs能诱导3个受试肝癌细胞系产生典型的EMT样形态学变化及其分子标记物表达改变,表现为上皮标记物E-钙黏蛋白表达减少(P<0.05),间质标记物波形蛋白表达增加(P<0.05)。与此同时,tHSCs能促进培养的3个肝癌细胞系增殖和迁移。结论从人肝癌组织中分离的原代tHSCs活力和纯度高,可用于tHSCs的生物学功能实验研究。tHSCs在诱导肝癌细胞产生EMT样改变的同时,体外促进肝癌细胞的增殖和迁移。
Objective To establish a method for the isolation and culture of tumor-associated hepatic stellate cells (tHSCs) in human hepatocellular carcinoma (HCC) and to investigate the effect of tHSCs on the biological behavior of hepatoma cells. Methods The cell viability of tHSCs was detected by density gradient centrifugation from freshly excised human hepatocellular carcinoma tissue samples. The characteristics of activated α-smooth muscle actin were detected by immunofluorescence staining. The morphological changes of tHSCs were observed by light microscope After the serum-free culture of tHSCs, the conditioned medium was collected and co-cultured with hepatocellular carcinoma cell lines QGY-7701, BEL-7402 and SMMC-7721 respectively. The hepatic stellate cell line LX-2 was used as a control. MTT assay, The cell proliferation, migration and the expression of epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma cells were detected by Western blotting and Western blotting. Results The viability of tHSCs isolated from hepatocellular carcinoma was above 95% with a purity of nearly 100%. The cells could be subcultured and frozen in vitro. THSCs induced typical EMT-like morphological changes and changes in their molecular markers in 3 human hepatocellular carcinoma cell lines. The expression of epithelial marker E-cadherin decreased (P <0.05) and interstitial marker vimentin The expression increased (P <0.05). In the meantime, tHSCs can promote the proliferation and migration of three hepatoma cell lines cultured. Conclusion The primary tHSCs isolated from human hepatocellular carcinoma tissue have high activity and purity and can be used for the biological function of tHSCs. THSCs induce EMT-like changes in hepatoma cells and promote the proliferation and migration of hepatoma cells in vitro.