中文摘要：

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。

英文摘要：

Objective To establish a method for isolating and purifying tumor-associated hepatic stellate cells （tHSCs） from human hepatocellular carcinoma （HCC） tissues and to study their impact on the biological behaviors of HCC cells in vitro. Methods The tHSCs were isolated from the fresh human HCC tissue by collagenase/pronase digestion, followed by density gradient centrifugation using Nycodenze. The viability of the isolated cells was determined by trypan blue staining assay. The tHSCs were identified by immunocytochemical staining of α-SMA, a specific marker for activated hepatic stellate cells. The morphologic changes of the tHSCs were observed under microscope. The conditioned medium （CM） of tHSCs cultured in serum-free DMEM was collected, and then used for co-culture with HCC cell lines QGY-7701, BEL-7402, and SMMC-7721. CM of human hepatic stellate cell line LX-2 was used as control. Cell viability, proliferation, migration potential and EMT- related protein expression changes were assessed by using the MTT assay, wound migration assay and Western blotting analysis, respectively. Results The viability of isolated tHSCs was over 95%, with a purity about 100%, and the isolated tHSCs could be passaged and cryopreserved, tHSCs significantly induced typical EMT-like morphological changes and altered expression of molecular markers in the three HCC cell lines, with the epithelial marker E-cadherin significantly decreased （P〈0.05） and the stromal marker Vimentin significantly increased （P〈0. 05）. Meanwhile, tHSCs efficiently enhanced the proliferation and migration of HCC cells in vitro. Conclusion We have established a method for isolating and purifying tHSCs from human HCC tissues, which can help future study on tHSCs, tHSCs can enhance the proliferation and migration of HCCcells through inducing EMT-like phenotype.