肺癌相关间充质干细胞分离及生物学特征探讨

Biological characteristics of lung cancer-derived mesenchymal stem cells

目的目前,关于肿瘤来源间充质干细胞(mesenchymal stem cells,MSCs)的研究多是肝癌、卵巢癌、胃癌和乳腺癌等,肺癌来源MSCs报道较少。本研究目的在于建立分离人肺癌相关MSCs的方法,进而对肺癌相关MSCs的生物学特征进行初步探讨。方法采用组织块分离法分离人肺癌组织来源的MSCs,倒置显微镜下观察细胞形态,绘制细胞生长曲线。利用慢病毒感染的方法建立表达绿色荧光蛋白(CopGFP)的人肺癌细胞A549细胞株(A549-GopGFP)和H1299细胞株(H1299-GopGFP)。在体外,采用Brdu增殖实验分别检测肺癌MSCs对A549和H1299增殖的影响;划痕和Transwell实验分别检测肺癌相关MSCs对A549和H1299迁移和侵袭能力的影响。在体内,接种肺癌相关MSCs和A549细胞,观察肺癌MSCs对A549细胞生长的影响。结果成功建立组织块法分离人肺癌相关MSCs的方法。所分离的细胞呈成纤维样形态,并呈漩涡式生长;其CD73(99.8%)、CD90(99.8%)、CD105(57.1%)和CD166(98.0%)呈阳性表达,CD14、CD19、CD34、CD45和HLA-DR均呈阴性表达,且具有向成骨细胞、脂肪细胞和成软骨细胞分化的能力。体外,肺癌相关MSCs对A549细胞(t=0.246,P=0.818)和H1299细胞(t=0.331,P=0.757)增殖均无明显影响,但对A549细胞(t=3.377,P=0.028)和H1299细胞(t=7.107,P=0.002)的迁移有显著促进作用,也能显著增强A549细胞(t=6.170,P=0.004)和H1299细胞(t=13.16,P<0.001)的侵袭能力。体内实验表明,肺癌相关MSCs促进A549细胞早期成瘤。结论利用组织块法能够分离获得人肺癌相关MSCs,此细胞影响体内肺癌细胞的生长,显著提高体外肺癌细胞迁移和侵袭能力。

OBJECTIVE The tumor-associated mesenchymal stem cells（MSCs）from liver cancer,ovarian cancer,gastric cancer and breast cancer has been reported as far.However,the lung cancer-associated MSCs are little known.Our study aims to establish the isolation method and explore the biological characteristics of mesenchymal stem cells derived from lung cancer.METHODS Mesenchymal stem cells were isolated from lung cancer by tissue block culture technique.Cell morphology was observed by inverted phase contrast microscope and cell growth was valued by cell counting.Lentiviral particle transduction was used to establish stable lung cancer cell lines expressing CopGFP green fluorescent protein（A549-CopGFP and H1299-CopGFP）.In vitro study,the effect of lung cancer-derived MSCs on the proliferation,migration and invasion of tumor cells was determined by Brdu assay,scratch assay and transwell assay,respectively.In vivo study,lung cancer-derived MSCs and tumor cells were seeded simultaneously to observe the effects of lung cancer-derived MSCs on the growth of tumor cells in vivo.RESULTS The isolation method of lung cancer-derived MSCs was established successfully.The isolated cells presented fibroblastic morphology and vortex growth.The cells expressed CD73（99.8%）,CD90（99.8%）,CD105（57.1%）,CD166（98.0%）positively and CD14,CD19,CD34,CD45,HLA-DR negatively.Additionally,these cells exhibited multiple differentiated capability which could differentiate into osteoblast,fat cells and cartilage cells.The results of in vitro study showed that lung cancer-derived MSCs had no effect on the proliferation of A549 cells（t=0.246,P=0.818）and H1299（t=0.331,P=0.757）cells,but could increase the migration of A549 cells（t=3.377,P=0.028）and H1299 cells（t=7.107,P=0.002）,and invasion of A549 cells（t=6.170,P=0.004）and H1299 cells（t=13.16,P〈0.001）.The in vivo experiments showed that lung cancer-derived MSCs could promote the growth of tumor cells.CONCLUSION Lung cancer-derived MSCs are successfully isolated by tissue block culture and these cells have important role in the growth,migration and invasion of lung cancer cells.