小鼠肝癌H22诱导淋巴管生成的体外研究

Induction of lymphangiogenesis by mouse ascites hepatoma cell H22 in vitro

目的建立Balb/c鼠淋巴管生成体外研究模型,观察小鼠肝癌细胞系H22对淋巴管生成和淋巴管内皮细胞增殖的影响。方法弗氏不完全佐剂诱导Balb/c鼠腹腔良性淋巴管瘤,分离、分切后,于纤维蛋白凝胶内用H22细胞条件培养液培养,倒置显微镜以及透射电镜观察;消化凝胶培养基分离细胞,免疫组化方法观察其Flt-4、c-fos和iNOS的表达情况,组织化学方法研究培养液NO含量变化。结果倒置显微镜及电镜可观察到微淋巴管从淋巴管瘤块长入凝胶培养基内;H22细胞条件培养液可促进淋巴管生成,诱导淋巴管内皮细胞表达Flt-4、c-fos和iNOS,并分泌NO。结论 淋巴管立体培养方法可较为方便的用于体外研究淋巴管生成,H22细胞对淋巴管生成有促进作用,淋巴管生成与癌淋巴道转移的关系值得进一步研究。

Objective To construct lymphangiogenesis model of Balb/c mouse in vitro to observe the impact of mouse ascites hepatoma cell H22 on the lymphangiogenesis and lymphatic endothelial proliferation. Methods Intraperitoneal benign lymphangioma induced by incomplete Freund' s adjuvant in Balb/c mouse was harvested and embedded in fibrin gel to culture in vitro with conditioned medium (CM) of H22 cell. The gels, observed by inverted microscope and transmission electronmicroscope, were then digested to separate the cells. The expression of Flt-4, c-fos and iNOS in the cell and the content of NO in medium were studied by immunohistochemical and histochemical method. Results Lymphatic capillary generating from lymphangioma specimen in the gels was observed by inverted microscope and transmission electronmicroscope. H22 CM was observed to enhance growth of the vessels, induce expressions of Flt-4, c-fos and iNOS in the separated cells and increase content of NO in the medium. Conclusion The model is convenient for in vitro study of lymphangiogenesis. H22 cell can accelerate lymphangiogenesis, which warrants further study for the understanding of the role of lymphangiogenesis in metastasis.