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To investigate the apoptosis of Hep-A cells induced by hyperthermia combined with Nitric Oxide donor (Isosorbide dinitrate, ISDN) and its mechanism. The inhibitory effect on the growth of Hep-A cells was measured by MTT assay. Apoptosis of Hep-A cells was observed by electron microscopy and flow cytometry. The levels of Bcl-2 were detected with Western blot assay. It showed stronger antiproliferative ability in three experimental groups than that in control, and hyperthermia combined with ISDN group had better inhibitory effect than other groups (p0.05). With electron microscopy, marked changes of cell apoptosis were observed, including microvilli disappearance or reduction, cell shrinkage, chromatin condensation or margination and the presence of "apoptosis bodies". The apoptotic ratio induced by hyperthermia and ISDN group was higher than other groups, furthermore, the levels of Bcl-2 were decreased in three experimental groups. The present study indicated that hyperthermia combined with ISDN could induce apoptosis of Hep-A cells and be more effective than either hyperthermia or ISDN, which may be related to expression decreased Bcl-2.

The aim of the experiment is to establish two murine hepatocarcinoma (HepA cell line) substrains with different lymphatic metastatic ability and compare their biological characteristics. Two murine hepatocarcinoma substrains, HepA-H and HepA-L, were established by the cell cloning and the selection way of lymph node metastasis. After tumor cells were injected subcutaneously to foot pad of NIH mice, the popliteal lymph node metastatic rates of HepA-H and HepA-L were 83.3% and 16.7% respectively, which has statistical significance (p0.01). There were no metastatic lesions were found in other organs, such as lung, liver, spleen and kidney. In vitro, HepA-H showed a higher ability in growth, migration and adhesion than that of HepA-L. The establishment of two murine hepatocarcinoma substrains provides a useful animal model for the study of mechanism of the lymphatic metastasis.