| Objective: To investigate the inhibitory effect of hypoxia inducible factor-1α(HIF-1α) anti-sense oligodexynucleotide combined with chemotherapy no human laryngeal squamous cacinoma of nude mice, and provide the evidence of animal experiment for the laryngeal squamous cacinoma using gene treatment.Methods: 1. Nude mice model bearing laryngeal carcinoma was established. Balb/c nude mice 70, 3-4 week age, 12-18g in weight, all females were selected. Experiment group: 0.2ml 2×106 Hep-2 cells were injected into nude mice in the back. Control group: 0.2ml PBS was injected into nude mice in the back. These nude mice were put into the condition of SPF and were feed with water and food. When tumor reaches 8-10 mm in size, they were used experiment. 2. Twenty-five nude mice were divided into five groups: contrast group, 1mg/kg group, 2mg/kg group, 3mg/kg group and 4mg/kg group, and apply abdomen injection every 48h for five times. Nude mice were killed at 10 days. The tumor's weight was determined. Part of tumor was fixed in the 70% alcohol. Flow Cytometry (FCM) was used to detect cell apoptosis. 3. Twenty nude mice were divided into four groups: group A (HIF-1a S-PS-ODN transfection + chemotherapy), group B (HIF-1a AS-PS-ODN transfection + chemiotherapy), group C (chemiotherapy), group D(blank for constrol). 100μg DNA was injected into animals in the group A and B (lipofectamine2000: DNA=1:1). All groups were injected with DDP every 48h. The duration was 16 day. The tumor weight and volume were determined every two days. The nude mice were killed 24h after the last injection. Flow cytometry (FCM) was used to detect cell apoptosis.The expression of HIF-1a, P53 and VEGF was detected by immunocytochemical method of PV and flow cytometry.Results: 1. Formation of tumor begin from 48h after injection of 2×106 Hep-2 cells. It reaches about 10mm at three weeks. The rate of tumor formation was 95%.It was reported squamous cell carcinoma by pathologist. 2. Under different dosage of chemo-drug, as the dosage increases, tumor cell inhibition rate and apoptosis rate were promoted. At the dosage of 3mg/kg, reaction of the nude mice was slight, and it impact on tumor was evident. It showed that 3mg/kg was the appropriate chemotherapy dose. 3. Impact on the tumor by HIF-1a AS-PS-ODN transfection combined with chemotherapy. Tumor weight of group B (HIF-1αAS-PS-ODN transfection for therapy) was the smallest in all groups. Tumor inhibition rate (56.83±4.57%) of group B (HIF-1αAS-PS-ODN transfection for therapy) were significantly higher than that in other 3 control (p<0.01). Apoptosis rate(44.75±5.71%)of group B(HIF-1a AS-PS-ODN transfection for therapy) were significantly higher than that in other 3 control(p<0.01). The results of immunocytochemical experiment: HIF-1αprotein and VEGF protein were in cytoplasm in most laryngeal squamous cells in all groups, and P53 protein in nuclear was in laryngeal squamous cells in all groups. Expression of these proteins in Group B (HIF-1αAS-PS-ODN transfection for therapy) was significantly lower in statistic than that in other 3 group. The results of Flow cytometry (FCM) experiment: After transfected by HIF-1a AS-PS-ODN, the expression of HIF-1α, P53, VEGF protein in Hep-2 cells were restrained. Their expressions in Hep-2 cells of HIF-1a AS-PS-ODN group were significantly lower in statistic than that in other 3 control groups(HIF-1a S-PS-ODN transfection for constrol, DDP for constrol, blank for control) (p<0.01). The results of electron microscopy: The membrane of tumor cells in group B was observed. Chromosome was concentrated to nucleus membrane, some were like new moons. Mitochondria had vacuole, rough endoplasmic reticulum was degranulated. Cell was round in shape in group D, as to plasm was rich, cell membrane was ellipse or irregular rule, chromatin was rich, and isoformate was a llitle. There was a big nucleus.Conclusion: 3mg/kg is the appropriate chemotherapy dose. Hypoxia inducible factor-1 alpha (HIF-1α) anti-sense oligodexynucleotide combined with chemotherapy human laryngeal squamous cacinoma of nude mice. All therapy groups had effect of inhibition on tumor (HIF-1a S-PS-ODN transfection + chemiotherapy, HIF-1a AS-PS-ODN transfection + chemiotherapy, chemiotherapy). Treatment strategy in Group B was the most effective in inhibition of tumors.
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