| Objective: To investigate the inhibitory effect of hypoxia inducible factor-1α(HIF-1α) anti-sense oligodexynucleotide combine with radiation to human laryngeal squamous cacinoma of nude mice,and establish method of killing the laryngeal squamous cacinoma using gene treatment.Methods1 Nude mice modle bearing laryngeal cacinoma was established.Balb/c nude mice 80,3-4 week age,12-18g weight,female were selected.Experiment group:0.2ml 2×106 Hep-2 cell was injected into nude mice in the back.Constrol 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 Thirty nude mice were divided into six groups: constrol group,2Gy group,5Gy group,10Gy group,15Gy group and 20Gy group, and apply fractionated irradiation, irradiate 2Gy or 1Gy per 24h. Nude mice were killed in10 days. The tumor's weight were determinded. A part of tumor was fixed in the 70% alcohol. Flow cytometry(FCM) was used to detect cell apoptosis.3 Thirty nude mice were divided into five groups: group A (HIF-1a S-PS-ODN transfection for constrol ),group B(HIF-1a AS-PS-ODN transfection for therapy),group C(LP2000 for constrol),group D(radio for constrol),and group E(blank for constrol),100μg DNA was injected in the A and B group.(lipofectamine2000:DNA=1:1),all group was injected per 72h.The domation were 27 day. The weight and tumor's volume were determinded every three days.The nude mice was killed 24h after the last injection. Flow cytometry(FCM) was used to detect cell apoptosis.The expression of HIF-1a,P53,VEGF was detected by immunocytochemical method of PV and flow cytometry.Results1 Formation of tumor begin from 48h after injection of o.2ml 2×106 Hep-2 cell, it reaches at 10mm three weeks. Formation of tumor in 7 nude mice was bad, 6 had no tumor,4 were died , 2 had no tumor because they were injected of PBS. The rate of tumor formation was 95.71%.It was reported squamous cell cacinoma by pathology.2 The average of tumor inhibition rate were 0,2.841%,9.817%,19.201%,27.822% after radiotherapy by0Gy ,2Gy ,5Gy, 10Gy ,15Gy.( P<0.01).The average of Apoptosis rate were 12.474±3.009% ; 12.982±4.064% ;15.922±3.840% ; 19.768±2.602% ; 24.362±2.131%after radiotherapy by0Gy ,2Gy ,5Gy, 10Gy ,15Gy.Krushal-Wallis Test,mean rank were 11.10 16.40 10.50 4.00(Chi-square=16.209 P<0.01).because the nude mice were all died in 20Gy group.It showed that 10Gy group was the better radio dose.3.1 Tumor weight of group B(HIF-1αAS-PS-ODN transfection for therapy)was the best smallest in all group. Tumor inhibition rate(50.21±8.10%)of group B(HIF-1αAS-PS-ODN transfection for therapy) were significantly higher in statistic than that in other 4 control(p<0.05).3.2 Apoptosis rate(34.522±4.159%)of group B(HIF-1a AS-PS-ODN transfection for therapy) were significantly higher in statistic than that in other 4 control(p<0.05).3.3 The results of immunocytochemical experiment :HIF-1αprotein and VEGF protein in cytoplasm in most laryngeal squamous cells in all group, P53 protein in nuclear in laryngeal squamous cells in all group. Group B(HIF-1αAS-PS-ODN transfection for therapy) were significantly lower in statistic than that in other 4 group.3.4 The results of Flow cytometry(FCM) experiment : After transfected by HIF-1a AS-PS-ODN,the expression of HIF-1α,P53,VEGFprotein in Hep-2 cells were restrained,their expression in Hep-2 cells of HIF-1a AS-PS-ODN group were significantly lower in statistic than that in other 4 control group(HIF-1a S-PS-ODN transfection for constrol , LP2000 for constrol, radio for constrol, blank for control).( p<0.05)3.5 The results of electron micrscope: group A (HIF-1α S-PS-ODN transfection for constrol ),group B(HIF-1αAS-PS-ODN transfection for therapy),group C(LP2000 for constrol),group D(radio for constrol),and group E(blank for constrol)The membrane of cell in B groups sticked out at angle,chromosome was concentrated to nucleus membrane, some were like moons. Mitochondria had vacuole, rough endoplasmic reticulum was degranulated. Cell was round in E group,plasmid was rich, cell membrane was ellipse or no rule, chromatin was rich,isoformed was a llitle. There was a big nucleus. Mitochondria had little vacuole. rough endoplasmic reticulum was little degranulated. In D group had died nucleus fraction pieces. Mitochondria was local edema vacuole,mixture of crests or disappearance,part of membrane disappeared. A group and C group were all most the same as B group,but it's apoptosis was lighter than B group.Conclusion: Hypoxia inducible factor-1α(HIF-1α) anti-sense oligodexynucleotide combine with radiation treatment human laryngeal squamous cacinoma of nude mice. All therapy group had effect of inhibition tumor(HIF-1αS-PS-ODN transfection for constrol,HIF-1αAS-PS-ODN transfection for therapy,LP2000 for constrol,radio for constrol),Group B was the best effect of inhibition tumor. |
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