The Effect Of Hypoxia-Inducible Factor-1¦Á Specificsmall Interfering RNA On the Expression of HIF-1¦Á In The Retinas Of Diabetic Rats

CHEN Song. Liu yan.

Clinical college of ophthalmology, Tianjin Medical University, Tianjin Eye Hospital, Tianjin institute of ophthalmology , Tianjin 300020, China

Objective
To investigate the inhibitory effect of HIF-1¦Á specific siRNA recombinant plasmid on the expression of HIF-1¦Á in the retina of diabetic rats and discuss the effect of HIF-1¦Á on the mechanism of diseases of retinal neovascularization , provide the basic rationale of treatment of diseases of retinal neovascularization .

Method
1. HIF-1 ¦Áspecific siRNA was constructed.100 SD rats were randomly devided into diabetic group( 79 rats) and normal group( 21 rats). Then the diabetic group was randomly into control group, vector group and gene therapy group. Diabetic rats were made by streptozocin. After the model of diabetic retinopathy was tested by FITC-Dextran angiograthy, liposome with vector plasmid and HIF-1¦Á siRNA were injected into vitreous of the gene therapy group and liposome with vector plasmid were injected into vitreous of the vector group. Every group was divided into three subsets seperately: group A1-A3, B1-B3, C1-C3 and D1-D3 at 24, 48 and 72 hours after injection.

2. The eyes were removed after 24, 48 and 72 hours in different group. After peeling off retina of rats with careful manipulation under the microseope,real time RT-PCR and Immunohistochemistry were taken to examine the expression of the HIF-1¦Á in retina respectively at 24, 48 and 72 hours afer the injection of vitreous. Two sections with integrate retina structure were selected from each eye and 5 fields from each section were selected and photographed by random(¡Á200 times). Then we analyzed the pictures using Image-Pro Plus 6.0. Mean integrated optic density (IOD) was used as the main statistic index.

3. The results were input the spss11.5 statistic software. Different treatment groups and subsets of different time between identical group were analyzed by one-way ANOVA. Then using SNK test to compare each group, P<0.05 was regarded as the standard of statistical significance.

Result
1. STZ induced diabetic rats had lasting and stable hyperglycaemia, displaying with the obvious signs of ¡°polydipsia, polyphagia, hperdiuresis and loss of weigh¡±. There was significant difference of weigh and plasma glucose of rats between the two groups at different time points(P<0.05).

2. Vascular morphology and distribution were changed in the retina of diabetic rats and lose the normal distribution pattern by FITC-Dextran fluorescence image.Retinal vascular was disorder.Retinal neovascularization,fluorescein leakage and microaneurysms was showed in the FITC-Dextran fluorescence image of retina in diabetic rats.The model of diabetic retinopathy was conformed. Vascular morphology and distribution were good in the retina of normal rats by FITC-Dextran fluorescence image.

3. The expression of HIF-1¦Á mRNA in retina was increased significantly in the control and vector groups than normal group and decreased in the gene therapy group compared with control group at the same time(P£¼0.05). The difference between different subgroups of group D was significant at the 0.05 level analyzed by statistics software spss11.5 (F=9.437,P=0.002). Futher more, we compared each subgroup with SNK test, the difference between D1and D2, D1 and D3 were significant(P£¼0.05). The difference between D2and D3 had no statistical significance(P=0.749). The expression of HIF-1¦Á mRNA in retina was decreased significantly in the group of 48 hours and 72 hours than which of the 24 hours(P£¼0.05). The difference of HIF-1¦Á mRNA in the retina between the 48 hous and 72 hours was not significantly(P£¾0.05).

4. The protein of HIF-1¦Á mainly expressed in the retinal ganglion cell layer, inner plexiform layer, inner nuclear, outer plexiform layer in diabetic group with immunihistochemistry. There was little expression of HIF-1¦Á in the retina of normal group. The expression of HIF-1¦Á significantly decreased in the in the gene therapy group compared with control group. The difference between different subgroups of group D was significant at the 0.05 level analyzed by statistics software spss11.5 (F=26.373,P=0.000). Futher more, we compared each subgroup with SNK test, the difference between D1and D2, D1 and D3 were significant(P£¼0.05). The difference between D2and D3 had no statistical significance(P=0.215). The expression of HIF-1¦Á in retina was decreased significantly in the group of 48 hours and 72 hours than which of the 24 hours. The difference of HIF-1¦Á in the retina between the 48 hous and 72 hours was not significantly.

Conclusion
The expression of HIF-1¦Á in the retina of diabetic rats was inhibited significantly by intravitreal injection of HIF-1¦Á specific interfering RNA. HIF-1¦Á
siRNA can become an new therapy for diabetic retinopathy and retinal neovascularization.