Objective To observe the inhibition of LipofectamineTM2000 (LF2000)mediated pSUPER recombinant plasmid expressing small interference RNA targeting hypoxia-induced factor (HIF)-1alpha;(pSUPERsiHIF-1alpha;) on retinal neovascularization in mice. Methods pSUPERsiHIF-1alpha; recombinant plasmid was created. Forty-eight (seven-day-old) C57BL/6J mice were randomly divided into a normal group, the control group, empty vector group and gene therapy group with 12 mice in each group. Mice in the normal group were kept in normal room air, while the other three groups retinal neovascularization was induced by hypoxia. The mice in control group were not treated. The mice in the vector group received intravitreous injection of pSUPER and LF2000 (1 mu;l), and the gene therapy group received pSUPERsiHIF-1alpha; and LF2000 (1 mu;l)one day before being returned to normal room air．Fluorescent angiography was used to assess the vascular pattern. The proliferative neovascular response was quantified by counting the nuclei of new vessels extending from the retina into the vitreous in cross-sections．HIF-lalpha;and vascular endothelial growth factor (VEGF) levels in retinas were measured by immune histochemical staining method and reverse transeriptase-polymerase chain reaction (RT-PCR). Results Fluorescent angiography showed radial branching pattern vessels in the normal group and distorted large vessels, obstructed capillaries, many neovascular tuffs, fluorescence leakage in the peripheral retina in the control group and vector group. The gene therapy group demonstrated a significant reduction in neovascular tufts and fluorescence leakage compared with the control group and the vector group. The number of vascular cell nuclei extending breaking through the internal limiting membrane(ILM) of control group and vector group increased significantly compared with normal group (F=5850.016,P＜0.05), while obviously decreasing in the gene therapy group compared with control group (F=3012.469,P＜0.05). Immunohistochemical staining showed the expression of HIF-1alpha; protein in nucleus and VEGF protein in cytoplasm. The expression of HIF-1alpha; protein in retina was negative, while VEGF protein was weakly positive in normal group. The expression of HIF-1alpha; and VEGF protein were both positive in control group and vector group, while weakly positive in gene therapy group. The Results of RT-PCR showed that the expression of HIF-1alpha; mRNA in retina was increased significantly in control group and vector group as compared with normal group (F=3102.326,P＜0.05), while decreasing significantly in gene therapy group as compared with control group (F=3336.425,P＜0.05). Conclusion Retinal neovascularization in the mice is significantly inhibited by intravitreal injection of LF2000-mediated recombinant plasmid pSUPERsiHIF-1alpha;.
ObjectiveTo observe the role of Notch signaling pathway inhibitor in differentiation process of stem cells derived from retinal Müller cells into the ganglion cell. MethodsRetinas of Sprague Dawley rat at postnatal 10-20 days were dissociated from eye balls. The third passage of Müller cells was used in this experiment, which cultured by repeated incomplete pancreatic enzyme digestion method. The retinal Müller cells were induced in the serum-free dedifferentiation medium. The cell proliferation state was observed under an inverted microscope. The expression of the specific markers Nestin and Ki-67 of retinal stem cells was measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The positive rate of nucleus was detected by Edu. The retinal stem cells was divided into Gamma secretase inhibtor-I (GSI) group and control group, the rate of ganglion cells was counted by using immunofluorescence staining. ResultsThe cell proliferation had gathered to form a sphere. Immunofluorescence staining showed that the expressions of Nestin and Ki-67 were (92.94±6.48%) and (85.96±6.04%) respectively. Edu positive rate of nucleus was (82.80±6.65)%. RT-PCR and Western blot further confirmed the high expression of Nestin and Ki-67 in the cell spheres but not in the Müller cells. The positive rate of ganglion cells were (16.98±2.87)% and (11.17±0.71)% in GSI group and control group respectively, with the significant difference (t=3.210, P=0.002). ConclusionNotch signaling pathway is an important regulatory gene in stem cells differentiated into retinal ganglion cell.