Therapeutic Effect Of CNTF On Rd Mouse And It's Eukaryotic Expression

Retinitis pigmentosa (RP) is the kind of disease that photoreceptors and retinal pigment epithelial cells degenerate gradually. The clinical sign of RP is progressive visual field defect, and leaves only tiny island of central visual field which will be disappeared at last. Until now, RP is classified as an unpreventable blindness disease by WHO, and there is no remedy for it. The only medicines could be recommended are VitA and Chinese traditional medicine on clinical, and can not stop the progress of RP, and there is limited effect. Neurotrophic factor is a kind of cytokines that have multi biological activities to central peripheral nervous system. Lots of animal experiments showed that neurotrophic factors have neuroprotective effect, and these factors are promising medicals for RP.CNTF can be found in wide range of central and peripheral nervous system, and play a key role on surviving of traumatic neuron and regeneration of axon. In this research, we observed the therapeutic effect of CNTF vitreous cavity injection on rd mice and biological effect of CNTF gene expressing in ARPE-19 cells.Methods:Section I : postnatal 10 days (P10) rd mice were randomized into three groups, CNTF treated group, sham-surgery group and blank control group. CNTF and PBS were injected into the vitreous of the treatment group and the sham-surgery group respectively on P10. After anesthesia, ERG of rd mice were examined on postnatal 14, 18, 22 and 30days, and then the mice were killed by over dosage anesthetic. Transmission electron microscope, light microscope, measurement of thickness of the retina outer nuclear layer and TUNEL assay were carried out.Section II: RT-PCR was used to amplify the cDNA of mouse CNTF gene, and truncated CNTF cDNA was obtained by site-directed mutagenesis. The two types of CNTF gene were cloned into plasmid pTracer-CMV and transfected ARPE-19 cells by transfectamine 2000. Dot blotting was used to detect the expression of CNTF. MTT and flow cytometry apoptosis assay were performed to observe the biologicaleffect of CNTF expression in ARPE-19 cells. Results:1. It was observed that the retina outer nuclear layer of the treatment group was thicker than that of the other two groups under light microscope in P14, P18, P22 and P30. There were statistical significance (p<0.05).2. TUNEL positive cells could be found in the outer nuclear layer of retina of the rd mice.3. Transmission result showed that apoptosis was a major pathway of pathologic changes in rd mouse retina, and inflammation was also involved in, and the CNTF treated group retina ultra structure of rd mouse was better than PBS treated group.4. ERG examination implicated that the retina function of CNTF treated group is better than control group.5. Wild type and truncated CNTF gene were amplified by RT-PCR, and their eukaryotic expression plasmids were successfully constructed.6. After ARPE-19 cells transfected with two types of recombinant plasmids, the CNTF had been detected in the cells culturing supernatant.7. Though Secretome 2.0 prediction, it is possible that CNTF is a non-classical secretory protein.8. MTT showed that the numbers of the cells transfected with two types of CNTF gene are more than cells transfected with blank pTracer, and the number of cells transfected with truncated CNTF gene is more than transfected with wild type.9. Quantitive apoptosis assay implicated that CNTF could partially inhibit the apoptosis that induced by the cells culturing with serum free culture.Conclusion:Vitreous injection of CNTF could slow photoreceptors' reduction of the rd mice, and may have therapeutic action on the retinitis pigmentosa of rd mouse. The therapeutic effect may be taken effect via anti-apoptosis. The duration of therapeutic is relatively short compared with the course of RP, and which is because of CNTF degradation and reduction of its concentration. Incorporation with molecular biologytechniques, it is a promising remedy for RP that to construct a drug delivery system of long period and delayed releasing CNTF. The truncated CNTF may have more powerful therapeutic effect than wild type. Expression of two types of CNTF in ARPE-19 cells gets prepared for gene therapy research of retinitis pigmentosa.