CN107338223B - Method for inhibiting apoptosis of photoreceptor cells and application of SCF overexpression virus in preparation of drugs for inhibiting apoptosis of photoreceptor cells - Google Patents

Abstract

The invention relates to a method for inhibiting apoptosis of photoreceptor cells and application of SCF overexpression virus in preparation of drugs for inhibiting apoptosis of photoreceptor cells. Therefore, the SCF/KIT signal pathway can inhibit the apoptosis of the photoreceptor cells and can be used for preventing and treating the diseases related to the retinal degeneration.

Description

Method for inhibiting apoptosis of photoreceptor cells and application of SCF overexpression virus in preparation of drugs for inhibiting apoptosis of photoreceptor cells

Technical Field

The invention particularly relates to a method for inhibiting photoreceptor cell apoptosis and application of an SCF overexpression virus in preparation of a medicament for inhibiting photoreceptor cell apoptosis.

Background

Retinal degeneration is a heterogeneous disease with vision loss caused by progressive degeneration of retinal nerve cells, and the pathogenic factors of the retinal degeneration are complex and mainly include genetic factors and environmental factors. Epidemiological investigation shows that retinal degeneration is the leading factor of vision loss in China at present, and retinal photoreceptor cell apoptosis is one of the main reasons causing blindness and low vision in China.

As the molecular mechanism of the apoptosis of the photoreceptor cells is unknown at present, no effective prevention and treatment means for diseases related to retinal degeneration exists so far.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides a method for inhibiting photoreceptor cell apoptosis and application of an SCF overexpression virus in preparation of drugs for inhibiting photoreceptor cell apoptosis.

A method for inhibiting apoptosis of photoreceptor cell by activating SCF/KIT signal pathway.

The method comprises the following steps: increased expression in photoreceptor cells by SCF facilitates activation of the SCF/KIT signaling pathway.

SCF is a stem cell factor, also a ligand for KIT (KITL).

KIT is the tyrosine kinase receptor KIT, also known as c-KIT.

Application of the SCF overexpression virus in preparing a medicament for inhibiting apoptosis of photoreceptor cells.

The SCF overexpression virus is adeno-associated virus AAV 8-Scf.

The invention has the beneficial effects that: the invention provides application of an SCF/KIT signal channel in inhibiting apoptosis of photoreceptor cells and application of an SCF overexpression virus in preparing a medicament for inhibiting apoptosis of the photoreceptor cells. Therefore, the SCF/KIT signal pathway can inhibit the apoptosis of the photoreceptor cells and can be used for preventing and treating the diseases related to the retinal degeneration.

Drawings

FIG. 1 is a graph of the results of an assay of retinal mild damage induced by intense light in two-month-old C57BL/6J mice. (wherein, in FIG. 1, A is the immunofluorescence result of anti-GFAP retinal section; B is the H & E staining result of retinal paraffin section; and C is the statistical analysis chart of the thickness of retinal outer nuclear layer.)

FIG. 2 is a graph showing the results of analysis of changes in gene expression in retinas induced by photodamage. (wherein, A is a light-induced retinal damage model for gene chip analysis, B is a heat map of gene chip expression profile analysis of retinas under normal light conditions (Control) and high light treatment (LD), and C is an RT-PCR result for verifying gene chip results.)

FIG. 3 is a graph showing the results of the analysis of the expression of photo-damage induced KITL and the activation receptor KIT. (wherein A, C, D is the result of Western Blot assay for detecting the amounts of KITL, KIT and p-KIT proteins, respectively; B is the data for statistical analysis of KITL content; E is the data for statistical analysis of KIT and p-KIT protein content.)

FIG. 4 is a graph showing the results of the internode expression analysis of photoinduced KITL in photoreceptor cells. (wherein panels A, B are slice immunofluorescence results for anti-SCF in retinas under normal light conditions and under high light treatment; panels a, B are from magnification of white box areas of panels A and B, respectively; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; IS, photoreceptor inner node; OS, photoreceptor outer node.)

FIG. 5 is a graph of the results of assays of the activity of KIT in the Wps mutation blocking retina. (wherein A IS a Western Blot result chart (left) for detecting changes in phosphorylation levels of KIT, ERK and AKT after intraocular KITL injection and a data statistical chart (right). B IS an immunofluorescence chart of two-month-old WT and Wps homozygous mouse retina sections for anti-ACK 45 and AB 5506. left IS a schematic diagram of two-antibody recognition receptor KIT sites; white arrows are KIT positive signals.RGC, ganglion cell layer; IPL, inner lamina, INL, inner nuclear layer; ONL, outer nuclear layer; IS, inner photoreceptor segment; scale 50 μm.)

FIG. 6 is a graph of the results of analysis of the photoproduction of retinal damage promoted by the mutations in Wps. (wherein A is a light damage pattern diagram; B is an amplitude waveform diagram of ERG analysis; the amplitude diagram of the upper half is a diagram of the detection result of the ERG in dark adaptation standard, the amplitude diagram of the lower half is a diagram of the detection result of the ERG in bright adaptation cone reaction; C and D are respectively a diagram of the statistical result of the ERG data of the ERG in dark adaptation standard and the ERG in bright adaptation cone reaction; E is a diagram of immunofluorescence of a retina section resisting GFAP; Control group is normal light treatment; LD group is strong light treatment; RGC, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; scale is 50 μm; p is 0.01.)

FIG. 7 is a graph showing the results of analysis of the photoproduction of retinal degeneration facilitated by the mutations in Wps. (wherein A IS a graph showing the result of morphological analysis of H & E-stained retinal tissue; the upper IS a graph showing the result of analysis of the thickness of the retinal outer nuclear layer; the lower IS a graph showing the result of analysis of the thickness of Rhodopsin in the retina; the upper IS a graph showing the immunofluorescence result of a retinal section against Rhodopsin; the lower IS a graph showing the band (left) and the data statistics result (right) of the expression of Rhodopsin in the retina by Western Blot; C and D are graphs showing the immunofluorescence result of a retinal section against Opsin and the statistics result of the number of Opsin-positive cells, respectively; E IS a graph showing the result of analysis of retinal apoptosis by TUNEL; the left IS a graph showing the fluorescence staining result of a section of TUNEL; the right IS a graph showing the statistics result of the number of TUNEL-positive cells; Control group IS a normal light irradiation treatment; LD group IS a strong light irradiation treatment; ONL, outer nuclear layer; IS, photoreceptor cell inner node OS; ONL, photoreceptor cell outer segments. The scale is 50 μm; represents p < 0.05; represents p < 0.01. )

FIG. 8 is a graph showing that AAV-CMV-GFP virus can specifically infect retinal photoreceptor cells. (wherein A is AAV-CMV-GFP virus vector information picture, B is fundus picture after AAV-CMV-GFP virus is injected into Wps homozygote mouse, the left is brightfield fundus picture, the right is GFP green fluorescence fundus picture, C is retina section fluorescence picture after AAV-CMV-GFP virus infection, ONL, outer nuclear layer, RPE, retinal pigment epithelium, scale is 50 μm, white arrow is GFP positive signal.)

FIG. 9 is a graph showing the analysis results of the overexpression of SCF in the retina by AAV-CMV-Scf virus. (wherein A is AAV-CMV-Scf virus vector information picture, B is immunofluorescence picture of retinal slice anti-SCF after AAV-CMV-Scf virus infection.)

FIG. 10 is a graph showing the results of analysis of the effect of AAV-CMV-Scf virus-overexpressed SCF on light-induced retinal degeneration. (wherein A IS an H & E staining result chart for analyzing the change of retinal structure after light-induced retinal injury, B IS an immunofluorescence chart for analyzing opsin after light-induced injury (upper row) and TUNEL detection for apoptosis analysis (lower row). NL group IS normal light treatment, LD group IS strong light treatment, RGC, ganglion cell layer, INL, inner nuclear layer, ONL, outer nuclear layer, IS, inner photoreceptor segment, OS, outer photoreceptor segment.)

Detailed Description

Embodiment of the first section: the experimental animals were 2-month-old C57BL/6J black mice. The normal light (200 lux) and the strong light (15, 000 lux, Philips cold light source LED lamp) are adopted to continuously irradiate the breeding cage from top to bottom for 6 days or 18 days, and then the appropriate light-induced retinal injury model is established through H & E staining retinal histomorphology analysis and anti-GFAP immunofluorescence Muller glial cell reactivation analysis. Subsequently, the gene expression profiling chip is used to analyze the changes in gene expression induced by photodamage and screen candidate target genes of interest. Finally, the change condition of the candidate target gene expression induced by photodamage is verified through RT-PCR, Western blot, immunofluorescence and other experiments.

In the research, a wild type C57BL/6J mouse is combined with long-time strong light treatment to establish a slight light injury model, so that a longer time window is provided for subsequent analysis of light-induced gene expression; then, analyzing the change condition of retinal gene expression induced by light damage by using a gene chip, finally further verifying the expression of differential genes by using an RT-PCR (reverse transcription-polymerase chain reaction) experiment, and screening a series of candidate neuroprotection related molecules, wherein the candidate neuroprotection related molecules comprise an SCF/KIT signal channel; finally, both KITL and KIT were found to be expressed in mature retinas by WB as well as IF assays, and photodamage induced the expression of KITL and promoted an increase in the phosphorylation level of KIT. These results suggest that photodamage activates the KITL/KIT signaling pathway in the retina.

FIG. 1, immunofluorescence results show that under normal light conditions, GFAP positive signals only sporadically appear at the ganglion cell layer, whereas after 6 days of intense light continuous treatment (LD 6 days), GFAP positive signals are significantly enhanced at the ganglion cell layer and appear at the inner plexiform layer and the inner nuclear layer (FIG. 1A). H & E staining results showed that photoreceptor cell outer segments were slightly, but not very, degenerated in 6 days of light, whereas photoreceptor cell outer segments were very significantly degenerated in 18 days of light treatment, compared to retinal structures in normal light (fig. 1B). In the dorsal region about 300 to 700 μm from the optic nerve, the outer nuclear layer was significantly thinned after 18 days of light treatment, while in the ventral region, the thickness of the outer nuclear layer was not significantly different from that of the normal control group, although there was a certain thinning tendency in the region 300 to 600 μm from the optic nerve (fig. 1C). These results indicate that retinal damage is very evident after 18 days of light induction, while retinal damage is not very evident after 6 days of light induction.

FIG. 2, the results of the gene chip show that the expression of Gfap is significantly up-regulated in the light-induced retina, suggesting that the retina suffers from photo-damage; the gene expression of visual signal transduction related proteins such as Rhodopsin, Grk1 and Opn1mw is obviously reduced by light induction; cytokines Edn2, Fgf2, Cntf and corresponding receptors Lifr and the like are all induced by light to be obviously up-regulated, and meanwhile, downstream genes Stat3 and Socs3 of a signal channel Cntf/Lifr are all induced by light to be obviously up-regulated; in addition, stem cell factor (SCF, also known as a ligand for KIT, KITL) is also significantly upregulated in light-induced damaged retinas (fig. 2A). The results of RT-PCR experiments show that the expression of Gfap is obviously up-regulated in the light-induced retina, and the cytokine Kitl (KIT ligand, also called Stem cell factor, SCF) is also obviously up-regulated in the light-induced retina. These results show that light induces SCF expression in the retina.

FIG. 3, Western Blot results show that specific bands appear in the size regions of molecular weight about 40KD (m-KITL) and 18KD (s-KITL), respectively, and that the positive signal in the light-treated retina of each band is significantly stronger than that of the normal control group (m-KITL, 2.39 + -0.87 fold; s-KITL, 4.52 + -1.57 fold, n = 3) (FIG. 3A, B), indicating that light damage induces the retinal up-regulation of KITL protein expression. Furthermore, the level of KIT phosphorylation (4.38 ± 0.73 fold, n = 3) was significantly higher in the photodamaged retina than in the normal control group, suggesting that photodamage activates the KIT signaling pathway.

FIG. 4, immunofluorescence results show that KITL is expressed predominantly in photoreceptor internodes in the retina and is light induced to be highly expressed in internodes (FIG. 4).

Embodiment of the second section: in the study, 2-month-old Wps mutant mice were used, exogenous SCF was injected intraocularly, multiple stimulation time points were selected, and changes in phosphorylation levels of KIT in retinas of WT and Wps homozygous mice were analyzed to determine whether Wps mutation disrupted the response of KIT to SCF. Subsequently, based on the results of the study in example 1 section, where photodamage activated the KIT signaling pathway in the retina, this section utilized the 2-month-old Wps mutation and C57BL/6J mice in conjunction with a model of photoinduced retinal damage, and at 18 days post-photoinduced, the KIT signaling pathway was analyzed for function during LIRD by ERG, H & E staining, and TUNEL detection.

The results show that the Wps mutation not only abolishes the KIT response to ligand KITL, but also further demonstrates that adult retinas do have KIT expression and its downstream signaling pathways. Under the induction of strong light, the amplitude of a wave and b wave detected by ERG of the Wps homozygote mouse is obviously reduced compared with that of the C57BL/6J mouse, the retina photoreceptor cell layer is obviously degenerated, and a large number of apoptotic cells are generated. These results suggest that Wps mutations promote highlight-induced retinal degeneration.

FIG. 5, KIT phosphorylation levels were significantly elevated in WT mouse retinas both 0.5 hours and 1 hour after KITL (100 μ g/. mu.l) injection (0.5 h, 2.3. + -. 0.32; 1h, 2.5. + -. 0.17, n = 4), but not in Wps homozygote mice (0.5 h, 1.1. + -. 0.132; 1h, 1.06. + -. 0.14, n = 4) (FIG. 5A). Furthermore, changes in phosphorylation levels of ERK and AKT, downstream molecules of KIT, were similar to those of KIT phosphorylation, indicating that injection of KITL induced increased phosphorylation levels of ERK and AKT in the WT mouse retina, but not in the Wps homozygous mouse retina. Antibodies recognizing the extracellular immunoglobulin region of receptor KIT (ACK 45) detected positive signals in WT retina, distributed across ganglion cell layer, inner plexiform layer, inner nuclear layer and outer nuclear layer, respectively, but it was difficult to detect positive signals in Wps homozygous mouse retina, with a small amount of positive signals in the inner nuclear layer only, indicating that Wps reduced KIT binding to antibody ACK45 (fig. 5B). Antibody AB5506 recognizing the intracellular protein kinase domain of KIT detected positive signals in both WT and Wps homozygous mouse retinas (fig. 5B). These results suggest that the Wps mutation disrupts the activity of KIT, possibly blocking the binding of KIT to ligand KITL resulting in reduced activity.

FIG. 6, after the mice were subjected to intense light for 18 days, the amplitude of both a wave (215. + -.10. mu.V, n = 5) and B wave (562. + -.63. mu.V, n = 5) of the standard ERG of the WT mice was found to be significantly reduced compared to that of the normal light WT mice (a wave, 347. + -.63. mu.V; B wave, 853. + -. 136. mu.V; n = 5) by visual neuroelectrophysiological (ERG) (FIG. 6B, C), suggesting that photodamage induces WT retinal function degeneration. Although the amplitude of both the a-wave (400 ± 24 μ V, n = 5) and B-wave (893 ± 154 μ V, n = 5) of the standard ERG of the Wps homozygote mouse was not significantly different from the WT mouse under normal light conditions, the amplitude of the a-wave (131 ± 13 μ V, n = 5) and B-wave (229 ± 79 μ V, n = 5) of the standard ERG of the Wps homozygote mouse was more significantly reduced than the WT mouse after photodamage induction (fig. 6B, D). Furthermore, the photopic ERG results also show similar phenomena. Photodamage induced b-wave amplitude reduction in both WT and Wps homozygote mice, but more pronounced b-wave amplitude reduction in Wps homozygote mice. Under normal lighting conditions, GFAP positive signals only appeared in the ganglion cell layers of WT and Wps homozygous mice, whereas GFAP was significantly upregulated in both WT and Wps homozygous mouse retinas and more significantly upregulated in Wps homozygous mice after intense light injury (fig. 6E). These results all indicate that the injury of Wps homozygote mice is more pronounced after highlight treatment than WT mice, suggesting that the Wps mutation promotes light-induced retinal damage.

FIG. 7, a result of H & E histomorphometric analysis, shows that photodamage apparently caused thinning of the outer nuclear layer of retinas of both WT mice and Wps homozygote mice, particularly in the dorsal region of the retina. After light-induced injury, photoreceptor outer segments of the WT mouse retina were significantly shortened, whereas photoreceptor outer segments of the Wps homozygous mouse retina were almost completely absent (fig. 7A). Immunostaining analysis of rod outer segment specific marker molecule Rhodopsin showed that Rhodopsin expression was significantly reduced in WT mouse photoreceptor outer segments after photoinduced injury, while the reduction in photoreceptor outer segment expression was more significant in Wps homozygous mouse retinas (fig. 7B). The specific marker molecule Opsin of the cone cells is subjected to immunostaining analysis, and the results show that under the condition of normal illumination, the Opsin expressions in retinas of WT mice and Wps homozygote mice are in strip shapes, are clearly visible and are uniformly spaced; after the treatment with strong light, the Opsin expression in the retinas of the WT mice and the Wps homozygote mice changed from the original strip shape to a point shape, and the quantity was significantly reduced compared with the quantity under normal illumination, especially the Opsin reduction in the retinas of the Wps homozygote mice was more significant (fig. 7C, D). TUNEL assay results showed that under normal light conditions, there was almost no TUNEL positive signal in the retinas of WT mice and Wps homozygous mice, whereas after intense light treatment TUNEL positive cells were detectable in both the WT mice (26 ± 8, n = 5) and Wps homozygous mice (68 ± 10, n = 5) outer nuclear retinal layers, and there was significantly more TUNEL positive cells in the retinas of the Wps homozygous mice than in the WT mice (fig. 7E). These results demonstrate that highlight treatment did induce apoptosis of retinal photoreceptor cells, and that Wps mutations promoted highlight-induced photoreceptor cell apoptosis.

Embodiment of the third section: the function of SCF in the light-induced retinal degeneration process is analyzed by combining the AAV-mediated gene overexpression technology of the adeno-associated virus with intraocular injection. Firstly, constructing a plasmid AAV8-CMV-Scf virus vector, determining to-be-sequenced to obtain a correct vector, and then packaging the vector to obtain the vector with the titer of 1 multiplied by 10¹²The above viruses. Subsequently, 0.5. mu.l of virus solution was injected intraocularly under a stereomicroscope into albino mice (Abino), and SCF expression was examined two weeks later and light-induced injury experiments were performed. Finally, after 3 days of light-induced injury, H was used&E staining and TNUEL detection, analyzing the effect of SCF on retinal degeneration.

The result shows that the AAV8-CMV-Scf virus can successfully mediate the abnormal high expression of the SCF in the photoreceptor cells, and can inhibit the retina degeneration and the apoptosis of the photoreceptor cells of albino mice induced by intense light, which indicates that the SCF can inhibit the apoptosis of the photoreceptor cells, and is expected to be used for treating diseases related to the photoreceptor cell degeneration.

Fig. 8, two weeks after injection of the virus into the eyes, no significant difference was observed by OCT ophthalmoscopy in the fundus of the injected virus compared to the non-injected group, suggesting that the injection into the eyes did not cause significant changes in the fundus. Subsequent observation through the green fluorescence channel revealed a GFP positive signal throughout the retinas of the injected virus group, whereas no GFP positive signal was observed in the non-injected group (FIG. 8B), indicating that AAV8-CMV-GFP virus was able to successfully infect the entire retina. The results of retinal sections showed that the GFP positive signals were mainly distributed in the inner segments of the photoreceptor cells, and the adjacent RPE also had a small amount of GFP positive signals (FIG. 8C), indicating that AAV8-CMV-GFP virus mainly infects photoreceptor cells of the retina and successfully mediates gene overexpression.

FIG. 9 shows that in the AAV8-CMV empty vector injection group, the expression level of SCF in the retina was very low, whereas in the AAV8-CMV-Scf virus injection group, the expression level of SCF was not significantly upregulated in the ganglion cell layer and the inner nuclear layer, but was abnormally highly expressed in the outer nuclear layer of the retina, and was mainly distributed in the outer ganglion of the photoreceptor cells and the connection with the nerve synapse formed between the neuronal cells in the inner nuclear layer (FIG. 9B).

FIG. 10, H & E staining, shows that intense light induced significant retinal thinning in Abino mice, mainly manifested by thinning of the outer nuclear layer and loss of photoreceptor outer nodes, whereas the thickness of the retina was significantly thicker in contralateral injection of AAV-CMV-Scf adenovirus (FIG. 10A). Immunofluorescence results showed that intense light induced an abnormal distribution of the opsin Rhodopsin in the retinas of Abino mice with the appearance of a large number of apoptotic cells, however, in the retinas contralaterally injected with AAV-CMV-Scf adenovirus, the opsin Rhodopsin distribution was more normal and the number of apoptotic cells was significantly lower than in the retinas not injected with the virus group (fig. 10B). These results indicate that injection of AAV8-CMV-Scf was effective in inhibiting light-induced retinal degeneration and photoreceptor apoptosis.

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.

Claims (1)

1. The application of the SCF overexpression virus in preparing the medicine for inhibiting the apoptosis of the photoreceptor cells is characterized in that the expression of the SCF in the photoreceptor cells is used for increasing and activating an SCF/KIT signal path to inhibit the apoptosis of the photoreceptor cells; the SCF overexpression virus is adeno-associated virus AAV 8-Scf.

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