Use of JAM-C as a target in the preparation of a drug for preventing and / or treating proliferative vitreoretinopathy

Abstract

The present invention discloses the use of JAM-C as a target in the preparation of a drug for preventing and / or treating proliferative vitreoretinopathy. The present invention uses JAM-C as a target to develop, screen or prepare a drug for preventing and / or treating proliferative vitreoretinopathy. The drug can target JAM-C and upregulate the expression level of JAM-C, and can inhibit the proliferation, migration, contraction and EMT process of RPE cells, which has extremely important clinical significance for the prevention and treatment of proliferative vitreoretinopathy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of JAM-C as a target in the preparation of a drug for preventing and / or treating proliferative vitreoretinopathy. Background Art

[0002] Proliferative vitreoretinopathy (PVR) is a serious complication of rhegmatogenous retinal detachment. It also occurs after other ophthalmic diseases or ophthalmic treatments that can cause retinal damage and intraocular inflammation, and has become one of the main causes of blindness. Currently, surgery is still the main treatment for PVR. However, among patients who have successful surgery, only 40-80% of patients have a relatively ideal improvement in vision, and the recurrence rate is more than 25%. Therefore, there is still a lack of effective treatment for PVR.

[0003] The main pathology of PVR is the formation of a proliferative membrane in the vitreous cavity and on the surface of the retina, which shrinks and stretches to cause retinal detachment. Among them, the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is the core link in the development of PVR. After the retina is damaged, the blood-retinal barrier is also damaged, and RPE cells migrate to the vitreous cavity. Due to the loss of normal connections between cells, they undergo EMT, proliferate and acquire mesenchymal characteristics, and form an epiretinal membrane by secreting extracellular matrix and releasing chemical factors to recruit glial cells and fibroblasts. It can be seen that clarifying the EMT mechanism of RPE cells and their role in PVR is of great significance for the treatment of PVR.

[0004] It is currently known that cell / growth factors (such as PDGF, TGF-β, VEGF), transcription factors (Snail, Twist) and microRNA (miR-124, miR-29b) are involved in regulating the EMT process of RPE cells. In the existing technology, anti-inflammatory drugs (glucocorticoids), anti-cell proliferation agents (such as 5-fluorouracil, daunorubicin) and growth factor inhibitors (Conbercept) have been used as candidate drugs for the treatment of PVR for clinical trials, but their therapeutic effects are not ideal. Summary of the invention

[0005] The present invention provides the use of JAM-C as a target in the preparation of a drug for preventing and / or treating ophthalmic diseases. The present invention uses JAM-C as a target to develop, screen or prepare a drug for preventing and / or treating ophthalmic diseases. The drug can target JAM-C and upregulate the expression level of JAM-C, and can inhibit the proliferation, migration, contraction and EMT process of RPE cells, which has extremely important clinical significance for the prevention and treatment of ophthalmic diseases.

[0006] According to a first aspect of the present invention, there is provided use of JAM-C as a target in screening or preparing a drug for preventing and / or treating proliferative vitreoretinopathy.

[0007] Preferably, the drug is capable of upregulating the expression level of JAM-C.

[0008] Junctional adhesion molecule (JAM) belongs to the immunoglobulin superfamily and is involved in cell-to-cell adhesion, cell polarization, proliferation and migration. The JAM family includes molecules such as JAM-A, JAM-B, JAM-C, JAM-4, JAM-L, CAR and ESAM.

[0009] The present invention conducted a series of experiments to explore and clarify the mechanism of action of the junctional adhesion molecule JAM-C on PVR. The results showed that JAM-C expression was downregulated in clinical samples of PVR patients, and JAM-C overexpression could inhibit the proliferation, migration, contraction and EMT process of RPE cells. On the contrary, knocking down JAM-C would promote the EMT process of RPE cells. Therefore, the absence or low expression of JAM-C is an important driving factor for the occurrence and development of PVR.

[0010] Based on the above research results, the present invention uses the junctional adhesion molecule JAM-C as a target to develop, screen or prepare a drug for preventing and / or treating proliferative vitreoretinopathy. The drug can target JAM-C and upregulate the expression level of JAM-C, and can inhibit the proliferation, migration, contraction and EMT process of RPE cells, which has extremely important clinical significance for the prevention and treatment of proliferative vitreoretinopathy.

[0011] According to a second aspect of the present invention, there is provided use of an agent for promoting JAM-C expression in the preparation of a medicament for preventing and / or treating proliferative vitreoretinopathy.

[0012] Preferably, the drug comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient, wherein the active ingredient is an agent that promotes the expression of JAM-C.

[0013] Preferably, the agent promoting JAM-C expression includes an activator of the JAM-C gene or protein.

[0014] Preferably, the activator of the JAM-C gene or protein includes an adeno-associated virus vector AAV-JAM-C that overexpresses JAM-C.

[0015] According to a third aspect of the present invention, there is provided use of a reagent for detecting the expression level of JAM-C in the preparation of an auxiliary diagnostic reagent for proliferative vitreoretinopathy.

[0016] Preferably, the expression level of JAM-C is decreased in patients with proliferative vitreoretinopathy.

[0017] Preferably, the reagents for detecting the expression level of JAM-C include specific primers for detecting JAM-C and an ELISA kit.

[0018] According to a fourth aspect of the present invention, there is provided a medicament comprising an agent that promotes JAM-C expression as an active ingredient.

[0019] The present invention has the following beneficial effects:

[0020] The present invention has discovered the mechanism of action of JAM-C in PVR through research, and found that the expression of JAM-C in the retina of PVR patients is downregulated compared with the retina of healthy people. Therefore, this scheme uses JAM-C as a detection index, prepares a substance that can be used to detect JAM-C and applies it to the preparation of a diagnostic reagent or auxiliary diagnostic reagent for PVR, can diagnose PVR patients and use the expression level of JAM-C as a prognostic indicator to detect the condition of PVR patients, and has good clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The expression of JAM-C in the vitreous humor and retina of normal subjects and PVR patients.

[0022] Figure 2 The results show the effect of transforming growth factor TGF-β2 on the expression level of JAM-C and EMT markers in ARPE19 cells.

[0023] Figure 3 The results show the effect of knocking down JAM-C on the EMT process of RPE cells.

[0024] Figure 4 This is the model of the PVR mouse model construction process and the experimental results of fundus photography, OCT detection, and HE staining of the constructed PVR mouse model.

[0025] Figure 5These are the results of the effect of RPE-specific JAM-C knockout on the PVR mouse model.

[0026] Figure 6 The results show the effect of overexpression of JAM-C on the PVR mouse model. DETAILED DESCRIPTION

[0027] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0028] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the reagents and materials used are all commercially available unless otherwise specified.

[0029] Example 1 Expression of JAM-C in the vitreous humor and retina of healthy subjects and PVR patients

[0030] The purpose of this example is to explore the expression of the junctional adhesion molecule JAM-C in the retina of patients with proliferative vitreoretinopathy (PVR). Waste vitreous fluid removed during vitreous surgery was collected, of which the waste vitreous fluid removed during vitreous surgery in PVR patients was recorded as the PVR group, with a sample size of n=14, and the waste vitreous fluid removed during vitreous surgery in patients with macular holes and cataracts was recorded as the control group, with a sample size of n=8. After centrifugation of the vitreous fluid in the PVR group and the control group, the supernatant was collected and stored at -80°C for subsequent experiments.

[0031] The supernatant stored at -80°C was thawed on ice and tested by Elisa according to the manufacturer's recommended protocol (AE98563Hu, AMEKO, Shanghai, China). The mRNA samples of PVR proliferation membrane (GSE41019) in the GEO database were analyzed. The results are shown in Figure 1 shown.

[0032] Depend on Figure 1 As shown in A, the expression level of JAM-C in the vitreous humor of the PVR group was significantly lower than that of the control group. Figure 1 As shown in Figure B, the vitreous proliferative membrane of PVR patients (GSE41019) has a lower expression level of JAM-C compared with the retina of normal people. The above results indicate that JAM-C is downregulated in the clinical samples of PVR patients.

[0033] Example 2 Effects of transforming growth factor-β2-induced RPE cell EMT on the expression level of JAM-C and EMT markers

[0034] The purpose of this example is to use transforming growth factor TGF-β2 to induce retinal pigment epithelium (RPE) cells, so that RPE cells undergo epithelial-mesenchymal transition (EMT), and to detect the expression level of JAM-C and EMT markers in the induced RPE cells to explore whether the expression level of JAM-C is related to the EMT changes of RPE cells.

[0035] The RPE cells used in this example are ARPE19 cells (purchased from ATCC, Manassas, Virginia, USA). ARPE19 cells were cultured in DMEM / F12 medium containing 10 vol% fetal bovine serum (Corning, USA) and 1 vol% penicillin-streptomycin mixture (ScienCell, USA), and then trypsin (Gibco, USA) with a concentration of 0.25 vol% was used for cell digestion and subculture. In this study, ARPE19 cells with a subculture of less than 20 generations were selected for subsequent experiments.

[0036] In this study, different concentrations (2 ng / mL, 4 ng / mL) of TGF-β2 were added to induce the EMT process of RPE cells. ARPE19 cells were collected 48 hours after stimulation and Western Blot analysis was performed. The results are shown in Figure 2 shown.

[0037] Table 1 Antibodies and proteins

[0038]

[0039] The specific operation steps of Western Blot detection are as follows: add pre-mixed RIPA lysis buffer (R0010-100, Solarbio) containing protease / phosphatase inhibitors (Thermo Scientific) to the collected ARPE19 cells, scrape the cells with a scraper and collect them in a 1.5 mL EP tube, centrifuge after sufficient lysis and collect the supernatant, and use the BCA protein concentration assay kit (P0009, Beyotime) to determine the protein concentration on a microplate reader (Tecan Infinite® 200Pro, Switzerland). The sample loading system was prepared according to the protein concentration, and 50 μg of sample was loaded for SDS-PAGE electrophoresis and PVDF membrane (bio-rad) transfer. The membrane was blocked with 5% milk at room temperature for 1 hour, and then the primary antibody was added and incubated overnight at 4°C after rinsing. The next day, the primary antibody was discarded, and the membrane was washed 3 times with TBST, each time for 10 min, and the corresponding secondary antibody was added. Incubate at room temperature for 1 hour, and the protein was developed on the developer GBOX-CHEMI-XX8 (Syngene, Ballenger Creek, MD, USA) using luminescent liquids of different intensities (Pico, Thermo Scientific or Immobilon, Millipore). The internal reference protein HSP90 was used as the baseline value of each group, and the ratio of the experimental group to the control group was used as the experimental result and plotted. Among them, the antibodies and proteins used in the Western Blot detection in this embodiment are shown in Table 1.

[0040] Depend on Figure 2 It can be seen that with the increase of TGF-β2 stimulation concentration, the expression of JAM-C in ARPE19 cells showed a downward trend, while the expression of EMT markers such as FIBRONECTIN, VIMENTIN and N-CADHERIN showed an increasing trend ( p<0.05, p<0.01, p<0.001).

[0041] Example 3 Effect of knockdown of JAM-C on the EMT process of RPE cells

[0042] The purpose of this example is to further study the effect of interfering with JAM-C in Example 2 on the EMT of ARPE19 cells. In this example, Western Blot was first used to detect the expression levels of EMT markers and cell junction proteins after knocking down JAM-C to explore the effect of knocking down JAM-C on the EMT process of RPE cells. The proliferation activity of ARPE19 cells after knocking down JAM-C was also detected using CCK8 experiment. At the same time, ARPE19 cells after knocking down JAM-C were scratched and the scratch healing area of ​​ARPE19 cells was counted, as well as the glue contraction experiment and the glue area ratio was counted. The results are as follows Figure 3 As shown, the gel contraction experiment is designed to simulate the PVR membrane contraction characteristics in vivo.

[0043] The operation steps of CCK8 cell proliferation experiment are as follows: Before the experiment, ARPE19 cells were cultured at 5×10 3 The cells were seeded at a density of 10 cells / well in a 48-well plate and grown to 85% confluence the next day. The cells were knocked down with siJAM-C and siCTRL (negative control) and cultured for 72 hours. The culture medium was replaced with fresh DMEM / F12 medium, and CCK-8 (K1018-10000t, APExBio) solution was added at a volume ratio of 1:10. The cells were cultured in the incubator for another 4 hours, and the absorbance of the cells at 450 nm was measured using a microplate reader (Tecan Infinite® 200 Pro, Switzerland).

[0044] The steps of the cell scratch test are as follows: Before the experiment, ARPE19 cells were cultured at 1.5×10 5 The cells were inoculated at a density of 10 cells / well in a 6-well plate. The next day, the cells grew to 85% confluency, and the prepared siJAM-C and negative control siRNA were added to the corresponding wells. After transfection for 24 hours, the culture medium was replaced with serum-free DMEM / F12 (Corning) medium and 1µM mitomycin C (M5353-0.2ML, Sigma) was added for 1 hour. The cells in the middle of the well plate were scratched with a 200 µL pipette tip, washed twice with serum-free DMEM / F12 (Corning), and replaced with 2 mL of fresh serum-free DMEM / F12 medium. Photos were taken and recorded as the cell scratch state at 0 hours. After 48 hours of scratching, photos were taken again to record the scratch state. The scratch area at two time points was statistically analyzed using ImageJ (NIH, USA) software to calculate the difference in scratch repair between the two groups.

[0045] The steps of the gel contraction experiment are as follows: knock down siJAM-C and siCTRL (negative control) in advance using a 12-well plate or larger culture plate. After 24 hours, the knocked-down cells were digested and resuspended in serum-free DMEM / F12 medium. During this period, a 24-well plate was prepared and 1 mL of 1% BSA (B2064, Sigma) was added to the wells to be plated. The 24-well plate was placed in a 37°C incubator for 1 hour. The digested cells were counted and 2.5×10 cells were taken per well. 5 500 μL of gel contraction system containing ARPE19 cells, 2 mg / mL type I collagen (200110-10, Xiangbo Biotechnology), 0.025 N NaOH (1 times the volume of collagen) and ddH2O was prepared on ice. The 1% BSA in the 24-well plate was removed, and the mixed gel contraction system was added to the corresponding 24-well plate. After incubation in a 37°C incubator for 20 minutes, 500 μL of serum-free DMEM / F12 culture medium was added on top of the gel. Photos were taken after 0 hour and 48 hours, and the difference in gel contraction area between the experimental group and the control group was quantified using ImageJ software.

[0046] In this example, the siRNA target gene sequences involved are shown in Table 2.

[0047] Table 2 siRNA target gene sequences

[0048]

[0049] In this example, the steps for the siRNA-mediated knockdown experiment are as follows:

[0050] 1. In advance, seed ARPE19 cells into 6-well plates and culture to about 85% confluence.

[0051] 2. Prepare transfection reagent: Mix 30 pmol JAM-C siRNA or control siRNA (RiboBio) with 150 µL Opti-MEM (31985070, Gibco) to prepare solution A. Mix 6 µL liposome transfection reagent LipofectamineRNAiMAX (Invitrogen) with another 150 µL Opti-MEM to prepare solution B. Mix the prepared A and B separately and incubate at room temperature for 5 min. Then, slowly add solution A to solution B, mix well and incubate for 15 min.

[0052] 3. Add the prepared transfection reagent to the corresponding wells. After 48 hours of transfection, collect the cells for further experiments.

[0053] Depend on Figure 3As shown in A, compared with ARPE19 cells without JAM-C knockdown (siCTRL), knockdown of JAM-C (siJAM-C) in cultured ARPE19 cells revealed by Western Blot analysis that knockdown of JAM-C caused increased expression of EMT markers such as FIBRONECTIN, VIMENTIN, and N-CADHERIN, and decreased expression of cell junction proteins such as E-CADHERIN and ZO-1.

[0054] Depend on Figure 3 As shown in B, the CCK8 experiment results showed that the absorbance of ARPE19 cells at a wavelength of 450 nm increased significantly after JAM-C was knocked down ( p<0.001), indicating that knocking down JAM-C in ARPE19 cells can significantly promote the activity and proliferation ability of ARPE19 cells.

[0055] Depend on Figure 3 As shown in C (scale bar: 200 μm) and D, after knocking down JAM-C in ARPE19 cells, scratch treatment was performed and the scratch status was recorded at 0 h and 48 h. It was found that the scratch healing area increased significantly with the knockdown of JAM-C ( p<0.01), indicating that knocking down JAM-C in ARPE19 cells can significantly enhance the activity, proliferation and migration ability of ARPE19 cells.

[0056] Depend on Figure 3 As shown in E (scale bar: 10 μm) and F, after knocking down JAM-C in ARPE19 cells, the cells were subjected to a gel contraction experiment and photographed to record the gel contraction state 48 hours later. It was found that the gel area was significantly reduced ( p<0.05), indicating that the contractile ability of ARPE19 cells was significantly enhanced after knocking down JAM-C in ARPE19 cells, that is, the low expression of JAM-C is conducive to the contraction of PVR membrane.

[0057] The above results indicate that low expression of JAM-C in RPE cells is an important driving factor for the occurrence and development of PVR.

[0058] Example 4 Construction of PVR mouse model

[0059] In this example, the PVR mouse model was constructed according to the following steps: 8-week-old wild-type C57 mice (purchased from Jiangsu Jicui Experimental Animal Center) were selected, and Matrigel (1.5 μL / eye, 356231, BD) was first injected under the mouse retina to induce retinal detachment. One week later, RPE cells (1×10 4cells / 1µL / eye) to further induce the formation of PVR. Four weeks after the injection of RPE cells, the PVR mouse model was established. Fundus photography and optical coherence tomography (OCT) images were acquired using a small animal microscopy system (Phoenix, Pleasanton, CA, USA). Subsequently, the mice were anesthetized and killed, and the eyeballs of the mice were collected for subsequent experiments. The eyeballs of the collected mice were stained with HE. The results are shown in the figure. Figure 4 As shown, Figure 4 A in the figure is a schematic diagram of the construction process of the PVR mouse model. Figure 4 B (scale bar: 100 μm) shows the experimental results of fundus photography (left), OCT detection (middle) and HE staining (right) of the constructed PVR mouse model (PVR group). Among them, wild-type C57 mice were used as the control group for the construction of this model.

[0060] The steps of HE staining are as follows: the eyeballs of mice were collected and fixed with FAS eyeball fixative (G1109, Wuhan Saiweier Biotechnology Co., Ltd.) at room temperature overnight. The eyeballs were simply trimmed to remove the surrounding fascia tissue, dehydrated through gradient ethanol and embedded in paraffin. The eyeballs were sliced ​​into 5 μm thick slices and attached to slides, then dewaxed and hydrated, and finally H&E staining was performed. The slices were histologically observed and photographed under a microscope (LSM 710, Zeiss, Germany).

[0061] Depend on Figure 4 It can be seen that the PVR mice in the experimental group can form epiretinal membranes (red arrows) and subretinal membranes (yellow arrows), indicating that the PVR mouse model can be successfully constructed using the method provided in this example.

[0062] Example 5 Effect of JAM-C deficiency on PVR mouse model

[0063] In this example, the RPE-specific JAM-C knockout PVR mouse model was constructed according to the following steps:

[0064] 1. Construction of RPE-specific JAM-C knockout mice: This study will use two types of tool mice, Jam-c flox / flox mice were donated by Dr. Triantafyllos Chavakis of the National Institutes of Health Cancer Research Institute (PMID: 21593193), and BEST1-Cre mice were donated by Dr. Joshua L Dunaief of the University of Pennsylvania (PMID: 21212186). RPE-specific Jam-c-deficient mice were obtained by crossing Jam-c flox / flox mice with BEST1-Cre mice, and the genotype was further determined by PCR and sequencing.

[0065] 2. Construction of PVR mouse model: 8-week-old RPE-specific JAM-C knockout mice and non-knockout control mice were used for PVR modeling. Matrigel (1.5 µL / eye, 356231, BD) was first injected under the retina of the mice to induce retinal detachment. One week later, ARPE19 cells (1×10 4 cells / 1µL / eye, the specific preparation steps are as follows) to further induce the formation of PVR. Four weeks after the injection of RPE cells, the PVR mouse model was established.

[0066] 3. Construction of a cell model for shRNA-mediated JAM-C knockdown: ARPE19 cells were passaged into 6-well plates in advance, and when the confluence reached about 85% the next day, 1.5 µg of JAM-C shRNA plasmid and control shRNA plasmid (Sigma) were prepared into transfection systems using JetPEI (Polyplus, Illkirch) according to the manufacturer's recommended experimental steps. The prepared reagents were added to the corresponding well plates. After 3 days, puromycin (1 µg / mL, Beyotime) was added to screen the positive cells that had successfully transferred the shRNA plasmid, and the positive cells were collected for the corresponding intraocular injection experiments.

[0067] The sequences of the shRNA used in this example are shown in Table 3:

[0068] Table 3 shRNA target gene sequences

[0069]

[0070] The eyeballs of the RPE-specific JAM-C knockout PVR mouse model constructed above were collected, and one part was used for Western blot to detect the knockout efficiency of JAM-C, and the other part was frozen sectioned and immunofluorescence stained. At the same time, the RPE / retina thickness, the area of ​​the PVR membrane on the retina, and the area of ​​the PVR membrane under the retina in the staining results were statistically analyzed. The results are shown in Figure 5 As shown, the PVR mouse model with RPE-specific JAM-C knockout was the experimental group, and the C57BL6 / J mouse model after control intervention was the control group.

[0071] The specific operation of Western blot was as follows: the eyeballs of the above-mentioned PVR model mice were collected for dissection and the RPE-choroid-sclera complex was separated. RIPA lysis buffer (R0010-100, Solarbio) pre-mixed with protease / phosphatase inhibitors (ThermoScientific) was added to the complex. After sufficient lysis, the supernatant was centrifuged and collected. The protein concentration was determined on a microplate reader (Tecan Infinite® 200 Pro, Switzerland) using a BCA protein concentration assay kit (P0009, Bio-Time). The loading system was prepared according to the protein concentration, and 50 μg of the sample was loaded for SDS-PAGE electrophoresis and transfer to a PVDF membrane (bio-rad). The membrane was blocked with 5% milk at room temperature for 1 hour, rinsed, and then the primary antibody was added and incubated overnight at 4°C. The next day, the primary antibody was discarded and the membrane was washed 3 times with TBST. Each time for 10 min, add the corresponding secondary antibody, incubate at room temperature for 1 hour, and use different intensities of luminescent liquid (Pico, Thermo Scientific or Immobilon, Millipore) to develop the protein on the developer GBOX-CHEMI-XX8 (Syngene, Ballenger Creek, MD, USA). For data analysis, the internal reference protein α-TUBULIN was used as the baseline value of each group, and the ratio of the experimental group to the control group was used as the experimental result and plotted.

[0072] The specific operation of immunofluorescence staining is as follows: after the animal model is prepared, the animal is fully anesthetized and killed by cervical dislocation, the eyeball is carefully removed, a small incision is gently made in the cornea, and the eyeball is fixed with 4% paraformaldehyde (158127, Sigma) at room temperature for 2 hours, the fascia tissue around the eyeball is cleaned, and the cornea and lens are discarded, and the eyeball is dehydrated in 10%, 20% and 30% sucrose (V900116, Sigma) in sequence, each dehydration is performed at room temperature for 1 hour or at 4°C overnight until the eyeball sinks to the bottom of the tube, and after dehydration, the eyeball is embedded with OCT embedding medium (4583, SAKURA), and the embedded eyeball tissue is cut into 10 µm thick slices using a freezing microtome (LEICA), and the slices are attached to slides; before staining, the eyeball slices are fixed again with 4% paraformaldehyde (158127, Sigma) at room temperature for 30 min, washed three times and blocked with PBS containing 5% goat serum (NGS, D9663, Sigma), 1% BSA and 0.5% Triton X-100 at room temperature for 1 hour, then replaced with the prepared primary antibody dilution solution (anti-FIBRONECTIN, F3648, Sigma) and incubated overnight at 4°C. The next day, the primary antibody was discarded and the cells were washed three times with PBS, replaced with the corresponding secondary antibody and incubated at room temperature for 2 hours. Finally, DAPI (4',6-diamidino-2-phenylindole, D9542, Sigma) was added to stain the cell nucleus, washed and sealed, and images were acquired using a confocal fluorescence microscope LSM 710 (Zeiss, Germany).

[0073] The antibodies and proteins used in Western blot and immunofluorescence staining in this example are shown in Table 1.

[0074] Depend on Figure 5 As shown in A, in RPE cells (RPE65 is a marker of RPE), the expression of JAM-C in the RPE-specific JAM-C knockout PVR mouse model was almost completely lost, indicating that the RPE-specific JAM-C knockout was successful, and finally the RPE-specific JAM-C knockout mouse was successfully constructed.

[0075] exist Figure 5 In B (scale bar: 50 μm), DIC (left) shows the RPE layer and free ectopic melanin (red stars), and fluorescent photography (right) shows the distribution of PVR membrane (FIBRONECTIN, green) in the fundus. The red arrow and yellow arrow indicate the formation of the upper and lower retinal membranes, respectively, and DAPI (blue) indicates the cell nuclei.

[0076] Depend on Figure 5It can be seen from CE in the RPE that JAM-C deficiency aggravates the formation of PVR membranes on and under the retina, and JAM-C deficiency also leads to more melanin deposition in the vitreous cavity and increased RPE thickness.

[0077] The above results indicate that RPE-specific knockout of JAM-C aggravates the lesions of PVR mice, further proving that in vivo, the loss of JAM-C in RPE is an important factor in the occurrence and development of PVR.

[0078] Example 6 Effect of overexpression of JAM-C on PVR mouse model

[0079] In this example, a PVR mouse model overexpressing JAM-C was constructed according to the following steps:

[0080] 1. Construction of RPE-specific JAM-C overexpression mice: Four-week-old C57BL / 6J mice (purchased from Jiangsu Jicui Experimental Animal Center) were subretinally injected with AAV-JAM-C carrying an RPE-specific promoter, and AAV-CTRL was used as a control (2×10 10 vg / 1μL / eye) was injected, and the PVR model was established in mice 4 weeks later. AAV-JAM-C and AAV-CTRL were purchased from Heyuan Biotechnology (OBIO, Shanghai, China).

[0081] 2. Four weeks after the above injection, Matrigel (1.5 µL / eye, 356231, BD) was injected subretinaally to induce retinal detachment. One week later, RPE cells (1×10 4 cells / 1µL / eye, the specific preparation steps are as follows) to further induce the formation of PVR. Four weeks after the injection of RPE cells, the PVR mouse model overexpressing JAM-C was established.

[0082] 3. Construction of a cell model using Adv-mediated JAM-C overexpression: ARPE19 cells were passaged into 6-well plates in advance. When the confluence reached about 85% the next day, 2×10 9 Adv-JAM-C and Adv-CTRL of vp (viral particles) were added to the corresponding well plates. After 48 hours, the corresponding overexpressed cells were collected for intraocular injection experiments. Adv-JAM-C and Adv-CTRL were purchased from Vigene (Shandong, China).

[0083] The eyeballs of the PVR mouse model overexpressing JAM-C constructed above were collected, and one part was used for Western blot to detect the overexpression efficiency of JAM-C, and the other part was frozen sectioned and immunofluorescence stained. At the same time, the RPE / retinal thickness, the area of ​​the PVR membrane on the retina, and the area of ​​the PVR membrane under the retina in the staining results were statistically analyzed. The results are shown in the figure. Figure 6 As shown, the PVR mouse model overexpressing JAM-C was the experimental group, and the PVR model constructed by mice injected with AAV-CTRL was the control group.

[0084] Specific operation reference for Western blot and immunofluorescence staining Figure 5 shown.

[0085] exist Figure 6 In A, the JAM-C overexpression group showed a stronger JAM-C expression effect, indicating that JAM-C overexpression was successful, and finally the JAM-C overexpressing PVR mouse was successfully constructed.

[0086] exist Figure 6 In B (scale bar: 50 μm), DIC (left) shows the RPE layer and free ectopic melanin (red stars), and fluorescent photography (right) shows the distribution of PVR membrane (FIBRONECTIN, green) and corresponding cell nuclei (DAPI, blue) in the fundus. The red arrow indicates the epiretinal membrane, and the yellow arrow indicates the subretinal membrane.

[0087] Depend on Figure 6 CE in the Figure 3 showed that overexpression of JAM-C could significantly reduce the formation of PVR membranes on and under the retina in the PVR mouse model, and significantly alleviated the increase in RPE / retinal thickness and melanin deposition caused by PVR lesions. That is, JAM-C overexpression made the RPE thickness in the PVR mouse model thinner, and the formation of PVR membranes on and under the retina also appeared milder (Scar bar: 50 µm, n=6 / group, p<0.05, p<0.01).

[0088] The above results indicate that overexpression of JAM-C can alleviate the pathological changes in the PVR mouse model.

[0089] In summary, compared with the retina of healthy people, the expression of JAM-C in the retina of PVR patients is downregulated, and overexpression of JAM-C can significantly reduce the RPE EMT pathology of the PVR animal model. The present invention uses JAM-C as a target to develop, screen or prepare a drug for preventing or treating ophthalmic diseases. The drug can target JAM-C and upregulate the expression level of JAM-C, and can inhibit the proliferation, migration, contraction and EMT process of RPE cells, which has extremely important clinical significance for the prevention and treatment of ophthalmic diseases.

[0090] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention is described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the technical solutions of the present invention can be modified or equivalently replaced, but these modifications or replacements are all within the protection scope of the present invention.

Claims

1. Use of an agent for promoting JAM-C expression in the preparation of a drug for preventing and / or treating proliferative vitreoretinopathy; the agent for promoting JAM-C expression is an adeno-associated virus vector AAV-JAM-C that overexpresses JAM-C.

2. Application of reagents for detecting JAM-C expression in the preparation of auxiliary diagnostic reagents for proliferative vitreoretinopathy.

3. The use according to claim 2, characterized in that The expression level of JAM-C is decreased in patients with proliferative vitreoretinopathy.

4. The use according to claim 2, characterized in that The reagents for detecting the expression level of JAM-C include specific primers for detecting JAM-C and an ELISA kit.

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