RNA molecule for treating retinal vascular diseases and its application

Abstract

The present invention relates to the field of biopharmaceutical manufacturing technology, and in particular to an RNA molecule for treating fundus vascular diseases and its application. The present invention innovatively provides a new type of RNA molecule, a single-stranded RNA with a sequence as shown in SEQ ID NO.1, which can reduce the tube formation, migration, proliferation and sprouting of endothelial cells, thereby effectively inhibiting neovascularization, and can also reduce vascular leakage and inhibit inflammatory response, providing broad clinical application prospects for the treatment of fundus vascular diseases. In addition, the RNA molecule has high safety and few side effects, and is expected to replace existing drugs for treating fundus vascular diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceutical manufacturing, and in particular to an RNA molecule for treating fundus vascular diseases and an application thereof. Background Art

[0002] Fundus vascular diseases are a type of disease characterized by fundus vascular lesions. The incidence rate is increasing year by year and has become a global public health problem and socioeconomic burden. Fundus vascular diseases include proliferative diabetic retinopathy (PDR), wet age-related macular degeneration (AMD), retinopathy of prematurity (ROP), etc. These diseases have become the main cause of blindness and visual impairment in all age groups from infants to the elderly, seriously affecting the quality of life of patients.

[0003] The main pathological characteristics of fundus vascular diseases are: damaged vascular barrier, vascular occlusion without perfusion, pathological neovascularization and secondary bleeding leakage, which then lead to blindness. The pathogenesis of fundus vascular diseases is extremely complex, involving a variety of cytokines and regulatory mechanisms, such as biochemical-molecular biological mechanisms, inflammatory-immune mechanisms, oxidative stress theory, epigenetic changes, etc. These mechanisms are intertwined and influence each other, so it is often difficult to achieve the ideal therapeutic effect by inhibiting a single pathway.

[0004] At present, the main treatments for this type of disease include systemic treatment, fundus laser photocoagulation, intraocular injection of drugs, and vitrectomy. Among them, intraocular injection of anti-vascular endothelial growth factor (VEGF) drugs has become the mainstream method of anti-angiogenic treatment. Studies have shown that fundus vascular disease is actually a chronic inflammatory disease, and endothelial disorders mediated by inflammatory responses will further aggravate vascular disease. In clinical practice, meloxicam (COX-2 inhibitor) and etanercept (soluble TNF-α antagonist) have been shown to effectively inhibit the typical histopathological changes of diabetic retinopathy.

[0005] However, existing treatments mainly focus on advanced lesions and have many shortcomings and relative side effects. For example, some patients do not respond to anti-vascular endothelial growth factor (VEGF) drug treatment, and even experience side effects such as retinal atrophy. In addition, these treatments often only target one aspect of either neovascularization or anti-inflammatory infiltration, and lack effective strategies to address both at the same time. The singleness of the therapeutic target makes it impossible to fully cover the multiple pathological processes of the disease. Because the pathogenesis of fundus vascular diseases is complex, involving multiple links such as neovascularization and inflammatory infiltration, a single treatment method may not be able to effectively control the progression of the disease, thereby limiting the treatment effect. Summary of the invention

[0006] In view of this, the present invention proposes an RNA molecule for treating retinal vascular diseases and its application.

[0007] The RNA molecules provided by the present invention can reduce the tube formation, migration, proliferation and sprouting of endothelial cells, thereby effectively inhibiting angiogenesis, and the RNA molecules can reduce vascular leakage and inhibit inflammatory reactions. Therefore, the present invention has a significant effect in treating fundus vascular diseases. In addition, the medicine prepared using the RNA molecules provided by the present invention not only has the function of resisting angiogenesis, but also has an anti-inflammatory effect, providing a broad clinical application prospect for the treatment of fundus vascular diseases.

[0008] The RNA molecules provided by the present invention will not damage endothelial cells, retinal cells and tissues under normal physiological conditions at therapeutic concentrations, so the possibility of side effects during treatment is low. The RNA molecules are expected to replace existing drugs for treating fundus vascular diseases, thereby reducing patients' discomfort and the risk of complications.

[0009] The technical solution of the present invention is achieved in this way:

[0010] In a first aspect, the present invention provides an RNA molecule for treating fundus vascular diseases, wherein the RNA molecule is a single-stranded RNA with a sequence as shown in SEQ ID NO.1, and the RNA molecule is named tsRNA-Cys-5-0007.

[0011] The tsRNA-Cys-5-0007 is a derivative fragment of tRNA-Cys. The RNA molecule has a high degree of homology in humans and mice. In the tsRBase database, the human-derived number of the RNA molecule is hsa_tsr015263, and the mouse-derived number is mmu_tsr009568.

[0012] In a second aspect, the present invention provides an RNA mimic for treating retinal vascular diseases, wherein the RNA mimic is artificially chemically synthesized; the RNA mimic is single-stranded RNA (sequence as shown in SEQ ID NO.1) and / or double-stranded RNA, the sequence of one chain of the double-stranded RNA is shown in SEQ ID NO.1, and the sequence of the other chain is shown in the reverse complementary sequence of SEQ ID NO.1.

[0013] In a third aspect, the present invention provides the use of the RNA molecule or the RNA mimic in preparing a product, wherein the product is used to prevent, treat and / or alleviate retinal vascular diseases.

[0014] In a fourth aspect, the present invention provides the use of the RNA molecule or the RNA mimic in the preparation of a product, wherein the product is used to inhibit one or more pathological behaviors of endothelial cells in angiogenesis, migration, proliferation and sprouting.

[0015] In a fifth aspect, the present invention provides the use of the RNA molecule or the RNA mimetic in the preparation of a product, wherein the uses of the product include at least one of the following uses: (I) reducing fundus vascular leakage; (II) promoting absorption of inflammatory leakage; (III) reducing choroidal neovascularization; (IV) reducing vascular inflammation; (V) reducing inflammatory cell infiltration; (VI) protecting the structure of retinal cells and / or retinal tissue; (VII) protecting the function of retinal cells and / or retinal tissue.

[0016] In a sixth aspect, the present invention also provides a composition for treating retinal vascular diseases, the composition comprising at least one of the following components: (1) tsRNA-Cys-5-0007 and pharmaceutically acceptable excipients thereof; (2) the RNA mimic and pharmaceutically acceptable excipients thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 This is the flow cytometry detection result of tsRNA-Cys-5-0007mimic on the survival and apoptosis of HRVECs under normal physiological conditions, where the horizontal axis FITC-A represents the fluorescence intensity of AnnexinV labeled with FITC, and the vertical axis PE-A represents the fluorescence intensity of PI labeled with PE;

[0019] Figure 2 This is the PI / Calcein-AM double staining test result of tsRNA-Cys-5-0007mimic on the survival and apoptosis of HRVECs under normal physiological conditions, where PI is the red dead cell signal and Calcein-AM is the green live cell signal;

[0020] Figure 3 This is the result of HE staining of paraffin sections showing the effect of tsRNA-Cys-5-0007 mimic on the morphology of mouse retinal tissue under normal physiological conditions;

[0021] Figure 4This is the result of frozen section TUNEL staining test on the effect of tsRNA-Cys-5-0007mimic on the survival and apoptosis of mouse visual cells under normal physiological conditions, where blue is DAPI signal, green is TUNEL signal, and the DNase-1 treatment group is used as a positive control for apoptosis treated with DNase;

[0022] Figure 5 This is the result of the Matrigel tube formation experiment to detect the effect of tsRNA-Cys-5-0007 on the tube-forming ability of endothelial cells under VEGF-induced pathological conditions;

[0023] Figure 6 The results of the Transwell cell migration assay were used to detect the effect of tsRNA-Cys-5-0007 on the migration ability of endothelial cells under VEGF-induced pathological conditions;

[0024] Figure 7 This is the result of the EdU proliferation experiment to detect the effect of tsRNA-Cys-5-0007 on the proliferation ability of endothelial cells under VEGF-induced pathological conditions, where blue is the DAPI signal and green is the EdU proliferation signal;

[0025] Figure 8 This is the experimental result of 3D endothelial cell spheroid sprouting experiment to detect the effect of tsRNA-Cys-5-0007 on the sprouting ability of endothelial cells under VEGF-induced pathological conditions;

[0026] Fig. 9 This is the result of Evans Blue staining to detect the effect of tsRNA-Cys-5-0007 on retinal vascular leakage in STZ-induced diabetic retinopathy (DR) mice;

[0027] Fig.10 The results of the leukocyte stasis experiment to detect the effect of tsRNA-Cys-5-0007 on retinal vascular inflammatory infiltration in STZ-induced DR mice, where green is the vascular signal and the white arrow points to the inflammatory cell infiltration signal;

[0028] Fig.11 The results of IB4 staining of choroidal flat mounts to detect the effect of tsRNA-Cys-5-0007 on laser-induced choroidal neovascularization in mice, where the four green fluorescent signals (marked by white dotted lines) surrounding the optic disc are the laser-induced neovascularization areas. DETAILED DESCRIPTION

[0029] Currently, the therapeutic targets for treating retinal vascular diseases are single, which makes it impossible to fully cover the multiple pathological processes of the disease. They often only target one aspect, either neovascularization or anti-inflammatory infiltration, and lack effective strategies to deal with both at the same time.

[0030] In view of this, the present invention innovatively provides a new type of RNA molecule, which can reduce the tube formation, migration, proliferation and sprouting of endothelial cells, thereby effectively inhibiting the formation of new blood vessels, and can also reduce vascular leakage and inhibit inflammatory response, providing a broad clinical application prospect for the treatment of fundus vascular diseases. In addition, the RNA molecule has high safety and few side effects, and is expected to replace existing drugs for the treatment of fundus vascular diseases, thereby reducing patients' discomfort and the risk of complications.

[0031] To this end, the embodiments of the present application disclose at least the following technical solutions:

[0032] In a first aspect, the present invention provides an RNA molecule for treating fundus vascular diseases, wherein the RNA molecule is a single-stranded RNA with a sequence as shown in SEQ ID NO.1, and the RNA molecule is named tsRNA-Cys-5-0007.

[0033] In a second aspect, the present invention provides an RNA mimic for treating retinal vascular diseases, wherein the RNA mimic is artificially chemically synthesized; the RNA mimic is single-stranded RNA (sequence as shown in SEQ ID NO.1) and / or double-stranded RNA, the sequence of one chain of the double-stranded RNA is shown in SEQ ID NO.1, and the sequence of the other chain is shown in the reverse complementary sequence of SEQ ID NO.1.

[0034] In certain embodiments, the RNA of the present invention may be free of any chemical modification. In other embodiments, in order to enhance stability or other beneficial features, the RNA of the present invention (whether single-stranded RNA or double-stranded RNA) may be chemically modified. The nucleic acids characterized by the present invention may be synthesized and / or modified by methods widely established in the art, such as glycosyl modification (e.g., modification at the 2′ position or 4′ position) or replacement of sugars; base modification, such as replacement with more stable bases, removal of unstable bases or bases matching the expanded pairing library, removal of bases (non-basic nucleotides) or conjugated bases; protrusions and end modifications, such as 5′ end modifications (e.g., phosphorylation, conjugation modification, inversion bonds, etc.) or 3′ end modifications (e.g., conjugation modification, addition of DNA nucleotides, inversion bonds, etc.); and double helix structure modifications, such as modification or replacement of phosphodiester bonds.

[0035] In some preferred embodiments, the RNA mimics have chemical modifications.

[0036] In some preferred embodiments, the RNA molecule is a chemically modified single-stranded RNA.

[0037] In some preferred embodiments, the RNA mimic is a chemically modified double-stranded RNA.

[0038] In some preferred embodiments, the chemical modification includes one or more of sugar modification, phosphate chain modification, base modification, protrusion and end modification, and double helix structure modification.

[0039] In a third aspect, the present invention provides the use of the RNA molecule or the RNA mimic in preparing a product, wherein the product is used to prevent, treat and / or alleviate retinal vascular diseases.

[0040] In a fourth aspect, the present invention provides the use of the RNA molecule or the RNA mimic in the preparation of a product, wherein the product is used to inhibit one or more pathological behaviors of endothelial cells in angiogenesis, migration, proliferation and sprouting.

[0041] In a fifth aspect, the present invention provides the use of the RNA molecule or the RNA mimetic in the preparation of a product, wherein the uses of the product include at least one of the following uses: (I) reducing fundus vascular leakage; (II) promoting absorption of inflammatory leakage; (III) reducing choroidal neovascularization; (IV) reducing vascular inflammation; (V) reducing inflammatory cell infiltration; (VI) protecting the structure of retinal cells and / or retinal tissue; (VII) protecting the function of retinal cells and / or retinal tissue.

[0042] In some preferred embodiments, the fundus vascular disease includes one or more of retinal neovascularization-related diseases, corneal neovascularization-related diseases, iris neovascularization-related diseases and choroidal neovascularization-related diseases.

[0043] In some preferred embodiments, the retinal neovascularization-related diseases include one or more of diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, retinal periphlebitis, proliferative vitreoretinopathy, retinal vasculitis, hypertensive retinopathy, age-related macular degeneration, polypoidal choroidal vasculopathy, high myopic choroidal neovascularization and idiopathic choroidal neovascularization.

[0044] In a sixth aspect, the present invention also provides a composition for treating retinal vascular diseases, the composition comprising at least one of the following components: (1) tsRNA-Cys-5-0007 and pharmaceutically acceptable excipients thereof; (2) the RNA mimic and pharmaceutically acceptable excipients thereof.

[0045] In certain embodiments, the compositions disclosed in the present invention can be delivered by any suitable route of administration, including intravenous, intratumoral, intraocular, oral and parenteral. In certain embodiments, the compositions are delivered systemically (e.g., via oral or intravenous administration). In certain other embodiments, the compositions are delivered topically to the eye by injection (e.g., intravitreal) or by eye drops.

[0046] Actual dosage levels of the RNA molecules or RNA mimetics in the compositions can be varied so as to obtain an amount of the RNA molecule or RNA mimetic that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0047] The selected dosage level will depend upon a variety of factors including the activity of the specific agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the specific compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the specific compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0048] A physician with ordinary skills in the art can easily determine and prescribe the effective amount of the desired composition. For example, a physician or veterinarian can prescribe and / or administer the dosage of the medicament used in the pharmaceutical composition at a level lower than that required for achieving the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. Typically, the suitable daily dose of the RNA molecule or RNA mimics described in the present invention will be the amount of the RNA molecule or RNA mimics at the minimum dose that effectively produces the therapeutic effect. Such an effective dose will generally depend on the above-mentioned factors.

[0049] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0050] The tsRNA-Cys-5-0007mimic and tsRNA-Cys-5-0007agomir used in the embodiments of the present invention are both chemically modified single-stranded RNAs, and the sequences are shown in SEQ ID NO.1. Among them, tsRNA-Cys-5-0007mimic is methylated at the 3' end; tsRNA-Cys-5-0007agomir is methylated at the 3' end and cholesterol modified. Both tsRNA-Cys-5-0007mimic and tsRNA-Cys-5-0007agomir were commissioned to be synthesized by Guangzhou Ruibo Biotechnology Co., Ltd.

[0051] Example 1 Effects of RNA mimics of the present invention on HRVECs cell survival and apoptosis

[0052] In this embodiment, HRVECs are divided into three groups, namely, the treatment group tsRNA-Cys-5-0007mimic (mimic), the non-treatment group NC mimic (Negative control) and the Ctrl group (blank control): the treatment group uses lipofectamine3000 transfection reagent and the tsRNA-Cys-5-0007mimic of the present invention to transfect HRVECs (human retinal vascular endothelial cells); the non-treatment group uses NC mimic (disordered nucleotide sequence of the same length as tsRNA-Cys-5-0007mimic) to transfect HRVECs; the Ctrl group does not undergo any treatment. Then the cells are collected and apoptosis is detected to evaluate the effect of tsRNA-Cys-5-0007mimic on cell survival and apoptosis under normal physiological conditions. Specifically as follows:

[0053] 1. Experimental materials:

[0054] HRVECs were purchased from ATCC Cell Bank (ACBRI-181), ECM medium (Cat No: 1001) was purchased from ScienCell Biotechnology, USA, fetal bovine serum (Cat No: FBS500-s) was purchased from AusGeneX, Australia, lipofectamine3000 (Cat No: L3000015) was purchased from Thermo Fisher Scientific, USA, tsRNA-Cys-5-0007mimic was purchased from Guangzhou Ruibo Biotechnology Co., Ltd., and trypsin-EDTA (Cat No: 25200114) was purchased from ThermoFisher Scientific, USA. Annexin V-FITC apoptosis detection kit (Cat No: C1062M) was purchased from Bio-Tech Biotechnology, China.

[0055] 2. Experimental steps:

[0056] (1) HRVECs were cultured in ECM medium containing 10% fetal bovine serum, 1% endothelial cell growth factor, and 1% penicillin / streptomycin, and the cells were seeded in 6-well plates;

[0057] (2) The cells were divided into three groups: treatment group, non-treatment group and Ctrl group. When the cell confluence reached about 60%, the treatment group was cultured for 24 hours with a liposome complex reaction system mixture consisting of lipofectamine3000 and tsRNA-Cys-5-0007mimic (50nM); in the reaction system of the non-treatment group, tsRNA-Cys-5-0007mimic (50nM) was replaced with NC mimic (50nM); the Ctrl group was not treated in any way.

[0058] (3) Digest the cells with 0.25% trypsin and collect the cell pellet by centrifugation. Add Buffer, PI solution and FITC solution in sequence according to the instructions of the Annexin V-FITC Cell Apoptosis Detection Kit and incubate in the dark for 20 minutes.

[0059] (4) Detect the percentage of cell apoptosis using flow cytometry.

[0060] 3. Experimental results

[0061] like Figure 1 As shown, under normal physiological conditions, after tsRNA-Cys-5-0007 mimic treatment, PI flow cytometry results showed that the proportion of live cells in each group was greater than 98%, and no obvious early apoptosis, late apoptosis and dead cells were observed. tsRNA-Cys-5-0007 at therapeutic concentrations will not cause apoptosis of HRVECs under normal physiological conditions.

[0062] Example 2 Effects of RNA mimics of the present invention on HRVECs cell survival and apoptosis

[0063] In this example, the cell transfection treatment method is the same as in Example 1, and then the PI / Calcein-AM double staining method is used to detect the survival and apoptosis of HRVECs to evaluate the effect of tsRNA-Cys-5-0007 mimic on cell survival and apoptosis under normal physiological conditions. The details are as follows:

[0064] 1. Experimental materials:

[0065] PI (Cat No: 17517) and Calcein-AM (Cat No: 22003) were purchased from AAT Bioquest Biotechnology Company, USA, dimethyl sulfoxide (Cat No: D2650) was purchased from Sigma Biotechnology Company, USA, and PBS (Cat No: BL302A) was purchased from Biosharp Biotechnology Company, China.

[0066] 2. Experimental steps:

[0067] (1) HRVECs were seeded in a 24-well plate, and the grouping and cell transfection treatment methods were the same as the experimental steps (1) and (2) in Example 1;

[0068] (2) Preparation of PI and Calcein-AM stock solutions: Dissolve 1 mg PI in 1 mL ddH2O to prepare PI stock solution, and dissolve 1 mg Calcein-AM in 1 mL anhydrous dimethyl sulfoxide (DMSO) to prepare Calcein-AM stock solution, and store in a sealed freezer at -20°C;

[0069] (3) Aspirate the culture medium in the original culture dish, wash twice with preheated sterile phosphate buffered saline (PBS), add 1 mL of serum-free ECM to each dish, then add 2 μL of PI stock solution and 3 μL of Calcein-AM stock solution in sequence, and then place in a 37°C, 5% CO2 incubator for staining for 10 min;

[0070] (4) After 10 minutes, place the sample under an inverted fluorescence microscope for observation. Excite the sample at a wavelength of 490±10 nm to observe the yellow-green living cells. Then excite the sample at a wavelength of 545 nm to observe the red dead cells. Take photos and record the results.

[0071] 3. Experimental results

[0072] like Figure 2 As shown, under normal physiological conditions, after tsRNA-Cys-5-0007 mimic treatment, no red PI signal was observed in the PI / Calcein-AM double-stained cells. The results showed that tsRNA-Cys-5-0007 would not cause the death of HRVECs under normal physiological conditions at therapeutic concentrations.

[0073] Example 3 Effect of RNA mimics (RNA agomir) of the present invention on mouse retinal tissue morphology

[0074] The mice were divided into a wild-type group, a treatment group, and a non-treatment group. The wild-type group was not treated in any way. The mice in the treatment group were injected with tsRNA-Cys-5-0007agomir (mimic) into the vitreous cavity, and the mice in the non-treatment group were injected with Scragomir (Scramble control, negative control) into the vitreous cavity. Retinal sections were made and HE staining was performed to observe whether the structure of the mouse retina had pathological changes, so as to evaluate the effect of tsRNA-Cys-5-0007 on the morphology of the mouse retina under normal physiological conditions. The details are as follows:

[0075] 1. Experimental materials:

[0076] C57BL / 6J mice were purchased from the Model Animal Institute of Nanjing University, and hematoxylin and eosin stain (Cat No: BP-DL001) was purchased from Nanjing Senbeijia Biotechnology Co., Ltd.

[0077] 2. Experimental steps:

[0078] (1) The mice were divided into a wild-type group, a treatment group, and a non-treatment group. The wild-type group did not receive any treatment, while the mice in the treatment group and the non-treatment group were injected once a week with 1 μL of 1 mM tsRNA-Cys-5-0007agomir injected into the vitreous cavity of each eye. The control group was injected with an equal amount of Scr agomir.

[0079] (2) General anesthesia was induced by intraperitoneal injection of ketamine (80 mg / kg) and xylazine (4 mg / kg). The head, limbs and tail of the mouse were fixed with the abdomen facing upward. The hair on the chest and abdomen was removed. The skin at the xiphoid process was incised, the fascia was bluntly separated, the ribs were cut, and the chest cavity was exposed.

[0080] (3) After the left ventricle was perfused with saline, the eyeballs were immediately harvested for paraffin sectioning.

[0081] (4) Take out the paraffin sections and place them in a 65°C oven for 25 min; then dewax and hydrate them: xylene for 30 min, and anhydrous ethanol for 2 min.

[0082] (5) 95% ethanol for 2 min; 80% ethanol for 2 min; 70% ethanol for 2 min, ddH2O for 2 min.

[0083] (6) Incubate with hematoxylin for 5 min, wash with water to remove the stain, and then incubate with eosin for 2 min. Observe the staining under a microscope.

[0084] (7) Dehydrate with ethanol step by step until transparent, seal the slides with neutral gum, let dry and take photos.

[0085] 3. Experimental results

[0086] like Figure 3As shown, after intravitreal injection of tsRNA-Cys-5-0007agomir, the structure of the mouse retina did not undergo obvious pathological changes. The results showed that tsRNA-Cys-5-0007 would not affect the morphology of mouse retinal tissue under normal physiological conditions.

[0087] Example 4 Effects of RNA agomir of the present invention on survival and apoptosis of mouse retinal cells

[0088] The mice were divided into wild-type group, treatment group, non-treatment group and apoptosis positive control group (DNA enzyme treatment). The wild-type group was not treated. The mice in the treatment group were injected with tsRNA-Cys-5-0007agomir (mimic) into the vitreous cavity, the mice in the non-treatment group were injected with Scr agomir (Scramble control, negative control) into the vitreous cavity, and the mice in the apoptosis positive control group were injected with Dnase-1 (DNA enzyme) into the vitreous cavity. Retinal frozen sections were prepared, and retinal cell apoptosis was detected using a one-step TUNEL cell apoptosis detection kit and DAPI staining to confirm the effect of tsRNA-Cys-5-0007 on the survival and apoptosis of mouse retinal cells under normal physiological conditions. The details are as follows:

[0089] 1. Experimental materials:

[0090] C57BL / 6J mice were purchased from the Model Animal Institute of Nanjing University, and the one-step TUNEL apoptosis detection kit (Cat No: C1086) and DAPI staining solution (Cat No: G6210) were purchased from Bio-Tech Biotechnology Co., Ltd., China.

[0091] 2. Experimental steps:

[0092] (1) The mice were divided into wild-type group, treatment group, non-treatment group and apoptosis positive control group (DNA enzyme treatment). The wild-type group was not treated. The mice in the treatment group were injected with tsRNA-Cys-5-0007agomir (mimic) into the vitreous cavity of the eyes, the mice in the non-treatment group were injected with Scragomir (Scramble control, negative control) into the vitreous cavity of the eyes, and the mice in the apoptosis positive control group were injected with Dnase-1 (DNA enzyme) into the vitreous cavity of the eyes. The mice in the treatment group, non-treatment group and apoptosis positive control group were injected once a week, and 1 μL, 1 mM tsRNA-Cys-5-0007agomir, control group Scra agomir or Dnase-1 were injected into the vitreous cavity of each eye, respectively.

[0093] (2) General anesthesia was induced by intraperitoneal injection of ketamine (80 mg / kg) and xylazine (4 mg / kg). The head, limbs and tail of the mouse were fixed with the abdomen facing upward. The hair on the chest and abdomen was removed. The skin at the xiphoid process was incised, the fascia was bluntly separated, the ribs were cut, and the chest cavity was exposed.

[0094] (3) After the left ventricle was perfused with saline, the eyeballs were immediately harvested for frozen section preparation.

[0095] (4) Warm the sections at 25°C for 30 minutes and wash three times with PBS.

[0096] (5) Permeabilize with 0.5% TritonX-100 in PBS at room temperature for 5 min and wash three times with PBS.

[0097] (6) Add mixed dye solution (each 100 μL contains 5 μL TdT enzyme, 45 μL fluorescent labeling solution, and 50 μL TUNEL detection solution), incubate at 37°C in the dark for 60 min, and wash three times with PBS.

[0098] (7) The cell nuclei were labeled with 4',6-diamidino-2-phenylindole (DAPI) stain (DAPI stock solution: PBS = 1:1500).

[0099] (8) Observe and record using a fluorescence microscope.

[0100] 3. Experimental results

[0101] like Figure 4 As shown, after intravitreal injection of tsRNA-Cys-5-0007agomir, no obvious green signal was observed in the cells of the mouse retina. tsRNA-Cys-5-0007 at therapeutic concentrations would not cause apoptosis of mouse retinal cells under normal physiological conditions.

[0102] Example 5 Application of RNA mimics of the present invention in inhibiting endothelial cell tube formation under pathological conditions

[0103] In this example, HRVECs were divided into 4 groups for culture, namely, Ctrl group, VEGF group, NC mimic group and tsRNA-Cys-5-0007mimic group. VEGF was used to simulate the pathological environment of the other three groups of HRVECs except the Ctrl group. The cells of the NC mimic group were treated with NC mimic+lipofectamine3000 by the method in Example 1, and the cells of the tsRNA-Cys-5-0007mimic+lipofectamine3000 were treated to simulate the extracellular matrix environment, so that the cells formed a clearly visible vascular network on the matrix gel, and the angiogenesis of the cells on the matrix gel was observed to detect the effect of tsRNA-Cys-5-0007 on the tube formation ability of endothelial cells under VEGF-induced pathological conditions. The details are as follows:

[0104] 1. Experimental materials:

[0105] Matrigel Matrix Basement Membrance (Cat No: 356234) was purchased from Corning Incorporated, USA.

[0106] 2. Experimental steps:

[0107] (1) HRVECs were divided into four groups for culture, namely, Ctrl group, VEGF group, NC mimic group, and tsRNA-Cys-5-0007 mimic group. Except for the Ctrl group, the other three groups of HRVECs were cultured with 25 ng / mL VEGF recombinant protein to simulate the pathological environment.

[0108] (2) HRVECs were seeded in a 24-well plate. When the cell confluence reached about 60%, the cells in the NC mimic group were treated with NCmimic+lipofectamine3000 (50 nM), and the cells in the tsRNA-Cys-5-0007mimic group were treated with tsRNA-Cys-5-0007mimic+lipofectamine3000 (50 nM), and the mixed solution was cultured for 24 hours.

[0109] (3) Take out the aliquoted Matrigel Matrix from -20℃, put it into 4℃ crushed ice to melt, and pre-cool the 24-well plate and pipette tip to -20℃;

[0110] (4) Take out the matrix gel, 24-well plate, and pipette tip from the 4°C refrigerator and place them on crushed ice in a clean bench. Rinse the 24-well plate with cold PBS, add 50 μL of matrix gel to each well, avoid generating bubbles, and incubate at 37°C for 30 min.

[0111] (5) Collect the treated HRVECs and adjust the cell density to 8×10 4 Pieces / mL;

[0112] (6) Add 1 mL of cell suspension to a 24-well plate pre-coated with matrix gel, incubate at 37°C for 4 h, and observe and record under an inverted microscope.

[0113] 3. Experimental results

[0114] like Figure 5 As shown, VEGF was used to simulate the pathological environment, and the number of branches of vascular endothelial cells was reduced after treatment with tsRNA-Cys-5-0007 mimic. tsRNA-Cys-5-0007 significantly inhibited the angiogenesis ability of endothelial cells under VEGF-induced pathological conditions.

[0115] Example 6 Application of RNA mimics of the present invention in inhibiting endothelial cell migration under pathological conditions

[0116] In this example, the grouping and treatment methods of the cells in each group were the same as in Example 5, and then crystal violet staining and Transwell method were used to detect the cell migration to detect the effect of tsRNA-Cys-5-0007 on the migration ability of endothelial cells under VEGF-induced pathological conditions. The details are as follows:

[0117] 1. Experimental materials:

[0118] Crystal violet (Cat No: C805211) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0119] 2. Experimental steps:

[0120] (1)(2) The same experimental steps (1) and (2) as those in Example 5;

[0121] (3) Preparation of 0.2% crystal violet solution: weigh 0.08 g of crystal violet powder and dissolve it in 40 mL of PBS. Shake overnight and filter twice with filter paper before use for staining.

[0122] (4) The treated HRVECs were collected and resuspended in ECM without fetal bovine serum (FBS) and the cell density was adjusted to 1×10 5 Pieces / mL;

[0123] (5) Add 500 μL of ECM complete medium containing 10% FBS to the lower chamber of the 24-well plate, select a chamber with a pore size of 8.0 μm and place it in the 24-well plate, remove the bubbles between the chamber and the liquid, and add 200 μL of cell suspension to the upper chamber, taking care to avoid generating bubbles;

[0124] (6) Continue culturing in a CO2 incubator for 24 h, and gently wipe off the unmigrated cells in the upper chamber with a cotton swab;

[0125] (7) Add 500 μL of 4% paraformaldehyde to a clean well of a 24-well plate, place the chamber in it and fix it for 15 min, take out the chamber and invert it on the table, and air dry it for 20 min;

[0126] (8) Discard the fixative solution, add 500 μL of 0.2% crystal violet solution, and stain for 5 min;

[0127] (9) Discard crystal violet, add 500 μL PBS, and wash three times, 2 min each time;

[0128] (10) Gently wipe the upper chamber with a cotton swab to remove the crystal violet that has not passed through the upper chamber, and observe and record using an inverted microscope.

[0129] 3. Experimental results

[0130] like Figure 6 As shown, VEGF was used to simulate the pathological environment, and the number of endothelial cells migrating to the lower chamber was reduced after treatment with tsRNA-Cys-5-0007 mimic. The tsRNA-Cys-5-0007 mimic significantly inhibited the migration ability of endothelial cells under VEGF-induced pathological conditions.

[0131] Example 7 Application of RNA mimics of the present invention in inhibiting endothelial cell proliferation under pathological conditions

[0132] In this example, the grouping and treatment methods of the cells in each group were the same as in Example 5, and then the cell proliferation was detected using an EdU cell proliferation detection kit, and the cell nucleus was stained with DAPI to detect the effect of tsRNA-Cys-5-0007 on the proliferation ability of endothelial cells under VEGF-induced pathological conditions. The details are as follows:

[0133] 1. Experimental materials:

[0134] BeyoClick TM EdU-488 cell proliferation detection kit (Cat No: C0071S) was purchased from Bio-Tech Biotechnology Co., Ltd., China.

[0135] 2. Experimental steps:

[0136] (1)(2) The same experimental steps (1) and (2) as those in Example 5;

[0137] (3) When the cell density reaches more than 90%, discard the old culture medium, wash twice with PBS buffer, add 5-ethynyl-2′-deoxyuridine (EdU) working solution (EdU stock solution: complete culture medium = 1:1000) to the 24-well plate at a volume of 1 mL / well, and incubate for 3 h;

[0138] (4) After the incubation, discard the EdU working solution, add 1 mL of 4% paraformaldehyde to each well, and fix at room temperature for 15 min;

[0139] (5) Discard the paraformaldehyde, add 500 μL of washing solution (PBS containing 3% BSA) to each well, and wash three times, each time for 3-5 minutes;

[0140] (6) Discard the washing solution, add 500 μL of permeabilization solution (PBS containing 0.3% Triton X-100) to each well, and incubate at room temperature for 10-15 min;

[0141] (7) Discard the permeabilization solution and add 500 μL of washing solution to each well and wash 1-2 times for 3-5 min each time;

[0142] (8) Discard the washing solution and prepare the Click reaction solution according to the manufacturer's instructions. Add 200 μL of Click reaction solution to each well and incubate at room temperature in the dark for 30 min.

[0143] (9) Discard the reaction solution and add 500 μL of washing solution to each well and wash 1-2 times for 3-5 min each time;

[0144] (10) Discard the washing solution, add 200 μL DAPI to each well, and incubate at room temperature in the dark for 10 min;

[0145] (11) Discard the DAPI staining solution and add 500 mL of PBST to each well and wash three times, each time for 3-5 minutes.

[0146] (12) Observe and record using a fluorescence microscope.

[0147] 3. Experimental results

[0148] like Figure 7 As shown, VEGF was used to simulate the pathological environment, and the proliferation signal of vascular endothelial cells was reduced after treatment with tsRNA-Cys-5-0007 mimic. tsRNA-Cys-5-0007 significantly inhibited the proliferation ability of endothelial cells under VEGF-induced pathological conditions.

[0149] Example 8 Application of RNA mimics of the present invention in inhibiting endothelial cell spheroid sprouting under pathological conditions

[0150] In this example, the grouping and treatment methods of the cells in each group were the same as in Example 5, and then the 3D endothelial cell spheroid sprouting experiment of HRVECs was used to detect the effect of tsRNA-Cys-5-0007 mimic on the sprouting ability of endothelial cells under VEGF-induced pathological conditions. The details are as follows:

[0151] 1. Experimental materials:

[0152] Carboxymethylcellulose (Cat No: 419273) was purchased from Sigma Biotechnology, Inc., USA. Type I rat tail collagen (Cat No: 354236) was purchased from Corning Incorporated, USA.

[0153] 2. Experimental steps:

[0154] (1)(2) The same experimental steps (1) and (2) as those in Example 5;

[0155] (3) Preparation of methylcellulose stock solution: 3 g of methylcellulose was weighed and added to a 500 mL culture flask, and a clean magnetic stirring rotor was added. The flask was sterilized at 121°C for 20 min, and then 250 mL of ECM preheated to 60°C was added. The flask was stirred at room temperature for 20 min, shaken at 4°C overnight, and centrifuged at 5000 rpm for 30 min. The supernatant was collected for cell sphere culture.

[0156] (4) Collect transfected HRVECs and adjust the cell density to 2×10 4 / mL, mix the cell suspension with the methylcellulose stock solution (mixing ratio 4:1), and inoculate 25μL / drop on the cover of the culture dish, taking care to avoid bubbles;

[0157] (5) Incubate inverted in a CO2 incubator for 24 h. Use a pipette to aspirate 10 mL of PBS to gently wash away the suspended spheres. Transfer the liquid to a 15 mL centrifuge tube and centrifuge at 800 rpm for 3 min to precipitate the cell spheres. Discard the supernatant and add 2 mL of methylcellulose stock solution containing 20% ​​FBS and 2 mL of collagen stock solution. Mix carefully to avoid generating bubbles.

[0158] (6) The cell spheroid mixture was added into a 24-well plate at a volume of 1 mL / well and incubated in a CO2 incubator for 30 min to promote collagen polymerization.

[0159] (7) After collagen polymerization, add 200 μL of ECM to each well and continue incubation for 24 h.

[0160] 3. Experimental results

[0161] like Figure 8As shown, using VEGF to simulate the pathological environment, the total length of 3D cell sphere sprouts was reduced after tsRNA-Cys-5-0007 mimic treatment, and tsRNA-Cys-5-0007 mimic significantly inhibited the sprouting ability of endothelial cells under VEGF-induced pathological conditions.

[0162] Example 9 Application of RNA agomir of the present invention in inhibiting retinal vascular leakage in DR model mice

[0163] In this embodiment, a DR (diabetic retinopathy) model was first established by the STZ-induced method, wherein DR mice were intraperitoneally injected with STZ, and the normal control group (Wt group) was intraperitoneally injected with citric acid and sodium citrate buffer; after the DR model was successfully established, the DR mice were divided into 3 groups: a treatment group (tsRNA-Cys-5-0007agomir), a Scr agomir group, and a PBS group, wherein tsRNA-Cys-5-0007agomir was injected into the vitreous cavity of the DR mice in the treatment group, an equal amount of Scr agomir was injected into the Scr agomir group, and an equal amount of PBS was injected into the PBS group. Then, retinal blood vessel EvansBlue staining was performed, and retinal tissue sections were prepared and observed to detect the effect of tsRNA-Cys-5-0007agomir on retinal vascular leakage in DR model mice. Specifically as follows:

[0164] 1. Experimental materials:

[0165] C57BL / 6J mice were purchased from the Model Animal Institute of Nanjing University, STZ powder (Cat No: 2196GR001) was purchased from Beijing Lanjieke Technology Co., Ltd., and tsRNA-Cys-5-0007agomir was purchased from Guangzhou Ruibo Biotechnology Co., Ltd.

[0166] Evans Blue (Cat No: K29136) was purchased from Beijing Keerhui, and heparin (Cat No: 1170GR001) was purchased from Guangzhou Saiguo Biotechnology Co., Ltd.

[0167] 2. Experimental steps:

[0168] (1) Establishment of DR model: A DR (diabetic retinopathy) model was established by STZ-induced method, wherein DR mice were intraperitoneally injected with STZ, and the normal control group (Wt group) was intraperitoneally injected with citric acid and sodium citrate buffer. The specific steps are as follows:

[0169] a) C57BL / 6J mice (8-12 weeks) were selected for the experiment and fasted for 8-12 hours before modeling;

[0170] b) Dissolve STZ at a concentration of 1.5% in a citric acid / sodium citrate buffer solution at a pH of 4.5, store on ice, and use up within 30 minutes after preparation;

[0171] c) Intraperitoneal injection at a dose of 0.1 mL / 10 g per mouse;

[0172] d) Normal control mice were intraperitoneally injected with equal amounts of citric acid and sodium citrate only;

[0173] e) Random tail vein blood glucose was measured after 7 days. If blood glucose ≥16.7, the diabetes model was successfully established. Fasting blood glucose was tested monthly during the experiment. Each blood glucose was greater than 16.7 mmol / L. After continuous measurement for 3 months, the DR model was considered to be successfully established. After the DR model was successfully established, the DR mice were divided into 3 groups: treatment group (tsRNA-Cys-5-0007agomir), Scr agomir group and PBS group. Among them, tsRNA-Cys-5-0007agomir was injected into the vitreous cavity of the DR mice in the treatment group, the same amount of Scr agomir was injected into the Scr agomir group, and the same amount of PBS was injected into the PBS group. Injection intervention was performed once a week. 1μL, 1mMtsRNA-Cys-5-0007agomir, Scr agomir or PBS were injected into the vitreous cavity of each eye, respectively.

[0174] (2) Preparation of Evans Blue solution: Dissolve Evans Blue powder in sterile saline to a concentration of 30 g / L, add heparin reagent to an anticoagulant concentration of 400 U / mL, ultrasonically vibrate for 5 min, filter with a sterile filter (0.22 μm), and store at 4°C in the dark.

[0175] (3) General anesthesia was induced by intraperitoneal injection of ketamine (80 mg / kg) and xylazine (4 mg / kg). The head, limbs, and tail of the mouse were fixed with the abdomen facing upward. The hair on the inner thigh was removed. Under a surgical microscope, the skin was longitudinally incised along the inner thigh, the fascia was bluntly separated, and the femoral vein was exposed to avoid bleeding.

[0176] (4) Use a sterile insulin syringe to draw 10 mL (30 mg / kg) of pre-prepared Evans Blue solution. Avoid inhaling bubbles that may cause air embolism in mice. Inject slowly and closely observe changes in the mouse's vital signs. The circulation time is about 30 minutes.

[0177] (5) After adequate circulation, the animals were killed by dislocating the neck, and the eyeballs were removed completely and gently to avoid retinal rupture and bleeding, and fixed in 4% paraformaldehyde at room temperature for 30 min;

[0178] (6) After removing the anterior segment, lens, vitreous body and other tissues, the retina was gently and completely peeled off, spread flat on a glass slide, cut evenly into a four-leaf clover shape, and sealed;

[0179] (7) Observe the vascular morphology and vascular wall leakage under an inverted fluorescence microscope.

[0180] 3. Experimental results

[0181] like Fig. 9 As shown, in DR model mice, retinal vascular fluorescence leakage was reduced after tsRNA-Cys-5-0007agomir treatment, and tsRNA-Cys-5-0007 could alleviate retinal vascular leakage in STZ-induced DR mice.

[0182] Example 10 Application of RNA agomir of the present invention in inhibiting retinal vascular inflammation in DR model mice

[0183] In this example, the DR model was established in the same manner as in Example 9, and the Wt group, treatment group, Scr agomir group and PBS group were also set up, and the administration method was also the same as in Example 9. Then a leukocyte stasis experiment was performed, and retinal tissue sections were prepared by staining with ConA solution, and the number of leukocytes adhered to the retinal vascular wall was observed under an inverted fluorescence microscope to confirm the infiltration of inflammatory cells in the retinal blood vessels after tsRNA-Cys-5-0007agomir treatment. The details are as follows:

[0184] 1. Experimental materials:

[0185] Concanavalin A (ConA) perfusion solution (Cat No: FL-1001) was purchased from Vector Laboratories, USA.

[0186] 2. Experimental steps:

[0187] (1) Establishing DR model, setting up Wt group, treatment group, Scr agomir group and PBS group respectively, the method is the same as Example 9.

[0188] (2) General anesthesia was induced by intraperitoneal injection of ketamine (80 mg / kg) and xylazine (4 mg / kg). The head, limbs, and tail of the mouse were fixed with the abdomen facing upward. The hair on the chest and abdomen was removed. The skin at the xiphoid process was incised, the fascia was bluntly separated, the ribs were cut, and the chest cavity was exposed.

[0189] (2) Normal saline was perfused into the left ventricle to remove non-adherent leukocytes, and then 10 mL of Con A was perfused to stain the blood vessels;

[0190] (3) The animals were killed by cervical dislocation, and the eyeballs were gently and completely removed and fixed in 4% paraformaldehyde at room temperature for 30 min;

[0191] (4) After removing the anterior segment, lens, vitreous body and other tissues, the retina was gently and completely peeled off, spread flat on a glass slide, cut evenly into a four-leaf clover shape, and sealed;

[0192] (5) Observe and count the number of leukocytes adhering to the blood vessel wall under an inverted fluorescence microscope.

[0193] 3. Experimental results

[0194] like Fig.10 As shown, in DR model mice, the number of retinal vascular inflammatory cell infiltration was reduced after tsRNA-Cys-5-0007 agomir treatment, and tsRNA-Cys-5-0007 could alleviate retinal vascular inflammation in STZ-induced DR mice.

[0195] Example 11 Application of RNA agomir of the present invention in inhibiting angiogenesis in CNV model (choroidal neovascularization)

[0196] In this embodiment, laser induction was first used to establish a CNV model, and three groups were set up: a treatment group, a Scragomir group, and a PBS group. After the CNV model was successfully established, tsRNA-Cys-5-0007agomir was injected into the vitreous of mice in the treatment group, and equal amounts of Scragomir and PBS were injected into the Scragomir group and the PBS group, respectively. Then, retinal / choroidal tissue flat slides were prepared, and the area of ​​new blood vessels was observed by immunofluorescence (IB4) staining to detect the effect of tsRNA-Cys-5-0007agomir on the formation of new blood vessels in the CNV model. The details are as follows:

[0197] 1. Experimental materials:

[0198] C57BL / 6J mice were purchased from the Model Animal Institute of Nanjing University, OcuLight GLx laser system was purchased from Iridex, USA, and tsRNA-Cys-5-0007agomir was purchased from Guangzhou Ruibo Biotechnology Co., Ltd. Lectin GS-IB4 (Cat No: 121413) was purchased from Thermo Fisher Scientific, USA.

[0199] 2. Experimental steps

[0200] (1) Establish a CNV model. The steps are as follows:

[0201] a) C57BL / 6J mice (6-8 weeks) were selected for the experiment and local anesthesia was performed by tetracaine hydrochloride eye drops;

[0202] b) 0.5% tropicamide eye drops were applied to dilate the pupils, and the whiskers of the mice were removed to prevent interference with the laser energy;

[0203] c) Apply levofloxacin eye ointment on a transparent glass slide and cover it on the mouse cornea. Adjust the position and angle of the glass slide to facilitate observation of the fundus under a slit lamp. Use the OcuLight GLx laser system from Iridex, USA, to induce 4 laser spots at 3, 6, 9 and 12 points at a distance of 1-2 optic discs from the optic disc, and pay attention to avoid retinal blood vessels. The laser parameters are set as follows: wavelength 532nm, spot size 50μm, pulse time 70ms, power 140mW. The appearance of bubbles at the laser site indicates that Bruch's membrane is destroyed and the model is successful. The bleeding laser spots are not included in the study;

[0204] d) The transparent slide was removed and three groups were set up: treatment group, Scr agomir group and PBS group. After the CNV model was successfully established, the mice in the treatment group were injected with 1 μL of 1 mM tsRNA-Cys-5-0007agomir in the vitreous, and the Scr agomir group and PBS group were injected with equal amounts of Scr agomir and PBS, respectively, and the mice were placed on a rewarming table for recovery;

[0205] (2) On the 7th day after modeling, mice were killed by cervical dislocation, and the eyeballs were removed and fixed with 4% paraformaldehyde at room temperature for 45 min. The eyeballs were placed in a culture dish filled with PBS, and the muscles and fascia on the surface of the eyeballs were peeled off under a live microscope. The cornea, lens, vitreous body and other tissues were removed, and the retina / choroid were completely removed, placed on a slide, and cut into a four-leaf clover shape;

[0206] (3) The retina / choroid unfolded on the slide was fixed again with 4% paraformaldehyde for 30 min, washed three times with PBS for 5 min each time, blocked and permeabilized with PBS containing 5% BSA and 1% TritonX-100 at 37°C for 45 min, and incubated with GS-IB4 at 4°C overnight. The samples were observed and recorded under a fluorescence microscope.

[0207] 3. Experimental results

[0208] like Fig.11 As shown, the area of ​​choroidal neovascularization was reduced after tsRNA-Cys-5-0007agomir treatment, and tsRNA-Cys-5-0007 could alleviate choroidal neovascularization in laser-induced CNV mice.

[0209] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of an RNA molecule for treating fundus vascular diseases in preparing a product, characterized in that: The product is used to treat and / or alleviate fundus vascular diseases; the sequence of the RNA molecule is shown in SEQ ID NO.1; the RNA molecule is an unmodified single-stranded RNA molecule, or a chemically modified single-stranded RNA mimic; the fundus vascular disease is diabetic retinopathy or choroidal neovascularization disease.

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