WO2023024134A1 - 原纤维蛋白-2重组蛋白在制备预防或治疗原纤维蛋白-2缺陷型眼病的产品中的用途 - Google Patents

原纤维蛋白-2重组蛋白在制备预防或治疗原纤维蛋白-2缺陷型眼病的产品中的用途 Download PDF

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WO2023024134A1
WO2023024134A1 PCT/CN2021/115997 CN2021115997W WO2023024134A1 WO 2023024134 A1 WO2023024134 A1 WO 2023024134A1 CN 2021115997 W CN2021115997 W CN 2021115997W WO 2023024134 A1 WO2023024134 A1 WO 2023024134A1
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fbn2
group
fibrillin
recombinant protein
aav
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毕宏生
蒋文君
温莹
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山东中医药大学附属眼科医院
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    • AHUMAN NECESSITIES
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Definitions

  • the invention relates to the field of biopharmaceuticals, in particular to the use of fibrillin-2 recombinant protein in the preparation of products for preventing or treating fibrillin-2 deficient eye diseases.
  • Fibrillin-2 (FBN2) gene variation has been proven to be an important cause of early-onset macular degeneration (Early-onset macular degeneration, EOMD) and age-related macular degeneration (Age related macular degeneration, AMD),
  • EOMD early-onset macular degeneration
  • AMD age-related macular degeneration
  • FBN2 gene mutation was found to be autosomal dominant in AMD families.
  • AMD patients with FBN2 gene mutation had clinical manifestations of drusen; more and more people have AMD, and in severe cases, it will loss of vision.
  • the FBN2 protein is involved in the formation of microfibrils and is critical for the formation and remodeling of the ECM and is involved in the formation of elastic fibers in connective tissues throughout the body.
  • FBN2 protein expression or loss of function in the eyes of AMD patients with FBN2 gene mutations which changes its permeability, elastin fiber packaging, and flexibility, is an important reason for the induction of retinopathy, but FBN2 gene defects in retinopathy The mechanism of action in the development of the disease is not clear.
  • Adeno-associated virus has the advantages of wide host range, multiple serotypes, high titer, high safety, high accuracy, stable expression, and low immunogenicity, and has been widely used in In basic medical research and clinical trials, it is regarded as one of the most promising research gene therapy vectors.
  • Protein-targeted intervention is widely used in the treatment of ophthalmic diseases, such as antibody (vascular endothelial growth factor, VEGF), etc., but the research on targeted drugs for FBN2 gene-deficient retinopathy and myopia has not been reported yet. Therefore, this application provides a new strategy for the clinical diagnosis and treatment of FBN2 gene-deficient eye diseases.
  • ophthalmic diseases such as antibody (vascular endothelial growth factor, VEGF), etc.
  • the main purpose of the present invention is to provide the use of fibrillin-2 recombinant protein in the preparation of products for preventing or treating fibrillin-2 deficient eye diseases.
  • This application uses AAV as a carrier to carry out RNAi construction of gene defect eye diseases, and injects recombinant proteins for targeted intervention treatment, providing a new method for targeted treatment of retinopathy.
  • the research of this application found that the genetically deficient retinal disease model that can be constructed by RNAi using AAV as a carrier mainly manifests as drusen-like exudates in the fundus, rough reflection areas in retinal coherent optical tomography, and degeneration of the outer granular layer of the retina. Thin, these are the clinical manifestations of retinopathy.
  • the fundus exudation was reduced and the outer granular layer of the retina was thickened, which played a certain therapeutic effect on retinopathy.
  • the present invention provides a use of a fibrillin-2 recombinant protein in the preparation of products for preventing or treating fibrillin-2 deficient eye diseases.
  • the fibrillin-2 deficient eye disease is at least one selected from hereditary retinopathy, axial myopia and congenital macular degeneration.
  • the product is medicine, reagent or vaccine.
  • the present invention provides the use of fibrillin-2 recombinant protein to regulate the amplitude of electroretinogram waveform for non-therapeutic purposes;
  • the regulating the amplitude of the electroretinogram waveform is to restore the amplitude of the electroretinogram waveform to normal;
  • the electroretinogram waveform amplitude is selected from at least one of the dark-adapted rod response waveform amplitude, the dark-adapted maximum mixed response waveform amplitude, the dark-adapted oscillatory potential response waveform amplitude and the light-adapted cone cell response waveform amplitude.
  • the present invention provides the use of fibrillin-2 recombinant protein in reducing retinal exudation and fundus deposits for non-therapeutic purposes.
  • the present invention provides the use of fibrillin-2 recombinant protein to increase the thickness of retinal outer nuclear layer for non-therapeutic purpose.
  • the invention provides the use of fibrillin-2 recombinant protein to increase the expression of fibrillin-2 for non-therapeutic purposes;
  • the expression of fibrillin-2 is selected from the expression of fibrillin-2 gene or the expression of fibrillin-2 protein;
  • the expression of the fibrillin-2 gene is the expression of fibrillin-2 gene mRNA.
  • the present invention provides a product for the prevention or treatment of fibrillin-2 deficient ophthalmopathy, the product uses fibrillin-2 recombinant protein as the sole active ingredient, or contains fibrillin-2 recombinant protein Compositions.
  • the product is a drug, and the composition also includes pharmaceutical excipients;
  • the pharmaceutical excipient is at least one selected from diluents, excipients, fragrances and sweeteners;
  • the preparation of the drug is at least one of suspension, emulsion, solution, syrup or injection;
  • the drug is administered by infusion or injection;
  • the drug is administered by intravitreal injection.
  • the content of fibrillin-2 recombinant protein in the product is 0.01-5.0 ⁇ g/ ⁇ L; preferably, the content of fibrillin-2 recombinant protein in the product is 0.1-1.0 ⁇ g/ ⁇ L; preferably, the The content of fibrillin-2 recombinant protein in the product is 0.2-0.5 ⁇ g/ ⁇ L.
  • the present invention proves in the construction of the FBN2 gene-deficient eye disease model and the experiment of injecting FBN2 protein that the exogenous FBN2 recombinant protein can improve the retinopathy and myopic axial growth caused by the FBN2 gene defect.
  • the present invention can construct the mouse model of FBN2 gene-deficient eye disease by injecting the FBN2 gene interference vector AAV. Quenched type, as well as decreased mRNA and protein expression of FBN2.
  • the present invention can effectively treat FBN2 gene-deficient retinopathy by injecting exogenous FBN2 recombinant protein, reduce the generation of yellow-white drusen-like deposits in the fundus, increase ONL thickness, restore ERG waveform amplitude, and increase FBN2 mRNA and protein expression.
  • the present invention is the first method for constructing FBN2 gene-deficient animal model by intravitreal injection of FBN2 gene interference vector AAV, and for the first time found that intravitreal injection of FBN2 recombinant protein has a therapeutic effect on FBN2 gene-deficient eye disease.
  • the study of the present invention further proves that the FBN2 gene defect causes a series of pathophysiological changes in the occurrence and development of retinopathy and myopia, and that the intravitreal injection of exogenous FBN2 recombinant protein can make up for the endogenous deficiency of FBN2 in the FBN2 gene-deficient eye disease, so as to achieve
  • the role of treating diseases provides a new method for the next step of targeted gene intervention research and points out the research direction.
  • Figure 1 shows the results of scanning confocal ophthalmoscopy and optical coherence tomography after different treatments
  • Figure 2 shows the results of Rod-b, Max-a, OPs total, and Cone-b waveform amplitudes after different treatments
  • Figure 3 is the result of the axial length of the eye after different treatments; a: visual axial length diagram; b: statistical diagram of the axial length of the eye;
  • Figure 4 is the result of the mRNA expression of the FBN2 gene in the retina after different treatments
  • Fig. 5 is the ELISA detection result of FBN2 protein expression in the retina after different treatments
  • Fig. 6 is the western blot detection result of FBN2 protein expression in the retina after different treatments
  • mice 8-week-old C57BL/6J mice, SPF grade, body weight 21-23g, were used. Before enrollment, the mice were screened for eye health, excluding corneal disease, cataract, and scanning laser ophthalmoscope (Scanning laser ophthalmoscope, SLO), optical coherence tomography (OCT) and electroretinogram (Electroretinogram, ERG) were used to exclude retinal fundus diseases.
  • SLO scanning laser ophthalmoscope
  • OCT optical coherence tomography
  • ERG electroretinogram
  • FBN2 adenovirus-related interference vector first synthesize single-stranded DNA oligonucleotides, then produce double-stranded DNA oligonucleotides, and pass through the enzyme cutting sites contained at both ends (BamHI recognition sequence: 5'G ⁇ GATCC 3', EcoRI recognition sequence: 5'G ⁇ AATTC 3') and connect it directly to the RNA interference vector after enzyme digestion; the clone that is correctly aligned is considered to be successfully constructed.
  • AAV-293 cells were co-transfected, the virus was packaged, the original solution was collected, concentrated and purified to obtain a concentrated solution.
  • Extract the AAV vector with improved purity and no endotoxin use HG transgene reagent to co-transfect the constructed virus vector and plasmid into AAV-293 cells, add Enhancing buffer 10-12 hours after transfection, and then replace fresh medium after 8 hours, After 48 hours in the culture medium, the cells including the virus and the supernatant were collected after the cells were detached, then concentrated and purified to obtain the AAV virus with a titer of 1.21E+12VG/mL.
  • the amino acid sequence of the FBN-2 recombinant protein is:
  • FBN-2 recombinant protein is efficiently produced by the host-vector system of E. coli.
  • the buffer for diluting FBN-2 recombinant protein includes 20mM Tris, 150mM NaCL, 1mM EDTA, 1mM dithiothreitol DTT, 0.01% Sarcosyl, 5% trehalose and proclin300.
  • the original concentration of FBN-2 recombinant protein was 200 ⁇ g/ ⁇ L.
  • the normal control group was fed normally without any treatment; after enrollment, the normal group was fed normally, and the negative control group was injected with 3 ⁇ L negative virus stock solution (AAV inactivated virus stock solution) into the vitreous cavity of both eyes; AAV group, FBN2 ultra-low concentration group, FBN2 low concentration group 3 ⁇ L AAV virus was injected into the vitreous cavity of both eyes in the control group, the FBN2 medium concentration group, and the FBN2 high concentration group to establish the FBN2 gene-deficient eye disease model.
  • AAV inactivated virus stock solution AAV inactivated virus stock solution
  • the dose of FBN2 recombinant protein was 3 ⁇ L, and the amount of FBN2 recombinant protein injected in the FBN2 ultra-low concentration group, FBN2 low concentration group, FBN2 medium concentration group, and FBN2 high concentration group were 0.3 ⁇ g/eye, 0.75 ⁇ g/eye, and 1.5 ⁇ g/eye , 3 ⁇ g/eye, injected once a week, and injected 3 times in a row.
  • the specific operation of intravitreal injection includes: using ofloxacin eye drops (3 times/d) one day in advance to anti-inflammatory the eyes of animals in the negative control group, AAV group, and FBN2 injection group.
  • 10g ⁇ L -1 sodium pentobarbital solution 50mg ⁇ kg -1 was injected intraperitoneally for anesthesia to ensure normal breathing, and mydriatic drugs were added to dilate the pupils. Place the anesthetized mouse under the microscope, disinfect the area around the eyes with povidone-iodine, rinse off the surface with 0.9% normal saline after 1 min, and then dip it dry with a dry sterile cotton swab, and rinse with Orbudine hydrochloride Caine eye drops for ocular surface anesthesia.
  • the specific operation of scanning confocal ophthalmoscope examination for SLO includes: before the detection, anesthetize with intraperitoneal injection of 10 g ⁇ L -1 pentobarbital sodium solution (50 mg ⁇ kg -1 ), dilate the pupil, and administer ofloxacin Eye ointment was applied to the cornea, and the mice were fully anesthetized. Align the eyes of the mice with the lens of the scanning laser ophthalmoscope, adjust the position of the mice, and take at least 3 pictures for each group and save the pictures based on the complete eye map that can be captured on the computer screen. All operations are performed by the same researcher , and the test results are shown in Figure 1.
  • the retinal exudation and deposits in the FBN2 low-concentration group were significantly reduced, the exudation and deposits in the FBN2 ultra-low-concentration group and medium-concentration group were slightly reduced, and there was no significant change in the FBN2 high-concentration group.
  • the retinal exudation and deposits in the FBN2 low-concentration group were significantly reduced, the exudation and deposits in the FBN2 ultra-low-concentration group and the medium-concentration group were significantly reduced, and the exudation and deposits in the FBN2 high-concentration group were slightly reduced. Not obvious.
  • ERG detection is carried out in an absolutely dark environment. Before the test, the mice were placed in a dark environment for more than 8 hours for dark adaptation. Before the test, anesthetize with intraperitoneal injection of 10g ⁇ L -1 pentobarbital sodium solution (50mg ⁇ kg -1 ), dilate the pupil, and anesthetize the ocular surface with oxybucaine hydrochloride eye drops, and use oxyfluorine Shaxing ointment was applied to the cornea, and the mice were completely anesthetized.
  • the Rod-b wave values of normal control group, negative control group, AAV group, FBN2 ultra-low concentration group, FBN2 low concentration group, FBN2 medium concentration group and FBN2 high concentration group were (140.40 ⁇ 18.79 ⁇ V, 138.41 ⁇ 17.42 ⁇ V, 141.35 ⁇ 15.80 ⁇ V, 140.22 ⁇ 18.33 ⁇ V, 140.97 ⁇ 17.58 ⁇ V, 139.14 ⁇ 15.36 ⁇ V, 141.35 ⁇ 18.03 ⁇ V).
  • negative control group 27.18 ⁇ 4.21 vs. 138.91 ⁇ 18.93 ⁇ V; FBN2 ultra-low concentration group vs. negative control group: 28.32 ⁇ 3.78 ⁇ V vs. 138.91 ⁇ 18.93 ⁇ V ; FBN2 low concentration group vs negative control group: 22.26 ⁇ 4.02 ⁇ V vs 138.91 ⁇ 18.93 ⁇ V, FBN2 medium concentration group vs negative control group: 23.45 ⁇ 4.27 ⁇ V vs 138.91 ⁇ 18.93 ⁇ V, FBN2 high concentration group vs negative control group: 24.11 ⁇ 3.78 ⁇ V vs. 138.91 ⁇ 18.93 ⁇ V, P ⁇ 0.05).
  • negative control group 26.28 ⁇ 4.33 ⁇ V vs. 142.91 ⁇ 17.44 ⁇ V; FBN2 ultra-low concentration group vs. 3.67 ⁇ V versus 142.91 ⁇ 17.44 ⁇ V; FBN2 low concentration group versus negative control group: 32.95 ⁇ 5.67 ⁇ V versus 142.91 ⁇ 17.44 ⁇ V; FBN2 medium concentration group versus negative control group: 30.38 ⁇ 4.29 ⁇ V versus 142.91 ⁇ 17.44 ⁇ V; FBN2 high concentration group Compared with the negative control group: 27.55 ⁇ 1.79 ⁇ V vs. 142.91 ⁇ 17.44 ⁇ V, P ⁇ 0.05); the comparison trend of each group with the normal control group was consistent with that of the negative control group.
  • the FBN2 low concentration group had increased waveform amplitude (76.33 ⁇ 7.46 ⁇ V vs. 25.96 ⁇ 4.59 ⁇ V, P ⁇ 0.05); Compared with the AAV group, there was no significant change in the amplitude of the Rod-b waveform between the concentration group and the AAV group (FBN2 ultra-low concentration group vs. AAV group: 28.97 ⁇ 5.00 ⁇ V vs. 25.96 ⁇ 4.59 ⁇ V; ⁇ 4.59 ⁇ V; FBN2 high concentration group vs AAV group: 30.40 ⁇ 4.42 ⁇ V vs 25.96 ⁇ 4.59 ⁇ V, P>0.05).
  • the Rod-b waveform amplitude was significantly increased in the FBN2 low-concentration group and FBN2 medium-concentration group compared with the AAV group (FBN2 low-concentration group versus AAV group: 118.33 ⁇ 12.42 ⁇ V vs. 26.25 ⁇ 3.88 ⁇ V; FBN2 Medium concentration group vs. AAV group: 77.00 ⁇ 8.51 ⁇ V vs. 26.25 ⁇ 3.88 ⁇ V, P ⁇ 0.05), FBN2 ultra-low concentration group, FBN2 high Group vs. AAV group: 31.25 ⁇ 4.79 ⁇ V vs. 26.25 ⁇ 3.88 ⁇ V; FBN2 high concentration group vs.
  • AAV group 58.88 ⁇ 5.32 ⁇ V vs. 26.25 ⁇ 3.88 ⁇ V, P>0.05).
  • the negative control group there was no significant difference in the amplitude of Rod-b waveform in the FBN2 low concentration group (118.33 ⁇ 12.42 vs. 136.91 ⁇ 17.73 ⁇ V, P>0.05).
  • the amplitude of Rod-b waveform in the FBN2 high concentration group was significantly lower (AAV group vs. negative control group: 26.25 ⁇ 3.88 ⁇ V vs. 136.91 ⁇ 17.73 ⁇ V; FBN2 ultra-low concentration group vs. negative control group: 31.25 ⁇ 4.79 ⁇ V vs.
  • the Rod-b wave values of normal control group, negative control group, AAV group, FBN2 ultra-low concentration group, FBN2 low concentration group, FBN2 medium concentration group and FBN2 high concentration group were (-121.97 ⁇ 18.67 ⁇ V, -123.85 ⁇ 16.48 ⁇ V, -117.26 ⁇ 17.83 ⁇ V, -119.77 ⁇ 18.63 ⁇ V, -115.7 ⁇ 17.88 ⁇ V, -121.97 ⁇ 15.46 ⁇ V, -117.26 ⁇ 15.33 ⁇ V).
  • negative control group -17.90 ⁇ 2.79 ⁇ V Compared with -116.07 ⁇ 18.77 ⁇ V; FBN2 high concentration group vs. negative control group: -15.07 ⁇ 3.07 ⁇ V vs. -116.07 ⁇ 18.77 ⁇ V, P ⁇ 0.05); the comparison trend between each group and the normal control group was consistent with that of the negative control group.
  • negative control group -22.53 ⁇ 4.67 ⁇ V vs. -117.05 ⁇ 15.83 ⁇ V; FBN2 high concentration group vs. negative control group: -19.90 ⁇ 3.67 ⁇ V Contrast -117.05 ⁇ 15.83, P ⁇ 0.05); the comparison trend between each group and the normal control group was consistent with the comparison trend with the negative control group.
  • FBN2 ultra-low concentration group, FBN2 high concentration group compared with AAV group Max-a waveform amplitude had no significant change ( BN2 ultra-low concentration group compared with AAV group: -23.94 ⁇ 6.67 ⁇ V versus -19.82 ⁇ 3.85 ⁇ V; FBN2 high concentration group compared with AAV group: -24.54 ⁇ 5.44 ⁇ V versus -19.82 ⁇ 3.85 ⁇ V, P>0.05);
  • the AAV group, the FBN2 ultra-low concentration group, the FBN2 low concentration group, the FBN2 medium concentration group, and the FBN2 high concentration group had a lower Max-a waveform amplitude, (AAV group compared with the negative control group: -19.82 ⁇ 3.85 ⁇ V versus -118.29 ⁇ 17.42 ⁇ V; FBN2 ultra-low concentration group versus negative control group: -23
  • negative control group -27.24 ⁇ 6.67 ⁇ V vs. -118.29 ⁇ 17.42 ⁇ V; FBN2 high concentration group vs. negative control group: -24.54 ⁇ 5.44 ⁇ V vs. -118.29 ⁇ 17.42 ⁇ V, P ⁇ 0.05); The comparison trend of each group with the normal control group was consistent with the comparison trend with the negative control group.
  • the Max-a waveform amplitudes of the FBN2 low-concentration group and FBN2 medium-concentration group were increased compared with the AAV group (FBN2 low-concentration group vs. AAV group: -99.44 ⁇ 9.42 vs. -19.17 ⁇ 3.67 ⁇ V, FBN2 medium concentration group vs AAV group -65.27 ⁇ 8.67 ⁇ V vs -19.17 ⁇ 3.67 ⁇ V, P ⁇ 0.05); FBN2 ultra-low concentration group, FBN2 high concentration group compared with AAV group Max-a waveform amplitude had no significant change (FBN2 super Low concentration group and AAV group: -24.72 ⁇ 4.33 ⁇ V vs.
  • the FBN2 low concentration group had no significant difference in the Max-a waveform amplitude (the FBN2 low concentration group compared with the negative control group: -99.44 ⁇ 9.42 ⁇ V vs. -116.05 ⁇ 16.71 ⁇ V; the FBN2 medium concentration group vs negative control group: -65.27 ⁇ 8.67 ⁇ V vs.
  • AAV group, FBN2 ultra-low concentration group, FBN2 high concentration group were significantly lower than the negative control group Max-a waveform amplitude (AAV Group vs negative control group: -19.17 ⁇ 3.67 ⁇ V versus -116.05 ⁇ 16.71 ⁇ V; FBN2 ultra-low concentration group versus negative control group: -24.72 ⁇ 4.33 ⁇ V versus -116.05 ⁇ 16.71 ⁇ V; 30.57 ⁇ 5.88 ⁇ V versus -116.05 ⁇ 16.71 ⁇ V,); the comparison trend between each group and the normal control group was consistent with the comparison trend with the negative control group.
  • the OPs-total wave values of the normal control group, negative control group, AAV group, FBN2 ultra-low concentration group, FBN2 low concentration group, FBN2 medium concentration group, and FBN2 high concentration group were (234.95 ⁇ 19.33 ⁇ V, 226.28 ⁇ 20.21 ⁇ V, 230.61 ⁇ 19.47 ⁇ V, 231.45 ⁇ 20.22 ⁇ V, 232.11 ⁇ 18.67 ⁇ V, 226.58 ⁇ 21.03 ⁇ V, 233.62 ⁇ 19.45 ⁇ V).
  • the amplitude of OPs-total waveform in each group was flat and showed a extinguished type (AAV group vs.
  • negative control group 22.23 ⁇ 3.90 ⁇ V vs. 226.7 ⁇ 18.37 ⁇ V; FBN2 ultra-low concentration group vs. 3.44 ⁇ V versus 226.7 ⁇ 18.37 ⁇ V; FBN2 low concentration group versus negative control group: 29.73 ⁇ 4.03 ⁇ V versus 226.7 ⁇ 18.37 ⁇ V; FBN2 medium concentration group versus negative control group: 26.37 ⁇ 3.67 ⁇ V versus 226.7 ⁇ 18.37 ⁇ V; FBN2 high concentration group Compared with the negative control group: 25.39 ⁇ 4.85 ⁇ V vs. 226.7 ⁇ 18.37 ⁇ V, P ⁇ 0.05); the comparison trend between each group and the normal control group is consistent with the comparison trend with the negative control group.
  • the FBN2 low concentration group had no change in the amplitude of OPs total waveform (133.56 ⁇ 18.77 ⁇ V vs. 272.8 ⁇ 21.34 ⁇ V, P>0.05); AAV group, FBN2 ultra-low concentration group, FBN2 medium concentration group, FBN2 high concentration group Compared with the negative control group, the OPs-total waveform amplitudes in the AAV group were significantly flatter, (AAV group vs. negative control group: 25.08 ⁇ 8.76 ⁇ V vs. 272.8 ⁇ 21.34 ⁇ V; 272.8 ⁇ 21.34 ⁇ V; FBN2 medium concentration group vs. negative control group: 70.75 ⁇ 23.33 ⁇ V vs.
  • the Cone-b wave values of the normal control group, negative control group, AAV group, FBN2 ultra-low concentration group, FBN2 low concentration group, FBN2 medium concentration group, and FBN2 high concentration group were (72.33 ⁇ 8.03 ⁇ V, 72.61 ⁇ 9.64 ⁇ V, 69.88 ⁇ 9.59 ⁇ V, 72.46 ⁇ 10.33 ⁇ V, 74.53 ⁇ 10.67 ⁇ V, 72.61 ⁇ 11.44 ⁇ V, 69.88 ⁇ 9.82 ⁇ V).
  • optical coherence tomography OCT includes: before the detection, anesthetize with intraperitoneal injection of 10g L -1 pentobarbital sodium solution (50mg kg -1 ), dilate the pupil, and apply ofloxacin ointment to the Cornea, wait until the mouse is fully anesthetized.
  • Wear a corneal contact lens with a diameter of 3mm and a diopter of -25D for the mouse align the mouse's eyes with the scanning lens, adjust the position of the mouse and the distance between the lens, and take a tomogram with an accurate position on the computer screen.
  • Standard take at least 3 pictures for each group and save the images, take pictures and analyze the images, measure the axial length of the eye, and the results are shown in Figure 3.
  • the axial length of the negative control group was (3.29 ⁇ 0.09mm), the axial length of the AAV group and the FBN2 protein injection group was (3.31 ⁇ 0.11mm, 3.31 ⁇ 0.12mm), there was no statistical significance ( P>0.05).
  • the recombinant protein group was injected with FBN2 recombinant protein for 3 times with an interval of 9 days; at the same time, the same amount of Ringer's injection buffer was injected into the AAV group.
  • the axial length of AAV group was significantly longer than that of negative control group and normal control group (AAV group vs. negative control group: 3.68 ⁇ 0.10mm vs. 3.40 ⁇ 0.03mm; AAV group vs.
  • Tissue grinding take out the frozen retinal tissue, transfer it to liquid nitrogen, grind it with a glass rod, add 200 ⁇ l Trizol to grind it for 2 minutes, and repeat the grinding. (Note: The sample volume cannot exceed 10% of the volume of the Trizol reagent).
  • RNA precipitation transfer the upper transparent liquid to a 1.5mL EP tube, add 0.3mL isopropanol, blow and mix well, let stand at room temperature, and then centrifuge in a pre-cooled high-speed centrifuge at 4°C for 20min , 14500g;
  • RNA washing remove the supernatant, add 1ml of 75% ethanol prepared from 0.25mL of DEPC water and 0.75mL of absolute ethanol, and centrifuge in a pre-cooled high-speed centrifuge at 4°C for 20min, 14500g;
  • RNA redissolution Remove the supernatant, then place it in a pre-cooled high-speed centrifuge, centrifuge at 4°C for 20 minutes, 14500g, gently suck off excess liquid with a gun, dry for 3 minutes, add 30 ⁇ l DEPC water to dissolve RNA precipitation, pipetting and mixing;
  • OD260/OD280 of the RNA sample is between 1.8 and 2.0. It is meaningful to measure the purity and concentration of the RNA with a K5600 ultra-micro spectrophotometer. Take 1 ⁇ L of the RNA sample and divide it to reduce the risk of repeated freezing and thawing, and then reverse it. Record, and store the remaining RNA samples in aliquots at -80°C to avoid RNA degradation.
  • the primer sequences were synthesized by Shanghai Sangong, and ⁇ -actin was used as an internal reference.
  • the primer sequences are listed in Table 1.
  • RT-PCR instrument was used to detect the relative expression of FBN2 and ⁇ -actin mRNA in the mouse retina of each group.
  • Set up 3 duplicate wells for the FBN2 gene use a 96-well plate, the total volume is 20 ⁇ L, including: 8 ⁇ L cDNA template diluted 20 times, both upstream and downstream primers are 1 ⁇ L, Add 10 ⁇ L of SYBR Green I Master, seal the membrane, and centrifuge slightly.
  • the reaction conditions were: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 10s, annealing at 54°C for 30s, extension at 72°C for 30s, a total of 45 cycles, and finally cooling at 40°C for 10s.
  • the 2- ⁇ CT method was adopted, and ⁇ -actin was used as an internal reference.
  • the 2- ⁇ CT method was used to calculate the fold change of the gene expression level in the FBN2 antibody group and the PBS group relative to the normal control group, and the relative expression of FBN2 and ⁇ -actin mRNA in the mouse retina of each group was quantitatively analyzed. The results As shown in Figure 4.
  • the mRNA expression of FBN2 gene in the FBN2 low concentration group [(3.00 ⁇ 0.37) ⁇ 10 -3 ] was significantly higher than that in the AAV group [(1.48 ⁇ 0.60) ⁇ 10 -3 ] (P ⁇ 0.05); Ultra-low concentration group [(1.69 ⁇ 0.71) ⁇ 10 -3 ], FBN2 medium concentration group [(2.01 ⁇ 0.22) ⁇ 10 -3 ], FBN2 high concentration group [(1.51 ⁇ 0.37) ⁇ 10 -3 ] and AAV group [(1.48 ⁇ 0.60) ⁇ 10-3] There was no significant change in the mRNA expression of FBN2 gene (P>0.05).
  • the mRNA expression of FBN2 gene in the FBN2 low concentration group [(3.00 ⁇ 0.37) ⁇ 10 -3 ] had no significant change (P>0.05); the AAV group [(1.48 ⁇ 0.60) ⁇ 10 -3 ], FBN2 ultra-low concentration group [(1.69 ⁇ 0.71) ⁇ 10 -3 ], FBN2 medium concentration group [(2.01 ⁇ 0.22) ⁇ 10 -3 ], FBN2 high concentration group [(1.51 ⁇ 0.71) ⁇ 10 -3 ], FBN2 high concentration group [(1.51 ⁇ 0.71) ⁇ 10 -3 0.37) ⁇ 10 -3 ] compared with the negative control group [(3.22 ⁇ 0.30) ⁇ 10-3], the mRNA expression of FBN2 gene decreased significantly (P ⁇ 0.05); The comparison trend is consistent.
  • mice were killed by intraperitoneal injection of an excessive amount of 10 g ⁇ L -1 pentobarbital sodium solution, the eyeballs were removed with curved forceps, rinsed in normal saline, placed under a Zeiss microscope, cut along the corneoscleral limbus, and removed.
  • the retina was gently separated with an iris restorer and stored in a sterile, enzyme-free 1.5ml EP tube, quickly frozen in liquid nitrogen, and stored at -80°C for later use.
  • 6 retinal tissues were selected in each group and transferred in liquid nitrogen.
  • tissue lysate to the tissue, put it on ice, grind it with a glass rod, then absorb the supernatant of the pulverized homogenate, then use ultrasonic grinding, then centrifuge the homogenate according to the above centrifugal conditions, and absorb the supernatant Store at -80°C.
  • concentration of the sample was detected by the BCA method, and the expression of the FBN2 protein was analyzed by ELISA according to the instructions. Specific steps are as follows:
  • the protein expression of the FBN2 low concentration group (7.51 ⁇ 0.87 ⁇ g/ml) was significantly higher than that of the AAV group (4.21 ⁇ 0.79 ⁇ g/ml) (P ⁇ 0.05); the FBN2 ultra-low concentration group (4.58 ⁇ 0.80 ⁇ g /ml), FBN2 medium concentration group (6.12 ⁇ 0.77 ⁇ g/ml), FBN2 high concentration group (5.01 ⁇ 0.36 ⁇ g/ml) and AAV group (4.21 ⁇ 0.79 ⁇ g/ml) showed no significant change in protein expression (P> 0.05).
  • AAV group (4.21 ⁇ 0.79 ⁇ g/ml), FBN2 ultra-low concentration group (4.58 ⁇ 0.80 ⁇ g/ml), FBN2 medium concentration group (6.12 ⁇ 0.77 ⁇ g/ml), FBN2 high concentration group (5.01 ⁇ 0.36 ⁇ g/ml)
  • the protein expression decreased significantly (P ⁇ 0.05); the trend of each group compared with the normal control group was consistent with that of the negative control group.
  • the pockets are well marked, after pouring out the blocking solution, cut the strips, put them into the corresponding pockets according to their names, seal them, and add a primary antibody to mark all the strips, 2 drive away the air bubbles and seal them, and 3 make the corresponding antibodies in the pockets;
  • the FBN2 protein injection group was the low concentration FBN2 group with the best therapeutic effect.
  • the protein expression of the FBN2 gene in the retina had no significant change (P>0.05); The expression of gene protein decreased (P ⁇ 0.05).
  • the protein expression of FBN2 gene in the retina of the FBN2 injection group was slightly lower (P>0.05)); compared with the AAV group, the protein expression of the FBN2 gene in the retina was significantly increased (P ⁇ 0.05).

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Abstract

原纤维蛋白-2重组蛋白在制备预防或治疗原纤维蛋白-2缺陷型眼病的产品中的用途。通过用玻璃体腔注射FBN2基因干扰载体AAV构建FBN2基因缺陷型眼病小鼠模型的方法,首次发现玻璃体腔注射FBN2重组蛋白对FBN2基因缺陷型眼病有治疗作用。同时进一步证明了FBN2基因缺陷在眼组织视网膜病变和近视发展中一系列病理生理改变,以及玻璃体腔注射外源性FBN2重组蛋白可以弥补FBN2基因缺陷型眼病模型FBN2内源性缺失的问题,从而达到治疗疾病的作用,为一步靶向基因干预研究提供了新方法,并指明研究方向。

Description

原纤维蛋白-2重组蛋白在制备预防或治疗原纤维蛋白-2缺陷型眼病的产品中的用途 技术领域
本发明涉及生物制药领域,具体涉及原纤维蛋白-2重组蛋白在制备预防或治疗原纤维蛋白-2缺陷型眼病的产品中的用途。
背景技术
原纤维蛋白-2(Fibrillin-2,FBN2)基因变异已经被证明是早发型黄斑变性(Early-onset macular degeneration,EOMD)、年龄相关性黄斑变性(Age related macular degeneration,AMD)形成的重要原因,2014年在AMD家系中发现了FBN2基因突变为常染色体显性遗传,2016年发现FBN2基因突变的AMD患者有玻璃膜疣的临床表现;有越来越多的人患有AMD,严重情况下会丧失视力。FBN2蛋白参与形成微纤维并对ECM的形成和重塑至关重要,参与全身结缔组织中弹性纤维的形成。FBN2基因变异的AMD患者眼中的FBN2蛋白表达降低或者功能丧失,这改变了其渗透性、弹性蛋白纤维的包装、以及柔韧性,这是诱发了视网膜病变的重要原因,但是FBN2基因缺陷在视网膜病变疾病中发生发展的作用机制尚不明确。
腺相关病毒(Adeno-associated virus,AAV)具有宿主范围广、有多种血清型、滴度高、安全性高、准确性高、表达稳定、免疫原性低等优点,已被广泛地应用于基础医学研究和临床试验中,被视为最有前途的研究基因治疗载体之一。
蛋白靶向干预广泛用于眼科疾病的治疗,如抗体(vascular endothelial growth factor,VEGF)等,但是针对FBN2基因缺陷型视网膜病变和近视的靶向药物研究上尚未见报道。所以,本申请为FBN2基因缺陷型眼病的临床诊断和治疗提供新策略。
发明内容
针对现有技术中存在的缺陷,本发明的主要目的在于提供原纤维蛋白-2重组蛋白在制备预防或治疗原纤维蛋白-2缺陷型眼病的产品中的用途。
本申请通过AAV为载体进行RNAi构建的基因缺陷型眼病,通过注射重组蛋白进行靶向干预治疗,为视网膜病变的靶向治疗提供新的方法。本申请研究发现,通过AAV为载体进行RNAi可以构建的基因缺陷型视网膜疾病模型,主要表现为眼底出现玻璃膜疣样渗出物,视网膜相干光学断层扫描出现不光滑反射区,视网膜外颗粒层变薄,这些均为视网膜病变的临床表现。经过注射重组蛋白进行靶向干预治疗后,眼底渗出减少,视网膜外颗粒层增厚,对视网膜病变起到了一定的治疗作用。
一方面,本发明提供了一种原纤维蛋白-2重组蛋白在制备预防或治疗原纤维蛋白-2缺陷型眼病的产品中的用途。
进一步的,所述原纤维蛋白-2缺陷型眼病选自遗传性视网膜病变、轴性 近视和先天性黄斑变性中的至少一种。
进一步的,所述产品为药物、试剂或疫苗。
在一个实施方式中,本发明提供了原纤维蛋白-2重组蛋白在非治疗目的调控视网膜电图波形振幅的用途;
优选的,所述调控视网膜电图波形振幅为将视网膜电图波形振幅恢复至正常;
优选的,所述视网膜电图波形振幅选自暗适应视杆细胞反应波形振幅、暗适应最大混合反应波形振幅、暗适应震荡电位反应波形振幅和明适应视锥细胞反应波形振幅中至少一种。
在一个实施方式中,本发明提供了原纤维蛋白-2重组蛋白在非治疗目的减少视网膜渗出及眼底沉积物的用途。
在一个实施方式中,本发明提供了原纤维蛋白-2重组蛋白在非治疗目的增加视网膜外核层厚度的用途。
在一个实施方式中,原纤维蛋白-2重组蛋白在非治疗目的降低眼轴长度的用途。
本发明提供了原纤维蛋白-2重组蛋白在非治疗目的提高原纤维蛋白-2的表达的用途;
优选的,所述原纤维蛋白-2的表达选自原纤维蛋白-2基因的表达或原纤维蛋白-2蛋白的表达;
优选的,所述原纤维蛋白-2基因的表达为原纤维蛋白-2基因mRNA的表达。
另一方面,本发明提供了一种预防或治疗原纤维蛋白-2缺陷型眼病的产品,所述产品是以原纤维蛋白-2重组蛋白作为唯一活性成分,或包含原纤维蛋白-2重组蛋白的组合物。
进一步,所述产品为药物,所述组合物还包括药用辅料;
优选的,所述药用辅料选自稀释剂、赋形剂、香味剂和甜味剂中的至少一种;
优选的,所述药物的制剂是混悬剂、乳剂、溶液剂、糖浆剂或注射剂中的至少一种;
优选的,所述药物的给药方式为滴注或注射;
优选的,所述药物的给药方式为玻璃体腔注射。
进一步,所述产品中原纤维蛋白-2重组蛋白的含量为0.01~5.0μg/μL;优选的,所述产品中原纤维蛋白-2重组蛋白的含量为0.1~1.0μg/μL;优选的,所述产品中原纤维蛋白-2重组蛋白的含量为0.2~0.5μg/μL。
本发明的有益效果如下:
1、本发明在FBN2基因缺陷型眼病模型构建以及注射FBN2蛋白的实验中证明,外源性FBN2重组蛋白可以改善FBN2基因缺陷引起的视网膜病变和近视性眼轴增长。
2、本发明通过注射FBN2基因干扰载体AAV后,可以构建FBN2基因缺陷型眼病小鼠模型,主要表现为眼底黄白色似玻璃膜疣沉积物、ONL变薄、眼轴增长、ERG波形振幅降低呈熄灭型,以及FBN2的mRNA和蛋白表达降低。
3、本发明通过注射外源性FBN2重组蛋白可以有效治疗FBN2基因缺陷型视网膜病变,可以减少眼底黄白色似玻璃膜疣沉积物的产生、增加ONL厚度、恢复ERG波形振幅、以及增加FBN2的mRNA和蛋白表达。
4、本发明首次通过用玻璃体腔注射FBN2基因干扰载体AAV构建FBN2基因缺陷型动物模型的方法,且首次发现玻璃体腔注射FBN2重组蛋白对FBN2基因缺陷型眼病有治疗作用。本发明研究进一步证明了FBN2基因缺陷在视网膜病变和近视发生发展中一系列病理生理改变,以及玻璃体腔注射外源性FBN2重组蛋白可以弥补FBN2基因缺陷型眼病FBN2内源性缺失的问题,从而达到治疗疾病的作用,为下一步靶向基因干预研究提供了新方法,并指明研究方向。
附图说明
图1为经过不同处理后的扫描共聚焦检眼镜检查和光学相干断层扫描检测结果;
图2为经过不同处理后的Rod-b、Max-a、OPs total、Cone-b波形振幅的结果;
图3为经过不同处理后的眼轴长度的结果;a:视眼轴长度图;b:眼轴长度统计图;
图4为经过不同处理后的视网膜中FBN2基因的mRNA表达的结果;
图5为经过不同处理后的视网膜中FBN2蛋白表达的ELISA检测结果;
图6为经过不同处理后的视网膜中FBN2蛋白表达的western blot检测结果;
上述图中,*表示相同条件下的与正常对照组相比,在α=0.05水平下差异显著;&表示相同条件下的与阴性对照组相比,在α=0.05水平下差异显著;#表示相同条件下的与AAV组相比,在α=0.05水平下差异显著。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中使用的材料、试剂等,如无特殊说明,均可从商业途径得到。下述实施例中使用的FBN-2重组蛋白的纯度>97%。
实施例1
试验中使用8周龄C57BL/6J小鼠,SPF级,体质21-23g,入组前对小鼠进行眼部健康进行筛查,排除角膜病、白内障以及通过扫描激光检眼镜(Scanning laser ophthalmoscope,SLO)、光学相干断层扫描(Optical coherence  tomography,OCT)和视网膜电图(Electroretinogram,ERG)分别排除视网膜眼底疾病。
构建FBN2腺病毒相关干扰载体:首先合成单链DNA低聚核苷酸,然后产生双链DNA低聚核苷酸,再通过其两端所含酶切位点(BamHI识别序列:5'G^GATCC 3',EcoRI识别序列:5'G^AATTC 3')将其直接接上酶切后的RNA干扰载体上;比对正确的克隆即为构建成功。共转染AAV-293细胞,包装病毒,收集原液,浓缩并纯化后得到浓缩液。抽提高纯度、无内毒素的AAV载体,使用HG transgene reagent将构建好的病毒载体及质粒共转染进AAV-293细胞,转染10~12h后加Enhancing buffer,接着8h后更换新鲜培养基,接着培养基48h后,细胞脱落后收集包括病毒的细胞及上清,之后对其浓缩、纯化后得到滴度为1.21E+12VG/mL的AAV病毒。
FBN-2重组蛋白的氨基酸序列为:
Figure PCTCN2021115997-appb-000001
FBN-2重组蛋白为大肠杆菌的宿主-载体系统有效地生成,稀释FBN-2重组蛋白的缓冲液包括20mM的Tris,150mM的NaCL,1mM的EDTA,1mM的二硫苏糖醇DTT,0.01%的十二烷基肌氨酸钠,5%的海藻糖和proclin300。FBN-2重组蛋白的原始浓度为200μg/μL。
选取63只8周龄C57BL/6J小鼠,随机分为7组:正常对照组、阴性对照组、AAV组、FBN2超低浓度组(0.3μg/眼)、FBN2低浓度组(0.75μg/眼)、FBN2中浓度组(1.5μg/眼)、FBN2高浓度组(3.0μg/眼)(n=9只/组)。正常对照组正常饲养不做任何处理;入组后,正常组正常饲养,阴性对照组双眼玻璃体腔注射3μL阴性病毒原液(AAV灭活病毒原液);AAV组、FBN2超低浓度组、FBN2低浓度组、FBN2中浓度组、FBN2高浓度组均双眼玻璃体腔注射3μL AAV病毒进行FBN2基因缺陷型眼病造模。2周后,FBN2超低浓度组、FBN2低浓度组、FBN2中浓度组、FBN2高浓度组分别双眼玻璃体腔注射浓度为0.1μg/μL、0.25μg/μL、0.50μg/μL、1μg/μL,剂量为3μL的FBN2重组蛋白,FBN2超低浓度组、FBN2低浓度组、FBN2中浓度组、FBN2高浓度组分别注射的FBN2重组蛋白量为0.3μg/眼、0.75μg/眼、1.5μg/眼、3μg/眼,每周注射1次,连续注射3次。其中,玻璃体腔注射的具体操作包括:提前一天用氧氟沙星滴眼液(3次/d)对阴性对照组、AAV组、FBN2注射组动物进行眼部消炎。腹腔注射10g·L -1戊巴比妥钠溶液(50mg·kg -1)进行麻醉,保证正常呼吸,并滴散瞳药进行扩瞳。将麻醉的小鼠置于显微镜下,用聚维酮碘进行眼部周围消毒,1min后用0.9%生理盐水冲洗掉表面聚维酮碘,后用干燥无菌棉棒蘸干,用盐酸奥布卡因滴眼液进行眼表麻醉。用显微镜齿镊 夹住球结膜,用微量注射器(10L),在角巩膜缘后进针,夹住针孔30s,松开镊子滴用典必殊滴眼液,术后1d继续滴用典必殊(3次)。
对实验结果数据的分析采用SPSS21.0统计软件,检验合正态分布,验方差齐性,涉及实验均为多组实验,均采用独立样本t检验的方差分析;计量资料用x±S表示,方差分析组间差异,Levene检验各组各指标方差齐性,P<0.05认为差异具有统计学意义。
实施例2
FBN2重组蛋白每注射1次后,利用SLO检测眼底变化。
扫描共聚焦检眼镜检查SLO的具体操作包括:进行检测之前,先用腹腔注射10g·L -1戊巴比妥钠溶液(50mg·kg -1)进行麻醉,并扩瞳,并用氧氟沙星眼膏涂抹至角膜,待小鼠处于完全麻醉状态。将小鼠眼睛对准扫描激光眼底镜镜头,调整小鼠位置,以电脑显示屏可以拍到完整的眼地图为标准,每组取至少3张图片并保存图像,所有操作均由同一研究者进行,检测结果如图1所示。
由图1的结果可知,注射阴性病毒后,阴性对照组,与正常对照相比,视网膜眼底图无明显变化;注射AAV病毒后,AAV组和各FBN2注射组出现视网膜渗出,黄白色似玻璃膜疣沉积物。注射1次FBN2蛋白后,FBN2超低浓度组、FBN2低浓度组、FBN2中浓度组、FBN2高浓度组渗出及沉积物均无明显改变。注射2次FBN2蛋白后,FBN2低浓度组视网膜渗出及沉积物减少较明显,FBN2超低浓度组和中浓度组渗出及沉积物稍减少,FBN2高浓度组无明显改变。注射3次FBN2蛋白后,FBN2低浓度组视网膜渗出及沉积物明显减少,FBN2超低浓度组和中浓度组渗出及沉积物减少较明显,FBN2高浓度组渗出及沉积物稍减少变化不明显。
实施例3
FBN2重组蛋白每注射1次后,利用ERG分别检测检测视网膜功能。视网膜电图ERG的具体操作包括:ERG检测在绝对黑暗环境进行。检测前把小鼠置于黑暗环境8h以上,进行暗适应。进行检测之前,先用腹腔注射10g·L -1戊巴比妥钠溶液(50mg·kg -1)进行麻醉,并扩瞳,用盐酸奥布卡因滴眼液进行眼表麻醉,并用氧氟沙星眼膏涂抹至角膜,待小鼠处于完全麻醉状态。将动物置于操作台上,采用RETI port系统,将由金制的环状角膜电极(直径为3.00mm)固定在小鼠双眼角膜,将针状参考电极分别插入小鼠口中左右口角两侧肌肉丰厚处,吧针状接地电极缓慢扎入小鼠右上肢皮下肌肉,待检测屏上地线稳定后,开始记录脑电图Rod-ERG、Max-ERG、Ops total、明适应10min后进行Cone-ERG、Flick-ERG波形记录,以及各振幅变化,每个波形取3个数值,进行分析,结果如图2所示。
(1)暗适应视杆细胞反应(Rod-b)
入组时正常对照组、阴性对照组、AAV组、FBN2超低浓度组、FBN2 低浓度组、FBN2中浓度组、FBN2高浓度组Rod-b波值分别为(140.40±18.79μV、138.41±17.42μV、141.35±15.80μV、140.22±18.33μV、140.97±17.58μV、139.14±15.36μV、141.35±18.03μV)。
阴性AAV注射后,阴性对照组与正常对照相比,各时间点Rod-b波形振幅无明显变化(阴性AAV注射后:138.91±18.93μV对比139.78±16.66μV;FBN2注射1次:142.91±17.44μV对比140.97±16.09μV,FBN2注射2次:141.16±15.09μV对比141.29±19.72μV;FBN2注射3次:136.91±17.73μV对比145.38±18.31,P>0.05);AAV注射后,各组与阴性对照组相比Rod-b波形振幅均变平缓,呈熄灭型(AAV组对比阴性对照组:27.18±4.21对比138.91±18.93μV;FBN2超低浓度组对比阴性对照组:28.32±3.78μV对比138.91±18.93μV;FBN2低浓度组对比阴性对照组:22.26±4.02μV对比138.91±18.93μV,FBN2中浓度组对比阴性对照组:23.45±4.27μV对比138.91±18.93μV,FBN2高浓度组对比阴性对照组:24.11±3.78μV对比138.91±18.93μV,P<0.05)。
FBN2蛋白注射1次后,各组与AAV组相比Rod-b波形振幅无明显变化(FBN2超低浓度组对比AAV组:26.44±3.67μV对比26.28±4.33μV;FBN2低浓度组对比AAV组:32.95±5.67μV对比26.28±4.33μV;FBN2中浓度组对比AAV组:30.38±4.29μV对比26.28±4.33μV;FBN2高浓度组对比AAV组:27.55±1.79μV对比26.28±4.33μV,P>0.05);各组与AAV组比较,Rod-b波形振幅均变平缓,呈熄灭型(AAV组对比阴性对照组:26.28±4.33μV对比142.91±17.44μV;FBN2超低浓度组对比阴性对照组:26.44±3.67μV对比142.91±17.44μV;FBN2低浓度组对比阴性对照组:32.95±5.67μV对比142.91±17.44μV;FBN2中浓度组对比阴性对照组:30.38±4.29μV对比142.91±17.44μV;FBN2高浓度组对比阴性对照组:27.55±1.79μV对比142.91±17.44μV,P<0.05);各组与正常对照组比较趋势和与阴性对照组比较趋势一致。
FBN2蛋白注射2次后,FBN2低浓度组与AAV组相比,波形振幅增大(76.33±7.46μV对比25.96±4.59μV,P<0.05);FBN2超低浓度组、FBN2中浓度组、FBN2高浓度组与AAV组相比Rod-b波形振幅无明显变化,(FBN2超低浓度组对比AAV组:28.97±5.00μV对比25.96±4.59μV;FBN2中浓度组对比AAV组:39.79±6.17μV对比25.96±4.59μV;FBN2高浓度组对比AAV组:30.40±4.42μV对比25.96±4.59μV,P>0.05)。各组与阴性对照组相比,Rod-b波形振幅均明显较低,(AAV组对比阴性对照组:25.96±4.59μV对比141.16±15.09μV;FBN2超低浓度组对比阴性对照组:28.97±5.00μV对比141.16±15.09μV;FBN2低浓度组对比阴性对照组:76.33±7.46μV对比141.16±15.09μV;FBN2中浓度组对比阴性对照组:39.79±6.17μV对比141.16±15.09μV;FBN2高浓度组对比阴性对照组:30.40±4.42μV对比141.16±15.09μV,P<0.05);各组与正常对照组比较趋势和与阴性对照组比较趋势 一致。
FBN2蛋白注射3次后,FBN2低浓度组、FBN2中浓度组与AAV组相比,Rod-b波形振幅明显增大(FBN2低浓度组对比AAV组:118.33±12.42μV对比26.25±3.88μV;FBN2中浓度组对比AAV组:77.00±8.51μV对比26.25±3.88μV,P<0.05),FBN2超低浓度组、FBN2高浓度组与AAV组相比Rod-b波形振幅无明显变化(FBN2超低浓度组对比AAV组:31.25±4.79μV对比26.25±3.88μV;FBN2高浓度组对比AAV组:58.88±5.32μV对比26.25±3.88μV,P>0.05)。与阴性对照组相比,FBN2低浓度组Rod-b波形振幅差异无明显变化,(118.33±12.42对比136.91±17.73μV,P>0.05),AAV组、FBN2超低浓度组、FBN2中浓度组、FBN2高浓度组Rod-b波形振幅均明显较低(AAV组对比阴性对照组:26.25±3.88μV对比136.91±17.73μV;FBN2超低浓度组对比阴性对照组:31.25±4.79μV对比136.91±17.73μV;FBN2中浓度组对比阴性对照组:77.00±8.51μV对比136.91±17.73μV;FBN2高浓度组对比阴性对照组:58.88±5.32μV对比136.91±17.73μV,P>0.05);各组与正常对照组比较趋势和与阴性对照组比较趋势一致。
(2)暗适应最大混合反应(Max-a)
入组时正常对照组、阴性对照组、AAV组、FBN2超低浓度组、FBN2低浓度组、FBN2中浓度组、FBN2高浓度组Rod-b波值分别为(-121.97±18.67μV、-123.85±16.48μV、-117.26±17.83μV、-119.77±18.63μV、-115.7±17.88μV、-121.97±15.46μV、-117.26±15.33μV)。
阴性AAV注射后,阴性对照组与正常对照组相比,实验各时间点Max-a波无明显变化(阴性AAV注射后:-116.07±18.77μV对比-116.45±17.44μV;FBN2注射1次:-117.05±15.83μV对比-117.45±17.33μV;FBN2注射2次:-118.29±17.42μV对比-116.45±18.68μV;FBN2注射3次:-116.05±16.71μV对比-119.5±17.33μV,P>0.05);AAV注射后,各组与阴性对照组,Max-a波形振幅均变平缓,呈熄灭型(AAV组对比阴性对照组:-18.90±2.84μV对比-116.07±18.77μV;FBN2超低浓度组对比阴性对照组:-17.19±2.67μV对比-116.07±18.77μV;FBN2低浓度组对比阴性对照组:-15.65±2.35μV对比-116.07±18.77μV;FBN2中浓度组对比阴性对照组:-17.90±2.79μV对比-116.07±18.77μV;FBN2高浓度组对比阴性对照组:-15.07±3.07μV对比-116.07±18.77μV,P<0.05);各组与正常对照组比较趋势同与阴性对照组比较趋势一致。
FBN2蛋白注射1次后,各组与AAV组相比,FBN2超低浓度组、FBN2低浓度组、FBN2中浓度组、FBN2高浓度组视网膜电图波形振幅无明显变化,(FBN2超低浓度组对比AAV组:-19.49±2.33μV对比-18.40±3.33μV;FBN2低浓度组对比AAV组:-25.65±5.22μV对比-18.40±3.33μV;FBN2中浓度组对比AAV组:-22.53±4.67μV对比-18.40±3.33μV;FBN2高浓度组对比AAV组:-19.90±3.67μV对比-18.40±3.33μV,P>0.05);各组与阴性对照组比较,MAX-a波 形振幅均变平缓,呈熄灭型,(AAV组对比阴性对照组:-18.40±3.33μV对比-117.05±15.83μV;FBN2超低浓度组对比阴性对照组:-19.49±2.33μV对比-117.05±15.83μV;FBN2低浓度组对比阴性对照组:-25.65±5.22μV对比-117.05±15.83μV;FBN2中浓度组对比阴性对照组:-22.53±4.67μV对比-117.05±15.83μV;FBN2高浓度组对比阴性对照组:-19.90±3.67μV对比-117.05±15.83,P<0.05);各组与正常对照组比较趋势同与阴性对照组比较趋势一致。
FBN2蛋白注射2次后,FBN2低浓度组、FBN2中浓度组与AAV组相比,Max-a波形振幅略增大(FBN2低浓度组对比AAV组:-67.95±5.67μV对比-19.82±3.85μV;FBN2中浓度组对比AAV组:-27.24±6.67μV对比-19.82±3.85μV,P<0.05),FBN2超低浓度组、FBN2高浓度组与AAV组相比Max-a波形振幅无明显变化(BN2超低浓度组与AAV组相比:-23.94±6.67μV对比-19.82±3.85μV;FBN2高浓度组与AAV组相比:-24.54±5.44μV对比-19.82±3.85μV,P>0.05);各组与阴性对照组相比,AAV组、FBN2超低浓度组、FBN2低浓度组、FBN2中浓度组、FBN2高浓度组Max-a波形振幅低平,(AAV组对比阴性对照组:-19.82±3.85μV对比-118.29±17.42μV;FBN2超低浓度组对比阴性对照组:-23.94±6.67μV对比-118.29±17.42μV;FBN2低浓度组对比阴性对照组:-67.95±5.67μV对比-118.29±17.42μV;FBN2中浓度组对比阴性对照组:-27.24±6.67μV对比-118.29±17.42μV;FBN2高浓度组对比阴性对照组:-24.54±5.44μV对比-118.29±17.42μV,P<0.05);各组与正常对照组比较趋势同与阴性对照组比较趋势一致。
FBN2蛋白注射3次后,FBN2低浓度组、FBN2中浓度组与AAV组相比,Max-a波形振幅均增大(FBN2低浓度组对比AAV组:-99.44±9.42对比-19.17±3.67μV,FBN2中浓度组对比AAV组-65.27±8.67μV对比-19.17±3.67μV,P<0.05);FBN2超低浓度组、FBN2高浓度组与AAV组相比Max-a波形振幅无明显变化(FBN2超低浓度组与AAV组:-24.72±4.33μV对比-19.17±3.67μV;FBN2高浓度组与AAV组:-30.57±5.88μV对比-19.17±3.67μV,P>0.05)。FBN2低浓度组、FBN2中浓度组与阴性对照组相比,Max-a波形振幅无明显差异,(FBN2低浓度组对比阴性对照组:-99.44±9.42μV对比-116.05±16.71μV;FBN2中浓度组对比阴性对照组:-65.27±8.67μV对比-116.05±16.71μV;P>0.05),AAV组、FBN2超低浓度组、FBN2高浓度组较阴性对照组Max-a波形振幅明显低平(AAV组对比阴性对照组:-19.17±3.67μV对比-116.05±16.71μV;FBN2超低浓度组对比阴性对照组:-24.72±4.33μV对比-116.05±16.71μV;FBN2高浓度组对比阴性对照组:-30.57±5.88μV对比-116.05±16.71μV,);各组与正常对照组比较趋势同与阴性对照组比较趋势一致。
(3)暗适应震荡电位反应(OPs total)
入组时正常对照组、阴性对照组、AAV组、FBN2超低浓度组、FBN2低浓度组、FBN2中浓度组、FBN2高浓度组OPs-total波值分别为(234.95±19.33μV、 226.28±20.21μV、230.61±19.47μV、231.45±20.22μV、232.11±18.67μV、226.58±21.03μV、233.62±19.45μV)。
阴性AAV注射后,阴性对照组与正常对照组相比,实验各时间点OPs-total波无明显变化(阴性AAV注射后:229.28±19.67μV对比238.56±20.44μV;FBN2注射1次:226.7±18.37μV对比250.95±21.33μV;FBN2注射2次:229.28±21.33μV对比222.95±18.67μV;FBN2注射3次:272.8±21.34μV对比234.95±22.67μV,P>0.05)。AAV注射后,各组与阴性对照组相比OPs-total波形振幅均变平缓,呈熄灭型,(AAV组对比阴性对照组:19.56±2.97μV对比229.28±19.67μV;FBN2超低浓度组对比阴性对照组:19.96±3.03μV对比229.28±19.67μV;FBN2低浓度组对比阴性对照组:22.75±3.57μV对比229.28±19.67μV;FBN2中浓度组对比阴性对照组:21.56±4.05μV对比229.28±19.67μV;FBN2高浓度组对比阴性对照组:26.75±3.33μV对比229.28±19.67μV,P<0.05);各组与正常对照组比较趋势同与阴性对照组比较趋势一致。
FBN2蛋白注射1次后,各蛋白注射组与AAV组相比,FBN2超低浓度组、FBN2低浓度组、FBN2中浓度组、FBN2高浓度组OPs-total波形振幅无明显变化,(FBN2超低浓度组对比AAV组:25.48±3.44μV对比22.23±3.90μV;FBN2低浓度组对比AAV组:29.73±4.03μV对比22.23±3.90μV;FBN2中浓度组对比AAV组:26.37±3.67μV对比22.23±3.90μV;FBN2高浓度组对比AAV组:25.39±4.85μV对比22.23±3.90μV,P>0.05)。各组与阴性对照组相比,OPs-total波形振幅均平缓,呈熄灭型(AAV组对比阴性对照组:22.23±3.90μV对比226.7±18.37μV;FBN2超低浓度组对比阴性对照组:25.48±3.44μV对比226.7±18.37μV;FBN2低浓度组对比阴性对照组:29.73±4.03μV对比226.7±18.37μV;FBN2中浓度组对比阴性对照组:26.37±3.67μV对比226.7±18.37μV;FBN2高浓度组对比阴性对照组:25.39±4.85μV对比226.7±18.37μV,P<0.05);各组与正常对照组比较趋势同与阴性对照组比较趋势一致。
FBN2蛋白注射2次后,FBN2低浓度组与AAV组相比,OPs-total波形振幅变大,(107.03±18.03μV对比24.51±8.67μV,P<0.05),FBN2超低中浓度组、FBN2中浓度组、FBN2高浓度组OPs-total波形振幅无明显变化(FBN2超低浓度组对比AAV组35.03±9.55μV对比24.51±8.67μV;FBN2中浓度组对比AAV组44.91±14.53μV对比24.51±8.67μV,FBN2高浓度组对比AAV组30.18±5.44μV对比24.51±8.67μV,P>0.05)。各组与阴性对照组相比,OPs-total波形振幅均明显变平缓,(AAV组对比阴性对照组:24.51±8.67μV对比229.28±21.33μV;FBN2超低浓度组对比阴性对照组:35.03±9.55μV对比229.28±21.33μV;FBN2低浓度组对比阴性对照组:107.03±18.03μV对比229.28±21.33μV;FBN2中浓度组对比阴性对照组:44.91±14.53μV对比 229.28±21.33μV;FBN2高浓度组对比阴性对照组:30.18±5.44μV对比229.28±21.33μV,P<0.05);各组与正常对照组比较趋势同与阴性对照组比较趋势一致。
FBN2蛋白注射3次后,FBN2低浓度组与AAV组相比,OPs total波形振幅明显增大,(133.56±18.77μV对比25.08±8.76μV,P<0.05);FBN2超低浓度组、FBN2中浓度组、FBN2高浓度组与AAV组相比OPs-total波形振幅均无明显变化(FBN2超低浓度组对比AAV组:37.83±6.97μV对比25.08±8.76μV;FBN2中浓度组对比AAV组:70.75±23.33μV对比25.08±8.76μV;FBN2高浓度组对比AAV组:58.31±9.24μV对比25.08±8.76μV,P>0.05)。FBN2低浓度组与阴性对照组相比,OPs total波形振幅无变化(133.56±18.77μV对比272.8±21.34μV,P>0.05);AAV组、FBN2超低浓度组、FBN2中浓度组、FBN2高浓度组与阴性对照组相比,OPs-total波形振幅均明显平缓,(AAV组对比阴性对照组:25.08±8.76μV对比272.8±21.34μV;FBN2超低浓度组对比阴性对照组:37.83±6.97μV对比272.8±21.34μV;FBN2中浓度组对比阴性对照组:70.75±23.33μV对比272.8±21.34μV;FBN2高浓度组对比阴性对照组:58.31±9.24μV对比272.8±21.34μV,P<0.05);各组与正常对照组比较趋势同与阴性对照组比较趋势一致。
(4)明适应视锥细胞反应(Cone-b)
入组时正常对照组、阴性对照组、AAV组、FBN2超低浓度组、FBN2低浓度组、FBN2中浓度组、FBN2高浓度组Cone-b波波值分别为(72.33±8.03μV、72.61±9.64μV、69.88±9.59μV、72.46±10.33μV、74.53±10.67μV、72.61±11.44μV、69.88±9.82μV)。
阴性AAV注射后,阴性对照组与正常对照组相比,实验各时间点OPs-total波无明显变化(阴性AAV注射后:69.56±11.33μV对比71.21±10.17μV;FBN2注射1次:68.77±9.33μV对比75.21±9.47μV;FBN2注射2次:70.23±6.63μV对比77.22±8.93μV;FBN2注射3次:71.03±8.44μV对比77.31±9.03μV,P>0.05)。AAV注射后,各组与阴性对照组相比,Cone-b波形振幅均变平缓,呈熄灭型,(AAV组对比阴性对照组:3.89±2.82μV对比69.56±11.33μV,FBN2超低浓度组对比阴性对照组:4.77±2.63μV对比69.56±11.33μV,FBN2低浓度组对比阴性对照组:3.43±3.44μV对比69.56±11.33μV,FBN2中浓度组对比阴性对照组:3.68±2.51μV对比69.56±11.33μV,FBN2高浓度组对比阴性对照组:3.47±1.67μV对比69.56±11.33μV,P<0.05);各组与正常对照组比较趋势同与阴性对照组比较趋势一致。
FBN2蛋白注射1次后,各蛋白注射组与AAV组相比,Cone-b波形振幅无明显变化,(FBN2超低低浓度组对比AAV组:3.93±2.56μV对比3.03±2.02μV,FBN2低浓度组对比AAV组:6.21±2.67μV对比3.03±2.02μV,FBN2中浓度组对比AAV组:4.44±3.33μV对比3.03±2.02μV,FBN2高浓度组对比 AAV组:3.99±1.95μV对比3.03±2.02μV,P>0.05)。各组与阴性对照组比Cone-b波形振幅均变平缓,呈熄灭型,(AAV组对比阴性对照组:3.03±2.02μV对比68.77±9.33μV,FBN2超低浓度组对比阴性对照组:3.93±2.56μV对比68.77±9.33μV,FBN2低浓度组对比阴性对照组:6.21±2.67μV对比68.77±9.33μV,FBN2中浓度组对比阴性对照组:4.44±3.33μV对比68.77±9.33μV,FBN2高浓度组对比阴性对照组:3.99±1.95μV对比68.77±9.33μV,P<0.05);各组与正常对照组比较趋势同与阴性对照组比较趋势一致。
FBN2蛋白注射2次后,各蛋白注射组与AAV组相比,Cone-b波形振幅无明显变化,(FBN2超低浓度组对比AAV组:7.67±3.22μV对比3.67±2.97μV;FBN2低浓度组对比AAV组:21.18±8.48μV对比3.67±2.97μV;FBN2中浓度组对比AAV组:10.86±4.03μV对比3.67±2.97μV;FBN2高浓度组对比AAV组:8.05±3.33μV对比3.67±2.97μV,P>0.05)。各组与阴性对照组相比,Cone-b波形振幅均明显平缓,(AAV组对比阴性对照组:3.67±2.97μV对比70.23±6.63μV;FBN2超低浓度组对比阴性对照组:7.67±3.22μV对比70.23±6.63μV;FBN2低浓度组对比阴性对照组:21.18±8.48μV对比70.23±6.63μV;FBN2中浓度组对比阴性对照组:10.86±4.03μV对比70.23±6.63μV;FBN2高浓度组对比阴性对照组:8.05±3.33μV对比70.23±6.63μV,P<0.05);各组与正常对照组比较趋势同与阴性对照组比较趋势一致。
注射3次FBN2蛋白后,与AAV组相比,FBN2低浓度组Cone-b波形振幅明显增大,25.61±7.67μV对比3.17±2.11μV,P<0.05),FBN2超低浓度组、FBN2中浓度组、FBN2高浓度组Cone-b波形振幅均无明显变化(FBN2超低浓度组对比AAV组:8.55±5.42μV对比3.17±2.11μV,FBN2中浓度组对比AAV组:18.57±8.03μV对比3.17±2.11μV,FBN2高浓度组对比AAV组:10.66±4.43μV对比3.17±2.11μV,P>0.05)。各组与阴性对照组相比,Cone-b波形振幅均明显平缓,但FBN2超低浓度组的治疗效应最明显(AAV组对比阴性对照组:3.17±2.11μV对比71.03±8.44μV;FBN2超低浓度组对比阴性对照组:8.55±5.42μV对比71.03±8.44μV;FBN2低浓度组对比阴性对照组:25.61±7.67μV对比71.03±8.44μV;FBN2中浓度组对比阴性对照组:18.57±8.03μV对比71.03±8.44μV;FBN2高浓度组对比阴性对照组:10.66±4.43μV对比71.03±8.44μV,,P<0.05);各组与正常对照组比较趋势同与阴性对照组。
实施例4
FBN2重组蛋白每注射1次后,利用OCT检测眼轴长度。
光学相干断层扫描OCT的具体操作包括:进行检测之前,先用腹腔注射10g L -1戊巴比妥钠溶液(50mg kg -1)进行麻醉,并扩瞳,并用氧氟沙星眼膏涂抹至角膜,待小鼠处于完全麻醉状态。为小鼠配戴直径为3mm、屈光度为-25D的角膜接触镜,将小鼠眼睛对准扫描镜头,调整小鼠位置、和镜头距离,以电脑显示屏可以拍到位置准确的断层图,为标准,每组取至少3张图 片并保存图像,进行拍摄并图像分析,测量眼轴长度,结果如图3所示。sh性AAV/AAV注射后,阴性对照组眼轴长度为(3.29±0.09mm),AAV组和FBN2蛋白注射组眼轴长度为(3.31±0.11mm,3.31±0.12mm),无统计学意义(P>0.05)。对重组蛋白组注射3次FBN2重组蛋白,每次间隔9天;同时对AAV组注射等量的Ringer's注射缓冲液。注射完成后,AAV组眼轴长度明显大于阴性对照组、正常对照组(AAV组对比阴性对照组:3.68±0.10mm对比3.40±0.03mm;AAV组对比阴性对照组:3.68±0.10mm对比3.40±0.09mm,P<0.05);FBN2低浓度蛋白注射组眼轴显著低于AAV组(3.35±0.03mm对比3.68±0.10mm,P<0.05);FBN2低浓度蛋白注射组眼轴与阴性对照组、正常对照组比较无统计学意义(P>0.05)。
实施例5
实验时每组挑选6个视网膜组织。视网膜组织总RNA的提取和RT-PCR的反应按照以下方法进行。
1、总RNA的提取
(1)组织研磨:将冻存的视网膜组织取出,液氮中转移,用玻璃棒研磨,加入200μl Trizol研磨2min,重复研磨,待EP管中无可见的视网膜组织后,在加入500μl Trizol吹打混匀(注:样本样本量不能超过Trizol试剂体积的10%)。
(2)分离:静置5min,待核蛋白复合体彻底分离后,加入150μl氯仿(三氯甲烷),剧烈震荡数次,静置2-3min(15-30℃),后置于预冷的高速离心机中离心,4℃下转速20min,14500g,离心结束后,RNA存在于上层液体中;
(3)RNA沉淀:将上层透明液体转移至1.5mL EP管中,加入0.3mL异丙醇,吹打混匀,室温静置,后置于预冷的高速离心机中离心,4℃下转速20min,14500g;
(4)RNA洗涤:去上清,加入1ml由0.25mL和DEPC水0.75mL无水乙醇配制75%乙醇,后置于预冷的高速离心机中离心,4℃下转速20min,14500g;
(5)RNA再溶解:去上清,后置于预冷的高速离心机中离心,4℃下转速20min,14500g,用枪轻吸除多余液体,干燥3min后,加入30μl DEPC水,以溶解RNA沉淀,吹打混匀;
(6)RNA样本的OD260/OD280的数值在1.8-2.0间为有意义,于K5600超微量分光光度计测定RNA纯度和浓度取1μLRNA样品,进行分装,减少因反复冻融,之后进行反转录,将剩余的RNA样本分装保存在-80℃避免RNA降解。
2、第一链cDNA合成
(1)依次加入1μL Random 6mers、2μL RNase Free dH 2O、dNTP Mixture、1μL Oligo dT Primer和5μL RNA样本,混匀后离心;
(2)温育5min,65℃金属恒温锅;
(3)依次加入1μLPrimeScriptⅡRTase、0.5μLRNase Inhabitor、4μL 5×PrimeScriptⅡBuffer、4.5μL RNase Free dH 2O,混匀后离心;
(4)温育10min(30℃),后转移至42℃水浴锅60min;
(5)放入95℃金属恒温锅中,使反转录酶失活(5min)。
3、设计与合成引物
引物序列合成与由上海生工,β-actin为内参,引物序列见表1。
表1视网膜各分子引物序列表
Figure PCTCN2021115997-appb-000002
4、实时荧光定量PCR反应
采用
Figure PCTCN2021115997-appb-000003
II RT-PCR仪检测各组小鼠视网膜中FBN2、β-actin mRNA的相对表达量。FBN2基因设置3个复孔,使用96孔板,总体积为20μL,包括:稀释20倍后的8μL cDNA模板,上下游引物均为1μL,
Figure PCTCN2021115997-appb-000004
SYBR Green I Master加10μL,封膜,轻度离心。反应条件为:预变性95℃5min,变性95℃ 10s,54℃退火30s,72℃延伸30s,共45个循环,最后40℃冷却10s。采用2 -△△CT方法,以β-actin为内参。通过2 △△CT方法计算FBN2抗体组和PBS组中相对于正常对照组的基因表达水平倍数的变化,对各组小鼠视网膜中FBN2、β-actin mRNA的相对表达量进行定量分析,结果如图4所示。
由图8的结果可知,阴性AAV注射后,阴性对照组[(3.08±0.39)×10 -3]与正常对照组[(3.16±0.45)×10 -3]相比,FBN2基因的mRNA的表达均无明显变化(P>0.05)。AAV后注射,与阴性对照组[(3.16±0.45)×10 -3]比,AAV组[(1.36±0.24)×10 -3]、FBN2超低浓度组[(1.35±0.26)×10 -3]、FBN2低浓度组[(1.45±0.30)×10 -3]、FBN2中浓度组[(1.54±0.41)×10 -3]、FBN2高浓度组[(1.55±0.04)×10 -3],FBN2基因的mRNA表达均明显下降,(P<0.05)。
FBN2蛋白注射3次后,FBN2低浓度组[(3.00±0.37)×10 -3]FBN2基因的mRNA表达比AAV组[(1.48±0.60)×10 -3]明显增加(P<0.05);FBN2超低浓度组[(1.69±0.71)×10 -3]、FBN2中浓度组[(2.01±0.22)×10 -3]、FBN2高浓度组[(1.51±0.37)×10 -3]与AAV组[(1.48±0.60)×10-3]比较FBN2基因的mRNA表达均无明显变化(P>0.05)。
FBN2低浓度组[(3.00±0.37)×10 -3]FBN2基因的mRNA表达与阴性对 照组[(3.22±0.30)×10 -3]比较无明显变化(P>0.05);AAV组[(1.48±0.60)×10 -3]、FBN2超低浓度组[(1.69±0.71)×10 -3]、FBN2中浓度组[(2.01±0.22)×10 -3]、FBN2高浓度组[(1.51±0.37)×10 -3]与阴性对照组[(3.22±0.30)×10-3]比较FBN2基因的mRNA表达均明显下降(P<0.05);各组与正常对照组比较趋势同与阴性对照组比较趋势一致。
实施例6
实验结束后,腹腔注射过量10g·L -1戊巴比妥钠溶液处死小鼠,用弯镊摘除眼球放入生理盐水中涮洗一下,放在蔡司显微镜下,沿角巩膜缘剪开后去用虹膜恢复器轻轻分离出视网膜存于无菌无酶1.5ml EP管,液氮速冻,-80℃保存备用。实验时每组挑选6个视网膜组织,液氮中转移。在组织中加入组织裂解液,置于于冰上,使用玻璃棒进行研磨,然后将粉碎后的匀浆,吸取上清,再用超声粉碎,然后将匀浆按上述离心条件离心,吸取上清利-80℃保存。用BCA法检测样品浓度,并按照说明书进行ELISA检测、分析FBN2蛋白的表达。具体步骤如下:
(1)试剂盒室温平衡,60min;
(2)在标准品孔,按要求各加的50μL标准品;
(3)样品孔,各50μL加检测样品,空白孔加稀释液50μL;
(4)各孔加100μL HRP标记得检测抗体,封孔,37℃恒温60min;
(5)配洗涤液:稀释至1×;
(6)弃去液体后拍干,每孔350μL洗涤液,1min后拍干,重复5次;
(7)每孔加入各50μL底物(A、B),37℃孵孵育15min(避光);
(8)加入终止液50μL每孔;
(9)在450nm波长下测定各孔的OD值;
(10)根据标准品浓度梯度和OD值计算标准曲线计算视网膜中目的蛋白相对含量,结果如图5所示。
由图5的结果可知,阴性AAV注射后,阴性对照组(7.11±0.98μg/ml)与正常对照组(7.13±1.13μg/ml)相比,蛋白的表达无明显差异(P>0.05)。AAV后注射,AAV组(4.02±0.92μg/ml)、FBN2超低浓度组(4.21±0.96μg/ml)、FBN2低浓度组(4.23±0.89μg/ml)、FBN2中浓度组(4.30±0.39μg/ml)、FBN2高浓度组(4.00±0.70μg/ml)与阴性对照组(7.11±0.98μg/ml)比,蛋白表达均明显下降(P<0.05),各组与正常对照组比较趋势同与阴性对照组比较趋势一致。FBN2蛋白注射3次后,FBN2低浓度组(7.51±0.87μg/ml)蛋白表达比AAV组(4.21±0.79μg/ml)明显增加(P<0.05);FBN2超低浓度组(4.58±0.80μg/ml)、FBN2中浓度组(6.12±0.77μg/ml)、FBN2高浓度组(5.01±0.36μg/ml)与AAV组(4.21±0.79μg/ml)比较蛋白表达均无明显变化(P>0.05)。AAV组(4.21±0.79μg/ml)、FBN2超低浓度组(4.58±0.80μg/ml)、FBN2中浓度组(6.12±0.77μg/ml)、FBN2高浓度组(5.01±0.36μg/ml)与阴性对照组(7.70±0.94μg/ml)比较蛋白表达均明显下降(P<0.05);各组与正常对照组比较趋势同 与阴性对照组比较趋势一致。
实施例7
制备蛋白样品,将各组样品与SDS-PAGE蛋白上样缓冲液按体积比5:1充分混匀,每组取3个样品等比混合,随后100℃煮沸6min,待样品放冷至室温后,放于-80℃冰箱中备用。
1、配胶
(1)刷玻璃板:流洁精+水,蒸馏水冲洗,晾干/吹干;
(2)夹板固定,加入无水乙醇4-5ml测漏,5min后不满倒掉;
(3)配分离胶:①丙烯酰胺(30%);②10%APS:现配现用:0.1g过硫酸胺;③Tns-HCI-SDS:用前摇匀,SDS结晶析出;④APS TEMED:冰上操作;⑤配镁丙烯酰胺:APS TEMED放回4℃冰箱;
(4)灌注后,加元水乙醒压平液面,不可用水;
(5)静置45min(可长不可短);
(6)配5%积层胶,倒出乙醇、倾斜,滤纸吸干,加积层胶在玻璃板
顶部,按儒插入蓝梳子;
(7)静置45min,将梳子垂直向上,缓慢拔出,在中间部位依次加入Mark,样品。将两块矮板对内,放入电泳槽中,两板之间加入适量电泳液,置于吸水纸上测,若不漏继续加,接近低矮板时停止。
2、电泳与转膜:
(1)将稀释的电泳液倒入电泳槽(两端对内向上),放纸上测漏;
(2)若不漏水,放入盒加满电泳液,小心拔出梳子,正常组组织注意;
(3)取出样品离心12000r/min,放在冰上,预染分子蛋白等;
(4)上样Marker 5ul,蛋白样品抽取上层,不儒抽到底部沉淀①上样时,加样器斜面抵住后玻璃板,缓慢加样。②换样前,蒸馏水多次冲洗加样器;
(5)电流槽加满,注意可用上样器,以免样品被冲出,槽外加液设过铁丝;
(6)80v 30mh后转为100v 120min;
(7)转膜前将1×转膜液提前20min放入-80℃冰箱预冷;
(8)剪滤纸PVDF膜,将转膜液倒入托盘中泡滤纸,PVD放入甲醇中泡30m,后用三蒸水水冲干净;
(9)电泳结后,取出玻璃板,用切板将上层胶切掉,制下的胶取下放入托
盆中,注意正反与左右,回收电流液;
(10)黑板→单层海棉→三层滤纸→胶-PVDF膜,三层滤低-海绵-透明板一层一层辅;
(11)将板合好扣紧放入槽中,黑时黑,红对红,放入冰盆,加满转膜液;
(12)分清正负极后盖好,100v 120min,4℃冰盖好;200mA/100min;
(13)TBST:10×TBST+900ml水+500mlTween悬液摇2h;
(14)转膜后,左上角剪角后取下,角左右放置在封口袋中,倒入10ml封闭液,摇床1h.封闭完善,TBST清洗3×5min之后抗体卵孵育检测;
(15)转膜液回收,清洗盆子,夹板海绵,海绵,先自来水泡后蒸馏泡,取出后置于托盘晾干3/4TBST稀释倍数→说明书.稀释液:1/4封闭液;
(16)封闭过程中配一抗,做口袋;
(17)口袋标记好,封闭液倒出后剪条带,按名称放入对应口袋,封口,加一抗①条带均作标记,②赶走气泡封口,③口袋作相应抗体;
(18)各条带固定后置于摇床上,0.5h-11(宜长不宜短),放入4℃过夜。
3、配二抗显影
(1)膜放入摇床45min.最长不超过2h;
(2)TBST:4x8ml洗;
(3)做二抗口袋:配二抗稀释液,1/4封闭液.3/4TBST;
(4)条带置于口袋中,加二抗,赶气泡,摇床1h;
(5)配AB液,回收二抗;
(6)TBST洗条带,4×8min;TBS洗2×5min(防止TST干扰显影);
(7)AB液滴在干净的板上,条带正面接触,反应2min,进行曝光。保存条带,将显影后的条带用TBST洗2遍,10min,20min时间非限定,再由TBST洗一遍,然后TBS封存;检测结果如图10所示。
表2
Figure PCTCN2021115997-appb-000005
FBN2蛋白注射组为治疗效果最好的FBN2低浓度组。由图6的结果可知,阴性AAV注射后,阴性对照组与正常对照组相比,视网膜的FBN2基因的蛋白的表达无明显变化(P>0.05);AAV注射后,AAV组和FBN2注射组FBN2基因的蛋白的表达降低(P<0.05)。FBN2蛋白注射3次后,FBN2注射组与正常对照组相比,视网膜的FBN2基因的蛋白表达稍低(P>0.05));与AAV组相比视网膜的FBN2基因的蛋白表达显著升高(P<0.05)。
本说明书中未作详细描述的内容属于本领域专业技术人员公知的现有技术。以上所述仅为本申请的实施例而已,并不用于限制本申请。对于本领域技术人员来说,本申请可以有各种更改和变化。凡在本申请的精神和原理之内所作的任何修改、等同替换、改进等,均应包含在本申请的权利要求范围之内。

Claims (20)

  1. 原纤维蛋白-2重组蛋白在制备预防或治疗原纤维蛋白-2缺陷型眼病的产品中的用途。
  2. 根据权利要求1所述的用途,其特征在于,所述原纤维蛋白-2缺陷型眼病选自遗传性视网膜病变、轴性近视和先天性黄斑变性中的至少一种。
  3. 根据权利要求1或2所述的用途,其特征在于,所述产品为药物、试剂或疫苗。
  4. 原纤维蛋白-2重组蛋白在非治疗目的减少视网膜渗出及眼底沉积物的用途。
  5. 原纤维蛋白-2重组蛋白在非治疗目的调控视网膜电图波形振幅的用途。
  6. 根据权利要求5所述的原纤维蛋白-2重组蛋白在非治疗目的调控视网膜电图波形振幅的用途,其特征在于,所述调控视网膜电图波形振幅为将视网膜电图波形振幅恢复至正常。
  7. 根据权利要求5所述的原纤维蛋白-2重组蛋白在非治疗目的调控视网膜电图波形振幅的用途,其特征在于,所述视网膜电图波形振幅选自暗适应视杆细胞反应波形振幅、暗适应最大混合反应波形振幅、暗适应震荡电位反应波形振幅和明适应视锥细胞反应波形振幅中至少一种。
  8. 原纤维蛋白-2重组蛋白在非治疗目的增加视网膜外核层厚度的用途;
    原纤维蛋白-2重组蛋白在非治疗目的降低眼轴长度的用途。
  9. 原纤维蛋白-2重组蛋白在非治疗目的提高原纤维蛋白-2的表达的用途。
  10. 根据权利要求9所述的原纤维蛋白-2重组蛋白在非治疗目的提高原纤维蛋白-2的表达的用途,其特征在于,所述原纤维蛋白-2的表达选自原纤维蛋白-2基因的表达或原纤维蛋白-2蛋白的表达。
  11. 根据权利要求9所述的原纤维蛋白-2重组蛋白在非治疗目的提高原纤维蛋白-2的表达的用途,其特征在于,所述原纤维蛋白-2基因的表达为原纤维蛋白-2基因mRNA的表达。
  12. 一种预防或治疗原纤维蛋白-2缺陷型眼病的产品,其特征在于,所述产品是以原纤维蛋白-2重组蛋白作为唯一活性成分,或包含原纤维蛋白-2重组蛋白的组合物。
  13. 根据权利要求12所述的产品,其特征在于,所述产品为药物,所述组合物还包括药用辅料。
  14. 根据权利要求13所述的产品,其特征在于,所述药用辅料选自稀释剂、赋形剂、香味剂和甜味剂中的至少一种。
  15. 根据权利要求13所述的产品,其特征在于,所述药物的制剂是混悬剂、乳剂、溶液剂、糖浆剂或注射剂中的至少一种。
  16. 根据权利要求13所述的产品,其特征在于,所述药物的给药方式为滴注或注射。
  17. 根据权利要求16所述的产品,其特征在于,所述药物的给药方式为玻璃体腔注射。
  18. 根据权利要求12-17中任一项所述的产品,其特征在于,所述产品中原纤维蛋白-2重组蛋白的含量为0.01~5.0μg/μL。
  19. 根据权利要求18所述的产品,其特征在于,所述产品中原纤维蛋白-2重组蛋白的含量为0.1~1.0μg/μL。
  20. 根据权利要求18所述的产品,其特征在于,所述产品中原纤维蛋白-2重组蛋白的含量为0.2~0.5μg/μL。
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