WO2017012143A1 - Utilisation de gmfb, agent d'interférence de gmfb et utilisation de l'agent d'interférence de gmfb - Google Patents

Utilisation de gmfb, agent d'interférence de gmfb et utilisation de l'agent d'interférence de gmfb Download PDF

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WO2017012143A1
WO2017012143A1 PCT/CN2015/085945 CN2015085945W WO2017012143A1 WO 2017012143 A1 WO2017012143 A1 WO 2017012143A1 CN 2015085945 W CN2015085945 W CN 2015085945W WO 2017012143 A1 WO2017012143 A1 WO 2017012143A1
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gmfb
diabetic retinopathy
cells
rats
shgmfb
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徐国彤
吕立夏
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同济大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

Definitions

  • the invention relates to the use of the cytokine GMFB, in particular to the application of GMFB as a biomarker for early diagnosis of diabetic retinopathy and as a target for therapeutic intervention, the application of GMFB interfering agents and GMFB interfering agents.
  • DR Diabetic Retinopathy
  • DME diabetic macular edema
  • PDR proliferative diabetic retinopathy
  • DR has become a significant social burden and social problem worldwide.
  • DR was once thought to be a microvascular lesion of the retina, and microcirculatory damage is a classic hallmark of DR, but there is increasing evidence that neurodegeneration is an early event in the pathogenesis of DR and is involved in the development of microvascular abnormalities. Histologically neuronal apoptosis and reactive gliosis are the most important features of DR neurodegeneration.
  • DM donated eyes have not found any microcirculation abnormalities in ophthalmologic examination, but they have the characteristics of major neurodegeneration.
  • Retinal ganglion cells are the first cells to undergo apoptosis in DR; loss of RGC leads to thinning of nerve fiber layer, detected by OCT in DM patients or DM patients with mild DR, without any microangiopathy Patients with DM type I and type II found abnormalities in ERG (electroretinogram). Neuronal apoptosis is accompanied by changes in Muller glial cells. It is unclear which neuronal apoptosis and gliosis are the first events in DR. Studying the mechanisms of DR neurodegeneration and identifying mediators of neurodegeneration are essential for developing new therapeutic strategies. Early identification of neurodegeneration from a clinical perspective is necessary for the application of neuroprotective drugs.
  • GMFB Glial cell maturation factor beta
  • neurodegeneration GMFB is the first 17kd acidic cytoplasmic protein isolated and purified from bovine brain. It is highly conserved in evolution and is mainly produced by astrocytes in the central nervous system. It plays an important role in the growth, differentiation and regeneration of brain tissue, and its expression is up-regulated during development and is significantly reduced in adulthood.
  • the rat retinal GMFB is expressed only in Muller cells and is expressed from embryonic day 14 to adulthood.
  • GMFB knockout mice are resistant to experimental autoimmune encephalitis and the toxicity of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine).
  • the cytokines currently used for DR progression include the following four categories: (1) factors that regulate natural immunity: IL-1b, IL-10; (2) factors that regulate lymphocyte activation and differentiation: IL-6 and IL -12; (3) factors related to activation of macrophages: TNFa and TGFb (4) chemokines, such as MCP-1, SDF-1, etc., there is no report on the role of GMFB in DR.
  • the object of the present invention is to provide an application of GMFB, a GMFB interfering agent and a GMFB interfering agent in order to overcome the drawbacks of the prior art described above.
  • GMFB is expressed in the ganglion cell layer, the inner nuclear layer, and the retinal pigment epithelial cell layer of the rat retina, and the GMFB content can be detected in the vitreous on the first day of STZ-induced diabetes (DM) elevation of blood glucose. High, lasting until the fourth week, then gradually decreasing, can be used as a biomarker to detect the pathogenesis of DR.
  • DM STZ-induced diabetes
  • GMFB treatment of rat Muller cells caused a decrease in glutamine synthetase content, resulting in impaired function of Muller cells; treatment of photoreceptor cell line 661w with GMFB induced autophagy; subretinal injection in normal SD rats 3*10 ⁇ 6gc AAV2/8-GMFB virus, 6 weeks after injection, ERG amplitude decreased, the retinal nucleus thinned, photoreceptor cells were lost, and ganglion cells were apoptotic. This finding confirms for the first time that GMFB is up-regulated early in DR and mediates neuroretinal degeneration.
  • ERG detection can find a decrease in b-wave amplitude, suggesting visual impairment. It may be related to the damage of the neural retina caused by oxidative stress caused by hyperglycemia. After 4 weeks of subretinal injection of 3*10 ⁇ 6gc AAV2/8-shGMFB, the B wave of ERG was increased compared with the empty virus group, suggesting that visual function was protected.
  • GMFB as a biomarker for early diagnosis and progression of diabetic retinopathy
  • the biomarker refers to detecting the content of vitreous GMFB to evaluate the progression of DR.
  • GMFB as a therapeutic target for diabetic retinopathy
  • a possible mechanism of GMFB-mediated retinal degeneration is provided, which can down-regulate the expression of GMFB protein in the retina of DR rats by interfering techniques, thereby protecting visual function .
  • a GMFB interfering agent is provided, wherein the GMFB interfering agent is a substance that interferes with GMFB activity or down-regulates GMFB expression, and the GMFB interfering agent is a chemically synthesized shGMFB, or a vector containing shGMFB, wherein shGMFB is a small hairpin GMFB oligonucleotide (small hairpin GMFB, shGMFB).
  • a GMFB interfering agent for the preparation of a pharmaceutical composition for preventing, ameliorating or treating diabetic retinopathy.
  • the present invention detects the content of vitreous GMFB in STZ-induced DM rats by ELISA, and finds that GMFB maintains a high level in the early stage of experimental DM rats to a high level until 4 weeks after onset, and then gradually decreases. It was then found that in STZ-induced type 1 diabetes (TIDM) rats, down-regulation of GMFB expression by RNA interference can inhibit glialization, down-regulate GFAP expression (glibative markers), and reduce inflammatory factor secretion. Protect visual function. However, AAV2/8-mediated overexpression of GMFB can cause apoptosis of ganglion cells and impair visual function.
  • TIDM STZ-induced type 1 diabetes
  • GMFB can be used as a biomarker for the occurrence and development of DR, and GMFB can also be used as a target for early intervention of DR to protect visual function.
  • the present invention demonstrates for the first time that in STZ-induced type I diabetes mellitus (TIDM) rats, GMFB is significantly elevated in vitreous in the early stage of onset. It was confirmed for the first time that with the progression of DR, the content of GMFB gradually decreased, and DR progression could be detected dynamically. The expression of GMFB in the DR rats was confirmed for the first time, which could protect visual function. The mechanism by which GMFB mediates retinal degeneration is first demonstrated, including a decrease in glutamine synthase in Muller cells, resulting in ganglion cell death and induction of autophagy by photoreceptor cells.
  • TIDM STZ-induced type I diabetes mellitus
  • the present invention has the following advantages:
  • GMFB-mediated neurodegeneration was first confirmed, including the reduction of glutamine synthase in Muller cells, ganglion cell apoptosis and autophagic death of photoreceptor cells;
  • FIG. 1 Expression of GMFB in different nuclear layers of DR
  • Figure 2 Results of changes in GMFB concentration with the course of DR in the vitreous of STZ-induced TIDM rats;
  • FIG. 3a shGMFB subretinal injection of DR rats, GMFB and GFAP changes
  • FIG. 3b shGMFB subretinal injection of DR rats, B-wave results were detected by ERG at the fourth and sixth weeks after injection;
  • FIG. 3c shGMFB subretinal injection of DR rats, the expression of GFAP and GMFB were detected at the mRNA level at the fourth week after injection;
  • Figure 4a Changes in the outer layer of the skin after 6 weeks of AAV2/8-GMFB subretinal injection
  • Figure 4b Apoptosis of ganglion cells 6 weeks after AAV2/8-GMFB subretinal injection
  • FIG. 6 GMFB treatment 661w causes autophagy results.
  • 661w was purchased from ATCC and the medium was low sugar DMEM.
  • the rMC-1 cell line was prepared in a laboratory with high glucose DMEM containing 10% serum and 1% P/S.
  • the culture environment was 37 ° C, 5% CO 2 and 95% air.
  • the overexpression of GMFB was mediated by AAV2/8 as a vector, and the vector AAV2/8-GMFB was recorded.
  • the interference vector mediated by AAV2/8 as a vector was recorded as AAV2/8-shGMFB.
  • AAV2/8-shGMFB and AAV2/8-GMFB are commercially packaged viruses, purchased from Wuhan Vinos Biotechnology Co., Ltd., titer 10 ⁇ 9gc/ml, LC3 virus purchased from Hanheng Bio, rat GMFB ELISA The kit was purchased from elabscience.
  • Preparation of diabetic rats Male SD rats, 160-180 g, were used to starve the rats for 24 hours before the experiment. STZ (60mg/kg body weight) was injected intraperitoneally to induce DM. The normal control group was intraperitoneally injected with an equal volume of citric acid solution. After 24 hours, blood was taken from the tail and the blood glucose was lower than 250mg/dL. STZ. Blood glucose was measured for 3 consecutive days. Rats whose blood glucose exceeded 250 mg/dL for 3 consecutive days were identified as DM rats (rats with blood glucose below 250 mg/dL will be excluded).
  • Tissue obtained by laser cutting was collected in 0.5 ml Trizol lysate.
  • RNA reverse transcription the first strand of cDNA was obtained by Promega's M-MLV reverse transcriptase. The main steps are as follows:
  • RNA was mixed with 2 ⁇ L of Takara's reverse transcription reagent supermix, and then reverse transcription was performed at 37 degrees for 15 minutes and at 85 degrees for 5 seconds.
  • Reverse transcription procedure 15 minutes at 37 ° C, 5 seconds at 85 ° C, and then stored in a refrigerator at -20 ° C for later use.
  • the first strand of cDNA obtained by reverse transcription of RNA was used as a template, and primers were designed as shown in Table 1.
  • the SYBR Green real-time PCR assay kit from Tiangen was used to detect the expression of the target gene.
  • the PCR amplification conditions were as follows: denaturation at 94 ° C for 10 minutes, entering a cycle (95 ° C for 5 sec, 60 ° C for 60 sec) for a total of 40 cycles, and collecting the dissolution profile.
  • GMFB GMFB was down-regulated in the ganglion cell layer and RPE layer, and the expression in the inner and outer nuclear layers was gradually up-regulated, which was higher than that of DM.
  • the expression of GMFB in the kernel layer was higher than that of DM.
  • the type I diabetes model was prepared by STZ. The method was as follows. The vitreous of diabetic rats was collected at different time points. 6-8 samples were collected at each time point and collected separately. 1000g was centrifuged at 4 degrees for 20 minutes and then transferred to supernatant. Standby, perform ELISA test, the results are shown in Figure 2, Figure 2, Cont is normal control, PI After injection for postinjection, DM is diabetes. As can be seen from Figure 2, on the first day of blood glucose elevation, GMFB was found to increase significantly, then decreased slightly, and remained at a high level, gradually decreasing from the fourth week, in DM. In the eighth week of onset, it fell to a level close to the normal camera. The elisa kit for rat GMFB was purchased from Elabscience (catalogue number E-EL-Ro419C) according to the kit instructions. The main steps include:
  • Sample collection The rat vitreous was collected and centrifuged at 1000 ⁇ g for 20 minutes, and the supernatant was taken for detection.
  • Standard preparation Centrifuge at 10000 ⁇ g for 1 minute, add 1.0 mL of standard & sample dilution to the lyophilized standard, tighten the tube cover, let stand for 10 minutes, and invert it upside down several times until it is fully dissolved. After that, gently mix it with a pipette (concentration: 1000 ng/mL). Then dilute the dilution as needed (Note: Do not directly dilute in the well). It is recommended to prepare the following concentrations: 1000, 500, 250, 125, 62.5, 31.25, 15.625, 0 ng/mL, and the standard & sample dilutions are directly used as blank wells of 0 ng/mL. For example, prepare 500ng/mL standard product: Take 0.5mL 1000ng/mL of the above standard product into EP tube containing 0.5mL standard & sample dilution solution, mix well, and the other concentrations are similar.
  • Biotinylated antibody working solution Calculate the amount required for the next experiment (in 100 ⁇ L/well) before the experiment, and prepare 100-200 ⁇ L in the actual preparation. Concentrated biotinylated antibody (1:100) was diluted to a working concentration with biotinylated antibody dilution 15 minutes prior to use. Used on the same day.
  • Enzyme conjugate working solution Calculate the amount required for the next experiment (in 100 ⁇ L/well) before the experiment, and prepare 100-200 ⁇ L in the actual preparation.
  • the concentrated HRP enzyme conjugate (1:100) was diluted to the working concentration with the enzyme conjugate dilution 15 minutes before use. Used on the same day.
  • each reagent should be equilibrated to room temperature; when preparing the reagent or sample, mix thoroughly and avoid foaming as much as possible.
  • step 5 Discard the liquid in the well, dry it, and wash the plate 5 times.
  • the method is the same as step 3.
  • TMB substrate solution
  • a rat model of type I diabetes was prepared in the same manner as in Example 1.
  • GFAP glial fibrillary acidic protein
  • 4',6-diamidino-2-phenylindole is a fat-soluble fluorescent dye that stains the nucleus; Zsgreen is a green label, RPE is a retinal pigment epithelial cell, and ONL is the outer nuclear layer. INL is the inner nuclear layer, GCL is the ganglion cell layer, and merge is overlapping, overlapping green fluorescence, red fluorescence and blue fluorescence.
  • RNA is extracted by the method of Trizol cleavage.
  • the main steps are as follows:
  • RNA reverse transcription the first strand of cDNA was obtained by Promega's M-MLV reverse transcriptase. The main steps are as follows:
  • RNA was mixed with 2 ⁇ L of oligo d (T), placed in a 72 ° C water bath for 5 minutes, and immediately ice bathed for 2 minutes, then oligo d (T) was combined with the poly-A tail of RNA, slightly centrifuged. .
  • the reverse transcriptase was inactivated by placing at 70 ° C for 10 minutes.
  • the obtained cDNA was single-stranded in a refrigerator at -20 °C.
  • Reverse transcription procedure 30 minutes at 16 ° C, 30 minutes at 42 ° C, and immediately after 5 minutes at 85 ° C for 5 minutes on ice. It can then be stored in a refrigerator at -20 ° C for use.
  • the first strand of cDNA obtained by reverse transcription of RNA was used as a template, and primers were designed as in Table 1 of Example 1.
  • primers were designed as in Table 1 of Example 1.
  • the PCR amplification conditions were as follows: denaturation at 94 ° C for 10 minutes, entering a cycle (95 ° C for 5 sec, 60 ° C for 60 sec) for a total of 40 cycles, and collecting the dissolution profile.
  • ERG method APS automatic visual electrophysiological tester (APS-2000) was purchased from Chongqing Kanghua Technology Co., Ltd. One day before the visual electrophysiological function test, the DM rats were transferred to a dark room for dark adaptation. I started doing it the next day.
  • Rat preparation Rats were intraperitoneally injected with 2% sodium pentobarbital (1 mL/500 g body weight) for anesthesia, 1 ⁇ Shen Mianxin (0.1 ml/200 g) for eyeballs, and then a drop of 0.5% tropicamide Wuxi Shanhe Group (Jiangsu, China), a drop of 0.4% oxybuprocaine hydrochloride surface anesthesia (Eisai Co Ltd, Tokyo, Japan), each eye with a little conductive paste. Insert the electrode: the ground wire is connected to the tail of the rat, the negative electrode is connected between the two ears of the rat, and the positive electrode is connected to the cornea of both eyes. Be careful not to touch the eyelids and the sclera.
  • Rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (1 mL/400 g body weight) before injection, followed by a drop of 0.5% tropicamide (Wuxi Shanhe Group, Jiangsu, China), a drop of 0.4% opiate hydrochloride Due to topical anesthesia (Eisai Co Ltd, Tokyo, Japan).
  • a 30-gauge needle was used to make a channel 2 mm behind the temporal sclera, then a 33-gauge needle (Lot #440602, Hamilton, Reno, NV) and a 5 ⁇ L microsyringe (P/ N:7634-01/00, Hamilton, Reno, NV) AAV2-CMV-hEPO was injected. If the injection is successful, a small vesicular structure can be seen on the fundus. After a few minutes, the retina becomes flat and the vesicles disappear. If the vitreous hemorrhage or crystal damage is excluded, the experiment is excluded.
  • 3*10 ⁇ 6gc AAV2/8-GMFB was injected into the subretinal space of normal rats in the same manner as in Example 3.
  • the retina was separated 6 h after injection, frozen sections were taken, and DAPI staining was performed, and the thickness of the nuclear layer was measured by Photoshop.
  • the thickness of the nuclear layer was significantly thinner outside the virus injection zone, and the thickness of the nuclear layer was zero, which was the optic nerve head.
  • Frozen sections were subjected to DAPI staining for retinal thickness measurement and cell count analysis.
  • Retinal thickness measurements were performed under a microscope at 400 magnification. The measurement was performed at a distance of 1 mm from the optic nerve head and on both sides of the optic papilla, including: (1) outer membrane-internal membrane (OLM-ILM); (2) outer membrane-arc cell layer (OLM-GCL); (3) outer nucleus Layer-outer mesh layer (ONL-OPL); (4) kernel layer (INL); (5) outer core layer (ONL).
  • OLM-ILM outer membrane-internal membrane
  • OLM-GCL outer membrane-arc cell layer
  • ONL-OPL outer nucleus Layer-outer mesh layer
  • INL kernel layer
  • ONL outer core layer
  • Five retinal sections were taken from each eyeball, with 4 rats in each group.
  • Apoptosis was detected using the In Situ Cell Death Detection Kit and the assay was performed according to the protocol provided in the kit.
  • the positive control was performed by incubating the retina with Grade I DNase-I for 10 minutes at room temperature, and the negative control was only labeled with no enzyme solution.
  • the sample was incubated at 37 ° C for 1 hour, washed 3 times with PBS, and directly observed under a fluorescence microscope (Nikon, Yokohama, Japan) with an excitation wavelength of 450-490 nm.
  • the rat Muller cell line was cultured in DMEM-high glucose medium containing 10% serum and double antibody.
  • the recombinant GMFB dry powder was dissolved in sterile PBS solution to a final concentration of 500 ug/ml.
  • GMFB was added to serum-free DMEM-high glucose medium, cells were collected at different time points, total protein was extracted, and glutamine was extracted by ELISA. Detection of synthase (GS).
  • the mouse photoreceptor cell line was cultured in DMEM-low sugar medium containing 10% fetal bovine serum and double antibody, and RFP-GFP-LC3 adenovirus was purchased from Hanheng organism. 661w first infected with 661w with RFP-GFP-LC3 adenovirus (titer 10 ⁇ 10 pfu). One day after infection, 661 w was treated with serum-free medium containing GMFB.
  • GMFB treatment for 661w4 hours the cells began to appear red and green fluorescence, spotted, suggesting that LC3 translocated to the lysosomal membrane, suggesting autophagy, treatment for 8 hours, punctate fluorescence, autophagy Red-green fluorescence needs to be observed under a confocal microscope.

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Abstract

La présente invention concerne l'utilisation de GMFB en tant que biomarqueur pour le diagnostic précoce et la progression de la rétinopathie diabétique (DR), l'utilisation de GMFB en tant que cible thérapeutique pour la rétinopathie diabétique, un agent d'interférence de GMFB et l'utilisation de l'agent d'interférence de GMFB. Par comparaison à l'état de la technique, la présente invention confirme que chez des rats présentant un diabète de type I induit par la STZ (TIDM), la teneur en GMFB dans le corps vitré est considérablement augmentée à un stade précoce de la maladie. Une première confirmation que la teneur en GMFB est progressivement diminuée, avec la progression de l'état de la DR qui peut être détectée de manière dynamique; et une première confirmation que l'interférence de l'expression de GMFB chez un rat présentant une DR permet de protéger la fonction visuelle. Une première confirmation que le mécanisme de GMFB sert d'intermédiaire à la dégénérescence rétinienne, consistant à provoquer une diminution de la glutamine synthétase dans les cellules de Muller, entraînant la mort de cellules ganglionnaires, et induire l'autophagie des cellules photoréceptrices de l'œil.
PCT/CN2015/085945 2015-07-21 2015-08-03 Utilisation de gmfb, agent d'interférence de gmfb et utilisation de l'agent d'interférence de gmfb WO2017012143A1 (fr)

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CN110542758B (zh) * 2019-03-26 2021-06-04 同济大学 Gmfb作为糖尿病骨质疏松症的生物标记物的应用
CN110542759B (zh) * 2019-04-04 2021-06-29 同济大学 Gmfb作为糖尿病肾病的生物标记物的应用
CN112043833A (zh) * 2020-08-31 2020-12-08 同济大学 一种视网膜色素细胞rpe的自噬和凋亡的抑制剂及其应用
CN112195244B (zh) * 2020-09-16 2022-04-19 同济大学 Gmfb作为肝细胞肝癌生物标志物的应用
CN113577068A (zh) * 2021-07-30 2021-11-02 同济大学 小分子化合物在制备治疗gmfb介导疾病的药物中的应用

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* Cited by examiner, † Cited by third party
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CN113759127A (zh) * 2021-08-18 2021-12-07 同济大学 Gmfb作为胰岛素抵抗的生物标记物的应用
CN113759127B (zh) * 2021-08-18 2024-03-26 同济大学 Gmfb作为胰岛素抵抗的生物标记物的应用

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