WO2022041645A1 - 一种视网膜色素细胞rpe的自噬和凋亡的抑制剂及其应用 - Google Patents

一种视网膜色素细胞rpe的自噬和凋亡的抑制剂及其应用 Download PDF

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WO2022041645A1
WO2022041645A1 PCT/CN2021/073278 CN2021073278W WO2022041645A1 WO 2022041645 A1 WO2022041645 A1 WO 2022041645A1 CN 2021073278 W CN2021073278 W CN 2021073278W WO 2022041645 A1 WO2022041645 A1 WO 2022041645A1
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autophagy
inhibitor
apoptosis
gmfb
fas
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PCT/CN2021/073278
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French (fr)
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吕立夏
徐国彤
刘彩莹
王娟
张介平
高芙蓉
田海滨
金彩霞
徐晶莹
欧庆健
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同济大学
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    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

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  • the invention belongs to the technical field of biomedicine, in particular to an inhibitor of autophagy and apoptosis of retinal pigment cell RPE and its application.
  • Diabetic retinopathy is a specific microvascular complication of diabetes, which is chronic, progressive, and potentially harmful to vision. It is the leading cause of blindness in the working population and the elderly.
  • DR Diabetic retinopathy
  • PDR proliferative DR
  • VTDR vision threatening DR
  • Epidemiological surveys in China show that the incidence rates of DR and PDR in the Chinese diabetic population are 23% and 2.83%, respectively, and almost half of the patients with diabetes for more than 20 years will suffer from DR.
  • DR can be divided into NPDR and PDR according to the severity of lesions and the formation of new blood vessels.
  • NPDR neurodegenerative disease glaucoma
  • macular edema can cause vision loss.
  • symptoms such as retinal neovascularization, vitreous hemorrhage, and traction retinal detachment appear on the basis of non-proliferative lesions.
  • severe ischemia neovascularization of the iris and angle of the chamber may occur, and eventually it evolves into neovascular glaucoma.
  • the current main treatment methods include laser photocoagulation, vitrectomy, etc.
  • Anti-VEGF, anti-inflammatory drugs and PKC inhibitors also have certain therapeutic effects.
  • the current treatment methods are mostly aimed at the late stage of the disease, and all have certain side effects and prognostic risks, the research on the molecular mechanism and treatment methods in the early stage of the disease is imminent.
  • the purpose of the present invention is to provide an inhibitor of autophagy and apoptosis of RPE of retinal pigment epithelial cells and its application.
  • Antagonists of tumor necrosis factor superfamily receptor FAS can inhibit its activation as a receptor of GMFB. Downstream autophagy and apoptosis pathways, thereby reducing apoptosis and autophagy responses, and delaying the onset of diabetic retinopathy in the early stage.
  • the present invention provides an inhibitor of autophagy and apoptosis of retinal pigment epithelial cell RPE, and the inhibitor includes an antagonist of tumor necrosis factor superfamily receptor FAS.
  • the antagonist comprises Met.
  • the antagonist inhibits tumor necrosis factor superfamily receptor FAS as a receptor for glial cell maturation factor ⁇ to activate downstream autophagy and apoptosis pathways.
  • the present invention also provides the application of the above inhibitor in the preparation of a medicament for delaying and/or treating diabetic retinopathy in the early stage.
  • the present invention also provides a medicine for delaying and/or treating diabetic retinopathy, which comprises the above-mentioned inhibitor.
  • the present invention has the following beneficial effects: the present invention proves for the first time that in the early vitreous of DR, the glial cell maturation factor GMFB acts as a cytokine to affect the autophagy and apoptosis pathways of RPE cells, thereby accelerating DR disease process.
  • tumor necrosis factor superfamily receptor FAS can act as a receptor of GMFB to activate downstream autophagy and apoptosis pathways, while its antagonist Met can inhibit FAS and its downstream related Activation of the pathway reduces apoptosis and autophagy, thereby delaying the pathogenesis of diabetic retinopathy.
  • Figure 1 shows the effect of GMFB in the vitreous cavity on the retina of rats, in which A indicates that the b-wave amplitude of the electroretinogram of SD rats caused by intravitreal injection of GMFB decreased, and the retinal function was impaired; B and C indicate that the intravitreal injection of GMFB caused the retina of rats The increase of apoptosis level, the abnormal activation of autophagy in the early stage and the abnormal process of autophagy degradation, affect the normal process of autophagy flow;
  • Figure 2 shows that the extracellular treatment of GMFB in the cell model leads to an increase in the apoptosis level of ARPE19, the activation of the early stage of autophagy and the abnormality of the autophagic degradation process, which affect the normal process of autophagy flow;
  • Figure 3 shows the screening process of GMFB receptors, in which A and B indicate that GMFB receptors were screened in ARPE19 cell model using siRNA library, and it was found that siRNA of eight receptors such as FAS and TGFBR1 can reduce autophagosomes and autophagolysosomes It has the effect of inhibiting the abnormal autophagy caused by GMFB, in which the expression of FAS is high, and its transcription level increases with GMFB treatment; C indicates that the use of the FAS-specific antagonist Met can block the cell surface receptor FAS , compared with the control group Mutant, it can hinder the abnormal autophagy flow caused by GMFB, which proves that FAS is one of the cell membrane receptors of GMFB;
  • Figure 4 is a graph showing the relationship between the FAS receptor antagonist Met and GMFB, in which A indicates that in the cell model, the FAS receptor antagonist Met can inhibit the apoptosis and abnormal autophagy flow caused by GMFB; BD indicates that in the animal model, simultaneous injection Met, a FAS receptor antagonist, can inhibit retinal apoptosis and abnormal autophagic flow induced by GMFB injection into the vitreous cavity, and at the same time reduce the damage of GMFB to retinal function.
  • the present invention provides an inhibitor of autophagy and apoptosis of retinal pigment epithelial cell RPE, and the inhibitor includes an antagonist of tumor necrosis factor superfamily receptor FAS.
  • the tumor necrosis factor superfamily receptor FAS acts as the receptor of extracellular glial maturation factor beta (GMFB) to activate downstream autophagy and apoptosis pathways, and its antagonist will inhibit FAS and its downstream Activation of related pathways reduces apoptosis and autophagy, thereby delaying the pathogenesis of diabetic retinopathy.
  • the antagonists of the present invention preferably include Met.
  • the ARPE19 cell line treated with 0.025 ⁇ g/ ⁇ l GMFB can increase the expression of apoptosis-related protein cleaved-caspase3 and autophagy-related proteins LC3, ATG5, p62 in cells , showed abnormal autophagic flow, and the addition of the FAS receptor antagonist Met (10 ⁇ g/ ⁇ l) would inhibit its changes; in the in vivo model, injection of 0.2 ⁇ g GMFB into the vitreous cavity of SD rats can damage the normal physiology of the retina In contrast, the Met group showed significantly improved retinal physiology compared with the co-injected Mutant group, and showed attenuated levels of apoptosis and normal autophagic flux.
  • the present invention also provides the application of the above inhibitor in the preparation of a medicament for delaying and/or treating diabetic retinopathy at an early stage.
  • the present invention does not specifically limit the dosage form of the drug.
  • the medicament of the present invention preferably further includes pharmaceutically acceptable excipients.
  • the present invention also provides a medicine for delaying and/or treating diabetic retinopathy, which comprises the above-mentioned inhibitor.
  • the present invention also provides a method for delaying and/or treating diabetic retinopathy, which can be achieved by intravitreal injection of Met (5 mg/0.1 mL).
  • Met 5 mg/0.1 mL.
  • the specific dosage and frequency are subject to the clinical test results.
  • ARPE19 cells were purchased from ATCC in DMEM-F12 medium containing 10% serum and 1% P/S. The culture environment was 37°C, 5% CO 2 and 95% air.
  • mRFP-GFP-LC3 lentivirus (HB-AP210-0001) was purchased from Hanheng Company.
  • Synthetic GMFB protein (Cat: 13244-HNAE) was purchased from Sino Biological. Penicillin/Streptomycin (15140155) was purchased from Invitrogen.
  • the reverse transcription kit PrimeScript TM RT Master Mix was purchased from Takara Company, and the PCR kit SuperReal PreMix Plus (SYBR Green) was purchased from Tiangen Company.
  • the primers used were synthesized by Suzhou Jinweizhi Company.
  • Antibodies used were anti-Phospho-Bcl-2 (Ser70) (#2827), Cleaved Caspase-3 (Asp175) (#9664), LC3A/B (D3U4C) (#12741), Phospho-NF- ⁇ B p65 from CST Company (Ser536) (#3033) antibody, and ATG5 Antibody (10181-2-AP) from Proteintech, P62/SQSTM1 Antibody (18420-1-AP), FAS/CD95 Antibody (13098-1-AP), HRP- Conjugated beta Actin Antibody(HRP-60008), Cy3–conjugated Affinipure Goat Anti-Rabbit IgG(H+L)(SA00009-2).
  • Rat preparation 4W SD rats with a weight of about 140g were purchased from Slack Company and kept in the SPF room of Tongji University Animal Center. Rats were randomly divided into four groups, PBS control group, GMFB treatment group, GMFB-Met treatment group and GMFB-Mutant treatment group.
  • APS automatic visual electrophysiological examination instrument (APS-2000) was purchased from Chongqing Kanghua Technology Co., Ltd.
  • the ground wire is connected to the rat's tail
  • the negative electrode is connected between the ears of the rat
  • the positive electrode is connected to the corneas of the two eyes. Be careful not to touch the eyelids and sclera.
  • the b-wave amplitude of the GMFB treatment group was significantly lower than that of the PBS group, indicating that the rise of GMFB in the vitreous cavity would damage retinal function; while the GMFB-Met treatment group Compared with the GMFB-Mutant treatment group, the b-wave amplitude was larger, indicating that the FAS receptor antagonist Met can protect the retinal function damage caused by GMFB.
  • Detection of apoptosis or autophagy-related protein changes by Western-blot after drug treatment or injection The lysate was collected by scraping the cells into a new EP tube, incubated on ice for 30 min, centrifuged at 10,000 rpm for 15 min at 4°C, gently aspirated the supernatant into a clean centrifuge tube, and stored at -80°C for later use.
  • Absolute ethanol promotes gelation, so that the surface of the gel is in a straight line; leave it for about 1 hour until the gel is completely solidified. When there is a broken line between the absolute ethanol and the gel, the gel has solidified. Pour off the anhydrous on the gel. ethanol, blot dry with filter paper.
  • a 5% stacking gel (4ml) was prepared as follows: 30% polyacrylamide 0.67ml, ddH2O 2.7ml , 1M Tris (pH 6.8) 0.5ml, 10% SDS 0.04ml, 10% ammonium persulfate 0.04ml, TEMED0 .004ml; add APS and TEMED before pouring glue, mix well after adding, and pour glue quickly.
  • Protein Marker After the membrane transfer, you can refer to Protein Marker and transfer it to PVDF membrane as a sign of successful membrane transfer. You can also stain the membrane with 1 ⁇ Ponceau red dye solution, and then rinse off the unstained dye solution with water to see the stains on the membrane. protein.
  • Eyeball preparation Eyeball samples were collected from SD rats after drug treatment at 2w and 4w respectively. After SD rats were killed by dislocation, the eyeballs were carefully removed, and the optic nerve was preserved;
  • Dissection Dissect the eyeball along the upper edge of the corneal and scleral limbus under a dissecting microscope, carefully remove the cornea, iris and lens to form a complete optic cup, and pay attention to gentle operation to prevent retinal detachment;
  • Section preparation Take the frozen section sample of the eyeball obtained in Example 4 for immunofluorescence staining detection.
  • Fixation fix with 4% PFA for 10 minutes, and then wash with PBS for three times, 5 minutes each time.
  • Permeate the membrane permeate the membrane with 0.25% Triton-X100 for 15 minutes, and wash with PBS for 3 times, 5 minutes each time.
  • Blocking Block with 5% fetal bovine serum at room temperature for 1 h;
  • Secondary antibody wash 3 times with PBS, 5 min each time; add fluorescently labeled secondary antibody from the same species as the primary antibody diluted with 5% fetal bovine serum, or the reaction solution described in the TUNEL kit, put it in a wet box, Incubate at 37°C for 1 h in the dark.
  • Nuclei staining with DAPI wash 3 times with PBS, 5 min each time; stain the nucleus with 0.5 ⁇ g/ml DAPI for 1 min, and wash 3 times with PBS, 5 min each time.
  • B in Figure 1 indicates that red fluorescence, a marker of apoptotic cells, increased in the RPE layer after GMFB was injected into the vitreous body, that is, the apoptosis level of retinal pigment epithelial cells was increased;
  • D in 4 concurrent injection of the FAS receptor antagonist Met reduced the increase in GMFB-induced apoptosis levels.
  • mRFP-GFP-LC3 cell line was carried out in accordance with the instructions for use of Hanheng biological product HB-AP210-0001.
  • ARPE19 cells were infected with autophagy double-labeled adenovirus when the confluence rate was between 50 and 70%, and the medium was changed after 2 hours.
  • the expression of GFP and RFP was observed after 24 hours of infection.
  • the positive cells were screened and expanded with Puromycin to form stable expression. cell line.
  • mRFP is used to label and track LC3, and the weakening of GFP can indicate the fusion of lysosomes and autophagosomes to form autolysosomes.
  • the fluorescence is quenched and only red fluorescence is detected. After the microscope imaging, the yellow spots that appear after the red-green fluorescence passes through the merge indicate autophagosomes, and the red spots indicate autophagolysosomes.
  • siRNA transfection group 5 ⁇ l Lipo2000 was used in each well to transfect siRNA of different receptors, and GMFB treatment was performed after 24-48 h; for the FAS antibody treatment group, each well was blocked with 5 ⁇ l of antibody at 37°C for 2 h.
  • 7.1 ARPE19 cells were evenly plated in six-well plates and cultured to 70-80%, then treated with 0.01 ⁇ g/ ⁇ l GMFB.
  • RNA reverse transcription 20 ⁇ l system, 1000ng RNA, take 4 ⁇ l of Takara's reverse transcription reagent supermix, add ddH 2 O to make up to 20 ⁇ l. Place in a PCR machine at 37°C for 15 min, 85°C for 5s, and store at -20°C for later use.
  • the qPCR system is 20 ⁇ l: 2 ⁇ l of cDNA template, 1 ⁇ l of primers, 10 ⁇ l of 2 ⁇ PCR Mix, and 7 ⁇ l of ddH 2 O to detect the expression level of the target gene.
  • PCR amplification conditions denaturation at 94°C for 10min, enter the cycle (95°C for 5sec, 60°C for 60sec), a total of 40 cycles, and collect the melting curve.

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Abstract

一种视网膜色素上皮细胞RPE的自噬和凋亡的抑制剂及其应用，涉及生物医药技术领域。一种视网膜色素上皮细胞RPE的自噬和凋亡的抑制剂，所述抑制剂包括肿瘤坏死因子超家族受体FAS的拮抗剂Met。经验证，肿瘤坏死因子超家族受体FAS可以作为GMFB的受体激活下游自噬和凋亡通路，而其拮抗剂Met则将抑制FAS及其下游相关通路的激活，减少细胞凋亡及自噬反应，从而延缓糖尿病视网膜病变的发病过程，可用于制备发病早期延缓和/或治疗糖尿病视网膜病变的药物。

Description

一种视网膜色素细胞RPE的自噬和凋亡的抑制剂及其应用
本申请要求于2020年08月31日提交中国专利局、申请号为2020108928362、发明名称为“一种视网膜色素细胞RPE的自噬和凋亡的抑制剂及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物医药技术领域，具体涉及一种视网膜色素细胞RPE的自噬和凋亡的抑制剂及其应用。
背景技术
糖尿病性视网膜病变(DR)是糖尿病的一种特异性微血管并发症，具有慢性、进行性、潜在危害视力等特征，是工作人群及老年人中首要的致盲原因。全球有超过9300万例DR患者，其中1700万例患有增殖性DR(proliferative DR，PDR)，2800万患有威胁视力的DR(vision threatening DR，VTDR)。我国的流行病调查显示，DR与PDR在中国糖尿病罹患人群中的发病率分别为23％和2.83％，有20年以上糖尿病病程的患者几乎有一半会罹患DR。根据病变严重程度以及新生血管的形成状况，可将DR分为NPDR和PDR。NPDR的主要表现为眼底视网膜微血管瘤、点状和斑状视网膜出血、毛细血管闭塞、视网膜水肿等，其中黄斑区水肿可引起视力下降。而PDR则在非增生性病变的基础上，出现视网膜新生血管、玻璃体积血、牵拉性视网膜脱离等症状。缺血严重的病例可发生虹膜、房角新生血管形成，最终演变为新生血管性青光眼。
由于DR晚期严重损害视力以致不可恢复，所以及时防治十分重要。目前的主要治疗方式有激光光凝术、玻璃体切割术等，抗VEGF、抗炎症药物及PKC抑制剂等也有一定的治疗作用。但由于现行的治疗手段多针对发病晚期，且均有一定的副作用及预后风险，针对发病前期分子机制和治疗方法的研究迫在眉睫。
发明内容
有鉴于此，本发明的目的在于提供一种视网膜色素上皮细胞RPE的自噬和凋亡的抑制剂及其应用，肿瘤坏死因子超家族受体FAS的拮抗体可以抑制其作为GMFB的受体激活下游自噬和凋亡通路，从而减少细胞凋亡及自噬反应，在发病早期延缓糖尿病视网膜病变的发病过程。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种视网膜色素上皮细胞RPE的自噬和凋亡的抑制剂，所述抑制剂包括肿瘤坏死因子超家族受体FAS的拮抗剂。
优选的，所述拮抗剂包括Met。
优选的，所述拮抗剂抑制肿瘤坏死因子超家族受体FAS作为胶质细胞成熟因子β的受体激活下游自噬和凋亡通路。
本发明还提供了上述抑制剂在制备发病早期延缓和/或治疗糖尿病视网膜病变的药物中的应用。
本发明还提供了一种延缓和/或治疗糖尿病视网膜病变的药物，所述药物包括上述的抑制剂。
本发明与现有技术相比具有的有益效果是：本发明首次证实了在DR早期玻璃体中胶质细胞成熟因子GMFB作为一种细胞因子影响到RPE细胞的自噬和凋亡途径，进而加速DR的病变过程。本发明实施例在细胞模型和SD大鼠模型中，肿瘤坏死因子超家族受体FAS可以作为GMFB的受体激活下游自噬和凋亡通路，而其拮抗剂Met则将抑制FAS及其下游相关通路的激活，减少细胞凋亡及自噬反应，从而延缓糖尿病视网膜病变的发病过程。在体外模型中，使用0.025μg/μl的GMFB对ARPE19细胞系进行处理，可以升高细胞中凋亡相关蛋白cleaved-caspase3及自噬相关蛋白LC3、p62的表达，并显示出自噬流的异常，而加入FAS受体的拮抗剂Met(10μg/μl)则将抑制其变化；在体内模型中，向SD大鼠玻璃体腔注射0.2μg GMFB，可以损伤视网膜的正常生理功能，同时检测到与细胞实验同步的分子变化，而和同时注射Mutant组相比，Met组视网膜生理功能显著提高，并显示出凋亡水平的减弱和自噬流的正常进行。
附图说明
图1为玻璃体腔GMFB对大鼠视网膜的影响图，其中A表示玻璃体腔注射GMFB导致SD大鼠视网膜电图b波波幅下降，视网膜功能受损；B和C表示玻璃体腔注射GMFB导致大鼠视网膜凋亡水平升高，自噬前期的激活和自噬降解过程的异常，影响自噬流正常进行；
图2为细胞模型中GMFB胞外处理导致ARPE19凋亡水平升高，自噬前期的激活和自噬降解过程的异常，影响自噬流正常进行；
图3为GMFB受体的筛选过程，其中A和B表示使用siRNA文库在ARPE19细胞模型中筛选GMFB受体，发现FAS、TGFBR1等八种受体的siRNA可以减少自噬体和自噬溶酶体的数量，有抑制GMFB引起的自噬异常的作用，其中FAS的表达量较高，且随着GMFB处理其转录水平增加；C表示使用FAS特异性拮抗剂Met可对细胞表面受体FAS进行封闭，与对照组Mutant相比，可阻碍GMFB引起的自噬流异常，证明FAS为GMFB的细胞膜受体之一；
图4为FAS受体拮抗剂Met与GMFB的作用关系图，其中A表示细胞模型中，FAS受体拮抗剂Met可以抑制GMFB引起的凋亡和自噬流异常；B-D表示动物模型中，同时注射FAS受体拮抗剂Met，可以抑制玻璃体腔GMFB注射引起的视网膜凋亡和自噬流异常，同时减轻GMFB对视网膜功能的损伤。
具体实施方式
下面结合实施例和附图对本发明进一步说明。
本发明提供了一种视网膜色素上皮细胞RPE的自噬和凋亡的抑制剂，所述抑制剂包括肿瘤坏死因子超家族受体FAS的拮抗剂。
在本发明中，所述肿瘤坏死因子超家族受体FAS作为胞外胶质细胞成熟因子β(GMFB)的受体激活下游自噬和凋亡通路，而其拮抗剂则将抑制FAS及其下游相关通路的激活，减少细胞凋亡及自噬反应，从而延缓糖尿病视网膜病变的发病过程。本发明所述拮抗剂优选包括Met。在本发明实施例中，通过体外模型证实，使用0.025μg/μl的GMFB对ARPE19细胞系进行处理，可以升高细胞中凋亡相关蛋白cleaved-caspase3及自噬 相关蛋白LC3、ATG5、p62的表达，显示出自噬流的异常，而加入FAS受体的拮抗剂Met(10μg/μl)则将抑制其变化；在体内模型中，在SD大鼠玻璃体腔注射0.2μg GMFB，可以损伤视网膜的正常生理功能，同时检测到与细胞实验同步的分子变化，而和同时注射Mutant组相比，Met组视网膜生理功能显著提高，并显示出凋亡水平的减弱和自噬流的正常进行。
本发明还提供了上述抑制剂在制备发病早期延缓和/或治疗糖尿病视网膜病变的药物中的应用。本发明对所述药物的剂型并没有特殊限定。本发明所述药物中优选还包括药学上可接受的辅料。
本发明还提供了一种延缓和/或治疗糖尿病视网膜病变的药物，所述药物包括上述的抑制剂。
本发明还提供了一种延缓和/或治疗糖尿病视网膜病变的方法，可通过玻璃体腔注射Met(5mg/0.1mL)实现，具体剂量及次数以临床试验结果为准。
下面结合实施例对本发明提供的视网膜色素细胞RPE的自噬和凋亡的抑制剂及其应用进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实验试剂和耗材来源：
ARPE19细胞购自ATCC，培养基为DMEM-F12培养基含10％血清和1％P/S。培养环境是37℃、5％CO 2和95％空气。
mRFP-GFP-LC3慢病毒(HB-AP210-0001)购自汉恒公司。
人类细胞因子受体siRNA文库(ON-
Figure PCTCN2021073278-appb-000001
siRNA Library-Human Cytokine Receptors)购自GE Dharmacon。
人工合成GMFB蛋白(Cat:13244-HNAE)购自Sino Biological。Penicillin/Streptomycin(15140155)购自Invitrogen。
反转录试剂盒PrimeScript TMRT Master Mix购自Takara公司，PCR试剂盒SuperReal PreMix Plus(SYBR Green)购自天根公司。所用引物由苏州金维智公司合成。
所用抗体有来自CST公司的抗Phospho-Bcl-2(Ser70)(#2827)，Cleaved Caspase-3(Asp175)(#9664)，LC3A/B(D3U4C)(#12741)， Phospho-NF-κB p65(Ser536)(#3033)抗体，和来自Proteintech公司的ATG5 Antibody(10181-2-AP)，P62/SQSTM1 Antibody(18420-1-AP)，FAS/CD95 Antibody(13098-1-AP)，HRP-conjugated beta Actin Antibody(HRP-60008)，Cy3–conjugated Affinipure Goat Anti-Rabbit IgG(H+L)(SA00009-2)。
实施例1
SD大鼠玻璃体药物注射
1.1大鼠准备：4W约140g重的SD大鼠购自斯莱克公司，饲养在同济大学动物中心SPF房。大鼠随机分为四组，PBS对照组，GMFB处理组，GMFB-Met处理组，GMFB-Mutant处理组。
1.2玻璃体腔注射：注射前大鼠腹腔注射2％戊巴比妥钠(1ml/400g体重)进行麻醉，1×速眠新(0.1ml/200g)进行肌肉松弛，然后给予一滴0.5％托吡卡胺散瞳(Wuxi Shanhe Group，Jiangsu，China)，一滴0.4％盐酸奥布卡因表面麻醉(Eisai Co Ltd，Tokyo，Japan)。在体视显微镜下，先用1ml注射器自角膜缘后垂直进针扎一小孔，然后再用注射针由该孔向玻璃体腔内注入相应液体。每只眼的注射体积为8μl。
实施例2
SD大鼠视网膜电图ERG检测生理功能
2.1仪器：APS全自动视觉电生理检查仪(APS-2000)购于重庆康华科技有限公司。
暗适应：做视觉电生理功能检查前一天，把SD大鼠转移到暗房，进行暗适应。第二天开始做。
2.2大鼠的准备：向大鼠腹腔注射2％戊巴比妥钠(1ml/500g体重)进行麻醉，1×速眠新(0.1ml/200g)进行肌肉松弛，然后给予一滴0.5％托吡卡胺散瞳(Wuxi Shanhe Group，Jiangsu，China)，一滴0.4％盐酸奥布卡因表面麻醉(Eisai Co Ltd，Tokyo，Japan)，每只眼睛各涂一点导电膏。
插电极：地线接大鼠尾巴上，负极接大鼠两耳朵之间，正极接两眼睛角膜上，注意不要触到眼睑和巩膜上。
2.3数据采集：打开软件“视觉电生理图”，点“FERG”，再点1,2通道， 一个为左眼通道，另一个为右眼通道，点“设置”，刺激次数为2次，刺激频率为0.05Hz；依次点击刺激强度(1)-0.0006325(cd*s/m)、(5)-0.006325(cd*s/m)、(9)-0.06325(cd*s/m)，每次强度至少间隔2min。点“示波”，待波的基线平稳时，点击“采集”，等听到“嘀”的一声后，采集完毕，点击“保存”。再点击“设置”，修改文档号和刺激强度，依次类推。等所有强度做完后，换下一只大鼠。所有大鼠做完后，打开“文件”，进行标定，双击曲线，曲线变为白色，点击“标定”。标定完a波后，按空格键，标定b波。所有标定完毕，点击“打印”，保存为.PDF格式。点击左下角按钮，退出软件，关闭电脑，关闭放大器。
数据如图1中A和图4中C的代表波形图及统计图所示，GMFB处理组较PBS组b波波幅明显下降，说明玻璃体腔GMFB的上升将损伤视网膜功能；而GMFB-Met处理组和GMFB-Mutant处理组相比b波波幅较大，说明FAS受体拮抗剂Met能够对GMFB造成的视网膜功能损伤起保护作用。
实施例3
药物处理或注射后Western-blot检测凋亡或自噬相关蛋白变化水平3.1蛋白的提取：将细胞接种在六孔细胞培养皿中，经相应的处理后，每孔加150μl含有Protease Inhibitor Cocktail的RIPA裂解液，用细胞刮收集细胞到新的EP管中，放冰上孵育30min，10000rpm，4℃离心15min，轻轻吸出上清至干净的离心管中，-80℃保存备用。
3.2蛋白浓度的测定：蛋白定量采用BCA定量法，具体方法如下：配制标准蛋白溶液2μg/μl，-20℃保存备用；根据标准品和样品的数目配制工作液，试剂A和试剂B按50:1的体积比充分混匀，现配现用；工作液总体积＝(标准品个数+待测样品个数)×(2个复孔数)×(200μl工作液)。将标准品按0μl、1μl、2μl、4μl、8μl、16μl的体积加到酶标孔中，每孔加入200μl的BCA工作液，制作标准曲线；待测样品每孔加入2μl，再加入200μl的BCA工作液，37℃孵育30min；用多功能酶标仪测定560nm处的吸光度值。
3.3蛋白样品的准备：根据准备上样的蛋白量和蛋白浓度，加入5× loading buffer，混合后于100℃加热10min，使其充分的变性，4℃，12000rpm，离心2min，除去沉淀，-80℃贮存备用。
3.4 SDS-PAGE电泳：分离胶的制备：10％的分离胶(10ml)配制方法如下：30％聚丙烯酰胺3.3ml，ddH 2O 2.7ml，1M Tris(pH 8.8)3.8ml，10％SDS 0.1ml，10％过硫酸铵0.1ml，TEMED 0.004ml；灌胶前加APS和TEMED，加完后充分混匀，迅速灌胶。用移液枪将配制好的分离胶溶液缓慢加入到装配好的玻璃板中，分离胶溶液高度约为6cm，给浓缩胶留出1.5cm高的位置，加好后在凝胶顶部缓缓加入无水乙醇促进胶凝，使凝胶表面呈一条直线；放置1h左右直到凝胶完全凝固，当无水乙醇和凝胶之间有一条折线时说明凝胶已经凝固，倒掉胶上的无水乙醇，用滤纸吸干。制备5％的浓缩胶(4ml)方法如下：30％聚丙烯酰胺0.67ml，ddH 2O 2.7ml，1M Tris(pH 6.8)0.5ml，10％SDS 0.04ml，10％过硫酸铵0.04ml，TEMED0.004ml；灌胶前加APS和TEMED，加完后充分混匀，迅速灌胶。用移液枪将配制好的浓缩胶溶液缓慢加入分离胶的上层，直至玻璃板的边缘，然后小心插入梳子，注意不要产生汽包，放置约1h左右凝胶，凝胶之后拔出梳子，准备电泳。将准备好的蛋白样品和蛋白标记分子加入到加样孔中，防止气泡产生，使用Bio-Rad电泳仪，槽内加满电泳液以后即可开始电泳，电泳条件为：80V电压，30min，换120V电压，1h30min左右。电泳至溴酚蓝刚好跑出分离胶底部停止。
3.5转膜：SDS-PAGE凝胶电泳结束以后，将玻璃板撬开后小心分离出凝胶，切去浓缩胶的部分，保留分离胶，浸泡于去离子水中；剪取与凝胶大小相似的PVDF膜放入无水甲醇中浸泡2min；将切好的凝胶、滤纸以及事先用无水甲醇浸泡过的PVDF膜放入转膜平衡液中平衡10min；采用Bio-Rad的转膜系统，以夹三明治的方式，从黑面到白面的顺序依次为：黑面(负极)-海绵垫-滤纸-凝胶-PVDF膜-滤纸-海绵-白面(正极)，滤纸、凝胶和PVDF膜要对齐，彻底清除内部气泡，然后放入装满转膜液的转膜仪中，转膜条件为300毫安，2h；转膜槽置于冰浴中。转膜结束后可以参考蛋白Marker转上PVDF膜作为转膜成功的标志，也可将膜用1×丽春红染液染色，然后用水冲洗掉没染上的染液就可看到膜上的蛋白。
3.6免疫杂交反应：用1×PBST洗掉丽春红，配置5％的BSA闭液，室温封闭1h。加入用PBST稀释的一抗，4℃过夜；1×PBST洗膜3次，每次10min。加入PBST稀释的HRP标记的二抗，室温1h。1×PBST洗膜3次，每次10min。ECL化学发光采集结果，以Gapdh作为对照内参。
3.7数据统计分析：用Image J软件western条带的灰度分析，然后与对应的Gapdh进行相比，然后再利用Graphpad Prism软件进行统计分析及作图。
实验结果如图1中C、图2中A以及图4中A和B所示，其中图1中C和图2中A分别在动物和细胞模型上证明，胞外GMFB的注射或处理将升高凋亡水平，增加LC3II对LC3I的比例，ATG5的含量，以及自噬降解标志物p62的含量，说明GMFB将激活自噬的早期过程，并通过影响降解过程阻碍正常自噬流的进行。而图4中A和B则表明，相比于在GMFB处理的同时加入Met对照变异体Mutant，同时加入FAS受体拮抗剂Met组的凋亡水平较低，并且可以抑制由GMFB造成的自噬流阻碍。
实施例4
视网膜冰冻切片的准备
4.1眼球准备：药物处理后的SD大鼠分别在2w和4w的时间点收取眼球样本，SD大鼠脱臼处死后，仔细摘除眼球，注意保留视神经；
4.2固定：置于4％多聚甲醛固定过夜；
4.3解剖：在解剖显微镜下沿角巩膜缘上缘解剖眼球，小心去除角膜，虹膜和晶状体，形成完整的视杯，注意操作轻柔防治造成视网膜脱离；
4.4脱水：用30％的蔗糖过夜脱水；
4.5包埋：用组织包埋液OCT包埋4℃平衡过夜；
4.6液氮速冻：将眼球用液氮快速冷冻，冷冻之前使得眼球的位置尽量垂直居中。-80℃保存冰冻样品。
4.7切片：用冰切机按8μm的厚度进行连续切片，取过视神经乳头的切片。将切片放在-80℃保存，使用前吹干。
实施例5
视网膜冰冻切片的免疫荧光染色
5.1切片准备：取实施例4中所得的眼球冰冻切片样品进行免疫荧光染色检测。
5.2烤片：50℃烤片0.5h。
5.3固定：4％PFA固定10min，再用PBS清洗三次，每次5min。
5.4透膜：用0.25％Triton-X100透膜15min，PBS洗3次，每次5min。
5.5封闭：5％胎牛血清室温封闭1h；
5.6一抗：加5％胎牛血清稀释的相应一抗，置于湿盒内防止干燥，4℃孵育过夜(TUNEL实验可忽略)。
5.7二抗：PBS洗3次，每次5min；加5％胎牛血清稀释的荧光标记的与一抗同种属来源的二抗，或TUNEL试剂盒所述反应液，置于湿盒内，避光37℃孵育1h。
5.8 DAPI染核：PBS洗3次，每次5min；用0.5μg/ml DAPI染细胞核1min，PBS洗3次，每次5min。
5.9封片：用荧光封片剂封片。
5.10拍照观察：在显微镜下拍照观察。
结果如图1中B和图4中D所示，其中图1中B结果表示，玻璃体注射GMFB后凋亡细胞标志红色荧光在RPE层增加，即增加视网膜色素上皮细胞的凋亡水平；而图4中D所示，同时注射FAS受体拮抗剂Met可以降低GMFB诱导的凋亡水平的增加。
实施例6
mRFP-GFP-LC3细胞系的建立和siRNA转染、药物处理后自噬流变化检测
6.1 mRFP-GFP-LC3细胞系的建立按照汉恒生物产品HB-AP210-0001的使用指南进行。ARPE19细胞汇合率介于50～70％时感染自噬双标腺病毒并于2h后换液，感染24h后开始观察到GFP、RFP表达，对阳性细胞使用Puromycin进行筛选和扩大培养，形成稳定表达细胞系。mRFP用于标记及追踪LC3，GFP的减弱可指示溶酶体与自噬小体的融合形成自噬溶酶体，即由于GFP荧光蛋白对酸性敏感，当自噬体与溶酶体融合后GFP荧光发生淬灭，此时只能检测到红色荧光。在显微镜成像后红绿荧光通过 merge后出现的黄色斑点即指示自噬体，红色的斑点指示自噬溶酶体。
6.2将已建立的细胞系铺板于已加有coverslip的24孔板，培养至50～70％并贴壁。
6.3针对siRNA转染组，每孔使用5μl Lipo2000对不同受体的siRNA进行转染，24～48h后进行GMFB处理；针对FAS抗体处理组，每孔使用5μl抗体37℃封闭2h。
6.4 siRNA转染或抗体封闭后吸出原培养基，使用0.01μg/μl GMFB处理。
6.5 GMFB处理不同时间后使用PBS冲洗3次，4％PFA固定10min。
6.6观察与计数：在显微镜下拍照观察并记录自噬体和自噬溶酶体的数量。
结果如图2中B、图3中A和C所示，其中图2中B显示GMFB处理后merge图中黄色斑点和红色斑点均有增多，说明自噬体形成增多但自噬后期降解过程出现阻碍。图3中A显示通过siRNA文库筛选，发现FAS、TGFBR1等八种受体的siRNA可以减少自噬体和自噬溶酶体的数量，有抑制GMFB引起的自噬异常的作用；图3中C显示提前使用FAS拮抗剂Met进行封闭，可以减弱GMFB引起的自噬功能异常。
实施例7
GMFB处理后ARPE19中不同细胞受体的mRNA表达量
7.1 ARPE19细胞均匀铺板于六孔板并培养至70～80％后，使用0.01μg/μl GMFB处理。
7.2 GMFB处理不同时间后，使用PBS冲洗3次，每孔加入1ml Trizol进行吹打，并收集到离心管中。
7.3向Trizol收集样品中加入200μl氯仿，剧烈混匀后，以12000rpm的转速4℃离心10min。
7.4离心后，将上清转移到新的离心管中，注意不要取到中间的蛋白层，加入等体积的异丙醇。
7.5 12000转4℃离心10min，弃去上清，将沉淀用75％的乙醇洗1～2次。
7.6将沉淀室温干燥后，用适量的20μl DEPC水溶解。
7.7使用Nanodrop进行定量，并计算反转录所需的体积。
7.8 RNA反转录：20μl体系，1000ng的RNA，取4μl的Takara的逆转录试剂supermix，再加ddH 2O补到20μl。置于PCR仪中37℃15min，85℃5s，结束后-20℃保存备用。
7.9定量PCR
(1)将RNA反转录后得到的cDNA稀释4倍作为模板，设计引物如表1。
(2)利用天根公司的SYBR Green实时荧光定量PCR检测试剂盒，qPCR的体系为20μl：2μl的cDNA模板，1μl引物，10μl 2×PCR Mix，7μl ddH 2O，检测目的基因的表达量。PCR扩增条件：94℃变性10min，进入循环(95℃ 5sec，60℃ 60sec)，一共40个循环，并收集溶解曲线。
(3)数据分析，采用2 -△△Ct法进行处理，以Gapdh做为内参。
实验结果如图3中B所示，即在未处理状态下受体FAS的表达量大于其他受体，GMFB处理后FAS表达量出现上升后下降。
表1 q-PCR中所用到的引物信息
Name Sequence(5’-3’) SEQ ID NO
qhsa_TNFRSF11A_For CCGCCTAAGTGGAGATAAGGAAA 1
qhsa_TNFRSF11A_Rev AAGTTCATCACCTGCCCGCT 2
qhsa_TNFRSF12A_For CTGAGCCTGACCTTCGTG 3
qhsa_TNFRSF12A_Rev ACATTGTCACTGGATCAGCG 4
qhsa_TNFRSF4_For AAGCCTGGAGTTGACTGTG 5
qhsa_TNFRSF4_Rev GTCCTCACAGATTGCGTCC 6
qhsa_FAS_For AAGCTCTTTCACTTCGGAGG 7
qhsa_FAS_Rev GGGCATTAACACTTTTGGACG 8
qhsa_TNFRSF17_For CTTGCATACCTTGTCAACTTCG 9
qhsa_TNFRSF17_Rev TTAAGCTCAGTCCCAAACAGG 10
qhsa_CXCR5_For GGAGCCTCTCAACATAAGACAG 11
qhsa_CXCR5_Rev GGGAGGTGTCGTTATAGTTGTC 12
qhsa_LIFR_For GTTGCTCTGGACAAGTTAAATCC 13
qhsa_LIFR_Rev AAGTATCAGGCCCCTTTGAAG 14
qhsa_TNFSF12_For TCGCAGCCCATTATGAAGTTC 15
qhsa_TNFSF12_Rev GTGACTATAAACTCCCCGATCTG 16
qhsa_IL1RAPL1_For TCCGATTCCACACTTGATTCTC 17
qhsa_IL1RAPL1_Rev TTGATTCGAACAGGCTCTCC 18
qhsa_BEX3_For CTTGCCCCTAATTTTCGATGG 19
qhsa_BEX3_Rev ACTGCAGCTCCCTAAGTTTTC 20
qhsa_BRE_For TCTCAGTCACTTTGGCACAG 21
qhsa_BRE_Rev AATGTGAGAGTTGGCTGGTC 22
qhsa_FBF1_For GTGGGCAGAATCCAGATGAG 23
qhsa_FBF1_Rev GGTGTCTCTGGTATGTGAAGC 24
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (7)

  1. 一种视网膜色素细胞RPE的自噬和凋亡的抑制剂，其特征在于，所述抑制剂包括肿瘤坏死因子超家族受体FAS的拮抗剂。
  2. 根据权利要求1所述抑制剂，其特征在于，所述拮抗剂包括Met。
  3. 根据权利要求1或2所述抑制剂，其特征在于，所述拮抗剂抑制肿瘤坏死因子超家族受体FAS作为胶质细胞成熟因子β的受体激活下游自噬和凋亡通路。
  4. 权利要求1～3任一项所述的抑制剂在制备发病早期延缓和/或治疗糖尿病视网膜病变的药物中的应用。
  5. 一种延缓和/或治疗糖尿病视网膜病变的药物，其特征在于，所述药物包括权利要求1～3任一项所述的抑制剂。
  6. 一种延缓和/或治疗糖尿病视网膜病变的方法，其特征在于，向玻璃体腔内注射权利要求5所述药物。
  7. 根据权利要求6所述方法，其特征在于，所述药物中Met的含量为5mg/mL。
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CN114404421B (zh) * 2022-02-28 2023-04-28 同济大学 选择性自噬激活剂qx77在制备干预或治疗糖尿病视网膜病变的药物中的应用

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