WO2016206172A1 - MicroRNA-7在制备抗胶质化药物中的应用 - Google Patents

MicroRNA-7在制备抗胶质化药物中的应用 Download PDF

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WO2016206172A1
WO2016206172A1 PCT/CN2015/085946 CN2015085946W WO2016206172A1 WO 2016206172 A1 WO2016206172 A1 WO 2016206172A1 CN 2015085946 W CN2015085946 W CN 2015085946W WO 2016206172 A1 WO2016206172 A1 WO 2016206172A1
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microrna
gmfb
preparation
expression
drug
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徐国彤
吕立夏
张介平
田海滨
张敬法
王娟
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同济大学
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • 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

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  • the present invention relates to the use of a microRNA-7, and more particularly to the use of a microRNA-7 for the preparation of an anti-glial drug.
  • 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 were found to have abnormal ERG. 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. Recent studies have shown that GMFB is a pro-inflammatory factor that is closely related to human central nervous system degenerative diseases such as Alzheimer's disease and Parkinson's disease.
  • GMFB knockout mice are resistant to the effects of experimental autoimmune encephalitis and MPTP.
  • microRNA-7 is a molecular marker of neurons in the sensory nervous system that controls the gene expression of the sensory nervous system of the nematode; it is highly conserved among different species.
  • the retina is a specialized central nervous sensory organ, and microRNA-7 is expressed in the nuclear layers of the retina.
  • microRNA-7 mediates cortical development, ganglion exocytosis and inhibition of a-synuclein toxicity.
  • microRNA-7 also plays an important role in tumor metastasis and stem cell differentiation.
  • microRNA-7 is highly conserved from nematodes to human sequences, only 9 of the predicted Drosophila and human orthologs suggest that microRNA-7 may have a distinctly different role in mammals.
  • the role of microRNA-7 in the process of glialinization of the nervous system has not been reported, and no report on the regulation of GMFB by miRNA has been reported.
  • the object of the present invention is to provide an application of MicroRNA-7 in the preparation of anti-gelatinization in order to overcome the drawbacks of the prior art described above.
  • GMFB is a direct target protein of MicroRNA-7
  • MicroRNA-7 can reduce the expression of GMFB in the rat Muller cell line rMC-1.
  • MicroRNA-7 regulates GMFB expression, inhibits gliosis, and protects neurological function. This finding is the first to confirm that the molecular mechanism by which microRNA-7 inhibits gliosis and protects nerve function is related to the action of GMFB.
  • microRNA-7 in a gliosis-based neurodegenerative disease application.
  • MicroRNA-383 inhibits glialization by directly binding to the 3-UTR terminus of GMFB mRNA, thereby regulating expression of a GMFB protein.
  • the present invention utilizes bioinformatics analysis to find a potential microRNA that regulates the expression of GMFB, namely, MicroRNA-7; the possible interaction between microRNA-7 and GMFB is calculated by miRNAda, and microRNA-7 is directly bound to GMFB through molecular biology experiments. The 3'UTR, thereby inhibiting the expression of GMFB. It was then found that in RD1 mice and STZ-induced type 1 diabetes (TIDM) rats, MicroRNA-7 was able to inhibit gliosis, significantly down-regulate GFAP expression (glycosylated markers), and down-regulate GMFB expression.
  • TIDM STZ-induced type 1 diabetes
  • the anti-glial drug refers to an agent that increases the expression level of MicroRNA-7.
  • the reagent for increasing the expression level of MicroRNA-73 includes a microRNA-7 molecule, a composition of MicroRNA-7 molecule as an active substance, and a carrier containing MicroRNA-7.
  • the gliosis is a gliosis that occurs in a degenerative disease of the nervous system.
  • the present invention demonstrates for the first time that overexpression of MicroRNA-7 in a rat Muller cell line can downregulate GMFB.
  • MicroRNA-7 was first demonstrated to regulate the expression of GMFB protein by directly binding to the 3-UTR terminus of GMFB mRNA.
  • Overexpression of MicroRNA-7 promotes the degradation of GMFB RNA, resulting in the inability of Muller cells to activate; MicroRNA-7 was first demonstrated to be useful for anti-glial treatment of neurodegenerative diseases and to protect neuronal function.
  • the present invention has the following advantages:
  • MicroRNA-7 The mechanism of action of MicroRNA-7 was first confirmed, that is, the expression of GMFB protein was regulated by directly binding to the 3-UTR end of GMFB mRNA;
  • MicroRNA-7 has neuroprotective effects.
  • Figure 1 Effect of microRNA-7 on GMFB expression
  • Figure 1a shows the microRNA-7 phylogenetic tree, which is highly conserved in humans with only microRNA-7a;
  • Figure 1b shows that the microRNA-7 seed sequence is highly conserved
  • Figure 1c Support for 3'-UTR interaction of overexpressed microRNA-7 with GMFB by reporter gene analysis
  • Figure 1d Transfection of microRNA-7 in Muller cells, downregulation of GMFB at protein level
  • Figure 1e Transfection of microRNA-7 in Muller cells, downregulation of GMFB at mRNA levels
  • Figure 2 Retinal injection of RD1 mice and STZ-induced TIDM rats, AAV-microRNA-7 inhibits GFAP expression and inhibits gliosis.
  • Figure 2a shows inhibition of glialization of RD1 mice by microRNA-7
  • Figure 2b shows inhibition of gliosis of microRNA-7 in TIDM rats
  • Figure 2c shows the improvement of electroretinogram amplitude after microRNA-7 inhibition of gliosis in TIDM rats
  • Figure 2d Quantitative statistics showing the improvement in electroretinogram amplitude after microRNA-7 inhibition of gliosis in TIDM rats;
  • Figure 3a shows that protein level GMFB is down-regulated after microRNA-7 overexpression in vivo
  • Figure 3b shows the statistics of downregulation of protein levels of GMFB after overexpression of microRNA-7 in vivo
  • Figure 3c shows that GMFB immunofluorescence is attenuated after overexpression of RD1 mouse microRNA-7;
  • Figure 3d shows that GMFB immunofluorescence is attenuated after overexpression of microRNA-7 in TIDM rats
  • FIG. 4 Overexpression of GMFB causes gliosis and increased release of inflammatory factors
  • Figure 4a shows that GMFB overexpresses 2w, and GFAP immunological activity is significantly enhanced, suggesting gelatinization;
  • Figure 4b A plot showing the amplitude of the GMFB overexpressing 4w and 6w, electroretinogram
  • Figure 4c A statistical diagram showing the amplitude map of the electroretinogram of GMFB overexpressing 4w and 6w;
  • Figure 4d shows that GMFB overexpresses 6w, and the expression of retinal inflammatory factors is significantly increased.
  • HEK293T was purchased from ATCC.
  • 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.
  • AAV2/8-microRNA-7 was commercialized with a titer of 10 ⁇ 9 gc/ml.
  • the HEK293T cell line was transferred to the microRNA-7 plasmid and the psicheck-2 plasmid (containing the GMFB3'-UTR region) to detect luciferase activity, and the luciferase activity assay kit was purchased from Promega.
  • the cells were first placed in a six-well plate at a density of about 50% the day before.
  • the medium was changed to serum-free and antibiotic-free DMEM medium, then two sterile centrifuge tubes were added, 250 ⁇ L of serum-free antibiotic-free medium was added, and 5 ⁇ L of liposome and 4 ⁇ g were added respectively.
  • the plasmid (2 ⁇ g each of microRNA-7 and psicheck2 plasmid) was mixed and allowed to stand for 5 minutes.
  • the medium containing the liposome was added to the medium containing the plasmid, mixed, and evenly added to the cells after 30 minutes.
  • the cells were replaced with normal high-glucose DMEM medium, and transfected for 36 hours, and lysed for reporter gene detection.
  • Figure 1 See Figure 1 for the results.
  • a shows the microRNA-7 phylogenetic tree, which is highly conserved in humans with only microRNA-7a; in Figure 1, b shows the microRNA-7 seed sequence, which is highly conserved.
  • Figure 1c supports the 3'-UTR interaction of overexpressing microRNA-7 with GMFB by reporter gene analysis.
  • d transfection of microRNA-7 in Muller cells, downregulation of GMFB at the protein level.
  • Figure 1 e Transfection of microRNA-7 in Muller cells, downregulation of GMFB at mRNA levels.
  • GMFB antibody was purchased from Proteintech, fluorescent quantitative PCR was purchased from Tiangen Bio, and primer synthesis was provided by biotech.
  • microRNA-71ul was injected into the subretinal space, and GFAP immunofluorescence was detected 4 weeks after birth, suggesting that gliosis was inhibited.
  • microRNA-73ul was injected into the subretinal space after injection 4 GFAP immunofluorescence was detected weekly, suggesting that gliosis was inhibited.
  • Figure 2c shows the improvement of electroretinogram amplitude after microRNA-7 inhibition of TIDM rats
  • Figure 2d shows quantitative statistics showing the improvement of electroretinogram amplitude after microRNA-7 inhibition of TIDM rats. .
  • the upper layer of concentrated glue is prepared. Pour off the upper layer of water in step 1. After adding the concentrated gel, slowly insert the comb and let it stand for solidification.
  • the extracted protein is boiled and denatured, it is added to the gel hole according to the same sample amount according to the protein concentration, and the electrophoresis is stopped after a certain time at a voltage of 100 V, and the protein is transferred to Millipore PVDF by wet transfer. On the membrane.
  • RNA is extracted by the method of Trizol cleavage.
  • the main steps are as follows:
  • RNA reverse transcription the first strand of cDNA is 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.
  • Primers were designed using the first strand of cDNA obtained by reverse transcription of RNA as a template.
  • 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.
  • Preparation of diabetic rats Male SD rats, 160-180 g, were starved 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). In the large-diabetic rats with diabetes mellitus, miR-73ul was injected into the subretinal space. Four weeks after the injection, the retina was isolated, and the protein was extracted and subjected to Western blot.
  • microRNA-71 ul was injected into the subretinal space, and was taken at 4 weeks after birth, and an equal volume of PBL was injected as a control.
  • DM rats were carefully removed from the eyeball (as far as possible with optic nerve) and fixed in 4% paraformaldehyde for three hours. Under a dissecting microscope, cut the cornea 2 mm above the limbus, cut the cornea, and carefully remove the lens and iris. The remaining eyeballs were dehydrated for 30 hours with 30% sucrose. Place the eyeball in the tissue embedding agent, place the optic nerve on one side, be careful not to have air bubbles, and equilibrate overnight at 4 °C. The next day, move to the -80 °C refrigerator for spare, mark the place of the optic nerve head. The embedded eyeballs were taken out from the -80 ° C refrigerator, and serially sliced with a mark and an optic nerve head by a cryostat, and the section thickness was 10 ⁇ m.
  • Immunofluorescence assay Retinal sections were incubated with PBS for 10 minutes, 0.25% tritonx-100 was permeable for 10 minutes, and then washed 3 times with PBS for 5 minutes each time. After blocking with 1% BSA for 30 minutes at room temperature, with anti-mouse anti-GS (1:200), mouse anti-CRALBP (1:50), rabbit anti-Recoverin (1:500), rabbit anti-GMFB (1:200) Rabbit anti-GFAP (1:200) was incubated overnight at 4 °C (co-stained with mouse anti-mCherry antibody (1:1000-1:2000), respectively), without the primary antibody group as a negative control.
  • the cells were washed three times with PBS for 5 minutes/time, and incubated with secondary anti-mouse FITC (1:100) or anti-rabbit FITC (1:100) for one hour at room temperature. After the secondary antibody was discarded, it was incubated with 0.5 ⁇ g/mL of DAPI for 30 seconds and washed 3 times with PBS for 5 minutes/time. The results of the fluorescence were observed under an inverted fluorescence microscope after mounting with DAKO and adding a coverslip.
  • Figure 3d shows the results of immunofluorescence in diabetic retinopathy rats. It can be seen that in the microRNA-7 intervention group, GMFB staining is diffused. In the unintervention group, the GMFB fluorescence signal is located in the inner nuclear layer. Figures 3a-d support down-regulation of GMFB after microRNA-7 intervention.
  • AAV2/8-GMFB virus 3 ul was injected into the normal SDG rats by subretinal injection. After injection, electroretinogram (ERG), immunofluorescence staining, retinal-related genes and inflammatory factor-related genes were detected at different time points. Quantitative PCR analysis.
  • the immunofluorescence method was as in Example 3.
  • the quantitative PCR method was as in Example 2.
  • 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.

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Abstract

提供MicroRNA-7抑制神经系统胶质化的应用；在大鼠视网膜Muller细胞系中过表达MicroRNA-7，可降低GMFB表达，抑制炎症因子产生。MicroRNA-7通过直接与GMFB mRNA的3-UTR端结合，调控GFAP蛋白的表达。

Description

MicroRNA-7在制备抗胶质化药物中的应用 技术领域
本发明涉及一种MicroRNA-7的用途，尤其是涉及一种MicroRNA-7在制备抗胶质化药物中的应用。
背景技术
随着我国经济的高速发展和老龄化进程的加速，糖尿病(diabetes milletus，DM)的患病率正呈快速上升的趋势，成为继心脑血管疾病、肿瘤之后另一个严重危害人民健康的重要慢性非传染性疾病。世界卫生组织推测，在2025年中国糖尿病患者将达到3亿。糖尿病视网膜病变(Diabetic Retinopathy，DR)，简称糖网病，是糖尿病最常见的并发症，在DM患者中1/3发生DR，1/10发生致命性威胁视力的糖尿病黄斑水肿(diabetic macular edema,DME)或者增殖性糖网病(proliferative diabetic retinopathy,PDR)，严重影响患者生活质量，世界范围内DR已经成为显著的社会负担和社会问题。DR曾被认为是视网膜的微血管病变，微循环损害是DR的经典标志，但是越来越多的证据提示在DR的病理过程中神经变性是一个早期事件，并参与微血管异常的发展。组织学上神经元凋亡和反应性的胶质化是DR神经变性的最重要的特征。目前DM捐献眼在眼科检查没有发现任何微循环异常，但是已经具有主要的神经变性的特点。视网膜节细胞(RGC)是DR最先被检测的发生凋亡的细胞；RGC丢失导致神经纤维层变薄，在DM患者或者有轻微DR的DM患者通过OCT检测都检测到，在没有任何微血管病变的DM I型和II型病人发现ERG异常。神经元凋亡伴随着Muller胶质细胞的变化。目前尚不清楚神经元凋亡和胶质化哪一个在DR中是第一个发生的事件。研究DR神经变性的机制及鉴定在神经变性的介导者对于研发新的治疗策略是非常必要的。从临床角度早期鉴别神经变性对于基于神经保护的药物的应用是必须的。
DR神经变性的机制研究现状：介导DR神经变性的主要机制有细胞外兴奋毒性谷氨酸(Glutamate，Glu)集聚、氧化应激增加、视网膜分泌保护因子的减少及慢性炎症。Schellin SA等用光镜和电镜检测发现，DM早期Muller细胞核已发生改变，而视网膜血管内皮细胞、周细胞并未见明显病理改变，DM早期视网膜Muller细胞的超微结构和生理功能已发生变化，不仅影响早期DM患者神经细胞功能异 常(表现为视觉敏感度及色觉敏感度下降，视网膜振荡电位b波异常)，而且影响整个DR的进展过程。在研究介导DR神经变性的分子中，我们更关注Muller细胞产生的分子。
胶质细胞成熟因子beta(glia maturation factor beta,GMFB)与神经变性：GMFB最早从牛脑分离纯化的17kd酸性胞浆蛋白，进化上高度保守，在中枢神经系统主要由星形胶质细胞产生，对脑组织生长、分化和再生有重要的作用，其表达在发育期上调，成年明显降低。在大鼠视网膜GMFB仅在Muller细胞表达，从胚胎14天到成年均表达。新近研究显示GMFB是一种促炎因子，与人中枢神经系统退行性疾病密切相关，如阿茨海默病和帕金森病。GMFB基因敲除小鼠能够抵抗实验性自身免疫性脑炎和MPTP的毒性。
microRNA-7是感觉神经系统神经元的分子标记，控制线虫的感觉神经系统的基因表达；在不同的物种中高度保守。而视网膜是一种特化的中枢神经感觉器官，microRNA-7在视网膜各核层均有表达。在哺乳动物中枢神经系统和体外的细胞培养，microRNA-7能够介导皮层发育、神经节外生和抑制a-synuclein毒性。而且microRNA-7在肿瘤转移和干细胞分化过程中也有重要的作用。尽管microRNA-7从线虫到人类序列高度保守，但是在预测的果蝇和人的正系同源物只有9个，提示microRNA-7在哺乳动物中可能有截然不同的作用。目前对于microRNA-7在神经系统胶质化(例如糖尿病视网膜变性、光感受器细胞快速变性)的过程中的作用尚未见报道，也未见GMFB受miRNA调控的报道。
发明内容
本发明的目的就是为了克服上述现有技术存在的缺陷而提供一种MicroRNA-7在制备抗胶质化中的应用。
本发明的目的可以通过以下技术方案来实现：
本发明发现GMFB是MicroRNA-7的直接靶蛋白，在大鼠Muller细胞系rMC-1中MicroRNA-7可降低GMFB的表达。在小鼠光感受器快速变性的模型RD1和STZ-诱导的一型糖尿病模型MicroRNA-7调控GMFB表达，抑制胶质化，保护神经功能。这一发现首次证实MicroRNA-7抑制胶质化保护神经功能的分子机制与GMFB作用相关。
本发明的第一方面，提供了MicroRNA-7在胶质化为主的神经变性疾病的保护 应用。
本发明的第二方面，提供了MicroRNA-383通过直接结合GMFB mRNA的3-UTR端，调控GMFB蛋白的表达，从而抑制胶质化。
本发明利用生物信息学分析未发现调控GMFB表达的一个潜在microRNA，即MicroRNA-7；通过miRNAda计算出microRNA-7与GMFB可能存在相互作用，通过分子生物学的实验证明MicroRNA-7直接结合在GMFB的3’UTR，从而抑制GMFB的表达。然后发现，在RD1小鼠和STZ-诱导的I型糖尿病(TIDM)大鼠中，MicroRNA-7能够抑制胶质化，明显下调GFAP的表达(胶质化的标记物)，下调GMFB表达。而GMFB过表达又能够引起GFAP活化，引起胶质化，这说明MicroRNA-7通过GMFB参与到对胶质化反应的抑制。接下来，在TIDM大鼠当将GMFB干扰后，明显抑制GFAP表达，抑制胶质化。这些结果说明MicroRNA-7可能通过GMFB抑制胶质化。
所述的抗胶质化药物是指提高MicroRNA-7表达量的试剂。
所述的提高MicroRNA-73表达量的试剂包括：MicroRNA-7分子、MicroRNA-7分子作为活性物质的组合物、含有MicroRNA-7的载体。
所述的胶质化为在神经系统退行性疾病中发生的胶质化。
本发明首次证实在大鼠Muller细胞系中过表达MicroRNA-7，可下调GMFB。首次证实MicroRNA-7通过直接与GMFB mRNA的3-UTR端结合，调控GMFB蛋白的表达。过表达MicroRNA-7促进GMFB RNA降解，导致Muller细胞不能活化；首次证实MicroRNA-7可用于神经退行性疾病的抗胶质化治疗，保护神经元的功能。
与现有技术相比，本发明具有以下优点：
1)在大鼠Muller细胞中过表达MicroRNA-7，可以下调胶质化相关基因的表达；
2)首次证实了MicroRNA-7的作用机制，即通过直接与GMFB mRNA的3-UTR端结合，调控GMFB蛋白的表达；
3)过表达MicroRNA-7促进GMFB RNA降解，胶质化过程受到抑制；
4)过表达MicroRNA-7具有神经保护作用。
附图说明
图1：microRNA-7对GMFB表达的影响；
图1a：显示microRNA-7进化树，在人只有microRNA-7a，高度保守；
图1b：显示microRNA-7种子序列高度保守；
图1c：通过报告基因分析支持过表达microRNA-7与GMFB的3’-UTR相互作用
图1d：在Muller细胞转染microRNA-7，在蛋白水平下调GMFB；
图1e：在Muller细胞转染microRNA-7，在mRNA水平下调GMFB；。
图2：在RD1小鼠和STZ-诱导的TIDM大鼠视网膜注射，AAV-microRNA-7抑制GFAP的表达，抑制胶质化。
图2a：显示microRNA-7对RD1小鼠的胶质化抑制；
图2b：显示microRNA-7对TIDM大鼠的胶质化抑制；
图2c：显示microRNA-7对TIDM大鼠的胶质化抑制后视网膜电图波幅的改善；
图2d：显示microRNA-7对TIDM大鼠的胶质化抑制后视网膜电图波幅的改善的量化的统计；
图3：体内microRNA-7对GMFB负性调控；
图3a显示：体内microRNA-7过表达后，蛋白水平GMFB下调；
图3b显示：体内microRNA-7过表达后，蛋白水平GMFB下调的统计；
图3c显示：在RD1小鼠microRNA-7过表达后，GMFB免疫荧光减弱；
图3d显示：在TIDM大鼠microRNA-7过表达后，GMFB免疫荧光减弱；
图4：GMFB过表达引起胶质化，炎症因子释放增加；
图4a：显示GMFB过表达2w，GFAP免疫活性明显增强，提示胶质化；
图4b：显示GMFB过表达4w和6w，视网膜电图的波幅图；
图4c：显示GMFB过表达4w和6w，视网膜电图的波幅图的统计图；
图4d：显示GMFB过表达6w，视网膜炎症因子表达明显增高。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明。
以下实施例中，HEK293T购自ATCC。rMC-1细胞系由实验室制备，培养基为高糖DMEM，含10％血清和1％P/S。培养环境是37℃、5％CO2和95％空气。 AAV2/8-microRNA-7商品化，滴度10^9gc/ml。
实施例1
在HEK293T细胞系转入microRNA-7质粒和psicheck-2质粒(含有GMFB3’-UTR区域)，检测荧光素酶的活性，荧光素酶活性检测试剂盒购自Promega。
转染质粒时，采用的是Invitrogen公司的lipofectamine 2000脂质体，主要步骤如下：
1.前一天先将细胞按照50％左右的密度铺在六孔板里。
2.转染前将培养基换成无血清无抗生素的DMEM培养基，然后取两个灭菌的离心管，各加入250μL无血清无抗生素的培养基，再分别加入5μL的脂质体和4μg的质粒(microRNA-7和psicheck2质粒各2ug)，各自混匀后，静置5分钟。
3.将含有脂质体的培养基加入到含有质粒的培养基中，混匀，30分钟后均匀滴加到细胞里。
4.6小时后换成正常的高糖DMEM培养基继续培养，转染36小时，裂解进行报告基因检测。
结果参见图1。图1中a显示microRNA-7进化树，在人只有microRNA-7a，高度保守；图1中b显示microRNA-7种子序列，高度保守。图1c通过报告基因分析支持过表达microRNA-7与GMFB的3’-UTR相互作用。图1中d：在Muller细胞转染microRNA-7，在蛋白水平下调GMFB。图1中e：在Muller细胞转染microRNA-7，在mRNA水平下调GMFB。
图1中c可以看出，在HEK293中，MicroRNA-7过表达降低荧光素酶的活性。
在大鼠Muller细胞中，MicroRNA-7过表达对GMFB的影响。GMFB抗体购自Proteintech，荧光定量PCR购自天根生物，引物合成由生工提供。
结果见图1d，在蛋白水平过表达microRNA-7引起GMFB减少；结果见图1e在mRNA水平，GMFB表达也减少。
实施例2
在RD1大鼠出生一周，视网膜下腔注射microRNA-71ul，在出生后4周检测GFAP免疫荧光，提示胶质化受到抑制。
结果参见图2a，MicroRNA-7过表达使GFAP表达下降。
在STZ-TIDM大鼠发病当天，视网膜下腔注射microRNA-73ul，在注射后4 周检测GFAP免疫荧光，提示胶质化受到抑制。
结果参见图2b，MicroRNA-7过表达使GFAP表达下降。
图2c显示microRNA-7对TIDM大鼠的胶质化抑制后视网膜电图波幅的改善；图2d示显示microRNA-7对TIDM大鼠的胶质化抑制后视网膜电图波幅的改善的量化的统计。
Western Blot方法如下
1.配制SDS-PAGE所用的凝胶，首先配制下层分离胶，配制好后，立即缓缓加入到装备好的两层玻璃板中，加入适量双蒸水封闭。
2.待下层分离胶凝固后，开始配制上层浓缩胶。将第1步中的上层水倒掉，加入浓缩胶后，慢慢将梳子插上，静置等待凝固。
3.将提取的蛋白煮沸变性后，根据蛋白浓度按照相同的样品量加入到凝胶孔内，100V的电压下，一定时间后停止电泳，采用湿转的方法，将蛋白转移到Millipore公司的PVDF膜上。
4.转膜结束后，将PVDF膜取出，用3％的脱脂奶粉或BSA将膜在室温封闭1-2小时。
5.封闭结束后，将PVDF膜从脱脂牛奶或BSA中取出，用PBST清洗2次，吸干后，加入适量配制好的一抗稀释液，室温摇匀2小时，使一抗与目的蛋白结合。所用的一抗包括：anti-GFAP(Proteintech)，anti-GMFB(Proteintech)anti-actin(Proteintech)。
提取RNA采用的是Trizol裂解的方法，主要步骤如下：
1.将细胞用PBS缓冲液洗1-2次，按比例加入Trizol裂解液(例如六孔板的一个孔对应1mL裂解液)。
2.细胞离壁后，转移到1.5mL的离心管，加入五分之一体积的氯仿，剧烈混匀后，以12000rpm的转速4℃离心15分钟。
3.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，冰浴20分钟。
4.12000rpm的转速4℃离心15分钟，弃去上清，将沉淀用75％的乙醇洗1-2次。
5.将沉淀室温干燥后，用适量的DEPC水溶解。测浓度。
RNA反转录，cDNA第一条链是通过Promega公司的M-MLV反转录酶获得 的，主要步骤如下：
1.首先取1-2μg的RNA与2μL的oligo d(T)混合，72℃水浴放置5分钟，立即冰浴2分钟后，使oligo d(T)与RNA的poly-A尾巴结合，轻微离心。
2.加入1.25μL的dNTP(10μM)、1μL M-MLV反转录酶和0.5μL RNA酶抑制剂，用DEPC水将反应体积调节到25μL。42℃水浴放置1小时。
3.70℃放置10分钟使反转录酶失活。将得到的cDNA单链保存于-20℃冰箱。
引物见下表1。
表1用于检测基因的引物
Figure PCTCN2015085946-appb-000001
Figure PCTCN2015085946-appb-000002
反转录体系如表2：
表2反转录体系
试剂 用量(μL) 终浓度
5×M-MLV buffer 4 1×
dNTP(10μM) 0.75 0.375mM
miRNA RT primer(1μM) 1.2 60nM
M-MLV 0.2 20U
RNA 1 0.2-200ng
H2O 12.85  
反转录程序：16℃30分钟，42℃30分钟，85℃10分钟后立即置于冰上5分钟。然后可于-20℃冰箱保存备用。
定量PCR
将RNA反转录后得到的cDNA第一链作为模板，设计引物。利用天根公司的SYBR Green实时荧光定量PCR检测试剂盒，检测目的基因的表达量。PCR扩增条件如下：94℃变性10分钟，进入循环(95℃5sec，60℃60sec)，一共40个循环，并收集溶解曲线。
实施例3
糖尿病大鼠制备：采用雄性SD大鼠，160-180g，实验前先将大鼠饥饿24小时。单次腹腔内注射STZ(60mg/kg体重)来诱发DM，正常对照组腹腔注射等体积的柠檬酸溶液；24小时后断尾取血测血糖，血糖值低于250mg/dL的大鼠补充注射STZ。连续3天测血糖。将血糖连续3天超过250mg/dL的大鼠确定为DM大鼠(血糖低于250mg/dL的大鼠将被排除)。在明确糖尿病发病大大鼠进行视网膜下腔注射miR-73ul，注射后4周，分离视网膜，抽提蛋白，进行Western blot，方法同前。
在RD1大鼠出生一周，视网膜下腔注射microRNA-71ul，在出生后4周收养，注射等体积PBL作为对照。
免疫荧光：
组织切片的制备：DM大鼠小心摘除眼球(尽量有视神经存在)，于4％多聚甲醛中固定三个小时。在解剖显微镜下，沿着角巩膜缘上方2mm处剪口，把角膜剪掉，再小心移去晶状体和虹膜。剩余的眼球放入30％的蔗糖脱水3个小时。把眼球放入组织包埋剂包埋，视神经放在一侧，注意不要有气泡，4℃冰箱平衡过夜。第二天移至-80℃冰箱备用，视神经乳头的地方做个标记。把包埋的眼球从-80℃冰箱拿出后，用冰冻切片机经过所做的标记和视神经乳头进行连续切片，切片厚度为10μm。
免疫荧光检测：视网膜切片用PBS湿润10分钟，0.25％tritonx-100透膜10分钟后，用PBS清洗3次，每次5分钟。室温下用1％BSA封闭30分钟之后，与一抗小鼠抗GS(1:200)、小鼠抗CRALBP(1：50)、兔抗Recoverin(1:500)、兔抗GMFB(1：200)、兔抗GFAP(1：200)在4℃过夜孵育(分别与小鼠抗mCherry抗体(1:1000-1:2000)共染)，不加一抗组作为阴性对照。次日继续用PBS清洗3次，5分钟/次，室温下避光分别与二抗anti-mouse FITC(1:100)或anti-rabbit FITC(1:100)孵育一小时。弃除二抗后，用0.5μg/mL的DAPI孵育30秒，用PBS清洗3次，5分钟/次。用DAKO封片，加上盖玻片后于倒置荧光显微镜下观察荧光结果。
结果参见图3a，western blot显示在年龄匹配的野生型小鼠，GMFB条带很弱；对RD1未干预组视网膜相比，microRNA-7干预后，GMFB条带变弱；在DR(糖尿病视网膜病变)大鼠的视网膜，经microRNA-7干预后，GMFB条带变弱，对以样品中a-tubulin(作为上样对照)进行半定量分析，显示经过microRNA-7干预后与为干预组相比，有显著性差异，见图3b，支持microRNA-7对GMFB的负调控。图3c，是RD1小鼠在microRNA-7干预后4周，未干预视网膜可见GMFB免疫荧光在内核层明显，干预后，GMFB染色弥散，减弱。图3d是糖尿病视网膜病变大鼠免疫荧光结果，可见在microRNA-7干预组，GMFB染色弥散，在未干预组，GMFB荧光信号位于内核层。图3a-d支持microRNA-7干预后GMFB下调。
实施例4
将AAV2/8-GMFB病毒3ul经视网膜下腔注射至正常180g左右的SD大鼠，注射后在不同的时间点检测视网膜电图(ERG)、免疫荧光染色、和视网膜相关基因、炎症因子相关基因的定量PCR分析。
免疫荧光方法如实施例3.定量PCR方法如实施例2。
ERG方法：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。做视觉电生理功能检查前一天，把DM大鼠转移到暗房，进行暗适应。第二天开始做。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)让眼球突出，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。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。
结果显示在AAV2/8-GMFB病毒视网膜下腔注射2周，免疫荧光显示GFAP显著活化，GMFB表达增高，提示GMFB的重组病毒在视网膜有效感染，且诱导Muller细胞表达GFAP增强(图4a)。在视网膜下腔注射病毒第4周、6周分别进行ERG检测，注射后4周没有显著差异，但在注射后6周，B波波幅降低(图4b)，对降低的B波进行统计学分析，显示在第六周有显著性差异(图4c)，提示GMFB在视网膜过度表达能够引起视功能下降。在注射后第六周，收集视网膜样本，抽提RNA，进行逆转录和定量PCR，发现Muller细胞的相关(GFAP、Vim)升高，GS(Muller细胞特异的酶)降低，炎症因子(CHI3L1、TNFa、IL-1beta)显著增高，光感受器细胞的标记基因(Crx和RECV)显著降低，提示光感受器细胞的破坏(图4d)。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的改 进和修改都应该在本发明的保护范围之内。

Claims (9)

  1. MicroRNA-7在制备抗胶质化药物中的应用。
  2. 根据权利要求1所述的MicroRNA-7在制备抗胶质化药物中的应用，其特征在于，所述的胶质化指中枢神经退行性疾病的胶质化的病理过程，包括视网膜退行性疾病。
  3. 根据权利要求2所述的MicroRNA-7在制备抗胶质化药物中的应用，其特征在于，在胶质化为主的疾病中过表达MicroRNA-7，保护神经功能。
  4. 根据权利要求3所述的MicroRNA-7在制备抗胶质化药物中的应用，其特征在于，MicroRNA-7通过下调GMFB，调控Muller细胞的功能。
  5. 根据权利要求3所述的MicroRNA-7在制备抗胶质化药物中的应用，其特征在于，MicroRNA-7通过下调GMFB，延缓视网膜变性。
  6. 根据权利要求4或5所述的MicroRNA-7在制备抗胶质化药物中的应用，其特征在于，MicroRNA-7通过直接结合GMFB mRNA的3-UTR端，调控GMFB蛋白的表达，抑制Muller细胞胶质化。
  7. 根据权利要求6所述的MicroRNA-7在制备抗胶质化药物中的应用，其特征在于，MicroRNA-7通过下调GMFB，抑制胶质化标记物的表达，减少Muller细胞炎症因子的表达，减少神经元凋亡。
  8. 根据权利要求1至5中任一所述的MicroRNA-7在制备抗胶质化药物中的应用，其特征在于，所述的抗胶质化药物是指提高MicroRNA-7表达量的试剂。
  9. 根据权利要求8所述的MicroRNA-7在制备抗胶质化药物中的应用，其特征在于，所述的提高MicroRNA-7表达量的试剂包括：MicroRNA-7分子、MicroRNA-7分子作为活性物质的组合物、含有MicroRNA-7的载体。
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