WO2022148500A2 - 多聚嘧啶序列结合蛋白在制备脊髓损伤的修复药物中的应用 - Google Patents

多聚嘧啶序列结合蛋白在制备脊髓损伤的修复药物中的应用 Download PDF

Info

Publication number
WO2022148500A2
WO2022148500A2 PCT/CN2022/084398 CN2022084398W WO2022148500A2 WO 2022148500 A2 WO2022148500 A2 WO 2022148500A2 CN 2022084398 W CN2022084398 W CN 2022084398W WO 2022148500 A2 WO2022148500 A2 WO 2022148500A2
Authority
WO
WIPO (PCT)
Prior art keywords
spinal cord
ptb
binding protein
polypyrimidine
tract
Prior art date
Application number
PCT/CN2022/084398
Other languages
English (en)
French (fr)
Other versions
WO2022148500A3 (zh
Inventor
杨日云
潘静莹
包璟崟
夏盼慧
Original Assignee
南通大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 南通大学 filed Critical 南通大学
Publication of WO2022148500A2 publication Critical patent/WO2022148500A2/zh
Publication of WO2022148500A3 publication Critical patent/WO2022148500A3/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/203—Retinoic acids ; Salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • the invention belongs to the technical field of biomedicine, and in particular relates to the application of a polypyrimidine sequence-binding protein silencing agent combined with retinoic acid and purmorphamine in the preparation of a spinal cord injury repair medicine.
  • SCI Spinal cord injury
  • the regenerative capacity of the damaged neurons that have not been lost is also very limited; third, there will be a variety of inhibitory regeneration factors and inflammatory factors at the injury site, forming a chemical microenvironment that is unfavorable for axon regeneration. These unfavorable factors are coordinated with each other and ultimately lead to difficult tissue repair and functional reconstruction in SCI.
  • astrocytes When nerve tissue is injured, astrocytes will proliferate reactively and exhibit progenitor cell characteristics, indicating that they have a high degree of plasticity in cell transdifferentiation. If we manage to use reactive astrocytes that proliferate and have negative effects after SCI as target cells to directly induce reprogramming into neurons, or even motor neurons with corresponding functions in situ, we can achieve the goal of properly eliminating glia.
  • the purpose of scarring which can supplement the neurons lost due to SCI in situ, and at the same time improve the microenvironment of axon regeneration, should be a good method to solve the difficulties affecting repair and regeneration after SCI, and to replace the treatment and personality of SCI cells.
  • the idea of chemical medicine R&D has contributed to a new way of realization.
  • PTB is an RNA-binding protein and plays an important role in the induction and differentiation of neurons.
  • Current studies have found that silencing PTB can reprogram astrocytes into functional neurons and promote the corresponding disease model mice. function is restored.
  • Spinal cord injury is a common neurological trauma that is difficult to treat. After spinal cord injury, astrocytes reactively proliferate and form glial scars at the injury site, thereby inhibiting the regeneration of neurons and axons; at the same time, a large number of motor neurons are lost. , the limited regeneration ability of damaged neurons, all of which bring great difficulties to the repair of spinal cord injury.
  • the purpose of the present invention is to provide the application of a polypyrimidine sequence-binding protein silencing agent combined with retinoic acid and purmorphamine in the preparation of a spinal cord injury repair medicine.
  • the polypyrimidine sequence-binding protein silencing agent is selected from lentivirus-packaged shRNA-PTB (5'-GGGTGAAGATCCTGTTCAATA-3').
  • the present invention also provides a method for inducing the differentiation of spinal cord reactive astrocytes into motor neurons in vitro. Amines promote differentiation.
  • the present invention silences the polypyrimidine sequence binding protein (PTB) by virus in vitro, and at the same time, the small molecule retinoic acid (RA) and purmorphamine (PMA) related to the differentiation of motor neurons are added together to successfully make the spinal cord of mice reactive
  • RA small molecule retinoic acid
  • PMA purmorphamine
  • Figure 1 shows the establishment of a primary mouse spinal cord reactive astrocyte model.
  • A is the GFAP staining of primary mouse spinal cord astrocytes; the mRNA level (B) and protein level (C) of GFAP in the spinal cord astrocytes of mice treated with 10 ⁇ g/mL LPS for 24 h were significantly higher than those in the control group rise. scale bar: 200 ⁇ m.
  • Figure 2 shows the efficiency results of silencing PTB in shRNA-PTB lentivirus-infected mouse spinal cord reactive astrocytes.
  • the protein (A) and mRNA (B) levels of PTB were significantly decreased after shRNA-PTB lentivirus infection of cells for 2 days.
  • Figure 3 is a light microscope image of shRNA-PTB lentiviral reprogramming of reactive astrocytes in mouse spinal cord. scalebar: 200 ⁇ m.
  • Figure 4 is a fluorescent image of shRNA-PTB lentivirus-induced reprogrammed neurons in mouse spinal cord reactive astrocytes. scale bar: 200 ⁇ m.
  • Figure 5 is a fluorescence image of shRNA-PTB lentivirus combined with RA and PMA to reprogram reactive astrocytes into motor neurons in mouse spinal cord. scale bar: 200 ⁇ m.
  • Figure 6 shows the transdifferentiation rate of mouse spinal cord reactive astrocytes directly reprogramming motor neurons.
  • PTB Polypyrimidine Binding Protein
  • RA small molecule retinoic acid
  • PMA Purmorphamine
  • the invention silences PTB through virus in vitro, and at the same time, the small molecule retinoic acid and purmorphamine related to the differentiation of motor neurons are added together, and the reactive astrocytes of the mouse spinal cord are successfully reprogrammed into motor neurons.
  • the in vivo study of the reprogramming strategy of PTB combined with small molecules in the repair of spinal cord injury provides help, and then achieves better repair and functional reconstruction of spinal cord injury.
  • Spinal cord tissue was obtained from neonatal mice.
  • the spinal cord was placed in a petri dish filled with ice D-Hanks solution and rinsed twice. The meninges and blood vessels on the surface were carefully stripped off with microsurgical forceps. D-Hanks solution was rinsed 2-3 times and transferred to another.
  • a petri dish chop the spinal cord to a chyle shape, transfer the chopped spinal cord block in the petri dish into a 15 mL centrifuge tube, then add an equal amount of 0.25% trypsin, digest it in a 37 °C water bath for 15 min, and mix by pipetting every 5 min.
  • the medium was changed in full after every 2 days to remove the dead cell debris that did not adhere to the wall, so that the glial cells could fully grow. Observe under the microscope during each medium change. After about 1 week, the cells covered the bottom of the bottle, and then further purified and cultured . When the glial cells are cultured for 7 to 9 days, after the cells are covered with the bottom of the culture flask, place them on a constant temperature shaking culture bed at 37°C, with a rotation speed of 280 rmp/min, for 16 to 18 hours (the constant temperature culture bed is UV sterilized in advance).
  • LPS lipopolysaccharide
  • shRNA-PTB silences the PTB of reactive astrocytes in the spinal cord of mice
  • the lentivirus-packaged shRNA-PTB (sequence 5'-GGGTGAAGATCCTGTTCAATA-3'SEQ ID NO.1) produced by Shanghai Heyuan Biotechnology Co., Ltd. was infected with reactive astrocytes 2d, and real-time RT-PCR was used.
  • the mRNA and protein expression changes of PTB were detected by techniques such as Western blot to determine the silencing efficiency of shRNA-PTB.
  • the protein (A) and mRNA (B) levels of PTB were significantly decreased after shRNA-PTB lentivirus infected cells for 2 days, indicating that shRNA-PTB lentivirus could reduce the reactive astrocytes in mouse spinal cord in vitro PTB expression, and the silencing efficiency is about 50%
  • the packaged shRNA-PTB lentivirus was used to infect reactive astrocytes, and after 2 days, the infection medium was replaced with induction medium (N3/basal medium: Insulin, sodium selenite, Retinoic acid, putrescine, ChIR99021, SB431542, Db-cAMP, FGF-basic, GDNF), half-change medium for induction medium every 2 d, induction culture for 7 d, 14 d, 16 d, 21 d, 28 d; immunocytochemical technique was used to detect MAP2 pan-neuronal markers and ChAT movement Neuronal marker expression.
  • induction medium N3/basal medium: Insulin, sodium selenite, Retinoic acid, putrescine, ChIR99021, SB431542, Db-cAMP, FGF-basic, GDNF
  • the cell bodies of the shCtrl group were flat, and the shape was the same as that of astrocytes; while the cell bodies of the shPTB group gradually took on a spherical or pyramidal shape, with different numbers and lengths protruding from the cell bodies. protrusions, showing a neuron-like morphology.
  • shRNA-PTB lentivirus can gradually change the morphology of reactive astrocytes in mouse spinal cord to neuron-like, and directly reprogram them into mature neurons of MAP2+ and motor neurons of ChAT+.
  • shRNA-PTB lentivirus combined with RA and PMA to reprogram mouse spinal cord reactive astrocytes
  • the packaged shRNA-PTB lentivirus was used to infect reactive astrocytes, and after 2 days, the infection medium was replaced with induction medium (N3/basal medium + 1 ⁇ M RA + 0.5 ⁇ M PMA, that is, N3/basal per 1000 mL).
  • induction medium N3/basal medium + 1 ⁇ M RA + 0.5 ⁇ M PMA, that is, N3/basal per 1000 mL.
  • 1 micromolar RA and 0.5 micromolar PMA were added to the medium, and the medium was half-changed every 2 d, and the culture medium was induced for 7d, 14d, 16d, 21d, and 28d; immunocytochemical techniques were used to detect MAP2 pan-neural
  • the expression of meta-markers and ChAT motor neuron markers, and the transdifferentiation rate of motor neurons was calculated.
  • the invention Based on the current situation of spinal cord injury repair and the progress of inducing in situ reprogramming, the invention achieves silencing of PTB in vitro, and simultaneously adds small molecules RA and PMA, and finally successfully reprograms the reactive astrocytes of the spinal cord into motor neurons; It provides a theoretical basis for the in vivo application of the reprogramming strategy of PTB combined with small molecules, and strives to achieve a better effect of spinal cord injury repair and functional reconstruction.
  • the present invention combines the two issues of suppressing hyperproliferative reactive astrocytes and replenishing lost motor neurons, and designs a method that can not only replenish the number of motor neurons lost due to spinal cord injury in situ, but also Simultaneously reducing the number of reactive astrocytes activated and proliferating due to spinal cord injury to alleviate the rapid proliferation of glial scars and improve the chemical microenvironment for axon regeneration, ultimately promoting functional recovery after spinal cord injury. .

Abstract

本发明公开了多聚嘧啶序列结合蛋白沉默剂联合维甲酸、嘌***胺在制备脊髓损伤的修复药物中的应用，属于生物医药技术领域。本发明在体外通过病毒来沉默多聚嘧啶序列结合蛋白(PTB)，同时联合添加与运动神经元分化相关的小分子维甲酸(RA)和嘌***胺(PMA)，成功使小鼠脊髓反应性星形胶质细胞重编程为运动神经元，为进一步体内研究PTB联合小分子的重编程策略在脊髓损伤后修复中的作用提供帮助，进而实现更好的脊髓损伤修复和功能重建效果。

Description

多聚嘧啶序列结合蛋白在制备脊髓损伤的修复药物中的应用 技术领域
本发明属于生物医药技术领域，具体涉及多聚嘧啶序列结合蛋白沉默剂联合维甲酸、嘌***胺在制备脊髓损伤的修复药物中的应用。
背景技术
脊髓损伤(spinal cord injury，SCI)是一种致残率高且后果严重的中枢神经***损伤性疾病，严重损害患者运动功能，往往导致损伤部位以下的瘫痪。目前公认的SCI治疗有三大难点：一是迅速活化的反应性胶质细胞，尤其是星形胶质细胞会大量增生形成致密的胶质疤痕组织，使本身生长相对缓慢的轴突在延伸穿越损伤处时，遇到难以逾越的机械屏障及其分泌的抑制因子形成的化学屏障；二是部分受损的神经元会因坏死或凋亡而丢失，仅靠残留的少量神经干细胞无法足量补充丢失的神经元，而未丢失的受损神经元自身的再生能力也很有限；三是在损伤处会出现多种抑制再生因子和炎症因子，形成不利轴突再生的化学微环境。这些不利因素相互协调，最终导致SCI难以组织修复和功能重建。
神经组织损伤时，星形胶质细胞会大量反应性增生并表现出祖细胞特性，说明其在细胞转分化方面具有高度的可塑性。如果设法将SCI后大量增殖并带有负面影响的反应性星形胶质细胞作为靶细胞原位直接诱导重编程为神经元，甚至是具有相应功能的运动神经元，达到既能适当消除胶质疤痕，又能原位补充因SCI丢失的神经元的目的，同时改善轴突再生的微环境，应该是个一举多得的解决SCI后影响修复再生难点的好方法，并为SCI细胞替换治疗和个性化医药研发设想贡献了新的实现途径。
发明内容
PTB是一种RNA结合蛋白且在神经元的诱导和分化中扮演重要角色，目前有研究发现通过沉默PTB可以将星形胶质细胞重编程为有功能的神经元，并促进相应疾病模型小鼠的功能恢复。脊髓损伤是一种常见且治疗困难的神经创伤，脊髓损伤后星形胶质细胞发生反应性增生并在损伤处形成胶质疤痕，进而抑制神经元和轴突的再生；同时运动神经元大量丢失，受损神经元再生能力有限，这些都给脊髓损伤后修复带来了巨大难度。
本发明的目的是提供多聚嘧啶序列结合蛋白沉默剂联合维甲酸、嘌***胺在制备脊髓损伤的修复药物中的应用。
在本发明中，所述多聚嘧啶序列结合蛋白沉默剂选用的是由慢病毒包装的shRNA-PTB(5’-GGGTGAAGATCCTGTTCAATA-3’)。
另一方面，本发明还提供了一种体外诱导脊髓反应性星形胶质细胞向运动神经元分化的方法，该方法是先通过病毒沉默多聚嘧啶序列结合蛋白，然后采用维甲酸和嘌***胺促进分化。
本发明在体外通过病毒来沉默多聚嘧啶序列结合蛋白(PTB)，同时联合添加与运动神经元分化相关的小分子维甲酸(RA)和嘌***胺(PMA)，成功使小鼠脊髓反应性星形胶质细胞重编程为运动神经元，为进一步体内研究PTB联合小分子的重编程策略在脊髓损伤后修复中的作用提供帮助，进而实现更好的脊髓损伤修复和功能重建效果。
附图说明
图1为原代小鼠脊髓反应性星形胶质细胞模型的建立结果。A为原代小鼠脊髓星形胶质细胞的GFAP染色；10μg/mL LPS处理小鼠脊髓星形胶质细胞24h后GFAP的mRNA水平(B)和蛋白水平(C)相较于control组明显升高。scale bar:200μm。
图2为shRNA-PTB慢病毒感染小鼠脊髓反应性星形胶质细胞沉默PTB的效率结果。shRNA-PTB慢病毒感染细胞2d后PTB的蛋白(A)和mRNA(B)水平明显降低。shRNA-PTB慢病毒(C)感染细胞2d后的荧光显微镜图。scale bar:200μm。
图3为shRNA-PTB慢病毒重编程小鼠脊髓反应性星形胶质细胞的光镜图。scalebar:200μm。
图4为shRNA-PTB慢病毒诱导小鼠脊髓反应性星形胶质细胞重编程神经元荧光图。scale bar:200μm。
图5为shRNA-PTB慢病毒联合RA和PMA重编程小鼠脊髓反应性星形胶质细胞为运动神经元荧光图。scale bar:200μm。
图6为小鼠脊髓反应性星形胶质细胞直接重编程运动神经元的转分化率。
具体实施方式
多聚嘧啶序列结合蛋白(PTB)是一种RNA结合蛋白且在神经元的诱导和分化中扮演重要角色，有研究指出通过沉默PTB可以将中脑的星形胶质细胞重编程为多巴胺能神经元，同时PTB沉默也可以使小鼠视网膜直接重编程为神经节细胞。目前，PTB的“减法”重编程策略大多数都定位于脑和视网膜的研究，其在SCI修复中的作用未有相关报道。同时，有研究表明小分子维甲酸(retinoic acid,RA)参与诱导神经分化、运动神经元轴突的生长；嘌***胺(Purmorphamine，PMA)通过激活sonic hedgehog信号通路来参与神经发生和分化，促进间充质干细胞向运动神经元的分化。因此，脊髓损伤后是否可以较简便地通过星形胶质细胞的PTB沉默联合添加RA和PMA原位诱导重编程获得一定数量的运动神经元，值得进行深入研究。
本发明在体外通过病毒来沉默PTB，同时联合添加与运动神经元分化相关的小分子维甲酸和嘌***胺，成功使小鼠脊髓反应性星形胶质细胞重编程为运动神经元，为进一步体内研究PTB联合小分子的重编程策略在脊髓损伤后修复中的作用提供帮助，进而实现更好的脊髓损伤修复和功能重建效果。
下面结合附图和具体实施例对本发明作进一步详细说明，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照本领域常规条件。
实施例1
1、原代培养获得纯度较高的小鼠脊髓星形胶质细胞
脊髓组织取自新生小鼠。将脊髓置于盛有冰D-Hanks液的培养皿中冲洗2次，用显微外科镊子将表面的脊膜和血管仔细剥除干净，D-Hanks液冲洗2-3次，转移到另一培养皿中，剪碎脊髓至呈乳糜状，将培养皿中剪碎的脊髓块移入15mL离心管中，然后加入等量的0.25％胰酶，在37℃水浴锅中消化15min，每5min吹打混匀至无明显组织块，再加入两倍量的10％FBS完全培养基来终止酶的消化作用；1000rmp、离心5min，然后弃上清，收集细胞沉淀，再用5mL基础培养基重新悬浮细胞，重复离心2次；用10％FBS的完全培养基重悬细胞，并且吹打细胞液分布均匀，用200目筛网过滤，制成初细胞悬液。
将初细胞悬液接种到25cm2培养瓶中，37℃、5％CO 2培养箱中先倒置孵育20min，再轻轻翻转培养瓶，然后取出瓶中的细胞悬液，在除去成纤维细胞成分后；将细胞悬液1000rmp、离心5min后添加5mL含10％FBS完全培养基中，轻轻吹打均匀，细胞计数器计数；再用含10％FBS的完全培养基稀释成5×105/mL，取15mL接种在75cm2培养瓶中。然后每2天后全量换液一次，去除未贴壁的死细胞碎片，使胶质细胞充分生长，每次换液时在显微镜下观察，大约1周后细胞铺满瓶底，然后再进一步纯化培养。当胶质细胞培养至7～9天，细胞铺满培养瓶的底部后，放置恒温振荡培养床上37℃，转速280rmp/min，16～18h(事先将恒温培养床紫外灭菌消毒)。在振荡结束后取出培养瓶，并用75％酒精灭菌消毒；去除培养瓶里的细胞悬液后(其中主要是少突胶质细胞和小胶质细胞)，再用新鲜培养基冲洗两遍，剩余的底层细胞主要为星形胶质细胞。加入1mL的0.25％胰酶进行消化，前后左右轻轻摇动培养瓶，在显微镜下观察细胞胞体缩小变圆后，立即用10％FBS完全培养基终止消化，再用细胞刮子轻轻将细胞刮下，然后用无菌吸管收集悬浮细胞于15mL的离心管中，离心1000rmp/min，5min，重复2次，最后将细胞接种于75cm2培养瓶中培养。
为了保证细胞的活力和纯度，一般选择2～3代的脊髓星形胶质细胞用于后续的实验研究。
2、原代小鼠脊髓反应性星形胶质细胞模型的建立
选择2～3代的脊髓星形胶质细胞，用10μg/mL脂多糖(LPS)刺激细胞24h，通过实时RT-PCR和Western blot等技术检测LPS刺激后星形胶质细胞特异性的基因GFAP的表达变化，使细胞活化成反应性星形胶质细胞。
如图1所示，10μg/mL LPS处理小鼠脊髓星形胶质细胞24h后GFAP的mRNA水平(B)和蛋白水平(C)相较于control组(空白)明显升高，说明成功构建了原代小鼠脊髓反应性星形胶质细胞模型。
3、shRNA-PTB沉默小鼠脊髓反应性星形胶质细胞的PTB
将上海和元生物技术股份有限公司生产的由慢病毒包装的shRNA-PTB(序列5’-GGGTGAAGATCCTGTTCAATA-3’SEQ ID NO.1)，感染反应性星形胶质细胞2d，采用实时RT-PCR和Western blot等技术检测PTB的mRNA和蛋白的表达变化，确定shRNA-PTB的沉默效率。
如图2所示，shRNA-PTB慢病毒感染细胞2d后PTB的蛋白(A)和mRNA(B)水平明显降低，说明shRNA-PTB慢病毒在体外可降低小鼠脊髓反应性星形胶质细胞的PTB表达，且沉默效率达50％左右
4、shRNA-PTB慢病毒重编程小鼠脊髓反应性星形胶质细胞
将包装好的shRNA-PTB慢病毒感染反应性星形胶质细胞，2d后将感染培养液换成诱导培养基(N3/基础培养基：Insulin,sodium selenite,Retinoic acid,putrescine,ChIR99021,SB431542,Db-cAMP,FGF-basic,GDNF)，以后每2d进行诱导培养基的半换液，诱导培养7d、14d、16d、21d、28d；利用免疫细胞化学技术检测MAP2泛神经元标志物和ChAT运动神经元标记物表达情况。
如图3所示，对照组即shCtrl组细胞胞体扁平，形态同于星形胶质细胞；而shPTB组的细胞胞体则逐渐呈球形或锥体形，由胞体伸出数量不等、长短不一的突起，呈现出神经元样形态。
如图4所示，shRNA-PTB重编程16d的细胞为GFP/MAP2双阳性，而shCtrl组则仅为GFP阳性。
由此可知，shRNA-PTB慢病毒可逐渐使小鼠脊髓反应性星形胶质细胞的形态向神经元样转变，并直接重编程其为MAP2+的成熟神经元和ChAT+的运动神经元。
5、shRNA-PTB慢病毒联合RA和PMA重编程小鼠脊髓反应性星形胶质细胞
将包装好的shRNA-PTB慢病毒感染反应性星形胶质细胞，2d后将感染培养液换成诱导培养基(N3/基础培养基+1μM RA+0.5μM PMA，即每1000mL的N3/基础培养基中加入1微摩尔的RA和0.5微摩尔的PMA)，以后每2d进行诱导培养基的半换液，诱导培养7d、14d、16d、21d、28d；利用免疫细胞化学技术检测MAP2泛神经元标志物和ChAT运动神经元标记物表达情况，并统计运动神经元的转分化率。
如图5所示，shRNA-PTB+RA+PMA组和shRNA-PTB组重编程28d的细胞为ChAT阳性，而shCtrl组则仅为GFP阳性。
如图6所示，重编程28d后，shRNA-PTB+RA+PMA组将脊髓反应性星形胶质细胞直接重编程为ChAT阳性的细胞的比率显著高于shRNA-PTB组。
本发明基于脊髓损伤修复的现状、诱导原位重编程的进展，实现体外沉默PTB，同时联合添加小分子RA和PMA，最终成功将脊髓的反应性星形胶质细胞重编程为运动神经元；为PTB联合小分子的重编程策略的体内应用提供理论基础，争取达到更好的脊髓损伤修复和功能重建效果。
本发明将抑制过度增生的反应性星形胶质细胞与补充丢失的运动神经元这两个问题联合起来考虑，设计了一种既能原位补充因脊髓损伤丢失的运动神经元数量，又能同时减少因脊髓损伤而活化增殖的反应性星形胶质细胞的数量，以减轻胶质疤痕的快速增生并改善轴突再生的化学微环境，最终促进脊髓损伤后功能恢复的一举多得的方法。

Claims (3)

  1. 多聚嘧啶序列结合蛋白沉默剂联合维甲酸、嘌***胺在制备脊髓损伤的修复药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于：所述多聚嘧啶序列结合蛋白沉默剂为慢病毒包装的shRNA。
  3. 根据权利要求1所述的应用，其特征在于：所述维甲酸的浓度为1μM，嘌***胺的浓度为0.5μM。
PCT/CN2022/084398 2021-04-16 2022-03-31 多聚嘧啶序列结合蛋白在制备脊髓损伤的修复药物中的应用 WO2022148500A2 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110413331.8A CN113171369A (zh) 2021-04-16 2021-04-16 多聚嘧啶序列结合蛋白在制备脊髓损伤的修复药物中的应用
CN202110413331.8 2021-04-16

Publications (2)

Publication Number Publication Date
WO2022148500A2 true WO2022148500A2 (zh) 2022-07-14
WO2022148500A3 WO2022148500A3 (zh) 2022-08-25

Family

ID=76923511

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/084398 WO2022148500A2 (zh) 2021-04-16 2022-03-31 多聚嘧啶序列结合蛋白在制备脊髓损伤的修复药物中的应用

Country Status (2)

Country Link
CN (1) CN113171369A (zh)
WO (1) WO2022148500A2 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113171369A (zh) * 2021-04-16 2021-07-27 南通大学 多聚嘧啶序列结合蛋白在制备脊髓损伤的修复药物中的应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102899285A (zh) * 2011-07-29 2013-01-30 复旦大学 一种体外诱导胚胎干细胞分化成为神经细胞方法
CN104313025A (zh) * 2014-08-29 2015-01-28 南通大学 抑制中枢神经***损伤后胶质疤痕形成的siRNA及其重组载体与应用
GB2588307B (en) * 2018-04-11 2023-08-16 Univ California Reprogramming of non-neuronal cells into neurons and methods and compositions to treat neurodegenerative diseases and disorders
CN113171369A (zh) * 2021-04-16 2021-07-27 南通大学 多聚嘧啶序列结合蛋白在制备脊髓损伤的修复药物中的应用

Also Published As

Publication number Publication date
WO2022148500A3 (zh) 2022-08-25
CN113171369A (zh) 2021-07-27

Similar Documents

Publication Publication Date Title
JP2020097634A (ja) 神経変性を治療するための方法及び組成物
US9439931B2 (en) Administering umbilical cord blood-derived mesenchymal stem cells to treat nerve injury
CN111500578A (zh) 调控ADSCs成骨分化及组织再生Circ RNA-FTO及其应用
WO2022148500A2 (zh) 多聚嘧啶序列结合蛋白在制备脊髓损伤的修复药物中的应用
TWI397585B (zh) 細胞培養方法及其應用
JP2010503727A (ja) 皮膚由来の幹細胞を含有する神経疾患治療用の細胞治療剤
CN111344392B (zh) 一种细胞诱导的方法
WO2013071469A1 (zh) 使人的体细胞逆向分化产生自体视网膜干细胞和自体视网膜细胞的方法、试剂盒及用途
CN113943699B (zh) 对抗高糖损伤的脐带间充质干细胞诱导液、方法及应用
CN110302398A (zh) 一种含有Atoh7和/或Pou4f的组合物、其制备方法及医药用途
KR102322635B1 (ko) 말초신경양 미세조직 제조방법 및 이의 용도
CN115671136A (zh) M0或M1型Ly6C+CX3CR1+单核细胞来源巨噬细胞在治疗肝纤维化中的用途
KR102552191B1 (ko) 중간엽 줄기세포가 각막 상피세포로의 분화를 유도하는 무혈청 완전 배지
CN109745331A (zh) Tert激活剂在制备治疗神经损伤药物中的应用
KR20190130394A (ko) 전구세포 배양액 및 다층 그래핀 필름을 포함하는 줄기세포 분화 촉진용 조성물 및 이의 용도
Pradana et al. Expression of Growth Associated Protein-43 (GAP-43) and Brain-derived Neurotrophic Factor (BDNF) from Adipose Mesenchymal Stem Cells (ASC) with fresh frozen nerve scaffold in 5% hypoxic condition
US20060263876A1 (en) Neural crest stem cells and uses thereof
CN116656609A (zh) 一种骨髓神经嵴细胞分泌的胞外囊泡的制备方法及用途
Fortuna et al. Three-dimensional Collagen Scaffolds in Cultures of Olfactory Ensheathing Cells Used for Severed Spinal Cord Regeneration
CN114107297A (zh) 一种miRNA模拟物及其应用
CN117487753A (zh) 一种治疗型小胶质细胞的建立和应用
Zou et al. Linc-NSC affects cell differentiation, apoptosis and proliferation in mouse neural stem cells and embryonic stem cells in vitro and in vivo
CN116942691A (zh) miR-20b-5p在制备促进神经再生和修复神经损伤药物及神经移植物中的应用
CN113789301A (zh) 一种制备高纯度的诱导性多能干细胞来源的人脑多巴胺能神经元的方法
CN116144595A (zh) 一种促使msc分化为少突胶质前体细胞的方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22736661

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22736661

Country of ref document: EP

Kind code of ref document: A2