CN113171369A - 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 PDFInfo
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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、嘌吗啡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)和嘌吗啡胺(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
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、嘌吗啡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系统损伤性疾病,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、嘌吗啡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吗啡胺促进分化。
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)和嘌吗啡胺(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;10 μg/mL LPS处理小鼠脊髓星形胶质细胞24 h后GFAP的mRNA水平(B)和蛋白水平(C)相较于control组明显升高。scale bar: 200 μm。
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2 d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2 d后的荧光显微镜图。scale bar: 200 μm。
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;嘌吗啡胺(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吗啡胺,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15 mL离心管中,然后加入等量的0.25%胰酶,在37℃水浴锅中消化15 min,每5 min 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;1000 rmp、离心5 min,然后弃上清,收集细胞沉淀,再用5 mL 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25 cm2培养瓶中,37℃、5% CO2培养箱中先倒置孵育20 min,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5 min后添加5 mL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15 mL接种在75 cm2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280 rmp/min,16~18 h(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1 mL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15 mL的离心管中,离心1000 rmp/min,5min,重复2次,最后将细胞接种于75 cm2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10 μg/mL 脂多糖(LPS)刺激细胞24 h,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,10 μg/mL LPS处理小鼠脊髓星形胶质细胞24 h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2 d,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2 d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,2 d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2 d进行诱导培养基的半换液,诱导培养7 d、14 d、16 d、21d、28 d;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16 d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,2 d后将感染培养液换成诱导培养基(N3/基础培养基+1 μM RA+ 0.5 μM PMA,即每1000 mL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2 d进行诱导培养基的半换液,诱导培养7 d、14 d、16 d、21d、28 d;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28 d的细胞为ChAT阳性,而shCtrl 组则仅为GFP阳性。
如图6所示,重编程28 d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。
序列表
<110> 南通大学
<120> 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
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gggtgaagat cctgttcaat a 21
Claims (3)
1.多聚嘧啶序列结合蛋白沉默剂联合维甲酸、嘌吗啡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1 μM,嘌吗啡胺的浓度为0.5 μM。
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