CN110804593B - 一种诱导皮肤成纤维细胞直接向神经元转分化的小分子化合物组合及应用 - Google Patents
一种诱导皮肤成纤维细胞直接向神经元转分化的小分子化合物组合及应用 Download PDFInfo
- Publication number
- CN110804593B CN110804593B CN201910988806.9A CN201910988806A CN110804593B CN 110804593 B CN110804593 B CN 110804593B CN 201910988806 A CN201910988806 A CN 201910988806A CN 110804593 B CN110804593 B CN 110804593B
- Authority
- CN
- China
- Prior art keywords
- neurons
- transdifferentiation
- inducing
- small molecule
- compound combination
- Prior art date
- Legal status (The legal status 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 status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/0619—Neurons
-
- 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
-
- 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
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- 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
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/415—1,2-Diazoles
- A61K31/416—1,2-Diazoles condensed with carbocyclic ring systems, e.g. indazole
-
- 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
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4409—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 4, e.g. isoniazid, iproniazid
-
- 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
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
- A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
-
- 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
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
-
- 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
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/15—Transforming growth factor beta (TGF-β)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/35—Vasoactive intestinal peptide [VIP]; Pituitary adenylate cyclase activating polypeptide [PACAP]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/70—Enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/70—Enzymes
- C12N2501/73—Hydrolases (EC 3.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Neurology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Neurosurgery (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Materials For Medical Uses (AREA)
Abstract
本发明提供了一种诱导皮肤成纤维细胞直接向神经元转分化的小分子化合物组合,所述的小分子化合物组合包括:CHIR99021,Forskolin,LDN193189,SB431542,SP600125,VPA和Y27632,用于诱导皮肤成纤维细胞向神经元的转分化,修复长距离外周神经缺损及中枢神经损伤。本发明的转分化技术可以高效获得神经元,并将其移植到脊髓损伤的动物模型后获得显著的疗效,通过运动能力改善,皮层诱发电位恢复等指标得出,本发明为脊髓损伤在内的相关神经系统疾病的治疗开辟了新的途径。
Description
技术领域
本发明涉及生物技术和神经发育领域,具体涉及一种诱导成纤维细胞直接向神经元转分化的小分子化合物组合及其用途。
背景技术
由于终末分化的神经元不能再生,中枢神经系统损伤不可逆。尽管干细胞具有分化为包括神经元在内的多种神经细胞的潜能,干细胞疗法也被认为是一种很有前景的中枢神经系统损伤修复解决方案,但研究表明移植到受损组织中的神经干细胞更容易分化为星形胶质细胞或少突胶质细胞,很少分化为功能性神经元。此外,干细胞移植应用仍面临几个主要障碍,包括肿瘤形成、移植物排斥和伦理问题。因此,找到治疗神经损伤和神经系统疾病更合适的治疗方法至关重要。
神经科学和细胞生物学的发展已经为获得神经元提供了多样化的解决方案。与诱导干细胞分化相比,将体细胞直接重编程为不经过干细胞状态的神经元具有显著优势,如分化效率高,诱导周期短,无伦理问题,潜在致瘤风险低等。用于神经元重编程的主要方法包括小分子诱导、转录因子和或miRNA转导以及外泌体转运等,已被用于将体细胞转分化为相应神经疾病所需的各种神经元亚型。出于对临床应用的安全性考虑,基于小分子的重编程比转基因方法更具有优势,后者具有外源DNA序列基因组插入的风险。小分子是细胞可渗透的和非免疫原性的,易于合成、保存、标准化和操作,并且成本低。此外,它们的生物学效应快速且通常是可逆的,并且可以通过不同的浓度和组合精确控制。最近,越来越多的研究表明,纯化合物即可诱导各种体细胞向神经细胞转分化。皮肤成纤维细胞是组织工程的优秀种子细胞,具有来源丰富、分布广泛、体外扩增能力强和便于自体移植等优点。研究表明,通过小分子诱导可以将不同来源和不同种属的成纤维细胞重编程为神经元,甚至是功能性神经元亚型。总之,安全有效的小分子诱导的神经元重编程方法具有巨大的临床应用潜力。
脊髓损伤(SCI)是由创伤性损伤或非创伤性损伤(如感染和肿瘤)引起的一种严重且不可逆的病状。SCI后的永久性功能和神经功能缺损以及再生能力有限归因于许多继发性细胞和分子反应,例如神经胶质和神经元细胞死亡,轴突丧失,脱髓鞘,炎症,坏死腔,神经胶质瘢痕形成和神经营养因子缺乏。尽管已被广泛研究,但仍没有有效的临床治疗方法SCI。
一种有希望的治疗策略旨在通过细胞移植替代失去的神经元,并与宿主神经元重建新的神经元回路。
发明内容
要解决的技术问题:本发明的目的是提供一种诱导皮肤成纤维细胞直接向神经元转分化的小分子化合物组合,用于诱导皮肤成纤维细胞向神经元的转分化,用来修复长距离外周神经缺损及中枢神经损伤。
技术方案:一种诱导皮肤成纤维细胞直接向神经元转分化的小分子化合物组合,所述的小分子组合包括:CHIR99021(GSK-3抑制剂),Forskolin(腺苷酸环化酶激活剂),LDN193189(BMP信号抑制剂),SB431542(选择性ALK5 /TGF-βI型受体抑制剂),SP600125(可逆且具有ATP竞争能力的JNK抑制剂),VPA(组蛋白脱乙酰基酶1的抑制剂)和Y27632(ROCK-I和ROCK-II的ATP竞争性抑制剂)。
优选的,所述CHIR99021,Forskolin,LDN193189,SB431542,SP600125,VPA和Y27632的摩尔比为(0.1-3):(0.5-20):(0.01-0.25):(0.1-5):(0.1-10):(100-1000):(0.1-30)。
优选的,所述CHIR99021,Forskolin,LDN193189,SB431542,SP600125,VPA和Y27632的摩尔比为3:50:0.1:2:10:500:5。
优选的,所述小分子化合物组合用于将皮肤成纤维细胞转分化为神经元,或用于制备将皮肤成纤维细胞转分化为神经元的药物组合物。
上述的小分子化合物组合在诱导皮肤成纤维细胞直接向神经元转分化的应用。
优选的,所述皮肤成纤维细胞为人皮肤成纤维细胞。
有益效果:本发明的诱导皮肤成纤维细胞直接向神经元转分化的小分子化合物组合具有以下优点:
1、筛选获得的小分子组合CFLSSVY(CHIR99021,Forskolin,LDN193189,SB431542,SP600125,VPA和Y27632)以往并无报道,为首次证实该组合高效率诱导转分化效应,CFVLSSY诱导获得了较高的神经元阳性率(即Tubb3阳性率),在分化7天时,Tubb3阳性的细胞高达82.09±1.07%,分化14天后,Tubb3阳性细胞进一步增加,达到87.03±1.03%(图1和图3B)。此外,此小分子组合也能高效诱导小鼠和人的皮肤成纤维向神经元的转分化,为60%和50%左右(图3C),本发明筛选的CFLSSVY的诱导转化率较高,高于现有同等技术中的转化率;
2、本发明的转分化技术可以高效获得神经元,并将其移植到脊髓损伤的动物模型后获得显著的疗效,通过运动能力改善,皮层诱发电位恢复等指标得出,本发明为脊髓损伤在内的相关神经系统疾病的治疗开辟了新的途径。
附图说明
图1. 本发明诱导皮肤成纤维细胞直接向神经元转分化的小分子组合筛选,筛选出CFLSSVY;
图2. 筛选获得的小分子组合CFLSSVY诱导大鼠皮肤成纤维细胞(RDFs)分化方案示意图;
图3. 小分子组合CFLSSVY诱导大鼠、小鼠和人的皮肤成纤维转分化为神经元前成纤维特异性标志物Vimentin和S100A4以及转分化后神经元特异性标志物Tubb3的免疫荧光染色检测及Tubb3阳性细胞比例的统计;
图4. Tubb3启动子报告系统载体示意图以及转分化诱导后细胞中mcherry的表达情况及其流式细胞术检测;
图5. RDFs经诱导转分化14 d和28 d后,成熟神经元特异标志物NeuN和Synapsin-1的免疫荧光检测;
图6. CFLSSVY诱导RDFs转分化为神经元过程中成纤维相关基因及神经元相关基因的表达变化;
图7. RDFs转分化获得的神经元与载体3D丝素蛋白支架一起移植到大鼠受损脊髓促进后肢运动能力恢复,表现为移植化学诱导的神经元CiNs组(3D-SF-CiNs)Basso-Beattie-Bresnahan (BBB)运动功能评分显著高于对照组(sham,SCI和3D-SF),并在45°倾斜栅格测试视频的连续延时帧中显示了两个后肢之间的协调性,其中,Sham为只敲除椎板未伤及脊髓的假手术组,SCI为脊髓损伤不处理组,3D-SF为移植单纯丝蛋白支架组,3D-SF-CiNs为移植载有CiNs的丝蛋白支架组;
图8. 神经元组(3D-SF-CiNs)移植入大鼠脊髓缺损后皮层运动诱发电位与其他对照组相比潜伏期较短,电压幅度较大,指示恢复较好,其中,SCI为神经损伤不处理组,3D-SF为单纯丝蛋白组,3D-SF-CiNs为移植载有CiNs的丝蛋白支架组;
图9. 脊髓损伤后单独3D丝素蛋白支架和载着CiNs的3D丝素支架蛋白植入大鼠脊髓缺损后切片的H&E染色及各组脊髓空洞面积检测,其中,SCI为神经损伤不处理组,3D-SF为单纯丝蛋白组,3D-SF-CiNs为移植载有CiNs的丝蛋白支架组;
图10. 各处理组(SCI,3D-SF和3D-SF-CiNs)植入大鼠脊髓缺损后8周,纵向切面的NF-H和GFAP免疫荧光染色及其荧光面积及荧光强度统计。其中,损伤区域靠近头端(rostral),损伤区域中间(centre)和损伤区域靠近尾端(caudal);
图11. CiNs植入大鼠脊髓缺损8周后可见存活的诱导神经元(GFP标记),并同时表达NF-H,且与宿主中NF-H指示的神经元紧密相连,相互作用;此外,损伤区域NF-H和MBP的共定位代表移植后神经纤维的再髓鞘化。
具体实施方式
实施例1
高效诱导皮肤成纤维细胞向神经元转分化的小分子组合筛选
1细胞培养
1.1成纤维细胞的制备和接种
动物的使用符合美国国立卫生研究院《实验动物的护理和使用指南》,并由苏州大学动物护理和使用委员会批准。根据世界医学协会《道德守则》(赫尔辛基宣言)进行了涉及人类受试者的实验。按文献报道,从大鼠,小鼠或人类体表提取并制备了皮肤成纤维细胞。简要地说,从新生大鼠或小鼠的背部解剖了一块皮肤,经组织剪碎消化,贴壁培养获得RDFs(大鼠皮肤成纤维细胞)和MDFs(小鼠皮肤成纤维细胞)。在所有捐助者的书面知情同意下并经机构伦理委员会批准收集和使用人类样品,人类成人真皮成纤维细胞(HDFs)取自包皮环切手术后健康个体的新生儿包皮。将真皮的皮肤组织切成小块,并用胰蛋白酶消化。所有分离的成纤维细胞均在成纤维细胞培养基(杜尔贝科改良的Eagle培养基(DMEM)中添加10%FBS,0.1 mM非必需氨基酸和2 mM Glutamax)中培养,扩增2-3代再使用。通过对波形蛋白Vimentin和S100A4(> 95%)进行免疫染色来确定纯度(图3A)。
1.2化学诱导皮肤成纤维细胞向神经元细胞的转分化
当接种在PDL包被的35 mm培养皿中的皮肤成纤维细胞汇合到60%~70%时,用含有N3培养基(即DMEM/F12,25 μg/ml胰岛素,50 μg/ml脱铁转铁蛋白,30 nM亚硒酸钠,20 nM孕酮,100 nM腐胺,1%双抗)并加入50 μM抗坏血酸,20 ng/ml bFGF,0.5% N2和1% B27的神经元诱导培养基代替成纤维细胞培养基,向其中添加了含有不同小分子或者小分子化合物组合的诱导培养基(CHIR99021,3 μM;Forskolin,50 μM;LDN193189,0.1 μM;SB431542,2 μM;SP600125,10 μM;VPA,0.5 mM和Y27632,5 μM):
(1)CFV(CHIR99021,Forskolin和VPA)
(2)CFV+LDN(CHIR99021,Forskolin,VPA和LDN193189)
(3)CFV+SB(CHIR99021,Forskolin,VPA和SB431542)
(4)CFV+SP(CHIR99021,Forskolin,VPA和SP600125)
(5)CFV+Y(CHIR99021,Forskolin,VPA和Y27632)
(6)CFV+LSSY(CHIR99021,Forskolin,LDN193189,SB431542,SP600125,VPA和Y27632)
(7)LDN+SB(LDN193189和SB431542)
(8)LDN+SP(LDN193189和SP600125)
(9)LDN+Y(LDN193189和Y27632)
(10)SB+SP(SB431542和SP600125)
(11)SB+Y(SB431542和Y27632)
(12)SP+Y(SP600125和Y27632)
(13)LDN(LDN193189)
(14)SB(SB431542)
(15)SP(SP600125)
(16)Y(Y27632)
以上16种小分子化合物或小分子化合物组合诱导大鼠皮肤成纤维细胞8 d,每隔一天更换一次培养基。为了更进一步促进神经元成熟,将神经元诱导培养基替换为成熟培养基(神经诱导培养基加入20 ng/ml BDNF,20 ng/ml NT3和20 ng/ml GDNF,并加小分子CFS(CHIR99021,3 μM;Forskolin,50 μM和SP600125,10 μM)以进一步促进诱导的神经元存活。14天后,换成不含bFGF和化学物质的神经元成熟培养基。每隔一天更换一次培养基。(图2)不同的小分子组合诱导的效率各不相同,其中CFV+LSSY获得了较高的神经元阳性率(Tubb3阳性率)在7天时,Tubb3阳性的细胞高达82.09 ± 1.07%,14天后,Tubb3阳性细胞进一步增加,达到87.03 ± 1.03%(图1和图3B)。此外,此小分子组合也能高效诱导小鼠和人的皮肤成纤维向神经元的转分化,为60%和50%左右(图3C),在诱导人的皮肤成纤维向神经元的转分化的阳性率方面,本发明的组合与其他人报道化学诱导相比效率更高,因此能获得更多的功能细胞用于神经损伤修复。
实施例2
化学诱导的神经元(CiNs)的鉴定
2.1免疫荧光染色
将接种在2D-PDL或3D-SF的细胞在冷4%多聚甲醛(PFA)中固定30分钟,用PBS洗涤3次(每次10分钟),在室温1.5小时或在4℃过夜孵育一抗。一抗用稀释液(PBS /0.02%NaN3/3%牛血清白蛋白(BSA)/0.2% Triton X-100)稀释,并在以下工作浓度下用:小鼠单克隆抗体(mAb)抗微管蛋白β3(Tubb3)(1:500稀释; BioLegend),兔mAb抗突触蛋白1(1:200稀释;Cell Signaling Technologies)和兔mAb抗NeuN(1:500稀释; Cell SignalingTechnologies)。此后,将细胞用PBS洗涤3次(10分钟)。将二抗Alexa FluorTM 594山羊抗小鼠或兔IgG(H + L)(Invitrogen)在PBS/0.02%NaN3/3%BSA中按1:200稀释,并在黑暗中于室温下孵育细胞1小时。随后将细胞在PBS中清洗3次(10分钟),并用Hoechst 33258(1:100稀释; Sigma-Aldrich)对细胞核进行复染。皮肤成纤维经CFLSSVY诱导后,7天显示Tubb3阳性,14天表达成熟神经元特异标志物NeuN,28天,表达功能性神经元特有的突触相关蛋白Synapsin-1(图5)。
2.2 Tubb3启动子报告基因系统验证转分化的效率
将Tubb3(NM_139254.2)启动子融合荧光蛋白mCherry连接到慢病毒载体中,以指示Tubb3的表达和神经元的转分化效率。小分子诱导皮肤成纤维细胞后,通过共聚焦显微镜和流式细胞仪检测表达红色荧光蛋白mcherry的细胞比例(图4)。
2.3 RNA提取及实时定量PCR检测成纤维相关基因及神经元相关基因的表达变化
使用Trizol试剂提取总RNA。根据供应商推荐的程序,使用RevertAid FirstStrand cDNA合成试剂盒从1 μg总RNA中合成cDNA。通过使用表1中列出的引物及SYBRPremix Ex TaqTM进行实时定量PCR(RT-PCR)检测。所有样品一式三份进行处理。将平均值用于测量,并且将结果相对于GAPDH的表达标准化。
结果如图6,在转分化过程中,成纤维相关基因表达显著下降,神经元相关的基因表达呈现先升高后下降的趋势。
实施例3
移植化学诱导的神经元(CiNs)治疗脊髓损伤
3.1 丝蛋白微纳米纤维多孔支架3D-SF的构建
蚕丝经0.05%(w/v)煮沸脱胶后,分散于含有0.1%氯化钙(w/v)的98%甲酸中(蚕丝:溶液比1:50),蚕丝分散液置于-20℃下冷冻24 h,然后于75%乙醇中解冻处理1h,再用去离子水中充分洗涤,对得到的湿态丝蛋白支架进行冷冻干燥即得到三维丝蛋白微纳米纤维多孔支架,经辐照灭菌后用于生物实验。
3.2 3D-SF上皮肤成纤维细胞的诱导分化
皮肤成纤维细胞接种在3D-SF上,用含有N3培养基(即DMEM/F12,25 μg/ml胰岛素,50 μg/ml脱铁转铁蛋白,30 nM亚硒酸钠,20 nM孕酮,100 nM腐胺,1%双抗)并加入50 μM抗坏血酸,20 ng/ml bFGF,0.5% N2和1% B27的神经元诱导培养基代替成纤维细胞培养基,向其中添加了含有小分子化合物组合CFLSSVY的诱导培养基,诱导细胞8 d,每隔一天更换一次培养基。为了更进一步促进神经元成熟,将神经元诱导培养基替换为成熟培养基(神经诱导培养基加入20 ng/ml BDNF,20 ng/ml NT3和20 ng/ml GDNF,并加小分子CFS(CHIR99021,3 μM;Forskolin,50 μM和SP600125,10 μM)以进一步促进诱导的神经元存活。14天后,换成不含bFGF和化学物质的神经元成熟培养基,从而获得3D-SF-CiNs.
3.3 脊髓横断模型的建立
使用从中国苏州大学实验动物中心购买的成年雌性Sprague-Dawley大鼠(220-250 g)。动物的使用符合《美国实验动物护理和使用指南》国立卫生研究院,并由苏州大学动物护理和使用委员会批准。用4%水合氯醛(0.1 mL/kg,腹膜内)麻醉所有动物。麻醉后,将手术部位剃毛并消毒。使用细尖的咬骨钳在T9-T10水平进行了背椎板切除术。通过使用尖头镊子和微剪刀进行两个完整的切口,将T10水平的2 mm脊髓段完全切除。用明胶彻底止血后,将动物分为四组(每组n = 10):3D-SF组接受3D-SF(2毫米厚和3毫米直径的没有细胞的丝纤维支架束),3D-SF-CiNs组接受3D-SF与CiNs联合治疗,SCI组接受脊髓损伤而不进行移植,而Sham组接受椎板切除术作为对照。术后动物接受了广泛的术后护理,包括青霉素(50,000 U/kg/d)注射5天,以限制感染和每天两次人工排尿直至膀胱张力和功能恢复。(图7A)
3.4 行为评估
在开放视野中进行21分的Basso,Beattie和Bresnahan(BBB)运动评分,并由两名对处理不知情的检查员观察4分钟,以评估后肢运动功能,包括关节运动,踩踏能力,每只大鼠在受伤后第1天以及每周长达8周的协调性和躯干稳定性。对于45°倾斜网格攀爬测试,所有行为评估均被录像。简而言之,每只动物都必须使用前肢和后肢移动身体,以到达网格的顶部。每个治疗组每次至少测试八只大鼠,每次疗程测试三次。结果如图7 B和C显示,移植神经元组(3D-SF-CiNs)Basso-Beattie-Bresnahan (BBB)运动功能评分显著高于对照组(SCI和3D-SF),并在45°倾斜栅格测试的视频的连续延时帧中显示了两个后肢之间的协调性
3.5 皮层运动诱发电位(cMEP)
术后八周,使用BIOPAC MP150在所有大鼠(假手术组,SCI组,3D-SF组和3D-SFCiNs组n = 6)中记录cMEPs。系统评估脊髓的生理完整性和后肢运动功能的恢复。麻醉后,用牙钻在颅骨中暴露运动皮层,通过脑立体定位仪找到前囟后2毫米处,在中线左侧或右侧2毫米处。为了诱导皮层cMEP,将两个钢质刺激电极放置在运动皮层上的此坐标位置,将记录电极插入对侧腓肠肌深2 mm处,并将参比电极深处置于背侧皮肤中3毫米,cMEP通过经颅电刺激诱发,电流强度为30 V,持续时间为0.2 ms。从单个cMEP扫描(200次扫描)的值测量平均峰值幅度和潜伏期的平均值。如图8,移植神经元组(3D-SF-CiNs)移植入大鼠脊髓缺损后皮层运动诱发电位与对照组相比潜伏期较短,电压幅度较大,指示恢复较好。
3.6 组织处理,组织学染色和免疫荧光染色
所有大鼠均用4%水合氯醛(0.1 mL/kg,腹膜内)深度麻醉,并通过升主动脉灌注0.9% NaCl。在4℃下在4% PFA中固定48小时后,将脊髓节段在PBS中的30%(w/v)蔗糖溶液中脱水。去除脊膜后,将包含病变部位的脊髓切成2厘米长,并包埋在OCT化合物中。使用低温恒温器(Leica CM1900)切割厚度为25 μm的切片,并将其贴附在明胶包被的载玻片上进行免疫荧光和H&E(苏木精和曙红)染色。如进行免疫荧光染色,在含有5% BSA和0.3% Triton的PBS中于室温孵育1小时。切片孵育一抗:兔抗GFAP(1:200稀释; Cell SignalingTechnologies)抗体来指示神经胶质瘢痕,小鼠抗NF-H(1:200稀释; Cell SignalingTechnologies)抗体检测轴突再生和兔抗MBP(1:200稀释;Cell Signaling Technologies)以评估髓鞘形成。细胞核是使用Hoechst 33258(1:100稀释;Sigma Aldrich)进行标记。使用共焦显微镜进行荧光显微拍照。如图9-11,术后8周,H&E染色结果表明,化学诱导的神经元植入受损脊髓后,大鼠脊髓缺损部位的空洞面积显著降低,提示对组织再生具有促进作用。NF-H和GFAP的组织免疫荧光染色显示大鼠脊髓缺损部位NF-H阳性神经纤维面积显著增大、GFAP阳性胶质面积明显降低,表明良好的神经轴突再生及胶质瘢痕的抑制作用。此外,8周后仍可见存活的诱导神经元,并表达NF-H,且与宿主NF-H阳性的神经元紧密联系,损伤区域NF-H与MBP的共定位指示移植后神经纤维的再髓鞘化。
Claims (3)
1.一种诱导皮肤成纤维细胞直接向神经元转分化的小分子化合物组合,其特征在于,所述的小分子化合物组合为:CHIR99021,Forskolin,LDN193189,SB431542,SP600125,VPA和Y27632,其中所述CHIR99021,Forskolin,LDN193189,SB431542,SP600125,VPA和Y27632的摩尔比为3:50:0.1:2:10:500:5。
2.如权利要求1所述的小分子化合物组合在诱导皮肤成纤维细胞直接向神经元转分化的应用。
3.根据权利要求2所述应用,其特征在于:所述皮肤成纤维细胞为人皮肤成纤维细胞。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910988806.9A CN110804593B (zh) | 2019-10-17 | 2019-10-17 | 一种诱导皮肤成纤维细胞直接向神经元转分化的小分子化合物组合及应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910988806.9A CN110804593B (zh) | 2019-10-17 | 2019-10-17 | 一种诱导皮肤成纤维细胞直接向神经元转分化的小分子化合物组合及应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110804593A CN110804593A (zh) | 2020-02-18 |
CN110804593B true CN110804593B (zh) | 2021-09-03 |
Family
ID=69488502
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910988806.9A Active CN110804593B (zh) | 2019-10-17 | 2019-10-17 | 一种诱导皮肤成纤维细胞直接向神经元转分化的小分子化合物组合及应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110804593B (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105039258A (zh) * | 2015-07-03 | 2015-11-11 | 北京大学 | 将非神经元细胞重编程为神经元样细胞的方法和组合物 |
CN106399248A (zh) * | 2016-09-30 | 2017-02-15 | 浙江大学 | 一种诱导成纤维细胞转分化为神经细胞的方法 |
CN106635991A (zh) * | 2016-12-30 | 2017-05-10 | 东莞惠恩生物工程有限公司 | 人皮肤成纤维细胞诱导为神经细胞的方法与应用 |
-
2019
- 2019-10-17 CN CN201910988806.9A patent/CN110804593B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105039258A (zh) * | 2015-07-03 | 2015-11-11 | 北京大学 | 将非神经元细胞重编程为神经元样细胞的方法和组合物 |
CN106399248A (zh) * | 2016-09-30 | 2017-02-15 | 浙江大学 | 一种诱导成纤维细胞转分化为神经细胞的方法 |
CN106635991A (zh) * | 2016-12-30 | 2017-05-10 | 东莞惠恩生物工程有限公司 | 人皮肤成纤维细胞诱导为神经细胞的方法与应用 |
Non-Patent Citations (2)
Title |
---|
Direct Conversion of Normal and Alzheimer’s Disease Human Fibroblasts into Neuronal Cells by Small Molecules;Wenxiang Hu等;《Cell Stem Cell》;20150806;第17卷(第2期);摘要,第205页图1、右栏第一段,第207页左栏第1段,第211页左栏第1段 * |
Highly efficient direct conversion of human fibroblasts to neuronal cells by chemical compounds;Ping Dai等;《Journal of clinical biochemistry and nutrition》;20150401;第56卷(第3期);第167页右栏第1段 * |
Also Published As
Publication number | Publication date |
---|---|
CN110804593A (zh) | 2020-02-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Woerly | Restorative surgery of the central nervous system by means of tissue engineering using NeuroGel implants | |
Noble et al. | Precursor cell biology and the development of astrocyte transplantation therapies: lessons from spinal cord injury | |
EP2076588B1 (en) | Expansion method for adult stem cells from blood, particularly peripheral blood, and relative application in medical field | |
KR101686315B1 (ko) | 편도 유래 중간엽 줄기세포로부터 슈반 세포의 분화 방법 | |
Hu et al. | Transplantation of neural scaffolds consisting of dermal fibroblast-reprogrammed neurons and 3D silk fibrous materials promotes the repair of spinal cord injury | |
Liard et al. | Adult‐brain‐derived neural stem cells grafting into a vein bridge increases postlesional recovery and regeneration in a peripheral nerve of adult pig | |
Zhu et al. | Bioorthogonal DOPA-NGF activated tissue engineering microunits for recovery from traumatic brain injury by microenvironment regulation | |
Li et al. | Platelet-rich fibrin-induced bone marrow mesenchymal stem cell differentiation into osteoblast-like cells and neural cells☆ | |
Abdolahi et al. | Improvement of rat spinal cord injury following lentiviral vector-transduced neural stem/progenitor cells derived from human epileptic brain tissue transplantation with a self-assembling peptide scaffold | |
Shen et al. | Transplantation of adult spinal cord grafts into spinal cord transected rats improves their locomotor function | |
Vasyliev et al. | Effects of neural crest-derived multipotent stem cells on regeneration of an injured peripheral nerve in mice | |
Chen et al. | Regeneration of sciatic nerves by transplanted microvesicles of human neural stem cells derived from embryonic stem cells | |
US11622964B2 (en) | Method for destroying cellular mechanical homeostasis and promoting regeneration and repair of tissues and organs, and use thereof | |
JP2016210730A (ja) | 医薬組成物及びその製造方法並びに医薬品 | |
CN103127494A (zh) | 一种神经再生生物胶及其制备方法和用途 | |
Xie et al. | Co-transplantation of MRF-overexpressing oligodendrocyte precursor cells and Schwann cells promotes recovery in rat after spinal cord injury | |
CN110804593B (zh) | 一种诱导皮肤成纤维细胞直接向神经元转分化的小分子化合物组合及应用 | |
CN111484977B (zh) | 重编程产生功能性去甲肾上腺素能神经元的方法 | |
CN112795535A (zh) | 诱导间充质干细胞释放促进皮肤上皮细胞分化特定功能外泌体的组合物及其应用 | |
CN110656087B (zh) | 一种manf基因修饰的脐带间充质干细胞及其制备方法与应用 | |
Khazaei et al. | Generation of definitive neural progenitor cells from human pluripotent stem cells for transplantation into spinal cord injury | |
US20220370507A1 (en) | Endothelial and smooth muscle like tissue produced from urine cells and uses related thereto | |
Li et al. | Effect of valproic acid combined with transplantation of olfactory ensheathing cells modified by neurotrophic 3 gene on nerve protection and repair after traumatic brain injury | |
Kosykh et al. | Postnatal neural crest stem cells from hair follicle interact with nerve tissue in vitro and in vivo | |
CN111886016A (zh) | 治疗脊髓损伤的组合物和方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |