阅读说明：本技术 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 (Small molecular compound combination for inducing skin fibroblast to directly transdifferentiate towards neuron and application ) 是由 胡雅楠 张焕相 张锋 杨敏 何其圣 于 2019-10-17 设计创作，主要内容包括：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。(The invention provides a small molecule compound combination for inducing the direct transdifferentiation of skin fibroblasts to neurons, which comprises the following components in part by weight: CHIR99021, Forskolin, LDN193189, SB431542, SP600125, VPA and Y27632 are used for inducing transdifferentiation of skin fibroblasts to neurons and repairing long-distance peripheral nerve defects and central nerve injuries. The transdifferentiation technology of the invention can efficiently obtain neurons, and the neurons can obtain obvious curative effect after being transplanted to an animal model of spinal cord injury, and can be obtained by indexes such as motor ability improvement, cortex evoked potential recovery and the like.)

1. A combination of small molecule compounds for inducing transdifferentiation of dermal fibroblasts directly into neurons, said combination of small molecule compounds comprising: CHIR99021, Forskolin, LDN193189, SB431542, SP600125, VPA and Y27632.

2. The combination of small molecule compounds for inducing transdifferentiation of dermal fibroblasts directly into neurons according to claim 1, wherein: the molar ratio of CHIR99021, Forskolin, LDN193189, SB431542, SP600125, VPA and Y27632 is (0.1-3): (0.5-20): (0.01-0.25): (0.1-5): (0.1-10): (100-1000): (0.1-30).

3. The combination of small molecule compounds for inducing transdifferentiation of dermal fibroblasts directly into neurons according to claim 1, wherein: the molar ratio of CHIR99021, Forskolin, LDN193189, SB431542, SP600125, VPA and Y27632 is 3: 50: 0.1: 2: 10: 500: 5.

4. the combination of small molecule compounds for inducing transdifferentiation of dermal fibroblasts directly into neurons according to claim 1, wherein: the small molecule compound combination is used for transdifferentiating skin fibroblasts into neurons or is used for preparing a pharmaceutical composition for transdifferentiating skin fibroblasts into neurons.

5. Use of a combination of small molecule compounds according to claims 1-4 for inducing the direct transdifferentiation of dermal fibroblasts into neurons.

6. Use according to claim 5, characterized in that: the skin fibroblast is human skin fibroblast.

Technical Field

The invention relates to the field of biotechnology and neural development, in particular to a small molecular compound combination for inducing fibroblast to directly transdifferentiate towards neurons and application thereof.

Background

Central nervous system injury is irreversible due to the inability of terminally differentiated neurons to regenerate. While stem cell therapy has been considered as a promising solution for repair of central nervous system injury, despite the potential of stem cells to differentiate into a variety of nerve cells, including neurons, studies have shown that neural stem cells transplanted into damaged tissues are more likely to differentiate into astrocytes or oligodendrocytes, and rarely into functional neurons. In addition, stem cell transplantation applications still face several major obstacles, including tumor formation, graft rejection, and ethical issues. Therefore, it is important to find a more suitable therapeutic approach for treating nerve damage and nervous system diseases.

Developments in neuroscience and cell biology have provided diverse solutions to the acquisition of neurons. Compared with the induction of stem cell differentiation, the direct reprogramming of somatic cells into neurons which do not pass through the stem cell state has significant advantages, such as high differentiation efficiency, short induction period, no ethical problem, low potential tumorigenic risk and the like. The major methods for neuronal reprogramming include small molecule induction, transcription factor and or miRNA transduction, and exosome transport, among others, and have been used to transdifferentiate somatic cells into the various neuronal subtypes required for the corresponding neurological disease. For safety reasons for clinical applications, small molecule-based reprogramming is advantageous over transgenic approaches, which risk genomic insertion of foreign DNA sequences. Small molecules are cell permeable and non-immunogenic, easy to synthesize, store, standardize, and manipulate, and are low cost. In addition, their biological effects are rapid and often reversible, and can be precisely controlled by different concentrations and combinations. Recently, more and more studies have shown that pure compounds induce transdifferentiation of various somatic cells into neural cells. The skin fibroblast is excellent seed cell for tissue engineering and has the advantages of rich source, wide distribution, strong in vitro amplification capacity, convenient for autologous transplantation, etc. Research has shown that fibroblasts from different sources and species can be reprogrammed to neurons, even subsets of functional neurons, by small molecule induction. In a word, the safe and effective small molecule induced neuron reprogramming method has huge clinical application potential.

Spinal Cord Injury (SCI) is a serious and irreversible condition caused by traumatic or non-traumatic injuries such as infections and tumors. Permanent functional and neurological deficits and limited regenerative capacity following SCI are attributed to a number of secondary cellular and molecular reactions, such as glial and neuronal cell death, axonal loss, demyelination, inflammation, necrotic cavities, glial scarring and neurotrophic factor deficiency. Despite extensive research, there is still no effective clinical treatment method SCI.

One promising therapeutic strategy aims to replace lost neurons by cell transplantation and to reestablish new neuronal circuits with host neurons.

Disclosure of Invention

The technical problem to be solved is as follows: the invention aims to provide a small molecular compound combination for inducing the transdifferentiation of skin fibroblasts directly to neurons, which is used for inducing the transdifferentiation of the skin fibroblasts to the neurons and used for repairing long-distance peripheral nerve defects and central nerve injuries.

The technical scheme is that the small molecule compound combination for inducing the direct transdifferentiation of skin fibroblasts to neurons comprises CHIR99021 (GSK-3 inhibitor), Forskolin (adenylate cyclase activator), LDN193189 (BMP signal inhibitor), SB431542 (selective ALK 5/TGF- β I type receptor inhibitor), SP600125 (reversible JNK inhibitor with ATP competitive capacity), VPA (inhibitor of histone deacetylase 1) and Y27632 (ATP competitive inhibitor of ROCK-I and ROCK-II).

Preferably, the molar ratio of CHIR99021, Forskolin, LDN193189, SB431542, SP600125, VPA and Y27632 is (0.1-3): (0.5-20): (0.01-0.25): (0.1-5): (0.1-10): (100-1000): (0.1-30).

Preferably, the molar ratio of CHIR99021, Forskolin, LDN193189, SB431542, SP600125, VPA and Y27632 is 3: 50: 0.1: 2: 10: 500: 5.

preferably, the small molecule compound combination is used for transdifferentiating skin fibroblasts into neurons or is used for preparing a pharmaceutical composition for transdifferentiating skin fibroblasts into neurons.

The small molecule compound combination is applied to inducing the skin fibroblast to directly transdifferentiate to the neuron.

Preferably, the skin fibroblasts are human skin fibroblasts.

Has the advantages that: the small molecular compound composition for inducing the direct transdifferentiation of the skin fibroblasts to the neurons has the following advantages:

1. the small molecule combination CFLSSVY (CHIR 99021, Forskolin, LDN193189, SB431542, SP600125, VPA and Y27632) obtained by screening has not been reported in the past, and for the first time, the combination has high efficiency induced transdifferentiation effect, and CFVLSSY has high neuron positive rate (namely Tubb3 positive rate), when differentiated for 7 days, Tubb3 positive cells reach 82.09 +/-1.07%, and after 14 days of differentiation, Tubb3 positive cells are further increased to reach 87.03 +/-1.03% (fig. 1 and fig. 3B). In addition, the small molecule combination can also efficiently induce the transdifferentiation of skin fibroblasts of mice and human to neurons, which is about 60% and 50% (fig. 3C), and the induced transformation rate of the CFLSSVY screened by the invention is higher than that in the prior art;

2. the transdifferentiation technology of the invention can efficiently obtain neurons, and the neurons can obtain obvious curative effect after being transplanted to an animal model of spinal cord injury, and can be obtained by indexes such as motor ability improvement, cortex evoked potential recovery and the like.

Drawings

FIG. 1 shows that CFLSSVY is screened out by small molecule combinatorial screening for inducing the direct transdifferentiation of skin fibroblasts to neurons according to the present invention;

FIG. 2 is a schematic diagram of a scheme for inducing rat skin fibroblast (RDFs) differentiation by screening the obtained small molecule combination CFLSSVY;

FIG. 3 shows that the small molecule combination CFLSSVY induces the immunofluorescence staining detection and the statistics of Tubb3 positive cell proportion of the pre-neuron and S100A4 neuron specific markers of the transdifferentiation of the skin fibroblasts of rats, mice and humans and Tubb3 neuron specific markers after the transdifferentiation;

fig. 4.Tubb3A promoter report system vector schematic diagram, and the expression condition of mcherry in cells after transdifferentiation induction and flow cytometry detection thereof;

FIG. 5 shows that after RDFs are induced to transdifferentiate for 14 d and 28 d, the mature neuron specific markers NeuN and Synapsin-1 are subjected to immunofluorescence detection;

FIG. 6 shows the expression changes of fiber-forming related genes and neuron-related genes in the process of inducing RDFs to transdifferentiate into neurons by CFLSSVY;

FIG. 7, the neurons obtained by RDFs transdifferentiation and the carrier 3D silk fibroin scaffold are transplanted to the injured spinal cord of a rat together to promote the recovery of hind limb motor ability, which is represented by that the motor function score of the transplanted chemically induced neuron CiNs (3D-SF-CiNs) group Basso-Beattie-Bresnahan (BBB) is significantly higher than that of the control group (Sham, SCI and 3D-SF), and the coordination between two hind limbs is shown in the continuous delay frame of a 45-degree inclined grid test video, wherein Sham is a pseudo-operation group for knocking out only a vertebral plate without injury and the spinal cord, SCI is a spinal cord injury non-treatment group, 3D-SF is a transplanted pure silk fibroin scaffold group, and 3D-SF-CiNs is a silk fibroin scaffold group transplanted with CiNs;

FIG. 8 shows that the cortical motor-induced potentials after the neuron group (3D-SF-CiNs) is transplanted into the spinal cord defect of the rat have shorter latency and larger voltage amplitude compared with other control groups, which indicates better recovery, wherein SCI is a nerve injury non-treatment group, 3D-SF is a pure silk protein group, and 3D-SF-CiNs is a silk protein scaffold group loaded with the CiNs during transplantation;

FIG. 9H & E staining of slices after implantation of single 3D silk fibroin scaffolds and CiNs-loaded 3D silk fibroin scaffolds into rat spinal defects after spinal cord injury and detection of area of spinal cavities in each group, wherein SCI is a nerve injury non-treatment group, 3D-SF is a pure silk fibroin group, and 3D-SF-CiNs is a silk fibroin scaffold group loaded with CiNs after transplantation;

FIG. 10 shows the immunofluorescence staining of NF-H and GFAP and the statistics of fluorescence area and fluorescence intensity of longitudinal sections of rats 8 weeks after the implantation of SCI, 3D-SF and 3D-SF-CiNs in each treatment group into spinal cord defects. Wherein the damaged area is close to the head end (rostral), the damaged area middle (centre) and the damaged area is close to the tail end (caudal);

FIG. 11. CiNs are implanted in rat spinal cord defects for 8 weeks with viable induced neurons (GFP-tagged) and co-expressing NF-H, tightly associated and interacting with NF-H-indicative neurons in the host; in addition, co-localization of NF-H and MBP in the lesion area represents remyelination of nerve fibers after transplantation.

Detailed Description