CN113151181B - Method for reprogramming adipose-derived stem cells into neurons and cell-adaptive hydrogel loaded with neurons for repairing spinal cord injury - Google Patents

Abstract

The invention realizes the conversion of ADSCs to neurons by reprogramming the Sox2 gene in the over-expressed ADSCs or silencing the expression of the PSEN1 gene of the ADSCs, or by reprogramming the Sox2 gene and silencing the PSEN1 gene expression in the ADSCs at the same time. The reprogramming method can improve the conversion rate of the ADSCs to the neurons, and a large number of neurons can be obtained. In addition, the invention also provides a hydrogel with improved cell activity, and the hydrogel can improve the efficiency of the transformation from the ADSCs to the neural stem cells.

Description

Method for reprogramming adipose-derived stem cells into neurons and cell-adaptive hydrogel loaded with neurons for repairing spinal cord injury

Technical Field

The invention relates to the technical field of gene editing, in particular to a method for reprogramming adipose-derived stem cells into induced neurons and application of cell-adaptive hydrogel loaded with the neurons in repairing spinal cord injury.

Background

Traumatic Spinal Cord Injury (SCI) is a Central Nervous System (CNS) disease with severe disease and poor prognosis, leading to neurological dysfunction below the damaged spinal cord segment, causing partial or total loss of sensory or motor ability, and leaving severe sequelae such as quadriplegia and intractable neuralgia.

Neural Stem Cell (NSC) transplantation is an important therapeutic approach for the repair of SCI and other neuronal diseases. Transplanted NSCs can be differentiated into nerve cells such as neurons and the like, replace damaged and degenerated spinal cord tissues and effectively promote SCI repair. However, NSCs are limited in source and difficult to obtain, so that it is currently urgently needed to find new seed cells to replace NSCs. Safford et al report that: adipose-derived stem cells (ADSCs) can be transdifferentiated into neuron-like cells, and the ADSCs may be an important source of stem cells with neural functions. The ADSCs are abundant in quantity, easy to separate and expand for culture, and can be used for damage repair without rejection reaction. However, the efficiency of conversion of ADSCs into neurons is currently low. Therefore, improving the efficiency of converting the ADSCs into neurons becomes an urgent technical problem in the field.

Disclosure of Invention

To solve the above technical problems, the present invention provides a method for transforming adipose-derived stem cells into neurons by reprogramming. In the method, the over-expression Sox2 gene is reprogrammed and the expression of the PSEN1 gene is inhibited, so that the neural stem cell gene expression program is transiently activated, and the ADSCs are converted into induced neurons (iNs).

The invention realizes the purpose of the invention through the following technical scheme:

in a first aspect: provided is a method for obtaining neurons by reprogramming adipose-derived stem cells, the method being any one of the following:

(a) After transfecting an over-expression Sox2 tool to the adipose-derived stem cells, continuously culturing with a Neurobasal nerve induction culture medium to obtain neurons; or

(b) Transfecting a tool for silencing or inhibiting PSEN1 expression into an adipose-derived stem cell, and continuously culturing with a Neurobasal nerve induction culture medium to obtain a neuron; or

(c) Transfecting a tool for over-expressing Sox2 and a tool for silencing or inhibiting PSEN1 expression into the adipose-derived stem cells, and continuously culturing with a Neurobasal nerve induction culture medium to obtain neurons.

The invention realizes the conversion of ADSCs to neurons by reprogramming Sox2 gene in over-expressed ADSCs or silencing and inhibiting the expression of PSEN1 gene, or by reprogramming and over-expressing Sox2 gene and silencing and expressing PSEN1 gene in ADSCs. The transformation rate of the ADSCs to the neurons can be improved by the reprogramming method, and a large number of neurons can be obtained.

Preferably, the means for over-expressing Sox2 is plasmid pcDNA3.1-Sox2.

Preferably, the tool for silencing or inhibiting PSEN1 expression is siRNA, and a target sequence of the siRNA for PSEN1 gene silencing is shown in SEQ ID NO. 1-3; preferably, the target sequence of the siRNA for PSEN1 gene silencing is shown in SEQ ID NO. 1; the sense strand sequence of the siRNA aiming at the target sequence shown as SEQ ID NO. 1 is shown as SEQ ID NO. 4, and the antisense strand sequence thereof is shown as SEQ ID NO. 5 or SEQ ID NO. 6; the sense strand sequence of the siRNA aiming at the target sequence shown as SEQ ID NO. 2 is shown as SEQ ID NO. 7, and the antisense strand sequence thereof is shown as SEQ ID NO. 8 or SEQ ID NO. 9; the sense strand sequence of the siRNA aiming at the target sequence shown as SEQ ID NO. 3 is shown as SEQ ID NO. 10, and the antisense strand sequence thereof is shown as SEQ ID NO. 11 or SEQ ID NO. 12; wherein, the 3' end of each sense strand and antisense strand in the siRNA sequence is subjected to pendant modification by UU or dTdT.

The knocking efficiency of the three siRNA target sequences is verified and compared through qPCR, and finally the knocking efficiency of the target sequence shown as SEQ ID NO. 1 is the best. Therefore, the sense and antisense strands of the siRNA of the target sequence as shown in SEQ ID NO. 1 are preferred as the best programming tool for knocking down PSEN1 expression. Wherein, the specific nucleotide sequence of PSEN1 is shown as SEQ ID NO. 13.

In a second aspect: provides the application of the gene Sox2 in the adipose-derived stem cells in a reprogramming tool for converting the adipose-derived stem cells into neurons.

Preferably, the means is plasmid pcDNA3.1-Sox2.

In a third aspect: provides the application of the gene PSEN1 in the fat stem cell in a reprogramming tool for converting the fat stem cell into the neuron.

Preferably, the tool is siRNA, and the specific nucleotide sequence of the tool is shown in SEQ ID NO 1-3. Preferably, the tool for inhibiting PSEN1 expression by silencing is siRNA, and the specific nucleotide sequence of the siRNA is shown as SEQ ID NO. 1.

In a fourth aspect: providing a hydrogel for treating spinal cord injury, wherein the hydrogel comprises: the hydrogel comprises the following components: 8% (w/v) gelatin, 10% (w/v) acrylated beta-cyclodextrin and concentration of 1X 10 ⁷ cells/ml ADSCs, and the balance phosphate buffer solution, wherein the ADSCs are treated with over-expressed SOX2 and silenced PSEN1.

Preferably, the ADSCs are treated by over-expressing SOx2 and silencing PSEN1, and specifically, the ADSCs are transfected with a tool for expressing the gene Sox2 and a tool for inhibiting the expression of the gene PSEN1.

The content of components in the hydrogel can influence the functional performance of the ADSCs, and the overhigh concentration of the gelatin and the acrylated beta-cyclodextrin can cause overhigh density of the hydrogel and is not beneficial to the loading of the ADSCs, so that the loading capacity of the ADSCs in the hydrogel is too low to achieve the treatment effect; the density of the hydrogel is too low due to too low concentration of the gelatin and the acrylated beta-cyclodextrin, the mechanical strength of the hydrogel is reduced, and the hydrogel has strong fluidity and cannot be fixed on a focus. Therefore, the invention has been found through a large amount of experimental studies to have the concentrationThe gelatin and the acrylic acid esterified beta-cyclodextrin can obtain hydrogel with optimal mechanical strength and rheological property to the maximum extent; in addition, the invention also optimizes the concentration of ADSCs of the hydrogel, the cell concentration is too low to achieve the treatment effect, 1 × 10 ⁷ cells/ml is the optimal concentration for hydrogel-supported ADSCs.

In a fifth aspect: provides the application of adipose-derived stem cells in the preparation of drugs or biological materials for treating spinal cord injury.

Preferably, the adipose stem cells are transfected with a SOX2 over-expression tool and a PSEN1 silencing tool.

Compared with the prior art, the invention has the following beneficial effects: the invention realizes the conversion of ADSCs to neurons by reprogramming Sox2 gene in over-expressed ADSCs or silencing and inhibiting the expression of PSEN1 gene, or by reprogramming and over-expressing Sox2 gene and silencing and expressing PSEN1 gene in ADSCs. The transformation rate of the ADSCs to the neuron cells can be improved by the reprogramming method, and a large number of neurons can be obtained. In addition, the invention also provides a hydrogel capable of improving the cell activity, and the hydrogel can improve the efficiency of transforming the ADSCs into the neural stem cells.

Drawings

FIG. 1 is a schematic diagram showing the result of the transient neural stem cell-like cell status in the reprogramming process of iNs by ADSCs. a: 3d, gene expression of neural stem cell markers in neural stem cell-like cells overexpressing Sox2 (internal reference is GAPDH). b: bright field images of neural stem cell samples formed after over-expression of Sox2. c: ADSC over-expressing Sox2, immunofluorescent staining pattern using EdU and DAPI labels. d: statistical plots of Sox2 over-expressed ADSCs, proliferating cells stained with EdU and DAPI-labeled immunofluorescence. Scale bar, 50 μm indicates p <0.001.

FIG. 2 is a schematic diagram of the modulation of signaling pathways by ADSCs during the development of neural stem cell-like cell states. a: a heatmap showing gene expression associated with neurogenesis. b: schematic representation of Notch signaling. c: schematic representation of the change in mRNA expression level of PSEN1 after treatment with three si-RNAs interfering with PSEN1. d: schematic representation of the change in protein expression levels of PSEN1 after treatment with three si-RNAs interfering with PSEN1.

Fig. 3 is a schematic diagram of induction of reprogramming of neural stem cells to iNs by simultaneously overexpressing Sox2 and knocking down PSEN1 gene (referred to herein as SIP treatment) in ADSCs. a: QPCR is a schematic diagram for detecting the gene expression level (internal reference gene is GAPDH) of neural stem cell marker Nestin. b: western Blot detection schematic diagram of protein expression levels of Nestin and Sox2 in ADSCs after SIP treatment. c: representative micrographs of SIP-treated ADSCs, stained with Nestin (green), sox2 (red) and DAPI, scale bar, 50 μm. d: schematic representation of protein expression levels of neuronal (iNs) markers GFAP and THBB 3; e: neuronal (iNs) markers GFAP and THBB3 were immunofluorescent stained, labeled using TUBB3 (green) and DAPI staining. Scale bar, 50 μm.

FIG. 4 is a schematic diagram showing the viability, proliferation and migration of cells of ADSCs treated with SIP in GelMA and HGM hydrogels. a: confocal micrographs (right, top and front view) of 3D distribution of DAPI-stained ADSCs (SIP) nuclei in GelMA and HGM hydrogels after incubation for 2 hours at 37 ℃, scale bar: 100 μm. b: statistical 3D distribution of DAPI-stained ADSCs (SIP) nuclei from the top of the gel in GelMA and HGM hydrogels. c: gelMA and HGM hydrogel loaded with ADSCs (SIP) is shown in the diagram of the proliferation capacity of CCK8 cells. d: and (3) fluorescent staining images of the cell viability of ADSCs (SIP) in HGM and GelMA hydrogels after in vitro culture for 1-7 days. e: statistical histogram of ADSCs (SIP) cell viability in HGM and GelMA hydrogels after 1-7 days of in vitro culture. f: after 1-14 days of culture, the cell viability fluorescence staining pattern of GelMA and HGM hydrogel loaded with ADSCs (SIP). g: after 1-14 days of culture, the gene expression of ADSCs (SIP) neural stem cell markers (nestin, sox 2) in HGM and GelMA hydrogels is shown schematically (the internal reference is GAPDH).

FIG. 5 is a schematic diagram showing that the transplanted dynamic hydrogel CaNeu-loaded ADSCs promote the electrophysiological and motor function recovery of rats after SCI. A: schematic diagram of experiment for transplanting CaNeu/ADSCs to rat SCI; B. c is a schematic diagram of hindlimb supporting capacity of each group of animals 8 weeks after spinal cord injury; c is 8 weeks after spinal cord injury, the hind limb support capacity of each group of animals was behaviorally scored for rat hind limb movement according to BBB score table, BBB score obtained for each group (/ P <0.05,/P <0.01 mean ± s.e.m, n = 9; d is an electrophysiological experiment schematic diagram of the transcranial electrical stimulation of the cerebral motor cortex of each group of animals after 8 weeks; e is a schematic diagram of hindlimb movement-induced potentials (MEPs) of animals in each group after 8 weeks, obvious MEPs appear in the CaNeu/ADSCs group, and the MEP of the SCI group is close to a baseline value; f is a statistical result diagram of the mean amplitude P-P values of the MEPs of each group (n.s. = no signature, # P <0.01, mean ± s.e.m, n = 5).

Detailed Description

In order to show technical solutions, purposes and advantages of the present invention more concisely and clearly, the technical solutions of the present invention are described in detail below with reference to specific embodiments. Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.

Example 1 preparation of neurons from adipose-derived Stem cells reprogramming to overexpress the SOX2 Gene

1. Obtaining and culturing adipose-derived stem cells (ADSCs)

Selecting 5 SD male rats of 90-100g randomly, and preparing surgical instruments (ophthalmic scissors, tweezers and the like);

carrying out intraperitoneal injection of pentobarbital sodium (3 percent, 50 mg/kg) for deep anesthesia, then carrying out sacrifice by adopting a cervical dislocation method, and soaking and disinfecting for 15min by using 75 percent ethanol;

thirdly, taking out adipose tissues on the epididymis by laparotomy under the aseptic condition, soaking the adipose tissues in PBS (phosphate buffer solution) containing 10% of double antibodies for 10min, and soaking the adipose tissues in PBS containing 5% of double antibodies for 5min;

fourthly, shearing fat tissues into a paste shape by using an ophthalmic scissors, performing shake digestion for 40min by using 0.1% collagenase at 37 ℃, stopping digestion by using 10% FBS-containing DMEM/F12, centrifuging for 10min at 1000r/min, and collecting cell precipitates;

fifthly, resuspending with DMEM/F12 containing 10% FBS, filtering with a 200-mesh screen, and adjusting the cell density to 1 × 10 ⁴ The culture medium is inoculated in a culture dish in each ml and placed in CO at 37 DEG C ₂ Culturing in a constant-temperature incubator; after 48h, the liquid is changed for the first time, then the liquid is changed every 3d, and after 7-9 d, the cells grow to be full of the bottom of the dish and then passage is carried out.

2. Tool for constructing over-expression Sox2

Selecting pcDNA3.1 plasmid as an empty vector, and constructing a prokaryotic expression vector for over-expressing Sox 2;

extracting the unloaded plasmid pcDNA3.1 by using a plasmid extraction kit;

thirdly, amplifying Sox2 by PCR, wherein the amplification primers are as follows:

Forward 5’-3’：GCGGAGTGGAAACTTTTGTCC；

reverse 5'-3': CGGGAAGCGTGTACTTATCCTT, and purifying the PCR product using a kit;

fourthly, connecting the purified PCR product, namely the target fragment Sox2, with a vector plasmid pcDNA3.1;

fifthly, using escherichia coli DH5 alpha competent cells to transform the ligation product, then adding the ligation product into LB culture medium containing Amp, culturing for 1.5h at 37 ℃ at 150r/min, taking 500 mu l of bacterial liquid to coat an LB flat plate containing Amp, and culturing overnight at 37 ℃;

sixthly, selecting single bacteria, and performing shake culture for 8 hours in 20ml of LB containing Amp;

keeping bacteria, storing in 50% glycerol and 50% bacteria solution at-80 deg.C;

extracting plasmids by using an endotoxin-free plasmid extraction kit, carrying out double enzyme digestion on the extracted plasmids by using BamHI and EcoRI, carrying out electrophoresis after enzyme digestion, and identifying whether the extracted plasmids contain target fragments; sending the plasmid which is verified to contain the target gene to a sequencing company for sequencing to further verify that the target gene Sox2 is inserted into the pcDNA3.1 plasmid, thereby obtaining the pcDNA3.1-Sox2 plasmid;

the plasmid obtained through the self-tapping is transfected into the ADSCs by using a lip3000 liposome transfection reagent, so that Sox2 is overexpressed in the ADSCs.

3. Neurons (induced neurons, iNs) are obtained.

After pcDNA3.1-Sox2 transfection of ADSCs for three days, the culture medium was changed to Neurobasal neural induction medium (BrainPhys) ^TM Neuronal Medium/BrainPhys ^TM Neural medium was purchased from beijing noro bio, brand: stem cell Technologies, cat # s: 05790, specification: 500 mL), and 10ng/mL FGF-2 was added thereto, followed by induction culture for 10 days.

Example 2 preparation of neurons from reprogrammed and silenced PSEN1 Gene-derived adipose-derived Stem cells

1. Obtaining and culturing adipose-derived stem cells (ADSCs)

The method of example 1 is used to obtain ADSCs and culture ADSCs.

2. Construction of a tool for Gene silencing inhibition of PSEN1 expression

The method includes designing three knocked-down target sequences according to a PSEN1 sequence, entrusting Ruibobo biology company to synthesize corresponding siRNA sense chains and antisense chains aiming at the three target sequences, and specifically obtaining the following information of the three target sequences and the sense chains and the antisense chains of the siRNA:

Si-PSEN1-001:

1 target sequence (5 '- > 3') of SEQ ID NO:1 GCAGCAGGCGTATCTCATT

4 sense strand (5 '- > 3') of SEQ ID NO: GCAGCAGGCGUAUCUCUCAUU

5 antisense strand (3 '- > 5') of SEQ ID NO CGUCGUCCGCAUAGUAA

6 antisense strand (5 '- > 3') of SEQ ID NO: AAUGAGAUACGCCUGCUGC

Si-PSEN1-002:

2 target sequence (5 '- > 3') of GGAACTTTCTGGGAGCATT

7 sense strand (5 '- > 3') of SEQ ID NO. GGAACUUUCUGGGGAGCAUU

SEQ ID NO 8 antisense strand (3 '- > 5')/CCUUGAAGACCCUCGUAA

9 antisense strand (5 '- > 3') of SEQ ID NO: AAUGCUCCCAGAAAAGUUCC

Si-PSEN1-003:

3 target sequence (5 '- > 3') of GGACCAACTTGCATTCCAT

10 sense strand (5 '- > 3') of GGACCAACUUUGCAUUCCAU

SEQ ID NO. 11 antisense strand (3 '- > 5'): CCUGGUUGAACGUAAGGUA

12 antisense strand (5 '- > 3') of SEQ ID NO AUGGAAUGCAAGUUGGCUCC

In the siRNA sequence, dTdT is used as a suspension modification at the 3' end of each sense strand and each antisense strand.

Performing qPCR verification on the knocking efficiency of the siRNAs of the three target sequences to select the best one with the knocking efficiency; among them, the most efficient knock-out is the target sequence (5 '- > 3') of SEQ ID NO. 1, GCAGCAGGCGTATCTCATT

And transfecting si-PSEN1 into the ADSCs by using a lip3000 liposome transfection reagent, thereby silencing PSEN1 in the ADSCs.

3. Acquired neurons (iNs)

After si-PSEN1 transfects ADSCs for three days, the culture medium is changed into Neurobasal nerve induction culture medium, 10ng/ml FGF-2 is added, and the induction culture is carried out for 10 days.

Example 3 preparation of neurons from adipose-derived stem cells reprogramming to overexpress SOX2 and silencing PSEN1 Gene

1. Obtaining and culturing adipose-derived stem cells (ADSCs)

The ADSCs are obtained and cultured by the method of the embodiment 1.

2. Tool for constructing over-expression Sox2

The tool for over-expression of Sox2 was constructed using the method described above in example 1.

3. Construction of a tool for Gene silencing inhibition of PSEN1 expression

The method of example 2 above was used to construct a means for gene silencing to inhibit PSEN1 expression.

4. Acquired neurons (iNs)

pcDNA3.1-Sox2 and si-PSEN1 were transfected into ADSCs using lip3000 liposomes, and after three days of treatment, neurobasal nerve induction medium was changed to medium, and 10ng/ml FGF-2 was added and induced for 10 days.

Example 4 detection of neural Stem cells or neurons obtained from assays

In order to verify that the neurons obtained by reprogramming the adipose-derived stem cells in examples 1 to 3 have characteristics of neurons, a series of detection means were used in this example for verification. The method comprises the following specific steps:

1. gene expression profiling of neural stem cell markers by q-PCR

As shown in FIG. 1-a, the neural stem cell-like cells of over-expressed Sox2 (example 1) showed higher expression levels of the neural stem cell markers Sox2, nestin, SOX1, and PAX6 than those of the control group. As shown in FIG. 2-c, the expression level of PSEN1 gene was significantly lower in ADSCs treated with siRNA (example 2) than in the control group. FIG. 3-a shows that the expression level of neural stem cell marker Nestin gene of ADSCs (example 3) treated with both over-expressed SOX2 and silenced PSEN1 gene is significantly increased compared to examples 1 and 2.

2. Western Blotting detection of protein expression of neural stem cell marker

Results the PSEN1 protein content in the siRNA-treated ADSCs (example 2) was significantly reduced, as shown in fig. 2-d. Three PSEN 1-siRNAs with different sequences were used to treat ADSCs. All three sirnas were effective in reducing PSEN1 mRNA levels as well as protein content compared to control (NC) sirnas, with the effect of siRNA001 sequence being most significant. FIG. 3-b shows that the neural stem cell markers SOX2 and Nestin of examples 1 to 3 have higher protein contents than those of the Control group (Control); in examples 1-3, the protein content of the neural stem cell markers SOX2 and Nestin of the ADSCs in example 3 is significantly higher than that in examples 1 and 2. FIG. 3-d shows that the neuronal markers GFAP and THBB3 of examples 1 to 3 are higher in protein content than the control group; in examples 1-3, the protein content of the neuronal markers GFAP and THBB3 of ADSCs in example 3 was significantly higher than that in examples 1 and 2.

3. Immunofluorescence staining assay

The results are shown in fig. 1-c and d, the cell proliferation rate of the ADSCs after over-expression of Sox2 is obviously higher than that of the control group, which indicates that the over-expression of Sox2 can significantly promote the proliferation of the ADSCs. FIG. 3-c shows that green indicates Nestin, red indicates Sox2, and the number of neural stem cells of examples 1-3 is significantly higher than that of the control group, and the number of neural stem cells of ADSCs of examples 1-3 is significantly higher than that of examples 1 and 2, compared with the control group. Fig. 3-e shows that although TUBB3 is represented by green color, the neuron numbers of examples 1 to 3 are significantly higher than those of the control group, and the neuron numbers of ADSCs in example 3 are significantly higher than those of examples 1 and 2 in examples 1 to 3, compared to the control group.

The above experimental results show that, although both over-expression of SOX2 and silencing PSEN1 can improve the expression level of neural stem cell markers Nestin and SOX2 genes and promote the transformation of ADSCs into neural stem cells, it is obvious that the simultaneous over-expression of SOX2 and silencing PSEN1 has a synergistic effect, can significantly improve the expression level of Nestin and SOX2 genes and further promote the transformation of ADSCs into neuronal cells, and the transformation efficiency is much higher than that of ADSCs treated by a single method (only silencing PSEN1 or only over-expressing SOX 2). Neuronal cells (iNs) obtained from ADSCs treated with SIP ("SIP" herein means simultaneous overexpression of SOX2 and silencing of PSEN 1) have neuronal properties with higher protein expression levels of the markers GFAP and THBB3 than those obtained from ADSCs treated with a single method.

EXAMPLE 5 hydrogel for treating spinal cord injury and method for preparing the same

The present example provides a hydrogel HGM for treating spinal cord injury, the hydrogel comprising: the hydrogel comprises the following components: 8% (w/v) gelatin, 10% (w/v) acrylated beta-cyclodextrin and a cell concentration of 1X 10 ⁷ cells/ml ADSCs, the balance being phosphate buffered saline (0.01M, pH 7.2-7.4), wherein the ADSCs are treated with over-expressed SOX2 and silenced PSEN1.

The preparation method of the hydrogel comprises the following steps: dissolving gelatin and acrylated beta-cyclodextrin in phosphate buffer solution at 37 deg.C to produce a mixed solution of gelatin with a fixed concentration of 8% (w/v) and acrylated beta-cyclodextrin (Ac-beta-CD) of 10% (w/v), adding ADSCs cell suspension, incubating at 37 deg.C for 2 hr to obtain a cell concentration of 1 × 10 ⁷ cells/ml, then initiator I2959 at 0.05% (w/v) was added. The mixture was transferred to a PVC mould at 37 ℃, cooled to 25 ℃ and then exposed to UV light at 365nm (10 mW/cm) ² 10 min) to obtain supramolecular hydrogels, in which the PVC molds are cylindrical, with a diameter of 5 mm and a depth of 3 mm.

Example 6 Effect of hydrogel loaded with ADSCs on cells

1. The 3D distribution of ADSCs in GelMA and HGM hydrogels was examined after incubating the ADSCs of example 3 with GelMA (available from Suzhou Intelligent manufacturing institute, model: EFL-GM-60) and HGM hydrogels, respectively, at 37 deg.C for 2 hours. The results are shown in FIGS. 4 a-b: compared to GelMA hydrogel, the ADSCs core is further from the top of the HGM hydrogel, indicating that the ADSCs (SIP) are deeper inside the HGM hydrogel. (GelMA hydrogel used in this example was methacrylic anhydride-modified gelatin as a main component, and GelMA was a commercially available hydrogel and used as a control material herein).

2. Influence of GelMA and HGM hydrogel loaded with ADSCs on proliferation capacity of CCK8 cells

The influence of GelMA and HGM hydrogel loaded with ADSCs on the proliferation capacity of CCK8 cells is detected, and the result is shown in figure 4-c, and the GelMA and HGM gel have the function of promoting the proliferation of the CCK8 cells.

3. Detecting the influence of GelMA and HGM hydrogel loaded with ADSCs on the activity of CCK8 cells

The influence of GelMA and HGM hydrogel loaded with ADSCs on CCK8 cell activity is detected, and the result is shown in figures 4-d, e and f, after in vitro culture is carried out for 1-14 days, gelMA and HGM hydrogel loaded with ADSCs can remarkably improve cell activity, but at 14 days, the number of living cells of HGM hydrogel is higher than that of GelMA hydrogel.

4. Detecting the expression of NestinH and Sox2 genes which are neural stem cell markers in ADSCs (example 3) in GelMA and HGM hydrogel

qPCR is adopted to detect the gene expression condition of the neural stem cell marker, and FIG. 4-g shows the expression condition of the neural stem cell marker Nestin and Sox2 genes in ADSCs (example 3) in GelMA and HGM hydrogel, and the expression level of the Nestin and Sox2 genes in the HGM hydrogel is obviously higher than that of GelMA, which indicates that the HGM hydrogel is more suitable for loading ADSCs.

EXAMPLE 7 Effect of hydrogel on treatment of spinal cord injury

Animal experiments:

the animal grouping method includes: randomly selecting 50 female SD rats of 220-250 g, and classifying into sham (sham operation group), SCI (simple injury group), ADSCs (iNs) group, and CaNeu groupGroup (material group unloaded with ADSCs) and CaNeu/ADSCs group, total 5 groups, each group n =10, cell density 1X 10 ⁷ cells/ml。

And carrying out deep anesthesia on rats by injecting sodium pentobarbital (3 percent, 50 mg/kg) subcutaneously. A dorsal laminectomy of the 10 th thoracic vertebra (T9-T10) was removed to expose the spinal cord. 2mm of T9-T10 spinal cord tissue was excised, resulting in 2mm total transection injury, followed by material transplantation or cell injection (1X 10) ⁷ cells/ml), and layer-by-layer suture after operation. After surgery, all animals were injected with penicillin for 7 consecutive days with artificial urination each day until the animals recovered partial self-urination capacity.

As shown in fig. 5, the representative records of walking gait of animals 8 weeks after spinal cord injury showed that there was a difference in the hindlimb walking pattern of the control group, spinal cord injury group, ADSC treated group, caNeu treated group and ADSC hydrogel-loaded treated group (CaNeu/ADSCs) in fig. 5B, and it was evident that the hindlimb of the ADSC hydrogel-loaded treated group (CaNeu/ADSCs) was able to walk normally. The BBB scale in figure 5C measures motor recovery (Tukey's multiple comparison test in two-way anova: <0.05, <0.01 mean ± s.e.m, n =9 animals) results show that the BBB score was highest for the ADSC loaded hydrogel treated group (CaNeu/ADSCs). FIG. 5E shows that CaNeu/ADSC group rats showed significant MEP response, while SCI group rats showed baseline levels of MEP. Mean MEP amplitudes in the CaNeu/ADSC group of animals were shown to be significantly higher in 5F than in the other groups of animals (one-way anova and post-Tukey analysis; n.s. = no significance,. P <0.01, mean ± s.e.m, n =5 animals).

The results show that the hydrogel (CaNeu/ADSCs) can be successfully converted into neurons and repair spinal cord injury, thereby achieving the effect of treating central nervous system diseases.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> Zhongshan university affiliated seventh Hospital (Shenzhen)

<120> a method for reprogramming adipose-derived stem cells into neurons and cell-adaptive hydrogel loaded with the neurons

Repairing spinal cord injury

<130> 4.13

<160> 13

<170> PatentIn version 3.3

<210> 1

<211> 19

<212> DNA

<213> human

<400> 1

gcagcaggcg tatctcatt 19

<210> 2

<211> 19

<212> DNA

<213> human

<400> 2

ggaactttct gggagcatt 19

<210> 3

<211> 19

<212> DNA

<213> humna

<400> 3

ggaccaactt gcattccat 19

<210> 4

<211> 19

<212> RNA

<213> Synthesis

<400> 4

gcagcaggcg uaucucauu 19

<210> 5

<211> 19

<212> RNA

<213> Synthesis

<400> 5

cgucguccgc auagaguaa 19

<210> 6

<211> 19

<212> RNA

<213> Synthesis

<400> 6

aaugagauac gccugcugc 19

<210> 7

<211> 19

<212> RNA

<213> Synthesis

<400> 7

ggaacuuucu gggagcauu 19

<210> 8

<211> 19

<212> RNA

<213> Synthesis

<400> 8

ccuugaaaga cccucguaa 19

<210> 9

<211> 19

<212> RNA

<213> Synthesis

<400> 9

aaugcuccca gaaaguucc 19

<210> 10

<211> 19

<212> RNA

<213> Synthesis

<400> 10

ggaccaacuu gcauuccau 19

<210> 11

<211> 19

<212> RNA

<213> Synthesis

<400> 11

ccugguugaa cguaaggua 19

<210> 12

<211> 19

<212> RNA

<213> Synthesis

<400> 12

auggaaugca aguuggucc 19

<210> 13

<211> 1407

<212> DNA

<213> human

<400> 13

atgacagaga tacctgcacc tttgtcctac ttccagaatg cacagatgtc tgaggacagc 60

cactccagca gcgtccgtag ccagaatgac aatcaagaac ggcagcagca tcatgacaga 120

cagagacttg acaaccctga gtcgatatct aatggccgac cccagagtaa ctttaccagg 180

caggtgatag agcaagatga ggaggaagac gaagagctga cattgaaata tggagccaag 240

cacgtcatca tgctctttgt tcctgtgacc ctctgcatgg tcgttgtggt ggccaccatc 300

aagtcagtca gcttctacac ccggaaggat gggcagctaa tctatacccc attcacagaa 360

gacaccgaga ctgtaggcca gagagccctg cactcgatct tgaatgccgc catcatgatc 420

agtgtcatcg tcgttatgac catcctcctg gtggtcctgt ataagtacag gtgctacaag 480

gtcatccatg cctggcttat tgtttcatct ctgttgttgc tgttcttttt ttcattcatt 540

tacttagggg aagtattcaa gacctacaat gtcgccgtgg actatattac ggttgcactc 600

ctgatctgga attttggtgt ggtcgggatg attgccattc actggaaagg cccactccga 660

ctgcagcagg cgtatctcat tatgatcagt gccctcatgg ccctggtatt tatcaagtac 720

ctccctgaat ggaccgcatg gctcatcttg gctgtgattt cagtatatga tttggtggct 780

gttctgtgcc ccaaaggtcc acttcgtatg ctggtcgaaa cagctcagga aagaaatgag 840

actctctttc cagctcttat ctattcctca accatggtgt ggttggtgaa tatggctgaa 900

ggagacccag aagcccaaag gagggtaccc aaaaacccca agtatagcac acaaggaaca 960

gagagggaag agacacagga cactggcact gggagcgatg atggtggctt cagtgaggag 1020

tgggaggccc aaagagacag tcacctgggg cctcatcgct ccactcctga gtcaagagct 1080

gctgtccagg aactttctgg gagcatcctc actagtgaag acccggagga aagaggagta 1140

aagcttgggc tgggagattt cattttctac agtgttctgg ttggtaaggc ctcagcgacc 1200

gccagtgggg actggaacac aaccatagcc tgctttgtag ccatattgat cggcctgtgc 1260

cttacgttac tcctgctcgc cattttcaag aaagcgttgc cggcccttcc catctccatc 1320

accttcgggc tcattttcta ctttgccacg gattatctcg tgcagccctt catggaccaa 1380

cttgcattcc atcaatttta tatctag 1407

Claims (3)

1. A method of obtaining neurons by reprogramming adipose-derived stem cells, the method comprising:

transfecting a tool for silencing PSEN1 expression into an adipose-derived stem cell, and continuously culturing with a Neurobasal nerve induction culture medium to obtain a neuron;

the tool for silencing PSEN1 expression is siRNA, and a target sequence of the siRNA aiming at PSEN1 gene silencing is shown in SEQ ID NO 1-3; the target sequence of siRNA for PSEN1 gene silencing is shown as SEQ ID NO. 1; the sense strand sequence of the siRNA aiming at the target sequence shown as SEQ ID NO. 1 is shown as SEQ ID NO. 4, and the antisense strand sequence thereof is shown as SEQ ID NO. 5 or SEQ ID NO. 6; the sense strand sequence of the siRNA aiming at the target sequence shown as SEQ ID NO. 2 is shown as SEQ ID NO. 7, and the antisense strand sequence thereof is shown as SEQ ID NO. 8 or SEQ ID NO. 9; the sense strand sequence of the siRNA aiming at the target sequence shown as SEQ ID NO. 3 is shown as SEQ ID NO. 10, and the antisense strand sequence thereof is shown as SEQ ID NO. 11 or SEQ ID NO. 12.

2. The method of claim 1, wherein the 3' end of each of the sense and antisense strands in the siRNA sequence is pendently modified by UU or dTdT.

3. The application of the tool for silencing PSEN1 expression in preparing a reprogramming tool for converting adipose-derived stem cells into neurons is characterized in that the tool for silencing PSEN1 expression is transfected into the adipose-derived stem cells, and the neurons are obtained after the tool is continuously cultured by a Neurobasal nerve induction culture medium;

the tool for silencing PSEN1 expression is siRNA, and a target sequence of the siRNA aiming at PSEN1 gene silencing is shown in SEQ ID NO 1-3; the target sequence of siRNA for PSEN1 gene silencing is shown as SEQ ID NO. 1; the sense strand sequence of the siRNA aiming at the target sequence shown as SEQ ID NO. 1 is shown as SEQ ID NO. 4, and the antisense strand sequence thereof is shown as SEQ ID NO. 5 or SEQ ID NO. 6; the sense strand sequence of the siRNA aiming at the target sequence shown as SEQ ID NO. 2 is shown as SEQ ID NO. 7, and the antisense strand sequence thereof is shown as SEQ ID NO. 8 or SEQ ID NO. 9; the sense strand sequence of the siRNA aiming at the target sequence shown as SEQ ID NO. 3 is shown as SEQ ID NO. 10, and the antisense strand sequence thereof is shown as SEQ ID NO. 11 or SEQ ID NO. 12.

Images