CN110724669A

CN110724669A - Umbilical cord mesenchymal stem cell modified by Nrf2 gene, and preparation method and application thereof

Info

Publication number: CN110724669A
Application number: CN201810712604.7A
Authority: CN
Inventors: 李陶
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-06-29
Filing date: 2018-06-29
Publication date: 2020-01-24

Abstract

The invention relates to the technical field of stem cells, in particular to an umbilical cord mesenchymal stem cell modified by Nrf2 gene and a preparation method and application thereof. The invention discloses an umbilical cord mesenchymal stem cell modified by Nrf2 gene, wherein the umbilical cord mesenchymal stem cell modified by Nrf2 gene is an umbilical cord mesenchymal stem cell over-expressing Nrf 2. The resistance of the UCMSC modified by the Nfr2 gene to nerve injury is enhanced, the UCMSC can effectively antagonize oxidative stress, and the UCMSC modified by the Nfr2 gene can effectively promote the UCMSC to be converted into neural stem cells compared with the non-modified UCMSC. The UCMSC modified by Nfr2 gene is transplanted in the body of the dementia mouse, and the UCMSC modified by Nfr2 gene can obviously improve the learning and memory disorder of the dementia mouse, protect the oxidative damage of the brain tissue of the dementia mouse, reduce the activity of AChE and NO of the hippocampus in the brain of the dementia mouse, and inhibit the release of inflammatory factors in the hippocampus tissue in the brain of the senile dementia mouse. The invention provides technical support for preparing the medicine for treating the senile dementia.

Description

Umbilical cord mesenchymal stem cell modified by Nrf2 gene, and preparation method and application thereof

Technical Field

The invention relates to the technical field of stem cells, in particular to an umbilical cord mesenchymal stem cell modified by Nrf2 gene and a preparation method and application thereof.

Background

Alzheimer's Disease (AD) is a progressive neurodegenerative disease that manifests clinically as memory and cognitive dysfunction, loss of language and behavioral abilities, severe amnesia and impaired motor function later in the patient, ultimately leading to death. Because of the long course of disease and the lack of effective treatment, many AD patients are burdened by families and society. Therefore, the search for effective AD therapies has become an urgent issue in the medical community.

Due to the presence of the blood cerebrospinal fluid barrier, many drugs have difficulty entering nervous system tissues, resulting in a great limitation of traditional drug therapy for AD. A great deal of research is currently being conducted on the basic and clinical treatment of AD, mainly including general therapy, neurotransmitter replacement therapy, pathological intervention therapy, neurotrophic therapy, and stem cell therapy. In recent years, stem cell therapy has attracted attention as a result of intensive studies on AD. Research shows that the umbilical cord mesenchymal stem cells can effectively improve the learning and memory functions of the Alzheimer disease patients. In the research of the human umbilical cord mesenchymal stem cells on treating Alzheimer disease model mice, the umbilical cord mesenchymal stem cells can increase the recovery of normal neurons and neural stem cells in brain tissues through a way of regulating immune response, and differentiate and replace diseased nerve cells of Alzheimer disease, but the capacity of adult stem cells for differentiating into the neural stem cells in vivo is limited, and the expected treatment effect is difficult to achieve.

The nuclear factor E2 related factor 2 (nuclear factor erythroid 2-related factor 2, Nrf2) belongs to cap 'n' collagen (CNC) family in transcription factors, and is a classical nuclear transcription factor for regulating and controlling the anti-oxidative stress response of organisms, and Nrf2 induces the expression of anti-oxidative protein after activation, so that the anti-oxidative stress capability of the organisms is improved.

At present, the anti-senile dementia medicine is prepared by modifying umbilical mesenchymal stem cells by using Nrf2 genes, and no report is provided at home and abroad.

Disclosure of Invention

The invention aims to provide an umbilical cord mesenchymal stem cell modified by Nrf2 gene and a preparation method and application thereof, wherein the umbilical cord mesenchymal stem cell is modified by Nrf2 gene, so that the differentiation capability of the umbilical cord mesenchymal stem cell into a neural stem cell is improved, and the umbilical cord mesenchymal stem cell modified by Nrf2 gene is transplanted into a senile dementia mouse body, so that the umbilical cord mesenchymal stem cell has obvious improvement effect on learning and memory disorder, brain tissue oxidative damage, damage of choline system in hippocampus tissue and inflammatory factor in hippocampus tissue of the senile dementia mouse.

The specific technical scheme is as follows:

the invention provides an umbilical cord mesenchymal stem cell modified by Nrf2 gene, wherein the umbilical cord mesenchymal stem cell modified by Nrf2 gene is an umbilical cord mesenchymal stem cell over-expressing Nrf 2.

In the present invention, the umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

Preferably, the sequence of the Nrf2 gene has the sequence as shown in SEQ ID NO: 1, or SEQ ID NO: 1 by replacing one or more nucleotides.

Preferably, the umbilical cord mesenchymal stem cell modified by the Nrf2 gene is prepared by the following method:

1) constructing an Nrf2 gene recombinant plasmid vector;

2) transfecting the umbilical cord mesenchymal stem cells with the Nrf2 gene recombinant plasmid vector to obtain the umbilical cord mesenchymal stem cells modified by Nrf2 genes.

In the embodiment of the invention, after the umbilical cord mesenchymal stem cells are transfected by the Nrf2 gene recombinant plasmid vector, the umbilical cord mesenchymal stem cells which over-express the Nrf2 protein are obtained, namely, the umbilical cord mesenchymal stem cells modified by the Nrf2 gene.

Preferably, the vector of the Nrf2 gene recombinant plasmid vector is a lentiviral vector.

More preferably, the lentiviral vector is a pWPXL plasmid.

Preferably, the packaging plasmid of the lentiviral vector is the pMD2.G plasmid and the envelope plasmid of the lentiviral vector is the psPAX2 plasmid.

The invention also provides a preparation method of the umbilical cord mesenchymal stem cell modified by the Nrf2 gene, which comprises the following steps:

1) constructing an Nrf2 gene recombinant plasmid vector;

Preferably, the vector of the Nrf2 gene recombinant plasmid vector of step 2) is a lentiviral vector.

More preferably, the lentiviral vector is a pWPXL plasmid.

Preferably, the preparation method of the umbilical cord mesenchymal stem cells modified by the Nrf2 gene comprises the following steps:

1) taking the pWPXL plasmid and the Nrf2 gene PCR product, carrying out BamHI/MluI double enzyme digestion, connecting the double enzyme digested pWPXL plasmid and the Nrf2 gene PCR product by using T4DNA ligase after purification and recovery, and integrating the Nrf2 gene into the pWPXL plasmid to form a recombinant pWPXL-Nrf2 plasmid;

2) the target plasmid pWPXL-Nrf2, the packaging plasmid pMD2.G and the envelope plasmid psPAX2 are transfected and cultured to the umbilical cord mesenchymal stem cell of P3 generation, and the umbilical cord mesenchymal stem cell modified by Nrf2 gene is obtained.

The invention also provides application of the umbilical cord mesenchymal stem cell modified by the Nrf2 gene or the umbilical cord mesenchymal stem cell modified by the Nrf2 gene prepared by the preparation method in promoting differentiation of the mesenchymal stem cell into a neural stem cell.

Preferably, the above application comprises the steps of:

1) constructing an Nrf2 gene recombinant plasmid vector;

2) transfecting the umbilical cord mesenchymal stem cells with the Nrf2 gene recombinant plasmid vector to obtain Nrf2 gene modified umbilical cord mesenchymal stem cells;

3) inducing the Nrf2 gene modified umbilical cord mesenchymal stem cells to differentiate into neural stem cells.

Preferably, the vector of the Nrf2 gene recombinant plasmid vector of step 1) is a lentiviral vector.

Preferably, the lentiviral vector is a pWPXL plasmid.

Preferably, the step 3) induction is specifically: pre-induction is carried out first, and then induction is carried out.

More preferably, the pre-induction time is 24h and the induction time is 7 d.

Preferably, the application of the mesenchymal stem cells of the umbilical cord modified by the Nrf2 gene in promoting the differentiation of the mesenchymal stem cells into the neural stem cells is as follows:

2) transfecting and culturing a target plasmid pWPXL-Nrf2, a packaging plasmid pMD2.G and an envelope plasmid PSPAX2 to obtain umbilical cord mesenchymal stem cells of a P3 generation to obtain Nrf2 gene modified umbilical cord mesenchymal stem cells;

3) pre-inducing the umbilical cord mesenchymal stem cells modified by the Nrf2 gene for 24 hours, wherein the pre-inducing culture medium comprises the following steps: L-DMEM + 10% fetal calf serum +100 mug/L basic fibroblast growth factor, then inducing with induction medium for 7d, induction medium: L-DMEM + 0.1% dimethyl sulfoxide +2mmol/L tretinoin +100 mug/L basic fibroblast growth factor +100 mug/L epidermal growth factor +100 mug/L brain-derived neurotrophic factor to obtain the Nrf2 gene modified umbilical cord mesenchymal stem cell.

The invention also provides an application of the umbilical cord mesenchymal stem cell modified by the Nrf2 gene or the umbilical cord mesenchymal stem cell modified by the Nrf2 gene prepared by the preparation method in preparing a medicine for treating senile dementia.

The invention also provides a medicament comprising: the umbilical cord mesenchymal stem cell modified by the Nrf2 gene or the umbilical cord mesenchymal stem cell modified by the Nrf2 gene prepared by the preparation method.

The above medicine is preferably used for treating senile dementia.

The invention provides an umbilical cord mesenchymal stem cell modified by Nrf2 gene, which is an umbilical cord mesenchymal stem cell overexpressing Nrf2, and experimental data show that, on one hand, the resistance of UCMSC modified by Nrf2 gene to nerve damage is enhanced, and oxidative stress can be effectively antagonized, and on the other hand, UCMSC modified by Nrf2 gene can effectively promote UCMSC to be converted into neural stem cell compared with unmodified UCMSC. The UCMSC modified by the Nrf2 gene is transplanted in a dementia mouse, and the UCMSC modified by the Nrf2 gene can obviously improve the learning and memory disorder of the dementia mouse, protect the oxidative damage of brain tissues of the dementia mouse, reduce the activity of AChE and NO of hippocampus in the brain of the dementia mouse, and inhibit the release of inflammatory factors in the hippocampus tissues in the brain of the senile dementia mouse. The invention provides technical support for preparing the medicine for treating the senile dementia.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without inventive exercise.

FIG. 1 is an agarose gel electrophoresis of the Nrf2 gene provided in the first embodiment of the present invention;

FIG. 2 is an electrophoretogram of pWPXL-Nrf2 plasmid and pWPXL plasmid provided in the first embodiment of the present invention;

FIG. 3 is a fluorescent microscope image of UCMSC modified by Nrf2 gene at the P3 generation according to the second embodiment of the present invention;

FIG. 4 is an electrophoretogram of Nrf2 protein expression in UCMSC with P3 generation detected by Western blot provided by the second embodiment of the invention;

FIG. 5 is a diagram H according to the third embodiment of the present invention₂O₂Results of LDH percentage content after treating nerve cell PC12 are shown;

FIG. 6 is a graph showing the results of the percentage of LDH content of the nerve cells treated with PC12 according to A.beta.25-35 provided in the third embodiment of the present invention;

FIG. 7 is a fluorescent microscope result chart of UCMSC-Nrf2 neural induced differentiation in vitro provided in the fifth embodiment of the present invention;

FIG. 8 is a graph showing the results of the electric jump test latency after transplanting UCMSC-Nrf2 into an Abeta 25-35 model mouse according to a seventh embodiment of the present invention;

FIG. 9 is a graph showing the results of the number of electrical jump test errors after transplantation of UCMSC-Nrf2 into an A β 25-35 model mouse according to the seventh embodiment of the present invention;

FIG. 10 is a diagram showing the NO activity detection of A β 25-35 model mouse transplanted with UCMSC-Nrf2 according to the eighth embodiment of the present invention;

FIG. 11 is a graph showing AChE activity detection after transplantation of UCMSC-Nrf2 in A β 25-35 model mouse as provided in example eight of the present invention;

FIG. 12 is a graph showing the measurement of IL-6, TNF- α, and IL-1 β contents after UCMSC-Nrf2 transplantation in model A β 25-35 mice as provided in example eight.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Other embodiments, which can be derived by one of ordinary skill in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The umbilical cord mesenchymal stem cell modified by the Nrf2 gene, the preparation method and the application reagent thereof provided by the invention can be purchased from the market.

The umbilical cord mesenchymal stem cell modified by the Nrf2 gene and the preparation method and the application thereof are further explained below.

EXAMPLE construction of recombinant plasmid

1. 2ml of peripheral anticoagulation blood was collected from 16 cases (28.3. + -. 10.5 years old) of normal healthy persons, and the whole blood was mixed well. 2ml of uniformly mixed peripheral blood is taken, 12ml of erythrocyte lysate is added in 4h, the mixture is centrifuged for 5min at 1000rpm, the supernatant is discarded, the mixture is resuspended and washed by PBS, 1ml of Trizol lysate is added, and RNA is extracted. According to Takara

According to the operation instructions of the RT Master Mix reverse transcription kit, 500ng of total RNA is used for reverse transcription, the reaction system is 1 mul, the reaction conditions are 37 ℃ for 15min, 85 ℃ for 15s, and the reaction product is stored at-20 ℃ for later use. The reaction system is as follows:

Template RNA 1μl

RT enzyme Master Mix 2μl

DEPC ddH₂O 7μl

2. the primer Forward is adopted: 5'-cgcggatccgcgatggatttgat-3', Reverse: 5'-cgacgcgtcgagaaaaactag-3', using the cDNA obtained in step 1 as a template, performing conventional PCR amplification. A50. mu.l reaction system containing 25. mu.l of premix Extaq, 50pmol/L of each of the upstream and downstream primers, 0.1. mu.g of cDNA template, and 50. mu.l of water was supplemented. The reaction was carried out according to the following procedure: pre-denaturation at 94 deg.C for 3min, denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, and extension at 72 deg.C for 45s, and circulating for 30 times, and extension at 72 deg.C for 7 min. And (5) carrying out electrophoresis identification on 1% agarose gel.

3. Mu.g of pWPXL plasmid and the PCR product were digested with BamHI/MluI. After purification and recovery, the double digested pWPXL plasmid and the PCR product are connected by T4DNA ligase, namely, the Nrf2 gene is integrated into the pWPXL plasmid to form a recombinant pWPXL-Nrf2 plasmid. After 1% agarose gel electrophoresis and sequencing identification, the slow virus packaging kit is used for packaging slow viruses.

As shown in FIG. 1, the length of the product is about 1782bp, which is consistent with the length of the Nrf2 gene.

As shown in FIG. 2, the recombinant pWPXL-Nrf2 plasmid was approximately 1782bp larger than pWPXL, indicating that Nrf2 had integrated into the pWPXL plasmid.

Example two Nrf2 Gene modification of umbilical mesenchymal Stem cells

1. Taking 293T cells in logarithmic growth phase at 5X 10⁶Inoculating into 100mm cell culture dish, changing into 6ml fresh DMEM complete culture medium after 24h when cell fusion degree reaches 80% -90%, adding 0.5ml mixture of objective plasmid pWPXL-Nrf210 μ g, packaging plasmid pMD2.G 6.5 μ g and envelope plasmid psPAX23.5 μ g after 4h, and slowly adding 50 μ l CaCl₂And (4) lightly mixing the mixture for packaging the lentivirus. Collecting virus liquid after 48h and 72h of packaging, concentrating, and storing at-80 deg.C for use.

2. Collecting waste umbilical cord tissue from hospital, transferring the umbilical cord tissue into sterile collection bottle, storing at 2-8 deg.C, and separating Wharton's jelly within 12 h. Applying the separated Wharton's jelly

Culturing hMSC SFM culture solution in T75 culture bottle at 37 deg.C and 5% CO₂Cultured in an incubator. The whole amount of liquid is changed the next day, half amount of liquid is changed every 2-3 days later, and tissue block is scraped at day 9. Continuously culturing cells until the cell fusion degree reaches 80% -90%, removing culture medium in the culture flask, washing with normal saline for 2 times, adding 0.25% EDTA-pancreatin for digestion, adding equal volume of complete culture medium to stop digestion when the cells become round, collecting, centrifuging, re-suspending with appropriate amount of complete culture medium, adjusting cell density to 0.5 × 10⁵Passaging was performed per ml. When the fusion degree of the P1 generation cells reaches more than 90%, the cell density is adjusted to be 0.5 multiplied by 10⁵Passaging was performed per ml, followed by subculture to P3 according to this standard.

3. Taking P3 generation UCMSC at 1 × 10 per bottle⁶Inoculating each cell into a T175 culture flask, when the cells grow and fuse to 70-80%, transfecting UCMSC with virus titer with MOI of 60IU/ml, removing virus solution after 8h, replacing with fresh culture solution, and continuously culturing for 96 h. And (3) extracting the UCMSC proteins which are transfected and untransfected, and detecting the expression condition of the Nrf2 at the protein level by using a Western blot method.

As shown in fig. 3, the culture form of the P3 generation umbilical cord mesenchymal stem cells is long spindle type, similar to fibroblasts.

As shown in fig. 4, the expression level of Nrf2 protein in the UCMSC transfected with lentivirus-mediated Nrf2 gene was significantly higher than that in the untransfected UCMSC, indicating that Nrf2 gene was successfully expressed in UCMSC.

Example TriUCMSC-Nrf 2 resistance to neuronal cell injury

When cells are damaged or die, intracellular Lactate Dehydrogenase (LDH) is released into the cell culture fluid, and therefore LDH is an important indicator for detecting cell damage or death. The hydrogen peroxide model and the A beta 25-35 model of the nerve cell PC12 are used as nerve cell injury models. Taking nerve cell PC12 at 1 × 10⁵Density per well was seeded in 6-well plates and UCMSC (1 × 10) prepared in example two step 2 was added⁴Hole/bore). After 24h of culture the cells were divided into a: control, B: hydrogen peroxide group, C: UCMSC + H₂O₂Group, D: UCMSC-Nrf2+ H₂O₂Group, E: group A β 25-35, F: UCMSC + Abeta_25-35Group, G: UCMSC-Nrf2+ A beta 25-35 group. Adding H to holes of corresponding groups₂O₂And A β 25-35 at final concentrations of 200 μ M and 20 μ M, respectively, and after 12h of co-incubation, cell supernatants were collected and assayed for LDH according to the LDH kit instructions.

As shown in fig. 5 and 6, H₂O₂And a β 25-35 induced PC12, whereas levels of cellular LDH were significantly increased after pretreatment with UCMSC and UCMS-Nfr2, and showed a more significant decrease in the UCMSC-Nrf2 group.

The experimental results show that the resistance effect of UCMSC-Nrf2 on nerve cell injury is obviously superior to that of unmodified UCMSC.

Example four UCMSC-Nrf2 resistance to oxidative stress in nerve cells

Oxidative stress plays an important role in the pathological development of senile dementia, so we have a UCMSC-Nrf2 pair H₂O₂And the MDA content, SOD, CAT and GSH-PX activity influence after the A beta 25-35 induces the PC12 cells are analyzed and determined.

Taking nerve cell PC12 at 1 × 10⁵Density per well was seeded in 6-well plates and UCMSC (1 × 10) prepared in example two step 2 was added⁴Hole/bore). After 24h of culture, the cells were dividedIs A: control, B: hydrogen peroxide group, C: UCMSC + H₂O₂Group, D: UCMSC-Nrf2+ H₂O₂Group, E: group A β 25-35, F: UCMSC + Abeta_25-35Group, G: UCMSC-Nrf2+ A beta 25-35 group. Adding H to holes of corresponding groups₂O₂And Abeta 25-35, the final concentrations of which are respectively 200 mu M and 20 mu M, after incubation for 12h, collecting cell supernatant, and measuring OD values of MDA, SOD, CAT and GSH-Px in the cells and calculating the activities of the OD values according to the operation of MDA, SOD, CAT and GSH-Px kit instructions by a colorimetric method.

As shown in tables 1 and 2, H is compared with the blank control₂O₂And a β 25-35, the MDA content in PC12 cells increased significantly after induction, indicating that intracellular lipid peroxide accumulation resulted after induction. And after UCMSC and UCMSC-Nrf2 treatment, the MDA content is obviously reduced, and compared with the UCMSC (p is less than 0.05), the UCMSC-Nrf2(p is less than 0.01), the MDA content is more obviously reduced. Similarly, the relevant antioxidant enzymes (SOD, GSH-Px and CAT) were also assayed, whether H₂O₂The model is also an A beta 25-35 model, and the antioxidant enzyme activities of the UCMSC-Nrf2(p is less than 0.01) group are all higher than that of the UCMSC group.

The experiment results show that UCMSC-Nrf2 can effectively antagonize H₂O₂And oxidative stress caused by A beta 25-35.

TABLE 1 UCMSC-Nrf2 vs. H₂O₂Effect of MDA content and SOD, CAT, GSH-Px Activity after Induction of PC12 cells

Note: p < 0.05, p < 0.01.

TABLE 2 influence of UCMSC-Nrf2 on MDA content and SOD, CAT, GSH-Px activity after A beta 25-35 induces PC12 cells

Note: p < 0.05, p < 0.01.

Example five UCMSC-Nrf2 neural induced differentiation in vitro

Experiment was divided into 2 groups: a normal control group of UCMSC and a UCMSC-Nrf2 group. The two groups of cells were arranged at 5X 10⁴And/hole inoculation is carried out in a 24-hole plate paved with cell climbing sheets in advance, culture solution is supplemented to 1mL for culture, and in-vitro nerve induction is carried out after cells are completely attached to the wall after 24 hours. Pre-induction is carried out for 24 hours, and a pre-induction culture medium: L-DMEM + 10% fetal calf serum +100 mug/L basic fibroblast growth factor, then inducing with induction medium for 7d, induction medium: L-DMEM + 0.1% dimethyl sulfoxide +2mmol/L tretinoin +100 μ g/L basic fibroblast growth factor +100 μ g/L epidermal growth factor +100 μ g/L brain-derived neurotrophic factor.

As shown in FIG. 7, a small percentage of the cells began to develop elongation-like elongation 7 days after the neural induction in the UCMSC normal control group, and a large percentage of the cells developed elongation-like elongation 7 days after the neural induction in the UCMSC-Nrf2 group, and a part of the cells exhibited typical neuron-like morphology.

The experimental results show that the UCMSC-Nrf2 has stronger capability of differentiating into neural stem cells.

Example establishment of an animal model for six-stage Alzheimer's disease

40 Kunming mice (20-25g) were selected, 10 were randomly selected as sham surgery groups, and 30 were selected as treatment groups. After 500mg/kg of chloral hydrate is anesthetized by intraperitoneal injection, the three-dimensional brain positioning instrument is fixed, after the head is disinfected, shaved and prepared, the scalp is cut to expose a human-shaped seam or a cross-shaped seam. Referring to brain stereotactic map, using anterior halogen as center, 2.0mm behind the anterior halogen and 2.5mm lateral to the left or right side of midline, drilling skull with dental drill, exposing dura mater, perpendicularly inserting needle 2.0mm from brain surface with micro-syringe, and slowly injecting Abeta into brain tissue_25-35mu.L (1 g/L). Injecting for 5min, reserving needle for 5min, slowly withdrawing needle to prevent reflux, sealing skull with dental mud after injection, sterilizing and suturing skin with penicillin powder, performing the same operation on the sham operation group and the treatment group, and injecting 0.9% sterile normal saline in the same amount into bilateral hippocampus. Mice were placed in an incubator until awakened after surgery. Randomly dividing the treatment group of model mice into a model group, a UCMSC group and a UCMSC-Nrf2 group, and carrying out tail vein injection of 0.2ml of UCMSC or UCMSC-Nrf2 for treatment (the formula is shown in the specification)Concentration of 1x10⁶Pieces/ml), sham and model groups were replaced with equal volumes of saline. 2 times a week for 5 weeks. The mouse behavioural test was performed 5 weeks later, with the test being performed every day in the morning for the same period.

EXAMPLE seven mouse behavioural Observation- -electric diving platform test

And (4) carrying out an electric jump bench small test experiment by using the senile dementia animal model and the grouping established in the sixth embodiment. The experimental device for the electric diving platform test is a rectangular organic glass box with the size of 55cm multiplied by 11cm multiplied by 33cm, and is uniformly divided into 5 small boxes by opaque black plastic plates. The bottom is laid with a copper fence as a stimulating electrode. A rubber pad with the diameter of 4.5cm and the height of 4.5cm is placed at one corner of each small box to serve as a safety platform of a mouse escape electrode. During the experiment, the animals are placed in the box to adapt for 5min, then the 36V alternating current is conducted, and the animals are stimulated by the electric shock to normally respond by jumping to a safety platform to avoid the electric shock. Most animals may be stimulated to jump to the copper grid again or multiple times, and thus jump to the platform multiple times. And recording the frequency of the electric shock received by each mouse within 3min as the error frequency. And (5) testing after 24h, and recording the latency period of the first platform jump of the mouse and the number of times of electric shock (error number) of the mouse.

The electric jump bench test was used to evaluate the effect of cell transplantation therapy on spatial learning and memory impairment in mice caused by A β 25-35. Evaluation was performed 24h after training and study, and the number of errors per group of mice within 3min and the mean latency of each group were analyzed.

As shown in fig. 8 and 9, the mice in the a β 25-35 model group had a shorter mean latency and a significantly increased number of errors (P < 0.01) compared to the sham group; compared with the model group, the incubation periods of the UCMSC group and the UCMSC-Nrf2 group are obviously prolonged (P is less than 0.05, P is less than 0.01), the error times are also obviously reduced (P is less than 0.05, P is less than 0.01), and the treatment effect of the UCMSC-Nrf2 is obviously better than that of the UCMSC.

The experimental result shows that UCMSC-Nrf2 has obvious improvement effect on mouse learning and memory disorder caused by Abeta 25-35.

Example eight effects of the oxidative status in the brain of mice

After the completion of the behavioral experiments in example seven, all animals were rapidly sacrificed by decapitation after deep anesthesia with chloral hydrate. The brain tissue was rapidly peeled off on a petri dish on which ice cubes were placed to obtain hippocampus. A portion of the hippocampus was rinsed with cold physiological saline to remove blood. Wiping with filter paper, weighing, adding 4 deg.C physiological saline at a weight/volume ratio of 1:9 to obtain 10% tissue homogenate, centrifuging at low temperature (4000r/min) for 10min, and collecting supernatant as biochemical index for detection; the other half of the hippocampus was stored in liquid nitrogen for subsequent Western Blot experiments.

Taking a certain amount of supernatant of the homogenate of the hippocampal tissue, and respectively measuring the content of MDA in the hippocampal tissue of the mouse by a thiobarbituric acid (TBA) micro-method according to the operation instruction of the kit, wherein the MDA in the sample can be condensed with the TBA to form a red product, and the maximum absorption peak is at 532 nm. The principle of measuring the SOD activity by using a xanthine oxidase method is that under the aerobic condition, the xanthine oxidase can oxidize xanthine to generate superoxide anion free radicals, the superoxide anion free radicals oxidize hydroxylamine to form nitrite, the nitrite is purple red under the condition of a certain color developing agent, and the oxidation process is influenced by the SOD to reduce the generation of the nitrite, so that the GSH content can be detected by using a DTNB method through an ultraviolet spectrophotometer. The protein concentration was measured using BCA protein concentration assay kit (bicinchoninic acid protein assay kit).

As shown in table 3, the MDA level in the hippocampal tissue of the model group mice was significantly increased compared to the sham-operated group, indicating that there was a large accumulation of lipid peroxidation products in the hippocampal tissue of the model group mice. Compared with the model group, both the UCMSC group and the UCMSC-Nrf2 can obviously reduce the MDA content (P is less than 0.05 and less than 0.01), and the UCMSC-Nrf2 group is more obvious. The experiment detects SOD and GSH at the same time, and the result shows that compared with a sham operation group, the SOD and GSH activity in the mouse hippocampal tissue of a model group is obviously reduced, which shows that the oxidation resistance of an organism is reduced; compared with the model group, the UCMSC group and the UCMSC-Nrf2 can improve the activity of SOD and GSH to different degrees (P is less than 0.01). The increase in the UCMSC-Nrf2 group was more pronounced.

The experimental results show that UCMSC-Nrf2 can effectively protect senile dementia mouse brain tissue oxidative damage caused by Abeta 25-35.

TABLE 3 protection of UCMSC-Nrf2 against Abeta 25-35 induced oxidative damage to brain tissue in mice with Alzheimer's disease

Note: p < 0.05, p < 0.01.

EXAMPLE nine Effect on Acetylcholinesterase (AChE) and Nitric Oxide (NO) content in Hippocampus tissues

Impairment of the choline system plays a key role in the pathogenesis of senile dementia. Using the model and cohort of alzheimer's disease established in example six, levels of AChE and NO in hippocampal tissues in mouse brains were determined using commercially available kits. AChE and NO assays were tested in detail according to the kit procedures. The enzyme activity in the sample is calculated by measuring the absorbance of the product. The NO detection method adopts an acid reductase method to detect the content of NO in a sample.

As shown in fig. 10, AChE and NO activities were significantly increased in hippocampal tissues of the abeta 25-35 model group mice compared to the sham-operated group mice; both AChE and NO levels were significantly reduced when therapy was given consecutively to UCMSC and UCMSC-Nrf2, compared to the model group, especially the UCMSC-Nrf2 group.

The experimental results show that UCMSC-Nrf2 can effectively reduce the activity of hippocampal AChE and NO in the brain of the senile dementia mouse caused by Abeta 25-35.

Example ten mice detection of anti-inflammatory factors in hippocampal tissue

The animal model and the grouping of senile dementia established in example six are utilized. The contents of interleukin-6 (IL-6), tumor necrosis factor (TNF-alpha) and interleukin 1 beta (IL-1 beta) in the hippocampal tissues of mice were determined by a commercially available ELISA kit. The specific experimental procedures were performed according to the kit instructions.

As shown in FIG. 12, compared with the sham-operated group, the inflammation-related factors (TNF-alpha, IL-1 beta and IL-6) in the hippocampal tissues in the brain of the model group mice were all increased sharply (P < 0.01), which indicates that the bilateral hippocampal injection of Abeta 25-35 in the mice causes a certain neuroinflammation. Compared with the A beta 25-35 model group, the content of inflammation-related factors of the UCMSC and the UCMSC-Nrf2 group is obviously reduced. Of these, the inflammatory factor decreased most significantly in the UCMSC-Nrf2 group.

The experimental results show that UCMSC-Nrf2 can effectively inhibit the release of inflammatory factors in hippocampal tissues in brains of senile dementia mice.

The above-mentioned embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the same; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications and substitutions do not depart from the essence of the corresponding technical solutions.

Sequence listing

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gatgactgca tgcagctttt ggcgcagaca ttcccgtttg tagatgacaa tgaggtttct 360

tcggctacgt ttcagtcact tgttcctgat attcccggtc acatcgagag cccagtcttc 420

attgctacta atcaggctca gtcacctgaa acttctgttg ctcaggtagc ccctgttgat 480

ttagacggta tgcaacagga cattgagcaa gtttgggagg agctattatc cattcctgag 540

ttacagtgtc ttaatattga aaatgacaag ctggttgaga ctaccatggt tccaagtcca 600

gaagccaaac tgacagaagt tgacaattat catttttact catctatacc ctcaatggaa 660

aaagaagtag gtaactgtag tccacatttt cttaatgctt ttgaggattc cttcagcagc 720

atcctctcca cagaagaccc caaccagttg acagtgaact cattaaattc agatgccaca 780

gtcaacacag attttggtga tgaattttat tctgctttca tagctgagcc cagtatcagc 840

aacagcatgc cctcacctgc tactttaagc cattcactct ctgaacttct aaatgggccc 900

attgatgttt ctgatctatc actttgcaaa gctttcaacc aaaaccaccc tgaaagcaca 960

gcagaattca atgattctga ctccggcatt tcactaaaca caagtcccag tgtggcatca 1020

ccagaacact cagtggaatc ttccagctat ggagacacac tacttggcct cagtgattct 1080

gaagtggaag agctagatag tgcccctgga agtgtcaaac agaatggtcc taaaacacca 1140

gtacattctt ctggggatat ggtacaaccc ttgtcaccat ctcaggggca gagcactcac 1200

gtgcatgatg cccaatgtga gaacacacca gagaaagaat tgcctgtaag tcctggtcat 1260

cggaaaaccc cattcacaaa agacaaacat tcaagccgct tggaggctca tctcacaaga 1320

gatgaactta gggcaaaagc tctccatatc ccattccctg tagaaaaaat cattaacctc 1380

cctgttgttg acttcaacga aatgatgtcc aaagagcagt tcaatgaagc tcaacttgca 1440

ttaattcggg atatacgtag gaggggtaag aataaagtgg ctgctcagaa ttgcagaaaa 1500

agaaaactgg aaaatatagt agaactagag caagatttag atcatttgaa agatgaaaaa 1560

gaaaaattgc tcaaagaaaa aggagaaaat gacaaaagcc ttcacctact gaaaaaacaa 1620

ctcagcacct tatatctcga agttttcagc atgctacgtg atgaagatgg aaaaccttat 1680

tctcctagtg aatactccct gcagcaaaca agagatggca atgttttcct tgttcccaaa 1740

agtaagaagc cagatgttaa gaaaaactag 1770

<210>2

<211>589

<212>PRT

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>2

Met Asp Leu Ile Asp Ile Leu Trp Arg Gln Asp Ile Asp Leu Gly Val

1 5 10 15

Ser Arg Glu Val Phe Asp Phe Ser Gln Arg Arg Lys Glu Tyr Glu Leu

20 25 30

Glu Lys Gln Lys Lys Leu Glu Lys Glu Arg Gln Glu Gln Leu Gln Lys

35 40 45

Glu Gln Glu Lys Ala Phe Phe Ala Gln Leu Gln Leu Asp Glu Glu Thr

50 55 60

Gly Glu Phe Leu Pro Ile Gln Pro Ala Gln His Ile Gln Ser Glu Thr

65 70 75 80

Ser Gly Ser Ala Asn Tyr Ser Gln Val Ala His Ile Pro Lys Ser Asp

85 90 95

Ala Leu Tyr Phe Asp Asp Cys Met Gln Leu Leu Ala Gln Thr Phe Pro

100 105 110

Phe Val Asp Asp Asn Glu Val Ser Ser Ala Thr Phe Gln Ser Leu Val

115 120 125

Pro Asp Ile Pro Gly His Ile Glu Ser Pro Val Phe Ile Ala Thr Asn

130 135 140

Gln Ala Gln Ser Pro Glu Thr Ser Val Ala Gln Val Ala Pro Val Asp

145 150 155 160

Leu Asp Gly Met Gln Gln Asp Ile Glu Gln Val Trp Glu Glu Leu Leu

165 170 175

Ser Ile Pro Glu Leu Gln Cys Leu Asn Ile Glu Asn Asp Lys Leu Val

180 185 190

Glu Thr Thr Met Val Pro Ser Pro Glu Ala Lys Leu Thr Glu Val Asp

195 200 205

Asn Tyr His Phe Tyr Ser Ser Ile Pro Ser Met Glu Lys Glu Val Gly

210 215 220

Asn Cys Ser Pro His Phe Leu Asn Ala Phe Glu Asp Ser Phe Ser Ser

225 230 235 240

Ile Leu Ser Thr Glu Asp Pro Asn Gln Leu Thr Val Asn Ser Leu Asn

245 250 255

Ser Asp Ala Thr Val Asn Thr Asp Phe Gly Asp Glu Phe Tyr Ser Ala

260 265 270

Phe Ile Ala Glu Pro Ser Ile Ser Asn Ser Met Pro Ser Pro Ala Thr

275 280 285

Leu Ser His Ser Leu Ser Glu Leu Leu Asn Gly Pro Ile Asp Val Ser

290 295 300

Asp Leu Ser Leu Cys Lys Ala Phe Asn Gln Asn His Pro Glu Ser Thr

305 310 315 320

Ala Glu Phe Asn Asp Ser Asp Ser Gly Ile Ser Leu Asn Thr Ser Pro

325 330 335

Ser Val Ala Ser Pro Glu His Ser Val Glu Ser Ser Ser Tyr Gly Asp

340 345 350

Thr Leu Leu Gly Leu Ser Asp Ser Glu Val Glu Glu Leu Asp Ser Ala

355 360 365

Pro Gly Ser Val Lys Gln Asn Gly Pro Lys Thr Pro Val His Ser Ser

370 375 380

Gly Asp Met Val Gln Pro Leu Ser Pro Ser Gln Gly Gln Ser Thr His

385 390 395 400

Val His Asp Ala Gln Cys Glu Asn Thr Pro Glu Lys Glu Leu Pro Val

405 410 415

Ser Pro Gly His Arg Lys Thr Pro Phe Thr Lys Asp Lys His Ser Ser

420 425 430

Arg Leu Glu Ala His Leu Thr Arg Asp Glu Leu Arg Ala Lys Ala Leu

435 440 445

His Ile Pro Phe Pro Val Glu Lys Ile Ile Asn Leu Pro Val Val Asp

450 455 460

Phe Asn Glu Met Met Ser Lys Glu Gln Phe Asn Glu Ala Gln Leu Ala

465 470 475 480

Leu Ile Arg Asp Ile Arg Arg Arg Gly Lys Asn Lys Val Ala Ala Gln

485 490 495

Asn Cys Arg Lys Arg Lys Leu Glu Asn Ile Val Glu Leu Glu Gln Asp

500 505 510

Leu Asp His Leu Lys Asp Glu Lys Glu Lys Leu Leu Lys Glu Lys Gly

515 520 525

Glu Asn Asp Lys Ser Leu His Leu Leu Lys Lys Gln Leu Ser Thr Leu

530 535 540

Tyr Leu Glu Val Phe Ser Met Leu Arg Asp Glu Asp Gly Lys Pro Tyr

545 550 555 560

Ser Pro Ser Glu Tyr Ser Leu Gln Gln Thr Arg Asp Gly Asn Val Phe

565 570 575

Leu Val Pro Lys Ser Lys Lys Pro Asp Val Lys Lys Asn

580 585

Claims

1. An Nrf2 gene modified umbilical cord mesenchymal stem cell, wherein the Nrf2 gene modified umbilical cord mesenchymal stem cell is an umbilical cord mesenchymal stem cell over expressing Nrf 2.

2. The Nrf2 gene-modified umbilical cord mesenchymal stem cell according to claim 1, wherein the sequence of the Nrf2 gene has the sequence as shown in SEQ ID NO: 1, or SEQ ID NO: 1 by replacing one or more nucleotides.

3. The Nrf2 gene-modified umbilical cord mesenchymal stem cell according to claim 1, wherein the cell is prepared by the following method:

1) constructing an Nrf2 gene recombinant plasmid vector;

4. The preparation method of the umbilical cord mesenchymal stem cell modified by the Nrf2 gene as claimed in any one of claims 1 to 3, which is characterized by comprising the following steps:

1) constructing an Nrf2 gene recombinant plasmid vector;

5. The preparation method according to claim 4, wherein the sequence of the Nrf2 gene has the sequence shown in SEQ ID NO: 1, or SEQ ID NO: 1 by replacing one or more nucleotides.

6. Use of the Nrf2 gene-modified umbilical cord mesenchymal stem cell of any one of claims 1 to 3 or the Nrf2 gene-modified umbilical cord mesenchymal stem cell prepared by the preparation method of any one of claims 4 to 5 for promoting differentiation of mesenchymal stem cell into neural stem cell.

7. Use according to claim 6, characterized in that it comprises the following steps:

1) constructing an Nrf2 gene recombinant plasmid vector;

2) transfecting the umbilical cord mesenchymal stem cells with the Nrf2 gene recombinant plasmid vector to obtain umbilical cord mesenchymal stem cells modified by Nrf2 genes;

8. The use of claim 7, wherein the sequence of the Nrf2 gene has the sequence as set forth in SEQ ID NO: 1, or SEQ ID NO: 1 by replacing one or more nucleotides.

9. Use of the Nrf2 gene modified umbilical cord mesenchymal stem cell of any one of claims 1 to 3 or the Nrf2 gene modified umbilical cord mesenchymal stem cell prepared by the preparation method of any one of claims 4 to 5 in preparation of a medicament for treating senile dementia.

10. A medicament, comprising: the Nrf2 gene modified umbilical cord mesenchymal stem cell of any one of claims 1 to 3 or the Nrf2 gene modified umbilical cord mesenchymal stem cell prepared by the preparation method of any one of claims 4 to 5.