Application of human peroxiredoxin-5 in inducing differentiation of human umbilical cord mesenchymal stem cells into neural stem cells and induction method

Abstract

The present invention provides an application of human peroxiredoxin-5 in inducing the differentiation of human umbilical cord mesenchymal stem cells into neural stem cells and an induction method, and relates to the field of cell engineering technology. The present invention provides an application of hPRDX5 in inducing the differentiation of human umbilical cord mesenchymal stem cells into neural stem cells. It is confirmed by the examples that adding different concentrations of hPRDX5 to the complete medium of UC-MSCs has a certain degree of NSCs induction effect; adding hPRDX5 to the neural stem cell induction differentiation medium has a significant NSCs induction effect.

Description

Technical Field

[0001] The invention belongs to the technical field of cell engineering, and specifically relates to the application of human peroxiredoxin-5 in inducing the differentiation of human umbilical cord mesenchymal stem cells into neural stem cells and an induction method. Background Art

[0002] Neurodegenerative diseases are difficult to cure with conventional treatments. The number of neurons in a specific subpopulation of patients will gradually decrease, which will lead to specific nervous system dysfunction. For example, degeneration and necrosis of dopaminergic neurons will lead to impaired striatal function, thus causing Parkinson's disease. As far as current pharmacological and neurosurgical treatments are concerned, they can only temporarily relieve symptoms and cannot prevent the progression of neurodegenerative diseases.

[0003] Under certain culture conditions, stem cells can be induced to differentiate into nerve cells, thereby repairing damaged nerve tissue. They can also secrete neurotrophic factors to promote the proliferation and differentiation of surviving cells. Studies in recent years have shown that transplanting neural stem cells (NSCs) with regenerative capacity is an accurate and effective treatment method in the treatment of central nervous system diseases. Transplanted NSCs play a cell replacement and paracrine role in the patient's central nervous system. Clinical studies using NSCs to treat diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and spinal cord injury are also being carried out.

[0004] Generally speaking, there are two ways to obtain NSCs. One is to directly isolate and obtain them from the brain tissue of adults or fetuses, and the other is to differentiate embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to indirectly obtain NSCs. Autologous NSCs are stored in the subventricular zone after adulthood, and as they age, autologous NSCs will only maintain minimal regenerative capacity, so autologous NSCs are not the best choice for stem cell transplantation. The isolation technology of ESCs is still immature and often difficult to carry out due to ethical issues. In addition, its potential tumorigenicity is also difficult to ignore. Although the use of iPSCs as an alternative seems promising, the reprogramming process is not only time-consuming, but the purity of the iPSCs obtained is also controversial. Studies have shown that human mesenchymal stem cells (hMSCs) do not have tumorigenicity and ethical issues. hMSCs can be obtained from a variety of adult tissues such as bone marrow and fat, and have been widely studied. hMSCs can also be obtained from fetal tissues, including placenta, amniotic fluid, umbilical cord, etc., but when converting hMSCs into NSCs, there may be tissue compatibility issues and the expression of the neural stem cell marker (Nestin) is low. Summary of the invention

[0005] The purpose of the present invention is to provide an application of human peroxiredoxin-5 (hPRDX5) in inducing human umbilical cord mesenchymal stem cells to differentiate into neural stem cells and an induction method. Under the induction of hPRDX5, the induced cells express a neural stem cell marker (Nestin) with a significant increase.

[0006] The present invention provides the use of human peroxiredoxin-5 in inducing human umbilical cord mesenchymal stem cells (UC-MSCs) to differentiate into neural stem cells (NSCs).

[0007] The present invention also provides the use of human peroxiredoxin-5 combined with neural induction factor in inducing the differentiation of human umbilical cord mesenchymal stem cells into neural stem cells.

[0008] Preferably, the human peroxide reductase-5 includes recombinant human peroxide reductase-5, and the nucleotide sequence of the recombinant human peroxide reductase-5 is shown in SEQ ID NO.1.

[0009] Preferably, the human peroxide reductase-5 includes recombinant human peroxide reductase-5, and the amino acid sequence of the recombinant human peroxide reductase-5 is shown in SEQ ID NO.2.

[0010] The present invention also provides a method for inducing human umbilical cord mesenchymal stem cells to differentiate into neural stem cells, comprising inducing and culturing the human umbilical cord mesenchymal stem cells in an induction medium;

[0011] The induction medium contains human peroxide reductase-5;

[0012] The basal culture medium of the induction culture medium includes UC-MSCs complete culture medium or neural stem cell induction differentiation culture medium.

[0013] Preferably, the human umbilical cord mesenchymal stem cells include cultured human umbilical cord mesenchymal stem cells of generation P4 to P10, with a degree of fusion of 90%.

[0014] Preferably, the concentration of human peroxide reductase-5 in the induction medium is not less than 1-10 μg / mL.

[0015] Preferably, the UC-MSCs complete culture medium comprises DMEM / F12 culture medium containing 10% fetal bovine serum (FPS).

[0016] Preferably, the neural stem cell differentiation medium is based on DMEM / F12 as the basic medium and also includes N-2, B-27 TM , 20ng / mL epidermal growth factor (EGF) and 20ng / mL basic fibroblast growth factor (bFGF).

[0017] Preferably, the parameters of the induction culture include: 37° C., 5% CO 2 concentration and saturated humidity.

[0018] Beneficial effects: The present invention provides an application of hPRDX5 in inducing human umbilical cord mesenchymal stem cells to differentiate into neural stem cells. The examples show that adding different concentrations of hPRDX5 to the UC-MSCs complete culture medium has a certain degree of NSCs induction effect; adding hPRDX5 to the neural stem cell induction differentiation culture medium has a significant NSCs induction effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0020] Figure 1The growth status of UC-MSCs in complete medium (100×, scale bar = 200 μm); A: UC-MSCs cultured in UC-MSCs complete medium on the second day showed spindle or triangle-shaped colonies; B: UC-MSCs cultured in complete medium on the fifth day showed vortex-shaped growth, 100% fusion;

[0021] Figure 2 Flow cytometry was used to detect the surface markers of UC-MSCs; the positive phenotypes were CD29, CD73, CD90 and CD105; the negative phenotypes were CD19, CD34, CD45 and HLA-DR;

[0022] Figure 3 The results of UC-MSCs differentiation into osteoblasts, adipocytes and chondroblasts (100×, scale bar = 100 μm); A: differentiation into osteoblasts; B: differentiation into adipocytes; C: differentiation into chondroblasts;

[0023] Figure 4 Observation pictures of UC-MSCs differentiation into NSCs (100×, scale bar = 200 μm); A: Day 1 of UC-MSCs differentiation into NSCs; B: Day 3 of UC-MSCs differentiation into NSCs;

[0024] Figure 5 Figure 1 shows the immunohistochemical detection of Nestin-positive cells (100×, scale bar = 50 μm); A: blank control group; B: positive control group; C: positive control + 1 μg / mL hPRDX5 group;

[0025] Figure 6 The ratio of Nestin-positive cells was detected by immunohistochemistry; the uninduced group was the blank control group, and the standard induced group was the positive control group; * indicates P < 0.05, ** indicates P < 0.01;

[0026] Figure 7 Western blot was used to detect the relative expression of Nestin protein; the uninduced group in the figure was the blank control group, and the standard induced group was the positive control group;

[0027] Figure 8 Western blot was used to detect the relative expression of Nestin protein; the uninduced group was the blank control group, and the standard induced group was the positive control group; * indicates P < 0.05, and ** indicates P < 0.01. DETAILED DESCRIPTION

[0028] The present invention provides the use of human peroxiredoxin-5 in inducing human umbilical cord mesenchymal stem cells to differentiate into neural stem cells.

[0029] The present invention does not specifically limit the source of the hPRDX5, and preferably includes one or more of recombinant hPRDX5, a gene encoding the recombinant hPRDX5, a recombinant vector containing the gene, and a recombinant cell containing the recombinant vector. The nucleotide sequence of the recombinant hPRDX5 of the present invention is preferably as shown in SEQ ID NO.1, and the encoded amino acid sequence is preferably as shown in SEQ ID NO.2. The recombinant vector of the present invention is preferably composed of the above-mentioned gene and an expression vector, and the expression vector preferably includes pET28a (+). When constructing the recombinant vector, EcoRI and HindIII are preferably used as restriction sites to construct the target gene into a plasmid vector. The recombinant cell of the present invention preferably includes a monoclonal cell strain, and more preferably is obtained by transferring the above-mentioned recombinant vector into an expression host. The expression host of the present invention is preferably Escherichia coli BL21 (DE3). The present invention does not specifically limit the specific construction method of the recombinant vector and the recombinant cell, and preferably refers to Chinese patent CN110105442A.

[0030] The present invention also provides a method for inducing human umbilical cord mesenchymal stem cells (UC-MSCs) to differentiate into neural stem cells (NCSs), comprising inducing and culturing the human umbilical cord mesenchymal stem cells in an induction medium;

[0031] The induction medium contains human peroxide reductase-5;

[0032] The basal culture medium of the induction culture medium includes UC-MSCs complete culture medium or neural stem cell induction differentiation culture medium for induction culture.

[0033] The present invention does not specifically limit the source of the UC-MSCs. In the embodiment, it is preferably extracted from the umbilical cord of healthy newborns born by caesarean section at full term. The obtained primary umbilical cord mesenchymal stem cells (UC-MSCs, P0) are washed with PBS, and then digested with 0.125% (m / v) trypsin to obtain a cell suspension. The residual gel blocks are removed by a 100 μm filter, and the cells are passaged after centrifugation to obtain P1 (first generation cells). The UC-MSCs used for induction in the present invention are preferably cells obtained after passage of the above-mentioned P1 generation cells, and cells of P4 to P10 generations can be induced. In the embodiment, P4 generation cells are preferably induced. When the P4 cells grow to 90% fusion, they are digested with 0.125% trypsin, centrifuged at 1500rpm for 5 minutes, and the supernatant is discarded; then the cells are washed with PBS, and the cells are resuspended in the induction medium to adjust the cell number to 2×10 5 Pieces / cm 2In the embodiment of the present invention, MSCs obtained from the umbilical cord Wharton's jelly are used, which are relatively easy to obtain; have low immunogenicity, do not have tissue compatibility issues, and avoid transplant rejection and the use of immunosuppressants.

[0034] In the induction medium of the present invention, the concentration of human peroxide reductase-5 is preferably 1-10 μg / mL, more preferably 1-8 μg / mL, more preferably 1-3 μg / mL, and more preferably 1 μg / mL. The basal medium of the induction medium of the present invention can be of two types, namely, UC-MSCs complete medium or neural stem cell induction differentiation medium as the basal medium, and the UC-MSCs complete medium preferably includes DMEM / F12 medium containing 10% fetal bovine serum; the neural stem cell induction differentiation medium preferably uses DMEM / F12 as the basal medium and also includes N-2, B-27 TM , EGF and bFGF, and N-2 and B-27 TM The dosage of N-2 and B-27 is fixed, while the dosage of EGF and bFGF can be adjusted. For example, in the embodiment, the neural stem cell differentiation medium includes 20 ng / mL EGF and 20 ng / mL bFGF. TM The additives are preferably commercially available N-2 additive (100X) and B-27 TM Additive (50X), for example, in 48.5 ml of DMEM / F12 medium containing 10% fetal bovine serum, take 0.5 ml of N-2 additive and dilute it 100 times and then add it all, B-27 TM Take 1 mL of the additive and dilute it 50 times before adding it all.

[0035] The parameters of the induction culture of the present invention preferably include induction culture for 3 days under the conditions of 37° C., 5% CO 2 and saturated humidity.

[0036] To further illustrate the present invention, the application of human peroxiredoxin-5 provided by the present invention in inducing human umbilical cord mesenchymal stem cells to differentiate into neural stem cells and the induction method are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1

[0038] UC-MSCs culture and identification

[0039] 1. Extraction of primary UC-MSCs cells

[0040] Under sterile conditions, select a 15cm umbilical cord from a healthy newborn baby born by caesarean section at full term, place it in a special umbilical cord storage bottle, and perform cell extraction within 12 hours. Use 75% alcohol to simply sterilize the storage bottle and then place it in the clean bench. The umbilical cord needs to be washed twice with PBS to wash away the umbilical cord blood, and then washed with 75% alcohol for simple sterilization. To facilitate subsequent operations, use surgical scissors to cut it into small segments of 1 to 2 cm. After removing the two arteries and one vein of the umbilical cord, tear off the white Wharton's jelly from the inner epidermis of the umbilical cord and use surgical scissors to divide it into 1 to 2 mm 3 After all the Wharton's gel was separated, the Wharton's gel was concentrated into a 50 mL centrifuge tube and washed twice with PBS at 1500 rpm for 1 min. Then the Wharton's gel was concentrated into a 75 cm 2 In the culture flask, add 5-10 mL of complete culture medium (DMEM / F12 + 10% FBS) according to the number of gel blocks. In the early stage (1-2 days), the number of cells is small and has not yet grown to a large scale. Most of them are spindle-shaped or triangular ( Figure 1 In the later stage (5-7 days later), as the cells gradually proliferate, the space for cell growth in the culture flask decreases, and the cells are arranged in a spiral shape. This situation is generally called "fusion" ( Figure 1 (B). The medium was changed every 7 days. After about 10 to 14 days, fibroblast-like cells were found crawling out of the glue blocks attached to the wall. These cells are primary umbilical cord mesenchymal stem cells (UC-MSCs, P0). Most of the glue blocks were then blown off with PBS and kept for subsequent culture. The cell suspension was then obtained by digestion with 0.125% trypsin, and the residual glue blocks were removed with a 100μm filter. After centrifugation, the cells were passaged to obtain P1 (first generation cells).

[0041] 2. Identification of UC-MSCs surface markers

[0042] When the cells were cultured to P4, the cell density of UC-MSCs was counted and 1×10 6 Centrifuge the cells at 1000rpm for 2min, discard the supernatant, resuspend the cells with 2mL PBS, centrifuge and discard the supernatant. Resuspend the cells with PBS, set up control tubes and test tubes, and add CD73-FITC, CD29-APC, CD105-PE, CD90-FITC, CD34-FITC, HLA-DR-Percp, CD45-PE and CD19-APC antibodies respectively. Incubate at 4℃ in the dark for 30min, add 2mL PBS to wash the cells and wash away excess antibodies. Finally, resuspend the cells with 200μL PBS and detect them on a flow cytometer. P4 cells exhibit 4 negative phenotypes and 4 positive phenotypes. The positive phenotypes are CD29, CD73, CD90 and CD105, and the negative phenotypes are CD19, CD34, CD45 and HLA-DR( Figure 2 ), indicating that the cultured UC-MSCs cells meet the requirements of the International Society of Cell Therapy for the phenotype of UC-MSCs.

[0043] 3. Identification of UC-MSCs differentiation ability

[0044] The UC-MSCs cells cultured to P4 were identified for their ability to differentiate into osteoblasts, adipocytes, and chondroblasts. Osteoblast differentiation, adipocyte differentiation, and chondroblast differentiation were all induced using commercial kits from Saiye Biotechnology according to the kit instructions, and were identified using Alizarin Red staining, Oil Red O staining, and Alician Blue staining. Calcium nodules stained with Alizarin Red were observed in UC-MSCs cultured in osteogenic induction medium ( Figure 3 Oil Red O dye was observed to accumulate in intracellular lipid droplets in UC-MSCs cultured in adipogenic induction medium ( Figure 3 In the UC-MSCs cultured in chondrogenic induction medium, it was observed that the cells gradually turned into chondrocytes. After fixed sections were stained with Alcian blue, the acidic mucopolysaccharide in the cartilage tissue turned blue ( Figure 3 Middle C).

[0045] Example 2

[0046] Neural stem cell differentiation culture

[0047] 1) When P4 cells grew to 90% confluence, they were digested with 0.125% trypsin to obtain UC-MSCs. Centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. After washing the cells twice with PBS, the cells were counted.

[0048] 2) Resuspend the cells in the following 5 culture media and adjust the cell number to 2×10 5 Pieces / cm 2 , respectively inoculated at 25cm 2 In each bottle, three parallel controls were set up for each group, and 5 mL of cell suspension was added to each bottle.

[0049] The experiment was divided into 5 groups, and the culture media used were:

[0050] ① Complete medium group (blank control): DMEM / F12 medium supplemented with 10% FBS;

[0051] ②Complete medium group + 1 μg / mL hPRDX5 group: DMEM / F12 medium supplemented with 10% FBS and 1 μg / mL hPRDX5;

[0052] ③Complete medium group + 10 μg / mL hPRDX5 group: DMEM / F12 medium supplemented with 10% FBS and 10 μg / mL hPRDX5;

[0053] ④Neural stem cell positive control culture medium group (positive control): DMEM / F12, N-2 (100X dilution), B-27 TM (50X dilution), 20 ng / mL EGF, and 20 ng / mL bFGF;

[0054] ⑤ Neural stem cell positive control medium + 1 μg / mL PRDX5 group: DMEM / F12, N-2 (100X dilution), B-27 TM (50X dilution), 20 ng / mL EGF, 20 ng / mL bFGF, and 1 μg / mL hPRDX5.

[0055] 3) After 3 days of differentiation induction, the cells of the above 5 groups (a total of 15 bottles) were digested with 0.125% trypsin, resuspended with maintenance medium, and inoculated in 25 cm 2 Since the neurospheres contain a large number of cells, it is difficult to separate the neurospheres into single cell suspensions by digestion and mechanical blowing. Therefore, the five groups of cells were cultured in the maintenance medium for 3 days. After 3 days, 0.125% trypsin was used to digest the cells of the five groups (a total of 15 bottles) and the cells were collected for subsequent detection.

[0056] The experimental groups using complete culture medium (② and ③) had almost the same appearance as the blank control group (①) during the 3-day induction process. Figure 1 ), and finally presents a spiral fusion.

[0057] The experimental group using the neural stem cell positive control medium (⑤) had almost no change in overall growth compared to the positive control group. During the induction of NSCs, two types of cells will appear in the early stage. Most of the cells will aggregate into clusters. At this time, the clustered cells are still growing on the wall, and the internal cells can be clearly seen under a microscope. A small number of cells are scattered in a rhombus or triangle shape ( Figure 4 At the end of the induction period, the cell clusters that adhered to the wall gradually grew into semi-adherent cell clusters, which are generally called neurospheres. It is difficult to see the individual cells in the neurospheres under a microscope, and they are of different sizes and unevenly distributed. It is difficult to determine the number of neurospheres in different culture media ( Figure 4 (middle B).

[0058] Example 3

[0059] Identification of NSCs induced in Example 2

[0060] 1) After 6 days of culture, the five groups of cells were respectively mounted and then subjected to immunohistochemical staining. The blue part is hematoxylin staining of the cell nucleus, and the brown part is Nestin protein staining. The brown part of the blank control group is smaller, and there are almost no Nestin-positive cells ( Figure 5 In the positive control group (④) and the positive control + hPRDX5 group (⑤), the brown part is larger, indicating that there are more Nestin-positive cells ( Figure 5 B and C). Then use a panoramic scanner to scan, and the experimental results are:

[0061] The positive control group was significantly different from the blank control group (P < 0.05), and the Nestin-positive cell ratio of the positive control group was higher than that of the blank control group, indicating that the positive control group was successfully constructed. The cells cultured with 1 μg / mL hPRDX5 added to the neural stem cell induction medium were significantly different from the positive control group (P < 0.05), and the Nestin-positive cell ratio of the induction group with hPRDX5 added on the basis of the positive control group was the highest, indicating that hPRDX5 helps UC-MSCs differentiate into NSCs. After adding hPRDX5 to the complete culture medium (1 μg / mL hPRDX5 experimental group and 10 μg / mL hPRDX5 experimental group), compared with the Nestin-positive cell ratio of the blank control group, although there was a certain degree of upward trend, the difference between them was not statistically significant. This shows that hPRDX5 alone has limited ability to induce UC-MSCs to differentiate into NSCs ( Figure 6 and Table 1).

[0062] Table 1 Nestin positive cell ratio in different culture media (%)

[0063]

[0064]

[0065] 2) Western blot was used to further detect the expression of Nestin at the protein level. The experimental result was the ratio of the gray value of the Nestin band to the gray value of the internal reference protein (GAPDH) band, which was called the relative expression of Nestin protein. Figure 7 , Figure 8 And as shown in Table 2.

[0066] Comparing the blank control group, 1μg / mL hPRDX5 group (② and ⑤), 10μg / mL hPRDX5 group (③) and positive control group, it can be found that adding hPRDX5 to the blank control group has a certain promoting effect on the relative expression of Nestin protein, and it shows a certain concentration dependence. However, after statistical analysis, it was found that there was no significant statistical difference between the blank control group, 1μg / mL hPRDX5 group and 10μg / mL hPRDX5 group. The relative expression of Nestin protein in the positive control group and the positive control + 1μg / mL hPRDX5 group was significantly different (P < 0.05). After adding 1μg / mL hPRDX5 to the neural stem cell induction medium, the induced stem cells can express more Nestin, which indicates that hPRDX5 and neural induction factors can work together to better promote the differentiation of UC-MSCs into NSCs. This result is consistent with the previous detection of the Nestin-positive cell ratio in immunohistochemistry experiments.

[0067] Table 2 Relative expression of Nestin protein in different culture media

[0068]

[0069] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of human peroxiredoxin-5 combined with neural induction factor in the preparation of a reagent for inducing human umbilical cord mesenchymal stem cells to differentiate into neural stem cells, characterized in that: The human peroxide reductase-5 includes recombinant human peroxide reductase-5, and the amino acid sequence of the recombinant human peroxide reductase-5 is shown in SEQ ID NO.2; The neural induction factors include N-2, B-27 TM , epidermal growth factor and basic fibroblast growth factor.

2. The application according to claim 1, characterized in that: The nucleotide sequence of the gene encoding the recombinant human peroxide reductase-5 is shown in SEQ ID NO.

1.

3. A method for inducing human umbilical cord mesenchymal stem cells to differentiate into neural stem cells in vitro, characterized in that: The method comprises inducing and culturing human umbilical cord mesenchymal stem cells in an induction medium; The basal medium of the induction medium includes a neural stem cell induction differentiation medium; The neural stem cell differentiation medium is based on DMEM / F12 as the basic medium, and also includes N-2, B-27 TM , 20 ng / mL epidermal growth factor, 20 ng / mL basic fibroblast growth factor and 1-10 μg / mL recombinant human peroxide reductase-5; the nucleotide sequence of the gene encoding the recombinant human peroxide reductase-5 is shown in SEQ ID NO.

1.

4. The method according to claim 3, characterized in that: The human umbilical cord mesenchymal stem cells include cultured human umbilical cord mesenchymal stem cells of generations P4 to P10, with a fusion degree of 90%.

5. The method according to claim 3, characterized in that: The induction culture parameters include: 37° C., 5% CO 2 concentration and saturated humidity.

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