Application of purple sweet potato pigment in preparation of product for inducing neural differentiation of stem cells

Abstract

The invention discloses application of purple sweet potato pigment in preparation of a product for inducing neural differentiation of stem cells, and belongs to the technical field of biological medicine. The stem cells are mesenchymal stem cells. The mesenchymal stem cells are bone marrow mesenchymal stem cells. The neural differentiation is differentiation to acetylcholinergic neurons. The product for inducing neural differentiation of the stem cells comprises a medicine for inducing the stem cells to be differentiated into neurons or a culture medium for inducing the stem cells to be differentiated into the neurons. The induction method comprises the following steps: pretreating the stem cells with a growth factor mixed solution, and then adding purple sweet potato pigment to promote the stem cells to differentiate into neurons. The concentration of the purple sweet potato pigment is 50-100 [mu] g / mL. Researches find that the purple sweet potato anthocyanin can target Atg2b and inhibit autophagy development by increasing miR-223-3p, and then BMSCs are induced to be differentiated into cholinergic neurons.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of purple sweet potato pigment in preparing products for inducing neural differentiation of stem cells. Background Art

[0002] Mesenchymal stem cells (MSCs) are pluripotent stem cells widely present in various tissues, with strong self-renewal ability and multidirectional differentiation potential. MSCs are considered to have great application potential in nerve regeneration and repair, especially in the treatment of nerve damage, neurodegenerative diseases and other aspects of the research is gradually increasing. Mesenchymal stem cells can differentiate into nervous system cells such as neurons and glial cells under certain conditions. Therefore, promoting the differentiation of mesenchymal stem cells into neurons has become an important research direction. However, the differentiation of stem cells is a complex process regulated by multiple factors. Autophagy, as a key mechanism of cell self-cleaning and metabolic balance, plays an important role in the differentiation of stem cells. Moderate autophagy contributes to the growth and differentiation of stem cells, but excessive autophagy may inhibit their differentiation potential. Therefore, regulating autophagic activity has become an important field for studying how to promote neural differentiation of stem cells.

[0003] Purple sweet potato (purple sweet potato) is a root vegetable rich in nutrients and multiple bioactive ingredients. It is an annual herbaceous plant of the Convolvulaceae family. It is an excellent sweet potato variety cultivated by Japan. Its flesh is purple-red, and the pigment contained in it is anthocyanin, called purple sweet potato color (PSPC). Foreign countries have studied purple sweet potatoes earlier, starting around the mid-20th century. However, the structure of purple sweet potato has always been the focus of research by foreign scholars, and later they have studied more about its physiological functions and antioxidant activity. Japan has conducted some research on the extraction, identification and physical and chemical properties of PSPC. Domestic scholars Lu Guoquan and others began to conduct systematic research on the pigment in 1996, and have made significant progress. It is reported that acylated pigment molecules can improve the stability of the pigment, and purple sweet potato pigment molecules are acylated pigment molecules, so they are more stable and have broad application prospects. At present, the research on purple sweet potato pigment is mostly about its antioxidant activity. For example, "The Inhibitory Effects of Purple Sweet Potato Color on Hepatic Inflammation Is Associated with Restoration of NAD+ Levels and Attenuation of NLRP3 Inflammasome Activation in High-Fat-Diet-Treated Mice" (Xin Wang et al., Molecules 2017, 22,1315; doi:10.3390 / molecules22081315) reported that PSPC was used to treat hepatitis; "Purple sweet potato color attenuates domoic acid-induced cognitive deficits by promoting estrogen receptor-a-mediated mitochondrial biogenesis signaling in mice" (Jun Lu et al., Free Radical Biology & Medicine 52 (2012) 646-659) reported that PSPC alleviated kainate-induced cognitive deficits in mice by promoting the estrogen receptor-a-mediated mitochondrial biogenesis signaling pathway; however, there has been no report on the use of PSPC to induce neural differentiation of mesenchymal stem cells. Summary of the invention

[0004] In view of the above-mentioned prior art, the purpose of the present invention is to provide an application of purple sweet potato pigment in the preparation of an induced stem cell neural differentiation product. The present invention has found through research that purple sweet potato anthocyanins can inhibit the development of autophagy by increasing miR-223-3p, targeting Atg2b, and then inducing mesenchymal stem cells (MSCs) to differentiate into cholinergic neurons, thus opening up a new use of purple sweet potato pigment.

[0005] To achieve the above object, the present invention adopts the following technical solution: The invention provides application of purple sweet potato pigment in preparing a product for inducing neural differentiation of stem cells.

[0006] Preferably, the stem cells are mesenchymal stem cells.

[0007] Preferably, the mesenchymal stem cells are bone marrow mesenchymal stem cells.

[0008] Preferably, the neural differentiation is differentiation into acetylcholinergic neurons.

[0009] Preferably, the product of inducing stem cell neural differentiation includes a drug for inducing stem cell differentiation into neurons or a culture medium for inducing stem cell differentiation into neurons.

[0010] Preferably, the induction method is: pretreating stem cells with a growth factor mixture, and then adding purple sweet potato pigment to promote the differentiation of stem cells into neurons.

[0011] Preferably, the growth factor mixture includes 50 μg / mL nerve growth factor β-NGF, 100 μg / mL fibroblast growth factor bFGF and 100 μg / mL epidermal growth factor EGF.

[0012] Preferably, the concentration of the purple sweet potato pigment is 50-100 μg / mL.

[0013] Preferably, the differentiation time is 5 to 10 days.

[0014] Beneficial effects of the present invention: (1) Through research, the present invention found that purple sweet potato anthocyanins can inhibit the development of autophagy by increasing miR-223-3p, targeting Atg2b, and thereby inducing bone marrow mesenchymal stem cells (BMSCs) to differentiate into cholinergic neurons.

[0015] (2) The present invention expands the application field of purple sweet potato anthocyanins, provides a potential new method for the clinical use of autologous stem cells to treat neurodegenerative diseases, and provides a theoretical basis for the clinical application of stem cells, which has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1: Study on the mechanism of PSPC inhibiting cell autophagy, where (a) is the effect of PSPC treatment on LC3 punctate aggregation; green is GFP-LC3, red is MAP2, and the lower left corner is an enlarged view of LC3 in the cell pointed by the white arrow; (b) is the number of cells with LC3 punctate aggregation; (c) is the number of LC3 dots in each cell; **p<0.05, *p<0.01, the above experiments are representative of three replicates; Figure 2 : Interfering with cell autophagy can affect the PSPC-mediated neural differentiation process of BMSCS, where (a) shows the effect of inhibiting autophagy on the neuronal marker MAP2 and cell morphology, and (b) shows the effect of enhancing autophagy on the neuronal marker MAP2 and cell morphology; Figure 3 : Autophagy negatively regulates PSPC-mediated BMSCs neural differentiation process; (a) shows the expression changes of neuronal markers ChAT, NSE and autophagy-related proteins and mTOR signaling pathway-related proteins after 3-MA blocking autophagy or rapamycin inducing autophagy; (b) shows the statistical analysis results of the grayscale values ​​of ChAT and NSE protein bands; (c) shows the statistical analysis results of the grayscale values ​​of autophagy-related proteins p62, beclin-1 and LC3 protein bands; (d) shows the statistical analysis results of the grayscale values ​​of p-mTOR and p-4EBP1 protein bands in the mTOR signaling pathway; **p<0.05, *p<0.01, the above experiments are representative of three replicates; Figure 4 :The miRNAs that change when PSPC induces BMSCs to differentiate into neural tissues; Figure 5 : Effects of transfection of miR-223-3p mimics and inhibitors on ChAT mRNA; Figure 6 : Effects of transfection with miR-223-3p mimics and inhibitors on Atg2b mRNA; Figure 7 : Cell survival rate of BMSCs treated with grape seed anthocyanins and purple sweet potato pigments for 1, 2, and 3 days; Figure 8 :PSPC induced MSCs to differentiate into neuron-like cells and increased the expression of the neuronal marker MAP2 protein; (a) shows the morphological changes of MSCs after treatment with PSPC and GSPE; (b) shows the expression of the neuronal marker microtubule-associated protein MAP2 by immunofluorescence; (c) shows the fluorescence intensity of MAP2; (d) shows the quantification of MAP2-positive cells; data are shown as mean ± SD, **p<0.01, compared with the ctr group; Fig. 9:Purple sweet potato pigment induces neural differentiation of MSCs; (a) is the mRNA level of nestin in MSCs treated with purple sweet potato pigment or grape seed anthocyanin for 3 and 6 days; (b) is the mRNA level of MAP2 in MSCs treated with purple sweet potato pigment or grape seed anthocyanin for 3 and 6 days; (c) is the mRNA level of NSE in MSCs treated with purple sweet potato pigment or grape seed anthocyanin for 3 and 6 days; (d) is the protein band of MAP2 and NSE in MSCs treated with purple sweet potato pigment or grape seed anthocyanin for 8 days; *p<0.05, compared with the ctr group; Fig.10 : Gene expression of neuronal subtype markers in mesenchymal stem cells treated with purple sweet potato pigment or grape seed anthocyanin for 6 days; Fig.11 : After 8 days of treatment of mesenchymal stem cells with purple sweet potato pigment, the protein level of cholinergic neuron marker ChAT increased; (a) is the protein band of cholinergic neuron marker ChAT in each group; (b) is the protein level of cholinergic neuron marker ChAT in each group; Fig.12 Acetylcholine stimulates PSPC-treated cells to produce Ca 2+ Influx phenomenon; (a) is the change of acetylcholine added to BM-MSCs treated with PSPC observed under laser scanning confocal microscopy. As time goes by, the Ca 2+ The fluorescence intensity increases and gradually returns to the resting level (arrow-marked cells). The lower left corner indicates the time (in seconds) after the addition of neurotransmitters. (b) Effects of various neurotransmitters on Ca2+ expression in the CTR group. 2+ concentration; (c) Effect of various neurotransmitters on Ca concentration in the GSPE group 2+ concentration; (d) The effects of various neurotransmitters on Ca concentration in the PSPC treatment group. 2+ The arrows indicate the time of adding neurotransmitters, Ach is acetylcholine, GABA is γ-aminobutyric acid, L-Glu is L-glutamate, and DA is dopamine. DETAILED DESCRIPTION

[0017] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0018] As introduced in the background technology section, although PSPC is a kind of natural anthocyanin, the composition of anthocyanins from different sources is also very different. The patent with application number CN202410795740.2 discloses a method for preparing neural stem cell membrane vesicles loaded with anthocyanins and its application in drugs for preventing and treating spinal cord injuries. Anthocyanins extracted from grapes are used to repair damaged neurons instead of inducing stem cell differentiation. Compared with other anthocyanins extracted from strawberries, purple cabbage and perilla, PSPC has higher stability and stronger antioxidant capacity; research on PSPC at home and abroad is still in its early stages.

[0019] Based on this, the purpose of the present invention is to provide the use of purple sweet potato pigment in the preparation of induced stem cell neural differentiation products. PSPC can inhibit brain inflammation, weaken oxidative stress, resist neuronal apoptosis, and thus improve the cognitive ability of mice, effectively preventing the onset and deterioration of Alzheimer's disease.

[0020] Autophagy is a "self-eating" phenomenon in cells. It forms autophagosomes by encapsulating intracellular organelles and proteins to be degraded, and fuses with lysosomes to form autophagolysosomes to degrade the encapsulated contents, thereby achieving cell homeostasis and organelle renewal. Since abnormal protein accumulation or amyloidosis is the main cause of neurodegenerative diseases, the regulatory role of autophagy in neurodegenerative diseases has become a research hotspot in recent years. In the neurites of the brains of AD patients, a large number of autophagic vesicles are abnormally aggregated, and the acidification of autophagic lysosomes in AD is abnormal. Existing studies have shown that during the differentiation of MSCs into neurons, the level of cell autophagy increases, and inhibiting autophagy can inhibit neural differentiation. However, there have been repeated reports on the negative role of autophagy in neural differentiation. For example, retinoic acid can activate cell autophagy, inhibit mTOR activity, and then induce mouse neuroblastoma to differentiate into neurons. These data reveal the dual regulatory role of autophagy in neural differentiation. MicroRNA (miRNAs) are a class of non-coding single-stranded RNA molecules with a length of about 18-25 nucleotides encoded by endogenous genes. They can complementarily bind to the 3' uncoding region (3'UTR) of the target gene mRNA, leading to the breakage and degradation of the target gene mRNA. More and more studies have shown that miRNAs are involved in neural differentiation, neural fate determination, neural migration, etc. The present invention uses PSPC to induce mouse bone marrow mesenchymal stem cells to differentiate into neurons, and identifies their differentiation types. The whole transcriptome sequencing technology is used to screen the miRNA that plays a role, and determine its target gene, which is associated with cell autophagy, to explore the molecular mechanism of PSPC-induced BMSCs neural differentiation, and to provide a scientific basis for discovering new treatment methods for neurodegenerative diseases. The present invention finds that miR-223-3p is expressed in PSPC-induced BMSCs neural differentiation. Overexpression of miR-223-3p can promote neural differentiation, and inhibition of miR-223-3p can inhibit neural differentiation, indicating that miR-223-3p plays an important role in PSPC-induced BMSCs neural differentiation. Further mechanism studies have shown that miR-223-3p can target the autophagy-related gene Atg2b, and preliminary Q-PCR experimental results also prove this theory. That is, purple sweet potato anthocyanins can increase miR-223-3p, target Atg2b, inhibit the development of autophagy, and then induce BMSCs to differentiate into cholinergic neurons.

[0021] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0022] The purple sweet potato pigment (PSPC) of the present invention was purchased from Qingdao Pengyuan Natural Pigment Research Institute (Qingdao, China).

[0023] Its components were studied in "The Inhibitory Effects of Purple Sweet Potato Color on Hepatic Inflammation Is Associated with Restoration of NAD+ Levels and Attenuation of NLRP3 Inflammasome Activation in High-Fat-Diet-Treated Mice" (Xin Wang et al., Molecules 2017, 22, 1315; doi:10.3390 / molecules22081315): HPLC analysis showed that the main components were acylated anthocyanins (>90%), including peonidin-3-O-(6-O-caffeoyl-2-O-β-D-pyranoglucoside)-5-O-β-D-glucoside, peonidin-3-O-(2-O-(6-O-caffeoyl-β-D-pyranoglucoside)-6-O-caffeoyl-β-D-pyranoglucoside)-5-O-β-D-pyranoglucoside, peonidin-3-O-(2-O-(6-O-feruloyl-β-D-pyranoglucoside)-6-O-caffeoyl-β-D-pyranoglucoside)-5-O-β-D-pyranoglucoside and cyanidin-3-O-(6-O-p-coumaryl)-β-D-pyranoglucoside, and the remaining components were other flavonoids.

[0024] Grape seed anthocyanins were purchased from Shandong Pingju Biotechnology Co., Ltd.

[0025] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0026] Example 1: Stem cell induction Mouse bone marrow mesenchymal stem cells were added to α-MEM medium (α-MEM medium + 10% fetal bovine serum) containing growth factors (growth factors include 50 μg / mL nerve growth factor β-NGF, 100 μg / mL fibroblast growth factor bFGF and 100 μg / mL epidermal growth factor EGF.) and cultured in an incubator at 37°C for 3 days to obtain pre-treated stem cells. Purple sweet potato pigment was then added to a concentration of 75 μg / mL to induce differentiation of mouse bone marrow mesenchymal stem cells, and cholinergic neurons were obtained after 8 days.

[0027] Example 2: Mechanism exploration In order to study whether autophagy is involved in the neural differentiation process induced by PSPC, the second generation BMSCs (mesenchymal stem cells, from mouse bone marrow) were digested and inoculated into six-well plates. After 1 day of adherent culture, the cells were treated according to the following groups. The normal group (nor) was cultured only with α-MEM medium (same as in Example 1); the control group (ctr): that is, the growth factor induced group, the growth factor was added to the α-MEM medium containing 10% fetal bovine serum (same as in Example 1); the purple sweet potato anthocyanin treatment group (PSPC): on the basis of the control group culture medium (the growth factor in Example 1 was added to the α-MEM medium containing 10% fetal bovine serum), different concentrations of purple sweet potato anthocyanin aqueous solution were added, and the concentration gradient of purple sweet potato anthocyanin was set to: 50 μg / mL, 100 μg / mL, and immunofluorescence staining was performed after 72 hours of drug treatment to detect the distribution and relative expression of microtubule-associated protein 2 (MAP2) and autophagy-related protein LC3.

[0028] (1) PSPC negatively regulates cell autophagy, thereby inducing neural differentiation of BMSCs.

[0029] Depend on Figure 1 It can be seen that in the process of PSPC-induced BMSCs differentiation into neurons, the punctate distribution of LC3 decreased and gradually tended to diffuse distribution, and the number of LC3 punctate distribution in MAP2-positive cells decreased significantly. There were more LC3 punctate aggregations in the cells of the nor group, and MAP2-positive cells were almost non-existent, indicating that the autophagic activity of this group of cells was high and the efficiency of cell neural differentiation was very low. The punctate aggregation of LC3 in the ctr group cells was slightly less than that in the nor group, and a small number of MAP2-positive cells appeared, indicating that the autophagic activity of this group of cells was slightly reduced, and a small number of cholinergic neurons were produced. The number of LC3 punctate aggregations in cells treated with 50 μg / mL and 100 μg / mL PSPC was significantly reduced, and a large number of cells expressed MAP2, proving that PSPC treatment would reduce the autophagic activity of cells and enhance the efficiency of bone marrow mesenchymal stem cells to differentiate into neural cells.

[0030] To further analyze the role of autophagy in the process of PSPC-induced neural differentiation, the second generation BMSCs were digested and inoculated into six-well plates. After 1 day of adherent culture, the autophagy inhibitor 3-methyladenine (3-MA) ​​at a concentration of 5mM or the autophagy inducer rapamycin at a concentration of 10nM was added to the groups for pretreatment for 2 hours. Then the cells were treated with drugs according to the above groups, and morphological observation and immunofluorescence staining of MAP2 were performed after 72 hours. At the same time, protein samples were collected for immunoblot analysis. Since 3-MA and rapamycin are both soluble in DMSO, the solvent was set as the control solvent, that is, DMSO with the same volume as 3-MA and rapamycin was added to the cells for pretreatment, named "solvent control". Depend on Figure 2 It can be seen that after pre-treatment of cells with the autophagy inhibitor 3-MA, MAP2 protein had a higher expression level, and the positive cell rate was higher than that of the normal control group, especially the cells in the PSPC group showed a stronger red fluorescence, indicating that the expression of MAP2 protein increased significantly after inhibiting autophagy, suggesting that inhibiting autophagy can enhance the ability of PSPC to induce neural differentiation of stem cells. After pre-treatment of cells with the autophagy inducer rapamycin, it was found that the intensity of red fluorescence was very low, indicating that the expression of MAP2 protein was very low after inducing autophagy, and bone marrow mesenchymal stem cells did not successfully differentiate into neural cells. Observation of cell morphology shows that after 3-MA inhibited autophagy activity, stem cells induced by PSPC were differentiated into typical neuron-like cells, while rapamycin-induced autophagy activity limited the differentiation of stem cells, and the cells remained long spindle-shaped. The above data confirm that PSPC induces neural differentiation of BMSCs by negatively regulating autophagy.

[0031] Western blot was further used to analyze the effect of autophagy interference on PSPC-induced BMSCs neural differentiation. After 3 days of autophagy interference, cells were lysed with RIPA lysis buffer to extract total protein. Protein concentration was determined using a BCA protein quantification kit (Thermo Fisher, A55864). The protein was transferred to a polyvinylidene fluoride membrane (PVDF) by electrophoretic transfer (Bio-Rad Laboratories, Inc., Hercules, CA). The PVDF membrane was incubated with primary antibodies against ChAT (Proteintech, 20747-1-AP), NSE (Proteintech, 10149-1-AP), p62 (Cell Signaling Technology, 5114), beclin-1 (Cell Signaling Technology, 3495), LC3 (Cell Signaling Technology, 2775), p-2448 mTOR (Cell Signaling Technology, 2971), mTOR (Cell Signaling Technology, 2972), p-4EBP1 (Thermo Fisher, 700238), 4EBP1 (ThermoFisher, MA5-32481), or β-actin (Proteintech, 66009-1-Ig) at 4°C overnight. After washing three times with PBS, secondary antibodies (goat anti-rabbit-HPR, Proteintech, SA00001-2 or goat anti-mouse-HRP, Proteintech, SA00001-1) with horseradish peroxidase were added and incubated for 1 h at 37 ° C. ECL enhanced chemiluminescence reagent (Thermo Fisher, 32209) was used to detect related proteins.

[0032] Depend on Figure 3It can be seen that 3-MA can accelerate PSPC-induced neural differentiation after inhibiting autophagy, while rapamycin has the opposite effect. In the solvent control treatment, when autophagy was not interfered with, after PSPC treatment, the expression of neural markers increased, while the level of beclin-1 decreased, the level of ubiquitin-binding protein (p62) increased, and the conversion of autophagy-related protein LC3 was inhibited, indicating that PSPC can reduce the level of autophagy during neural differentiation. The expression of p62 protein increased significantly after PSPC and 3-MA treatment (p<0.01), and the expression decreased after rapamycin treatment. The expression of autophagy-related protein beclin-1 and LC3B-I / II conversion increased after rapamycin treatment, verifying the functions of autophagy inhibitors and inducers. In addition, the expression of acetylcholine transferase recombinant protein ChAT was significantly increased under the co-treatment of 3-MA and PSPC compared with the solvent control treatment, and the expression was significantly decreased after rapamycin treatment. The expression trends of neuron-specific enolase NSE and ChAT were consistent. They are an acidic protease unique to neurons and neuroendocrine cells, indicating that inhibiting autophagy can increase the expression of neuronal markers, inducing autophagy can reduce the expression of neuronal markers, and autophagy negatively regulates the PSPC-mediated neural differentiation process of BMSCs.

[0033] (2) miR-223-3p is involved in PSPC-induced neural differentiation of BMSCs To further explore the mechanism of PSPC inhibiting autophagy and inducing neural differentiation of BMSCs, whole transcriptome sequencing was performed on bone marrow mesenchymal stem cells (BMSCs) treated with PSPC, and the changes in miRNA were analyzed in detail. It was found that PSPC could change the expression of multiple miRNAs in cells, including upregulation of miR-146a-5p, miR-223-3p and downregulation of miR-92a-5p, miR-222-5p, miR-152-5p, miR-148b-5p and miR-148a-5p, etc. Subsequently, RNA was extracted from cells treated with PSPC for 1, 2, and 3 days, and the expression of miRNA was detected.

[0034] Depend on Figure 4 It can be seen that PSPC can upregulate miR-223-3p and inhibit the levels of miR-92a-5p, miR-222-5p, miR-152-5p, miR-148b-5p and miR-148a-5p, and the results are consistent with the results of whole transcriptome sequencing.

[0035] By analyzing the results of miRNA, it was found that miR-223-3p had the largest upregulation, and TargetScan and other software predicted that miR-223-3p might target autophagy-related genes Atg2b (gene ID: 314415) and Atg9a (gene ID: 363254), so miR-223-3p was selected for further study in subsequent experiments. The miR-223-3p mimics and miR-223-3p inhibitors (mimics purchased from Guangdong Ruibo Biotechnology Co., Ltd., catalog number: miR10000892-1-5; inhibitors purchased from Guangdong Ruibo Biotechnology Co., Ltd., catalog number: miR20000892-1-5) were transfected into BMSCs in a concentration gradient manner to detect the effect of overexpression or silencing of miR-223-3p on PSPC induced differentiation.

[0036] BMSCs were seeded in a 24-well plate, with 30,000 cells planted in each well. The next day, transfection was performed, and the transfection method for each well was as follows: Tube A: Dissolve the miR-223-3p mimic or inhibitor in 50 μL Opti-MEM (reduced serum medium), gently blow 3-5 times to mix, and let stand at room temperature for five minutes. At the same time, a control group was set up, and the non-miR-223-3p control mimic and the control inhibitor of miR-223-3p (control mimic purchased from Guangdong Ruibo Biotechnology Co., Ltd., catalog number: miR1N0000001-1-5; control inhibitor purchased from Guangdong Ruibo Biotechnology Co., Ltd., catalog number: miR2N0000001-1-5) were dissolved in Opti-MEM according to the above method as a control experiment. Tube B: Dissolve 1 μL LipofectamineTM 3000 transfection reagent in 50 μL Opti-MEM, gently blow 3-5 times to mix, and let stand at room temperature for five minutes. The two were mixed and allowed to stand at room temperature for 20 minutes. 100 μL of the transfection complex was added to the serum-free α-MEM medium (Thermo Fisher, 12561056) in the well plate, and the well plate was shaken to mix and then cultured in an incubator for 6 hours. Serum (nor), growth factor (ctr, same as in Example 1) and purple sweet potato pigment (PSPC, final concentration of 50 μg / mL) were added in groups, and samples were collected after 72 hours.

[0037] Depend on Figure 5 It can be seen that the expression of ChAT increased after cells were transfected with miR-223-3p mimics, that is, the effect of PSPC inducing neural differentiation was enhanced after transfection with miR-223-3p. However, ChAT decreased after transfection with inhibitors, and the miR-223-3p inhibitor blocked the induction of PSPC. The above results show that PSPC can induce BM-MSCs by enhancing the expression of miR-223-3p Differentiation towards neurons, reducing the expression of miR-223-3p prevented the neural differentiation of BM-MSCs.

[0038] (3) miR-223-3p targets Atg2b TargetScan and other target gene analysis software were used to predict the potential target genes of miR-223-3p, and it was found that miR-223-3p may target the autophagy-related gene Atg2b. After transfecting miR-223-3p mimics and inhibitors into BMSCs, the cells were grouped and treated with drugs (drug concentration was 50 μg / mL), and the grouping was the same as that of Example 2 (2), and the influence of Atg2b in this process was detected. Figure 6 It can be seen that the expression of Atg2b was very low after transfection of miR-223-3p mimics, and the expression of Atg2b was significantly increased after transfection of miR-223-3p inhibitors. These results indicate that during the process of PSPC-induced neural differentiation of BMSCs, miR-223-3p can target Atg2b to block the autophagy process and enhance the efficiency of PSPC-induced neural differentiation of BMSCs.

[0039] Test example (1) Relative expression of MAP2 protein The experiment was divided into blank control group, grape seed anthocyanin group, and purple sweet potato pigment group; the test concentration of the grape seed anthocyanin group and the purple sweet potato pigment group was 75 μg / mL, and an equal amount of clean water was added to the blank control group.

[0040] The second generation BMSCs cells were prepared into a single cell suspension using α-MEM medium (Thermo Fisher, 12561056) containing 10% fetal bovine serum at a density of 1×10 5cells / mL, and the cells were seeded into 6-well plates. After the mesenchymal stem cells adhered to the wall, the original culture medium was removed, and the α-MEM culture medium containing the growth factors in Example 1 and 10% fetal bovine serum was replaced, and the differentiation induction experiment was started according to the above grouping. Blank control group (ctr): the same amount of water as the purple sweet potato pigment was added to the culture medium; grape seed anthocyanin group (GSPE): 75 μg / mL of grape seed anthocyanin aqueous solution was added to the culture medium; purple sweet potato pigment treatment group (PSPC): 75 μg / mL of purple sweet potato anthocyanin aqueous solution was added to the culture medium. This time was recorded as day 0. During the experiment, the cell morphology of each group was observed every day and photographed and recorded under an inverted microscope. New differentiation culture medium was replaced every 2 days (blank control group replacement: α-MEM culture medium + 10% fetal bovine serum + growth factors in Example 1, GSPE group replacement: α-MEM culture medium + 10% fetal bovine serum + growth factors in Example 1 + GSPE; PSPC group replacement: α-MEM culture medium + 10% fetal bovine serum + growth factors in Example 1 + PSPC). On days 1, 2, and 3, the cell status was detected by CCK-8 assay, and on day 3, the relative expression of MAP2 protein was detected by immunofluorescence staining.

[0041] according to Figure 7 and Figure 8 As shown in the results, grape seed anthocyanins and purple sweet potato pigments have good biocompatibility with MSCs, but only purple sweet potato pigments can increase the protein expression of neuronal marker MAP2 in BMSCs.

[0042] (2) Experimental grouping was the same as above. Total RNA of BMSCs cultured for 3 and 6 days was extracted from each group. The expression of neural-specific markers such as nestin, neuron-specific enolase (NSE), and microtubule-associated protein 2 (MAP2) was detected by real-time quantitative polymerase chain reaction (RT-qPCR), and semi-quantitative analysis was performed. RNA was reverse transcribed into cDNA, amplified by qPCR, and the Cq value of each gene in each group was analyzed. On the 8th day, cells were lysed with RIPA lysis buffer to extract total protein. Protein concentration was determined using a BCA (Bicinchoninic Acid) protein quantification kit (Thermo Fisher, A55864). The protein was transferred to a polyvinylidene fluoride membrane (PVDF) by electrophoretic transfer (Bio-Rad Laboratories, Inc., Hercules, CA). The PVDF membrane was incubated with primary antibodies against MAP2 (Proteintech, 17490-1-AP), NSE (Proteintech, 10149-1-AP), or Nestin (Proteintech, 19483-1-AP) at 4°C overnight. After washing three times with PBS, secondary antibodies with horseradish peroxidase (Proteintech, SA00001-2) were added and incubated for 1 hour at 37°C. The relevant proteins were detected using enhanced chemiluminescence reagent (Thermo Fisher, 32209).

[0043] according to Fig. 9 As shown, compared with the control group and the grape seed anthocyanin treatment group, purple sweet potato pigment significantly increased the expression of neural differentiation-related genes and proteins, confirming that purple sweet potato pigment can induce BMSCs to differentiate into neurons.

[0044] (3) Experimental grouping was the same as above. Total RNA of BMSCs cultured for 6 days was extracted from each group. The expression of neural specific markers such as acetylcholine (ChAT), glutamate receptor (GluR), N-methyl-D-aspartate receptor 1 (NMDA1), γ-aminobutyric acid receptor α1 (GABRA1), and dopamine receptor D2 (DRD2) was detected by real-time quantitative polymerase chain reaction (RT-qPCR), and semi-quantitative analysis was performed. RNA was reverse transcribed into cDNA, and then amplified by qPCR, and the Cq value of each gene in each group was analyzed.

[0045] according to Fig.10 As shown, purple sweet potato pigment can significantly increase the markers of acetylcholinergic neurons, but has no obvious effect on the expression of markers of other subtypes of neurons, indicating that the type of neurons induced by purple sweet potato pigment-induced mesenchymal stem cell differentiation is acetylcholinergic neurons.

[0046] On day 8, cells were lysed with RIPA lysis buffer to extract total protein. Protein concentration was determined using a BCA protein quantification kit (Thermo Fisher, A55864). The membrane was transferred to a polyvinylidene fluoride membrane (PVDF) by electrophoretic transfer (Bio-Rad Laboratories, Inc., Hercules, CA). The PVDF membrane was incubated with a primary antibody against ChAT (Proteintech, 20747-1-AP) at 4°C overnight. After washing three times with PBS, a secondary antibody with horseradish peroxidase (Proteintech, SA00001-2) was added and incubated for 1 hour at 37°C. The relevant proteins were detected using an enhanced chemiluminescence reagent (Thermo Fisher, 32209). A calcium ion fluorescent probe (Fluo-4 AM) was used to evaluate the responsiveness of cells to neurotransmitters after culturing at a concentration of 75 μg / mL on day 8 in each group. Acetylcholine neurotransmitter was used to stimulate cells, and laser confocal microscopy was used to monitor the cells in real time.

[0047] according to Fig.11 and Fig.12 As shown, the acetylcholinergic neurons induced by purple sweet potato pigments have electrophysiological activity and are functional neurons.

[0048] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Application of purple sweet potato pigment in the preparation of induced stem cell neural differentiation products.

2. The use according to claim 1, characterized in that: The stem cells are mesenchymal stem cells.

3. The use according to claim 2, characterized in that: The mesenchymal stem cells are bone marrow mesenchymal stem cells.

4. The use according to claim 1, characterized in that: The neural differentiation is differentiation into acetylcholinergic neurons.

5. The use according to claim 1, characterized in that: The product for inducing neural differentiation of stem cells includes a drug for inducing stem cells to differentiate into neurons or a culture medium for inducing stem cells to differentiate into neurons.

6. The use according to claim 1, characterized in that: The induction method comprises: pretreating stem cells with a growth factor mixture, and then adding purple sweet potato pigment to promote the differentiation of stem cells into neurons.

7. The use according to claim 6, characterized in that: The growth factor mixture includes 50 μg / mL nerve growth factor β-NGF, 100 μg / mL fibroblast growth factor bFGF and 100 μg / mL epidermal growth factor EGF.

8. The use according to claim 6, characterized in that: The concentration of the purple sweet potato pigment is 50-100 μg / mL.

9. The use according to claim 6, characterized in that: The differentiation time is 5 to 10 days.

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