Umbilical cord mesenchymal stem cell for overexpressing ATG5 as well as preparation method and application of umbilical cord mesenchymal stem cell
By constructing umbilical cord mesenchymal stem cells overexpressing ATG5, they enhance their antioxidant stress ability, and solve the problem of oxidative stress stimulation inhibiting MSC function, achieving better treatment effects for acute colitis.
Patent Information
- Application Number
- CN202410118518.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-29
- Publication Date
- 2025-07-29
AI Technical Summary
Continuous oxidative stress stimulation inhibits the function of mesenchymal stem cells (MSCs), resulting in reduced MSC treatment effects and limits its application.
Umbilical cord mesenchymal stem cells overexpressing ATG5 were constructed. Adenovirus particles were prepared and transfected with umbilical cord mesenchymal stem cells to enhance their antioxidant stress ability.
It improves the antioxidant ability of umbilical cord mesenchymal stem cells in a hypoxic oxidative stress environment and significantly improves the therapeutic effect of acute colitis.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an umbilical cord mesenchymal stem cell overexpressing ATG5, a preparation method thereof, and an application thereof. Background Art
[0002] Mesenchymal stem cells (MSCs) are a type of stem cells with self-renewal ability and differentiation ability. They have a variety of biological functions, including immunomodulatory function, paracrine function, etc., and have the characteristics of immunomodulatory ability and low immunogenicity. They have a good alleviating effect in a variety of diseases and are applied to anti-inflammatory, treatment of autoimmune diseases, degenerative diseases, etc.
[0003] With the development of MSC transplantation technology, MSCs have been widely applied in a variety of disease models. However, during the transplantation process, only a small number of MSCs survive at the transplantation site. The main reason is the oxidative stress damage in the microenvironment, the imbalance between reactive oxygen species (ROS) and the antioxidant system in MSCs. Continuous oxidative stress stimulation may inhibit the function of MSCs, resulting in a reduction in the therapeutic effect of MSCs and limiting the application of MSCs. Summary of the Invention
[0004] The present invention discloses an umbilical cord mesenchymal stem cell overexpressing ATG5, a preparation method thereof, and an application thereof, and solves the problem that continuous oxidative stress stimulation currently inhibits the function of MSCs, resulting in a reduction in the therapeutic effect of MSCs and limiting the application of MSCs.
[0005] A preparation method of an umbilical cord mesenchymal stem cell overexpressing ATG5, the method comprising the following steps:
[0006] Step 1: Insert the target gene ATG5 into a tool vector plasmid, and after digestion with enzymes, amplification of the target gene, and plasmid extraction, obtain a plasmid carrying the ATG5 gene;
[0007] Step 2: Co-transfect a host cell with the plasmid carrying the ATG5 gene obtained in Step 1 and a packaging plasmid carrying an adenovirus genome to prepare an adenovirus particle;
[0008] Step 3: Transfect the adenovirus particle obtained in Step 2 into umbilical cord mesenchymal stem cells to obtain a genetically engineered umbilical cord mesenchymal stem cell overexpressing ATG5.
[0009] Further, the transcript number of the target gene ATG5 in Step 1 is NM_001146.
[0010] Further, the vector number of the tool vector plasmid in Step 1 is GV135, and the element sequence is CMV-MCS-EGFP.
[0011] Furthermore, in step 2, the adenoviral genome in the packaging plasmid carrying the adenoviral genome lacks E1 and E3.
[0012] Furthermore, in step 2, the host cell is HEK293 cell.
[0013] Furthermore, in step 2, the MOI index of the adenovirus particles is 100 - 200.
[0014] Furthermore, in step 3, the transfection time is 48 hours.
[0015] The genetically engineered umbilical cord mesenchymal stem cells overexpressing ATG5 prepared by the above preparation method.
[0016] Furthermore, the umbilical cord mesenchymal stem cells overexpressing ATG5 have the ability of antioxidant stress.
[0017] Application of an umbilical cord mesenchymal stem cell overexpressing ATG5 in the preparation of a preparation for treating inflammatory bowel disease.
[0018] The present invention discloses an umbilical cord mesenchymal stem cell overexpressing ATG5, a preparation method and an application thereof. The present invention uses Ad-cre recombinant adenovirus to infect mouse bone marrow MSCs of the ATG5 Cre-LoxP system, and shows that after ATG5 knockout, the proliferation of mouse bone marrow MSCs can be significantly inhibited. In addition, it is found that in a hypoxic oxidative stress environment, some antioxidant genes in umbilical cord MSCs are inhibited; by infecting umbilical cord MSCs with an ATG5 overexpression recombinant adenovirus, it is found that the oxidative stress damage of MSCs caused by hypoxia can be protected, and the oxidative stress ability of cells can be improved. The umbilical cord mesenchymal stem cells overexpressing ATG5 constructed by the present invention are injected into an acute colitis animal model, and have a stronger effect of improving colitis symptoms. The above results provide a theoretical basis for intervening autophagy to enhance MSC function and applying it to disease treatment in the future. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 For the isolation and identification of mouse bone marrow MSCs; wherein Figure A is a schematic diagram of the isolation and culture of bone marrow MSCs, Figure B is the morphology of the isolated passage 3 mouse bone marrow MSCs under an optical microscope (scale bar: 200 μm), Figure C is a representative flow cytometry diagram of the identification of positive surface markers of mouse bone marrow MSCs, and Figure D is a representative flow cytometry diagram of the identification of positive surface markers of mouse bone marrow MSCs;
[0020] Figure 2Knockout and identification of the ATG5 gene induced by Cre adenovirus; in which Panel A shows the GFP green fluorescence photographs of bone marrow MSCs isolated from ATG5 cre-LoxP system mice after infection with Ad GFP control virus and Ad cre adenovirus (scale bar: 200 μm), Panel B shows the detection of ATG5 mRNA expression levels in cells by qRT-PCR after adenovirus infection; Panel C shows the detection of ATG5 protein levels in cells by WB after adenovirus infection; all data are expressed as mean ± standard deviation, **p<0.01, compared with the MSC Ad GFP control group;
[0021] Figure 3 Detection of the proliferation ability of mouse bone marrow MSCs after ATG5 gene knockout; in which Panel A shows the schematic diagram of the proliferation generations of the MSC group, the MSC + control adenovirus Ad GFP group, and the MSC + cre adenovirus group at 0 h, 48 h, and 72 h detected by the Dye670 staining method, with different colors representing the cell proliferation generations, and Panel B shows the proliferation coefficient obtained by Modfit LT5 analysis; all data are expressed as mean ± standard deviation, ***p<0.001, compared with the MSC Ad GFP control group;
[0022] Figure 4 Construction and expression identification of an adenovirus overexpressing ATG5; in which Panel A shows the GFP green fluorescence photographs of umbilical cord MSCs after infection with GFP control adenovirus and an adenovirus overexpressing ATG5 (scale bar: 500 μm), Panel B shows the detection of the infection efficiency of the adenovirus overexpressing ATG5 at 100 and 200 MOI by flow cytometry, Panel C shows the detection of ATG5 mRNA expression levels by qRT-PCR after adenovirus infection, and Panel D shows the detection of ATG5 protein levels in cells by WB after adenovirus infection; all data are expressed as mean ± standard deviation, ***p<0.001, compared with the Ad GFP control group;
[0023] Figure 5 Detection of the antioxidant ability of umbilical cord MSCs after overexpression of ATG5; in which Panel A shows the detection of the expression levels of antioxidant genes such as CAT, SOD1, Nrf2, HO-1, and GCLC in MSCs cultured in normoxia and hypoxia environments by qRT-PCR, Panel B shows the content of glutathione peroxidase (GSH-Px) in the MSC culture supernatant, Panel C shows the content of catalase (CAT) in MSC cells, Panel D shows the content of catalase (CAT) in the MSC culture supernatant, and Panel E shows the detection of the hydroxyl radical scavenging ability in the MSC culture supernatant; all data are expressed as mean ± standard deviation, *p<0.05, ***p<0.001, ns: no significant difference, compared with the AdGFP control group in the ATG5 overexpression group;
[0024] Figure 6For the therapeutic effect of overexpressing ATG5 in umbilical cord MSCs on the acute colitis model, Figure A is a schematic diagram of the modeling process and treatment method, Figure B is the relative body weight change trend of each group of mice during the modeling process every day, Figure C is the colon photo of each group of mice, Figure D is the statistical result of the colon length of each group of mice, Figure E is the Disease Activity Index (DAI) score of each group of mice, Figure F is the pathological photo of colon HE staining of each group of mice, Figure G is the content of peripheral blood serum inflammatory factors (IL-6, IL-1β, TNF-α, G-CSF, IFN-γ, KC) in each group of mice. All data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ns: no significant difference, compared with the MSC-GFP control group and the DSS model group, and compared with the MSC-ATG5 overexpression group and the DSS model group. Detailed implementation mode
[0025] The present invention provides a method for isolating and culturing mouse bone marrow MSCs. By isolating and culturing bone marrow MSCs of ATG5 cre-LoxP system mice and infecting them with Ad cre adenovirus to achieve ATG5 gene knockout, the proliferation ability of bone marrow MSCs after ATG5 gene knockout was detected. Overexpressing ATG5 in umbilical cord MSCs was found to have stronger antioxidant stress ability and better therapeutic effect in the DSS-induced acute colitis model of mice.
[0026] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
[0027] Example 1
[0028] I. Materials and methods.
[0029] 1. Method for isolating and culturing bone marrow MSCs of ATG5 Cre-LoxP system mice.
[0030] (1) In this experiment, 6-8-week-old C57BL / 6 ATG5 Cre-LoxP system mice that had been identified were taken. After decapitating and sacrificing the mice, they were placed in 75% alcohol for about 10 minutes.
[0031] (2) Use sterile scissors and forceps to isolate the intact femur and tibia of the hind limbs and remove the muscle tissue.
[0032] (3) Place the femur and tibia into a 1.5 mL Ep tube, add 0.2% type I collagenase and cut them into pieces with scissors. Then add the same volume of α-MEM medium (BI, Israel), and place it in a 37°C constant temperature shaker at 200 r / min for 2 hours.
[0033] (4) Centrifuge at 1200 rpm for 5 minutes, discard the supernatant, add 5 mL of α-MEM medium, and supplement it with 10% fetal bovine serum (Procell, Wuhan), 5% platelet lysate (BI, Israel), 1% ITS solution (Gibco, USA), and 1% penicillin / streptomycin solution (Procell, Wuhan). Inoculate the cells into a T25 culture flask and culture them in an incubator at 37°C with 5% CO2. Replace the medium every 2 - 3 days. When the cell density reaches 90% (usually 7 days), passage the cells and continue the culture. Passage the cells to P3 generation for subsequent experiments.
[0034] 2. Identification of surface markers of mouse bone marrow MSCs.
[0035] The positive markers of mouse bone marrow MSCs are Sca-1, CD29, and CD44; the negative markers are CD34, CD11b, and CD45. After culturing mouse bone marrow MSCs to P3 generation, digest and count the cells, and label the flow antibodies Sca-1 PE, CD29 PE, CD44 PE, CD11b APC, CD45 APC, and CD34 FITC under light protection. After antibody labeling, detect them on a flow cytometer and analyze the expression using FlowJo V10 software.
[0036] 3. Preparation of ATG5 gene knockout mouse bone marrow MSCs.
[0037] Construct a recombinant adenovirus expressing cre recombinase and a GFP control adenovirus, and infect the bone marrow MSCs isolated from ATG5 Cre-LoxP system mice. The multiplicity of infection (MOI) is 100. Harvest the cells 48 hours after adenovirus infection of MSCs for detecting the expression efficiency of ATG5 at the mRNA and protein levels.
[0038] 4. Analysis of the proliferation ability of mouse bone marrow MSCs after ATG5 gene knockout.
[0039] After mouse bone marrow MSCs are infected with Ad cre and Ad GFP adenoviruses, after identifying stable knockout of ATG5, stain the cells according to the instructions of Dye670 dye, and seed the cells into a 6-well plate. Collect the cells at 0 h, 48 h, and 72 h respectively, perform fluorescence detection using a flow cytometer, and analyze the proliferation coefficient using Modfit LT5 software.
[0040] 5. Detection of the antioxidant stress ability of MSCs overexpressing ATG5 in a hypoxic environment.
[0041] Preparation of umbilical cord mesenchymal stem cells overexpressing ATG5:
[0042] Step 1: Insert the target gene ATG5, the transcript number of the target gene ATG5 is NM_001146, human, into the tool vector plasmid, the tool vector plasmid number is GV135, and the element order is CMV-MCS-EGFP. After digestion, target gene amplification and plasmid extraction, an adenovirus shuttle plasmid carrying the ATG5 gene is obtained;
[0043] Step 2: Mix the adenovirus shuttle plasmid carrying the ATG5 gene obtained in Step 1 with the packaging plasmid DNA solution carrying the adenovirus genome (E1 and E3 deleted) and DMEM medium (Gibco, USA) to a fixed volume of 50 μL, then add 10 μL of Lipofectamine 2000 (Invitrogen, USA) to form a DNA / Lipofectamine 2000 mixture. Co-transfect HEK293 cells with the DNA / Lipofectamine 2000 mixture. After the cells show cytopathic effects, collect the cells and supernatant, and repeatedly freeze-thaw 3 times to obtain the adenovirus particles from the supernatant; the MOI index of the adenovirus particles is 100 and 200;
[0044] Step 3: Transfect the adenovirus particles obtained in Step 2 into umbilical cord mesenchymal stem cells to obtain genetically engineered umbilical cord mesenchymal stem cells overexpressing ATG5.
[0045] Identify the prepared umbilical cord mesenchymal stem cells overexpressing ATG5. After the identification is completed, culture one group of MSCs transfected with the control adenovirus in a normal oxygen environment, one group of MSCs transfected with the control adenovirus in a 1% oxygen environment, and one group of MSCs transfected with the adenovirus overexpressing ATG5 in a 1% oxygen environment. After 48 h, collect the cells and detect the expression of antioxidant genes such as CAT, SOD1, Nrf2, HO-1, and GCLC in the cells by real-time quantitative PCR; take the cell culture supernatant to detect the contents of glutathione peroxidase (GSH-Px) and catalase (CAT); grind the cells to detect the content of catalase (CAT) in the cells; take the cell culture supernatant to detect the hydroxyl radical scavenging ability of the cells.
[0046] 6. Therapeutic effect of umbilical cord MSCs overexpressing the ATG5 gene on acute colitis in mice.
[0047] Eight-week-old male C57BL6 / N mice were acclimated in the animal house for 3 - 5 days before modeling. The modeling method was to add 2.5% DSS to the drinking water for the mice to drink freely. 100 mL was added for every 5 mice in each cage, and it was changed every 2 days; DSS was continuously administered for 6 days, and then changed to pure water for 3 days. Umbilical cord MSCs were infected with Ad GFP control adenovirus and Ad ATG5 overexpressing ATG5 adenovirus respectively. Cells were collected 48 h after infection for model treatment. The experiment was set up with a normal control group, a DSS modeling group, a DSS modeling + MSC Ad GFP group, and a DSS modeling + MSC Ad ATG5 group. There were 5 mice in the control group and the model group, and 10 mice in each of the other groups. MSCs were injected into the tail veins of the mice on the 3rd and 5th days after the start of modeling, with 1×10 6 cells injected per mouse each time.
[0048] Observation indicators: The body weight of the mice was weighed every day, and the fecal traits and fecal occult blood conditions of the mice were observed for integral evaluation. The mice were euthanized at the end point of modeling (the 10th day), and the peripheral blood of the mice was taken and plasma was separated for subsequent detection of inflammatory factors; the colon of the mice was taken to measure the length and photographed, and the intestinal tissue was quickly frozen in liquid nitrogen for subsequent mechanism research; the fecal occult blood test was performed on the colon contents according to the kit instructions, and the remaining contents were quickly frozen in liquid nitrogen for subsequent intestinal microbiota detection.
[0049] II. Results.
[0050] 1. Isolation and identification of mouse bone marrow MSCs.
[0051] The flow chart of the isolation and culture of mouse bone marrow MSCs is as Figure 1 shown in A. After isolation and culture, a large number of cells were observed to adhere and grow after 3 days. The cells were spindle-shaped and formed colonies ( Figure 1 B). P3-generation cells were labeled with antibodies, and the surface markers of bone marrow MSCs were detected by flow cytometry. It was identified that Sca-1, CD29, and CD44 were positive, and the positive rates were 99.2%, 99.9%, and 92.1% respectively ( Figure 1 C); CD11b, CD34, and CD45 were negative, and the positive rates were 0.75%, 0.091%, and 6.28% respectively ( Figure 1 D). The ratio of positive to negative surface markers met the identification criteria for mouse bone marrow MSCs.
[0052] 2. Knockout of the ATG5 gene in mouse bone marrow MSCs by the ATG5 cre-LoxP system.
[0053] After isolation and culture of ATG5 cre-LoxP system mouse bone marrow MSCs, P3 cells were infected with Ad GFP control adenovirus and Ad cre adenovirus respectively. After 48 hours of infection, GFP green fluorescence was observed under a fluorescence microscope ( Figure 2 A). Cells infected with adenovirus were separately extracted for RNA and protein, and after identification by real-time quantitative PCR and detection by Western blot (WB), knockout of the ATG5 gene was successfully achieved at the mRNA level and protein level ( Figure 2 B-C).
[0054] 3. Knockout of the ATG5 gene significantly inhibited the proliferation of bone marrow MSCs.
[0055] After knockout of the ATG5 gene in MSCs by infecting with Ad cre adenovirus, Dye670 staining showed that the number of cell proliferation generations and the number of cells were significantly lower than those in the control group ( Figure 3 A). After knockout of the ATG5 gene, the proliferation rate of MSCs was significantly inhibited, and the proliferation index of the ATG5 gene knockout group was significantly lower than that of the control group ( Figure 3 B).
[0056] 4. Construction and identification of an overexpression vector of the ATG5 gene in MSCs.
[0057] An overexpression recombinant adenovirus vector of ATG5 was constructed. The transcript of ATG5 (human) is: NM-004849. Ad GFP control adenovirus and Ad ATG5 overexpression adenovirus were respectively used to infect human umbilical cord mesenchymal stem cells, and the MOI was set at 100 and 200. Under the condition of 100 MOI, GFP green fluorescence was observed under a fluorescence microscope after 48 hours of adenovirus infection ( Figure 4 A); Cells infected with Ad ATG5 overexpression adenovirus for 48 hours were respectively collected at 100 MOI and 200 MOI for flow cytometry to detect the proportion of GFP ( Figure 4 B). The adenovirus infection efficiency at 100 MOI reached 78.7%, and the adenovirus infection efficiency at 200 MOI reached 92.8%. Cell RNA was collected and the expression of ATG5 was detected by real-time quantitative PCR. The mRNA expression of ATG5 was significantly increased compared with the control group at the multiplicity of infection of 100 MOI and 200 MOI. Cell proteins were collected for WB detection, and it was found that the protein expression of ATG5 was significantly increased compared with the control group at the multiplicity of infection of 100 MOI and 200 MOI.
[0058] 5. The antioxidant stress ability of MSCs with overexpression of ATG5 was improved in a hypoxic environment.
[0059] Compared with umbilical cord MSCs cultured under normal oxygen conditions, the antioxidant genes such as CAT, SOD1, and Nrf2 of MSCs cultured in a hypoxic environment were all inhibited. Similarly, in umbilical cord MSCs overexpressing ATG5 in a hypoxic environment, the antioxidant genes such as CAT and GCLC were significantly increased, and SOD1, Nrf2, HO-1, etc. showed an increasing trend( Figure 5 A).
[0060] Compared with the normoxia group, the contents of glutathione peroxidase (GSH-Px) and catalase (CAT) in the cell culture supernatant of umbilical cord MSCs cultured in a hypoxic environment decreased, and the intracellular catalase (CAT) and hydroxyl radical scavenging ability decreased; after overexpressing ATG5, the contents of glutathione peroxidase (GSH-Px) in the cell culture supernatant, and the contents of catalase (CAT) in the supernatant and cells were all significantly increased, and the hydroxyl radical scavenging ability of the cells showed an increasing trend( Figure 5 B).
[0061] 6. MSCs overexpressing ATG5 have a stronger therapeutic effect on acute colitis.
[0062] Figure 6 A is a schematic diagram of the modeling process of acute colitis in mice; the body weight of mice showed a downward trend on the 6th day after DSS modeling, and the body weight of the DSS model group decreased the most on the 9th day, while the body weight of the MSC treatment group overexpressing ATG5 decreased the least( Figure 6 B); Figure 6 C is a representative colon photo of each group; the colon length statistics of each mouse in each group are shown in 6D. The colon length of the MSC treatment group overexpressing ATG5 was significantly different from that of the DSS model group; the disease activity index (DAI) obtained from the colon length, fecal occult blood, and fecal character scores showed that the score of the MSC treatment group overexpressing ATG5 was significantly lower than that of the model group( Figure 6 E); the colon tissue was subjected to HE staining pathological analysis. The colon villus structure of the DSS model group was damaged and a large number of inflammatory cells aggregated. After treatment with MSCs and MSCs overexpressing ATG5, the villus structure of the colon tissue returned to normal( Figure 6 F); the serum separated from the peripheral blood of each group was used for the detection of inflammatory factors. After DSS modeling, the concentrations of inflammatory factors such as IL-6, G-CSF, and KC increased significantly; after treatment with MSCs, the concentrations of inflammatory factors including IL-6, IL-1β, TNF-α, G-CSF, and KC decreased significantly( Figure 6 G).
[0063] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.
Claims
1. A method for preparing umbilical cord mesenchymal stem cells overexpressing ATG5, characterized in that, The method includes the following steps: Step 1: Insert the target gene ATG5 into the tool vector plasmid. After digestion with enzymes, amplification of the target gene, and plasmid extraction, a plasmid carrying the ATG5 gene is obtained; Step 2: Mix the plasmid carrying the ATG5 gene obtained in Step 1 with the packaging plasmid carrying the adenovirus genome, and co-transfect the host cell to obtain adenovirus particles; Step 3: Transfect the adenovirus particles obtained in Step 2 into umbilical cord mesenchymal stem cells to obtain genetically engineered umbilical cord mesenchymal stem cells overexpressing ATG5.
2. The preparation method according to claim 1, wherein In Step 1, the transcript number of the target gene ATG5 is NM_001146.
3. The preparation method according to claim 1, characterized in that, In Step 1, the vector number of the tool vector plasmid is GV135, and the element sequence is CMV-MCS-EGFP.
4. The preparation method according to claim 1, wherein In Step 2, the adenovirus genome in the packaging plasmid carrying the adenovirus genome lacks E1 and E3.
5. The preparation method according to claim 1, characterized in that, In Step 2, the host cell is HEK293 cell.
6. The preparation method according to claim 1, characterized in that In Step 2, the MOI index of the adenovirus particles is 100 - 200.
7. According to the preparation method described in claim 1, characterized in that, In Step 3, the transfection time is 48 hours.
8. Genetically engineered umbilical cord mesenchymal stem cells overexpressing ATG5 prepared by the preparation method according to any one of claims 1 to 7.
9. The umbilical cord mesenchymal stem cells overexpressing ATG5 according to claim 8, characterized in that, The umbilical cord mesenchymal stem cells overexpressing ATG5 have the ability to resist oxidative stress.
10. Use of umbilical cord mesenchymal stem cells overexpressing ATG5 in the preparation of a preparation for treating inflammatory bowel disease.