CN113633775A - Application of agent for over-expressing phosphofructokinase in preparation of drugs for delaying cell senescence - Google Patents
Application of agent for over-expressing phosphofructokinase in preparation of drugs for delaying cell senescence Download PDFInfo
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Abstract
The invention relates to an application of a reagent for over-expressing phosphofructokinase in preparing a medicament for delaying cell senescence, belonging to the technical field of genetic engineering. The invention provides an application of a reagent for over-expressing phosphofructokinase in preparing a medicament for delaying cell senescence. The invention discusses about the influence of PFKM on the glycolysis level of MSCs to regulate cell senescence, organically connects PFKM with MSCs senescence for the first time, provides experimental basis for clarifying a regulation mechanism of stem cell senescence, is beneficial to maintaining the number and functions of stem cells, and has important significance for delaying body senescence and preventing and treating various aging diseases.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to application of a reagent for over-expressing phosphofructokinase in preparation of a medicament for delaying cell senescence.
Background
The average life of people in modern society is gradually prolonged, and the social problem of aging population is generated[1]Aging of individuals is one of the important factors that contribute to the aging of the population. Aging (Aging) is a complex phenomenon caused by natural rules, the Aging of the body is regulated and controlled by multiple factors and multiple genes, the proliferation and differentiation capacity and the physiological function of cells gradually decline along with the passage of time, and the steady maintenance and regeneration capacity are weakened[2]. The incidence of aging-related diseases such as diabetes and cardiovascular and cerebrovascular diseases is also gradually increased along with aging of individuals[3]The mortality rate increases. Therefore, aging has become an important problem to be solved urgently in the field of life science and the field of society, and research on aging is concerned by more researchers.
The normal life activities of the body require a dynamic balance of cell senescence death elimination and neogenesis cell growth supplementation, and the regeneration capacity of human tissues depends on the capacity and potential of stem cells to replace damaged tissues or cells. Stem cells are a special cell population with strong self-renewal capacity and multi-differentiation potential in the body. In regenerative medicine, adult stem cells are used for the treatment of aging-related diseases, such as autologous stem cell transplantation and tissue, organ regeneration and repair in patients[4]. However, stem cells, like somatic cells, undergo morphological changes during life, undergo various degrees of deterioration in their proliferation and differentiation abilities, and are difficult to escape from the aging host, and thus aging of stem cells affects the aging of the whole body and the occurrence of various aging diseases[5]. Mesenchymal Stem Cells (MSCs) are the basis of cell therapy of many diseases, are used as adult stem cells which are researched earlier and deeply, have the advantages of strong activity, low immunogenicity, no ethical dispute and the like,widely applied to clinical and basic research. Successful MSCs treatment requires large scale long time in vitro cell culture[6]However, normal bone marrow samples are difficult to obtain, sufficient bone marrow MSCs at a specific age stage cannot be obtained, and after a limited number of in vitro subcultures, morphological changes occur in cells, the proliferation and differentiation capacity thereof are also reduced to various degrees, and MSCs are aged[7]. The aging of stem cells greatly restricts the clinical application of stem cells, and the curative effect of autologous stem cell transplantation of patients is reduced. Many progressive metabolic changes and functional decline are accompanied by cellular aging[8]However, there is currently no method for obtaining large quantities of "young" MSCs by reversing cellular senescence.
[1]Martineau A,Plard M.Successful aging:analysis of the components of a gerontological paradigm[J].Geriatrie Et Psychologie Neuropsychiatrie De Vieillissement,2018,16(1):67-77.
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[3]Dodig S,Cepelak I,Pavic I.Hallmarks of senescence and aging[J].Biochem Med(Zagreb),2019,29(3):030501.
[4]Cianflone E,Torella M,Biamonte F,et al.Targeting Cardiac Stem Cell Senescence to Treat Cardiac Aging and Disease[J].Cells,2020,9(6).
[5]Sahin E,Depinho RA.Linking functional decline of telomeres,mitochondria and stem cells during ageing[J].Nature,2010,464(7288):520-528.
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Disclosure of Invention
The invention aims to provide application of a reagent for over-expressing phosphofructokinase in preparing a medicament for delaying cell senescence. The invention discusses about the influence of PFKM on the glycolysis level of MSCs to regulate cell senescence, organically connects PFKM with MSCs senescence for the first time, provides experimental basis for clarifying a regulation mechanism of stem cell senescence, is beneficial to maintaining the number and functions of stem cells, and has important significance for delaying body senescence and preventing and treating various aging diseases.
The invention provides an application of a reagent for over-expressing phosphofructokinase in preparing a medicament for delaying cell senescence.
The invention also provides application of the reagent for over-expressing the phosphofructokinase in preparing a medicine for improving the glycolysis level of cells.
The invention also provides application of the agent for over-expressing the phosphofructokinase in preparing a medicament for promoting cell proliferation and differentiation.
The invention also provides application of a reagent for over-expressing phosphofructokinase in preparing a medicament for up-regulating one or more than two of the following indexes of cells, wherein the indexes comprise: glucose consumption, lactate level, ATP content and ECAR level.
The invention also provides application of the agent for over-expressing the phosphofructokinase in preparing a medicament for down-regulating the expression of senescence-associated beta galactosidase.
The invention also provides the use of an agent which overexpresses phosphofructokinase in the manufacture of a medicament for down regulating the expression of senescence-associated factors in a cell, including P16INK4a。
Preferably, the cells comprise mesenchymal stem cells.
Preferably, the mesenchymal stem cells comprise senescent mesenchymal stem cells.
The invention also provides an improved senescent mesenchymal stem cell, which is capable of overexpressing phosphofructokinase myotype.
The invention also provides a construction method of the improved aged mesenchymal stem cells, which comprises the following steps: infecting aged mesenchymal stem cells with lentiviruses carrying a phosphofructokinase gene; the MOI value is 50-250, and the infection time is 48-72 h.
The invention provides an application of a reagent for over-expressing phosphofructokinase in preparing a medicament for delaying cell senescence. The invention aims to research that glycolysis level of MSCs can be up-regulated after artificial modification of PFKM in MSCs, and aging of MSCs is delayed by further promoting glycolysis through PFKM. The method utilizes the coding mRNA to regulate and control the fate of the cells, has certain feasibility in technology, changes the characteristics of the cells from the gene level, and can regulate and modify the cell self and the microenvironment around the cell self, thereby achieving the better treatment purpose.
Specifically, primary cells are extracted from bone marrow of 1-2 month old male Wistar rats, and in vitro subculture is carried out by using a full bone marrow wall attachment method to obtain early generation MSCs (EPMSCs, P1-P3) and late generation MSCs (LPMSCs, P10-P12). By morphological observation (cell morphology characteristics, cell surface area and aspect ratio), senescence-associated-beta-gal (SA-beta-gal) staining and senescence-associated factor P16INK4aTo establish replicative senescence MSCs. The glycolysis level of the aged MSCs is obviously reduced by detecting glucose consumption, ATP content, lactic acid and ECAR level.
The slow virus carrying PFKM is used to infect aged MSCs with good state, the MOI value is 50-250, and the infection lasts 48-72 h. The transfection efficiency is detected by a fluorescence microscope and RT-qPCR, the efficiency is the best when the virus is transfected for 72h, and the expression of PFKM can be obviously up-regulated by about 2.5 times. The glycolytic levels of the improved senescent MSCs were found to be significantly upregulated by cellular glucose consumption, ATP content, lactate and ECAR levels.
By using aged MSCs carrying PFKM and having good lentivirus infection state, the morphological characteristics, SA-beta-gal staining and aging related factor P16 are observedINK4aThe PFKM is found to provide theoretical basis and experimental scheme for obtaining a large amount of young MSCs by in vitro culture by up-regulating glycolysis level of aging MSCs and delaying aging of MSCs, thereby deepening the understanding of the aging potential mechanism of MSCs and being beneficial to the exploration of new targets for preventing and treating aging-related diseases.
Test results show that the rat late generation MSCs (LPMSCs, P10-P12) have obvious shape aging, and the expression level of PFKM is obviously lower than that of early generation MSCs (EPMSCs, P1-P3); the glycolysis level of the cells is detected, and the glucose consumption, ATP content, lactic acid and extracellular acid secretion rate of the aged MSCs are obviously reduced. By artificially increasing the expression level of PFKM in senescent MSCs, the glycolysis level of senescent MSCs, such as glucose consumption, ATP production, lactic acid and ECAR level, can be up-regulated. After PFKM expression is improved, glucose is more effectively catalyzed and converted into glucose-6-phosphate, the sugar metabolism direction in cells is determined, and the sugar metabolism level in aged MSCs is remodeled.
Drawings
FIG. 1a shows the cell morphology of EPMSCs and LPMSCs under an inverted phase contrast microscope according to the present invention;
FIG. 1b shows the results of the morphological statistical analysis of EPMSCs and LPMSCs cells according to the present invention;
FIG. 1c shows the results of SA- β -gal staining and quantitative analysis provided by the present invention;
FIG. 1d shows the real-time fluorescent quantitative PCR detection of EPMSCs and senescence-associated factor P16 in LPMSCs according to the present inventionINK4aResults of mRNA expression levels;
FIG. 2a shows the results of detecting the glucose consumption of EPMSCs and LPMSCs according to the present invention;
FIG. 2b is the results of the detection of the extracellular fluid lactate secretion levels of EPMSCs and LPMSCs according to the present invention;
FIG. 2c shows the ATP content detection results of EPMSCs and LPMSCs according to the present invention;
FIG. 2d shows the ECAR level detection results of EPMSCs and LPMSCs according to the present invention;
FIG. 2e is the result of real-time fluorescent quantitative PCR detection of the expression level of PFKM mRNA in EPMSCs and LPMSCs provided by the present invention;
FIG. 2f shows the result of detecting the expression level of PFKM proteins in EPMSCs and LPMSCs by Western Blot;
FIG. 3a is a graph showing the infection of senescent MSCs with different concentrations of lentivirus according to the present invention;
FIG. 3b is a graph showing the virus with the optimal MOI value infecting aged MSCs for 72h according to the present invention;
FIG. 3c is a graph showing that the expression of PFKM mRNA in MSCs is increased by multiple after the lentiviral transfection is detected by real-time fluorescent quantitative PCR provided by the present invention;
FIG. 3d is a graph showing that the expression of PFKM proteins in MSCs is increased by fold after the detection of lentivirus transfection by Western Blot provided by the present invention;
FIG. 4a is a graph showing the results of glucose consumption measurements of modified senescent MSCs according to the present invention;
FIG. 4b is a graph showing the results of the cellular lactate level assay of the improved senescent MSCs provided by the present invention;
FIG. 4c is a graph showing the results of measuring ATP levels in the modified aged MSCs of the present invention;
FIG. 4d is a graph showing the results of measuring cellular ECAR levels of the modified senescent MSCs provided by the present invention;
FIG. 4e is a graph showing the improved cell morphology of senescent MSCs under an inverted phase contrast microscope in accordance with the present invention;
FIG. 4f is a result of a cytomorphological statistical analysis of the modified senescent MSCs provided by the present invention;
FIG. 4g shows the results of cellular SA- β -gal staining and quantitative analysis of the modified senescent MSCs provided by the present invention;
FIG. 4h shows that the real-time fluorescent quantitative PCR provided by the present invention detects the senescence-associated factor P16 in the improved senescent MSCsINK4aResults of mRNA expression levels.
Detailed Description
The invention provides application of a reagent for over-expressing Phosphofructokinase (PFKM) in preparation of a medicament for delaying cell senescence. In the invention, the nucleotide sequence of the gene of the phosphofructokinase is shown as SEQ ID NO. 1: ATGACCCATGAAGAGCATCATGAAGCCAAAACCCTGGGGATCGGCAAGGCCATCGCCGTGTTGACCTCTGGTGGAGATGCCCAAGGTATGAATGCTACGGTCAGGGCTGTGGTACGAGTTGGCATCTTCACCGGGCTCCGCGTCTTCTTTGTCCATGAGGGTTACCAAGGCCTGGTGGATGGTGGAGAGCACATCAGGGAGGCCACCTGGGAGAGCGTGTCCATGATGCTCCAGCTGGGAGGCACGGTGATTGGAAGTGCCCGATGCAAGGACTTCCGGGAGCGAGAAGGACGACTCCGAGCTGCCCACAACCTGGTGAAGCGGGGGATCACCAATCTGTGTGTCATCGGAGGCGATGGCAGCCTCACTGGGGCTGACACTTTTCGTTCAGAGTGGAGTGACTTATTGAATGACCTCCAGAAAGATGGGAAGATCACAGCCGAGGAGCGTACAAAGTCCAGCTACCTGAACATCGTTTTCCTGGTTGGCTCAATCGACAATGACTTCTGTGGCACTGATATGACCATTGGTACTGACTCTGCCCTGCACCGCATTGTAGAGATCGTGGACGCCATCACCACCACCGCTCAGAGCCACCAGAGGACATTTGTGTTAGAAGTGATGGGCCGCCACTGTGGATACCTGGCCCTTGTCACCTCTCTGTCGTGTGGGGCCGACTGGGTTTTCATTCCCGAGTGTCCGCCAGATGACGACTGGGAAGAACACCTTTGTCGCCGTCTCAGTGAGACAAGAACCCGTGGTTCTCGTCTCAACATCATCATTGTTGCTGAGGGTGCAATCGACAAAAACGGGAAGCCAATCACCTCAGAAGACATCAAGAACCTGGTGGTAAAGCGTCTTGGATATGATACCAGGGTCACTGTTCTGGGACATGTACAGAGGGGTGGGACACCATCAGCCTTTGACCGGATCCTGGGCAGCAGGATGGGTGTGGAAGCAGTGATGGCACTTTTGGAGGGGACCCCAGACACCCCAGCCTGTGTGGTGAGCCTCTCTGGTAATACGGCTGTGCGCCTGCCCCTCATGGAGTGTGTCCAGGTGACCAAAGACGTGACCAAGGCTATGGATGAGAAGAGATTTGATGAAGCCATTAAGCTGAGAGGCCGGAGCTTCATGAACAACTGGGAGGTATACAAGCTTCTAGCTCATGTCAGACCCCCAGTCTCTAAGGGCGGGTTGCACACGGTGGCTGTGATGAACGTGGGGGCCCCAGCTGCTGGAATGAATGCTGCAGTTCGCTCTACTGTGAGGATTGGCCTTATCCAAGGCAACCGAGTGCTGGTCGTGCATGATGGCTTTGAGGGTCTGGCCAAAGGTCAGATTGAGGAAGCTGGCTGGAGCTATGTTGGAGGCTGGACTGGTCAAGGTGGTTCCAAACTTGGTACTAAAAGGACTCTACCCAAGAAGAACCTGGAACAGATCAGTGCCAACATAACCAAGTATAACATCCAGGGCCTGGTTATCATTGGGGGCTTTGAGGCTTACACAGGGGGCTTGGAGCTGATGGAGGGCAGGAAGCAGTTTGATGAGCTCTGCATCCCATTTGTGGTCATTCCCGCCACGGTTTCCAATAATGTCCCAGGGTCAGACTTCAGCATCGGGGCTGACACAGCACTGAACACCATCTGCACGACCTGTGACCGAATCAAGCAGTCTGCAGCAGGCACCAAGCGGCGAGTGTTTATCATCGAGACGATGGGTGGTTACTGTGGCTATCTGGCCACCATGGCAGGACTGGCGGCTGGGGCTGATGCTGCCTACATTTTTGAGGAGCCCTTCACCATCCGAGATCTCCAGGTTAATGTTGAACATCTGGTGCAGAAGATGAAAACAACTGTGAAGAGAGGCCTGGTGCTGAGGAATGAGAAGTGCAACGAGAACTACACTACTGATTTCATTTTCAACCTGTACTCTGAGGAGGGGAAGGGCATCTTCGACAGCAGGAAGAACGTGCTTGGCCACATGCAGCAGGGTGGAAACCCAACTCCCTTTGACAGGAATTTTGCCACCAAGATGGGTGCTAAGGCTACGAATTGGATGTCTGGGAAAATCAAAGAGAGTTACCGTAATGGACGGATCTTTGCCAACACTCCCGACTCAGGTTGTGTTCTGGGGATGCGTAAGAGGGCCCTGGTCTTTCAGCCAGTGACTGAGCTGAAGGACCAGACAGATTTTGAGCACCGAATCCCCAAAGAACAGTGGTGGCTGAAGCTGAGGCCAATCCTCAAAATCCTGGCCAAGTACGAGATTGATCTGGACACCTCTGACCACGCCCACCTGGAGCACATTTCCAGGAAGCGGTCTGGAGAAGCTGCTGTC are provided. In the present invention, the cells preferably include Mesenchymal Stem Cells (MSCs). In the present invention, the mesenchymal stem cell preferably comprises an aging mesenchymal stem cell. Mesenchymal stem cells are the basis of cell therapy for many diseases and have become the main seed cells for stem cell tissue engineering. The normal bone marrow sample is difficult to obtain, sufficient bone marrow MSCs at a specific age stage cannot be obtained, the glycolysis level of the MSCs is reduced along with the increase of the in vitro culture times, the proliferation activity and the survival capability of the MSCs are weakened, the multidirectional differentiation potential is reduced, and the cells are aged. The aging of stem cells greatly restricts the clinical application of stem cells, and the curative effect of autologous stem cell transplantation of patients is reduced. The invention discusses about the influence of PFKM on the glycolysis level of MSCs to regulate cell senescence, organically connects PFKM with MSCs senescence for the first time, provides experimental basis for clarifying a regulation mechanism of stem cell senescence, is beneficial to maintaining the number and functions of stem cells, and has important significance for delaying body senescence and preventing and treating various aging diseases. The PFKM plays an important role in improving the cytological characteristics of the MSCs and delaying the senescence of stem cells. The agent for overexpressing phosphofructokinase in the present invention preferably comprises other cytokines, chemical molecules, etc. that increase the PFKM expression of MSCs by indirect action; other vectors carrying the PFKM gene such as plasmid, lentivirus, adenovirus or adeno-associated virus, or other microorganisms containing these vectors. All the reagents which artificially improve the intracellular PFKM level in the MSCs to modify the cytological characteristics of the MSCs and delay the senescence of stem cells are protected by the invention.
The invention also provides application of the reagent for over-expressing the phosphofructokinase in preparing a medicine for improving the glycolysis level of cells. In the present invention, the cells preferably include mesenchymal stem cells. In the present invention, the mesenchymal stem cell preferably comprises an aging mesenchymal stem cell. Overexpression of PFKM in MSCs is of key significance to the level of remodeling cell glycolysis. The invention protects the technologies and methods for up-regulating cellular glucose consumption, lactic acid level, ATP content, ECAR level and other indexes capable of reflecting glycolysis level by artificially improving the intracellular PFKM expression level in MSCs.
The invention also provides application of the agent for over-expressing the phosphofructokinase in preparing a medicament for promoting cell proliferation and differentiation. In the present invention, the cells preferably include mesenchymal stem cells. In the present invention, the mesenchymal stem cell preferably comprises an aging mesenchymal stem cell.
The invention also provides application of a reagent for over-expressing phosphofructokinase in preparing a medicament for up-regulating one or more than two of the following indexes of cells, wherein the indexes comprise: glucose consumption, lactate level, ATP content and ECAR level. In the present invention, the cells preferably include mesenchymal stem cells. In the present invention, the mesenchymal stem cell preferably comprises an aging mesenchymal stem cell.
The invention also provides application of the agent for over-expressing the phosphofructokinase in preparing a medicament for down-regulating the expression of senescence-associated beta galactosidase. In the present invention, the cells preferably include mesenchymal stem cells. In the present invention, the mesenchymal stem cell preferably comprises an aging mesenchymal stem cell.
The invention also provides the use of an agent which overexpresses phosphofructokinase in the manufacture of a medicament for down regulating the expression of senescence-associated factors in a cell, including P16INK4a. In the present invention, the cells preferably include mesenchymal stem cells. In the present invention, the mesenchymal stem cell preferably comprises an aging mesenchymal stem cell.
The cell factors and growth factors secreted by the cells after the PFKM is artificially and highly expressed in the MSCs and the active ingredients in exosomes are changed in quality and quality. Over-expression of PFKM promotes MSCs to secrete the cytokines, growth factors and exosomes, improves the glycolysis level of cells, promotes cell proliferation and differentiation, and further delays the aging of stem cells. Therefore, the invention protects all the substances and methods for separating and purifying the active ingredients such as cell factors, growth factors, exosomes and the like from the MSCs with high expression of PFKM and preparing the therapeutic agent for improving the glycolysis level of the MSCs and delaying the aging of the MSCs.
The invention also provides an improved senescent mesenchymal stem cell, which is capable of overexpressing phosphofructokinase myotype. In the present invention, the improved aging mesenchymal stem cell preferably includes an expression vector containing a PFKM nucleotide sequence. The invention improves the expression level of PFKM by introducing an expression vector containing PFKM nucleotide sequence into senescent MSCs. The experiment of the invention shows that the glycolysis level of the improved aging MSCs is increased, the morphology of the aging MSCs tends to be young, the dyeing positive rate of the aging-related beta galactosidase (SA-beta-gal) of the improved aging MSCs is reduced, and the aging-related factor P16INK4aThe expression is obviously reduced, the cell activity is increased, and the aging of the MSCs is effectively delayed. The human overexpression PFKM can remodel glycolysis level of aging MSCs, so that aging of mesenchymal stem cells is delayed, theoretical basis and experimental scheme are provided for obtaining a large amount of young MSCs by in vitro culture, understanding of aging potential mechanism of the MSCs is deepened, and the invention is beneficial to the exploration of new targets for preventing and treating aging related diseases.
The invention provides a construction method of an improved aging mesenchymal stem cell, which comprises the following steps: infecting aged mesenchymal stem cells with lentiviruses carrying a phosphofructokinase gene; the MOI value is 50-250, and the infection time is 48-72 h. That is, when the vector overexpressing phosphofructokinase is a lentivirus carrying a phosphofructokinase gene, the present invention is preferably constructed by the above-described method. The source of the lentivirus carrying the phosphofructokinase gene is not particularly limited, and conventional commercial products well known to the technicians in the field are adopted, for example, the lentivirus which is constructed by Shanghai Jikai gene medicine science and technology GmbH and stably expresses PFKM (the vector GV 358: Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin is used as a skeleton vector, and the PFKM gene is constructed on the vector by adopting a double enzyme digestion method (AgeI/AgeI enzyme digestion)), and the titer of the virus is 3E + 8. In the present invention, the MOI value is preferably 200, and the infection time is preferably 72 hours. In the embodiment of the invention, PFKM lentivirus infection gradient is preferably set to infect senescent MSCs according to MOI values of 50, 100, 150, 200 and 250, the MOI value with infection efficiency of more than 80% and minimal cytotoxicity is the optimal MOI value after 48h culture, the optimal MOI value is selected for subsequent experiments, an experimental group LV-PFKM is obtained after 72h culture, and a negative control group LV-Vector is obtained by infecting senescent MSCs in the same state with a control group lentivirus (GV358 Vector). The invention preferably adopts real-time fluorescent quantitative PCR (RT-qPCR) to detect the expression condition of PFKM in MSCs infected by lentivirus. The test results show that the optimal MOI value is 200 according to the invention, wherein the virus infection efficiency is more than 80% and the cytotoxicity is the minimum. Constructing MSCs with stable and high expression PFKM, detecting MSCs after lentivirus infection for 72h by real-time fluorescence quantitative PCR, and stably maintaining PFKM expression in an overexpression group to be about 2.5 times that of a negative control group
The use of the agent for overexpressing phosphofructokinase of the present invention for preparing a medicament for delaying cellular aging will be described in further detail with reference to the following specific examples, and the technical solutions of the present invention include, but are not limited to, the following examples.
Example 1
Establishing rat MSCs in vitro replication aging model
1.1 isolation of Primary cells
The rat is placed in a laboratory environment for 2-3 hours, the rat is adapted to the laboratory environment, the rat is killed by neck removal after anesthesia, the whole body is disinfected by iodophor, deiodinated by 75% alcohol, the steps are repeated for 3 times, the rat is rapidly separated to obtain bilateral humerus, femur and tibia, the bone is soaked in PBS containing 1% penicillin-streptomycin, and the rat is rapidly moved into a clean bench. Removing tissues such as muscles and fascia attached to the surfaces of bones in a super clean bench, and placing the bones in a complete culture solution; removing two ends of a bone by the rongeur, and repeatedly flushing a marrow cavity by using a 5ml syringe to suck culture solution until the marrow cavity becomes semitransparent. Gently sucking the bone marrow rinse solution with a 5ml pipette to a 40 μm cell filter screen on a 50ml centrifuge tube, centrifuging at room temperature for 5min, and filteringThe liquid after the reaction is 1500rpm, then the supernatant is discarded, the cell mass is resuspended, the cell mass is evenly mixed and then is transferred into a 10cm culture dish, and the culture dish is placed at 37 ℃ and 5% CO2Culturing in a cell incubator, changing the liquid half every other day, and then changing the liquid 2-3 days according to the cell state.
1.2 construction of MSCs in vitro replication aging model
Performing in vitro subculture on the primary MSCs to obtain MSCs of different generations, and performing aging assessment on the MSCs of each generation, including morphological observation and analysis of the MSCs, activity detection of aging-related beta-galactosidase (SA-beta-gal) and aging-related factor P16INK4amRNA expression detection, and constructing an MSCs in vitro replication aging model. Early-generation MSCs (EPMSCs, P1-P3) and late-generation MSCs (LPMSCs, P10-P12) were used to represent young and replicating senescent cells, respectively.
1.3 morphological Observation and analysis of MSCs
Observing EPMSCs and LPMSCs obtained by in-vitro subculture under a microscope, evaluating the growth condition and morphological characteristics of cells, randomly selecting about 20 fields to collect images, recording and calculating the surface area and the length-width ratio of single cells in the fields by using Cell Entry software, and performing statistical analysis.
1.4 senescence-associated beta-galactosidase (SA-beta-gal) Activity assay (cell senescence beta-galactosidase staining kit, Biyuntian)
Culturing EPMSCs and LPMSCs in a 6-well plate, when the cell growth density reaches 80-90%, washing with PBS for 3 times (3 min each time), adding 200 μ l of fixative into each well, fixing at room temperature for 10-15min, removing fixative, washing with PBS for 3 times (5 min each time). Adding 500 μ l of working solution into each well after washing, sealing the edge of the well plate with sealing film to prevent the working solution from volatilizing, and placing in a CO-free container2At 37 ℃ in an incubator overnight. Observing under an inverted phase contrast microscope on the next day, acquiring images under a random visual field, recording the number of blue-stained cells as the number of positive cells, and calculating the percentage of the number of the positive cells in the total number of the cells.
1.5 fluorescent quantitative PCR (RT-qPCR) for detecting aging-related factor mRNA expression level
(1) Extraction of cellular RNA
And when the cell growth density reaches 80-90%, sucking the culture solution, washing with PBS for 2-3 times, adding 1ml of Trizol into the culture dish, cracking at room temperature for 15min, repeatedly blowing until all cells are completely cracked, sucking all the liquid into 1.5ml of EP tube, centrifuging at 4 ℃, rotating at 12000rpm for 15min, and sucking the supernatant into another EP tube. Chloroform was added at 200. mu.l/ml Trizol, mixed by vigorous shaking, vortexed was stopped, and allowed to stand at room temperature for 15 min. Centrifuging at 4 deg.C at 12000rpm for 15min, sucking the upper aqueous phase into a new EP tube, adding isopropanol according to 500 μ l/ml Trizol, gently shaking, and standing at room temperature for 15 min. Centrifuging at 4 deg.C at 12000rpm for 15min, discarding supernatant, precipitating the bottom of the tube to obtain RNA, adding pre-cooled 75% ethanol 1ml, flicking the bottom of the tube to suspend the precipitate, centrifuging at 4 deg.C at 12000rpm for 15min, discarding supernatant, and drying at room temperature for 10min until it becomes semitransparent. Adding 10-20 μ l DEPC water to dissolve RNA, detecting RNA concentration and purity on machine, and optionally adding or subtracting DEPC water according to RNA concentration.
(2) RNA reverse transcription into cDNA (TransScript All-in-One First-Strand cDNA Synthesis Supermix for qPCR, TransGen Biotech)
The reverse transcription reaction procedures as shown in tables 1 and 2 were performed according to the kit instructions:
TABLE 1 reverse transcription reaction System
TABLE 2 reverse transcription reaction procedure
The synthesized cDNA can be directly subjected to qPCR (qPCR primer set is shown in Table 3, wherein beta-actin is internal reference) or stored in a refrigerator at-20 ℃.
TABLE 3 qPCR primer set
(3) RT-qPCR assay (TransStart Top Green qPCR SuperMix, TransGen Biotech)
The reaction system is prepared as shown in Table 4:
TABLE 4 reverse transcription reaction System
The reverse transcription procedure is shown in table 5:
TABLE 5 reverse transcription reaction procedure
CT values were measured and statistically analyzed.
EPMSCs and LPMSCs are obtained by in vitro subculture, and as shown in figure 1a, obvious morphological difference of two groups of cells is observed under a microscope, the EPMSCs have good growth state, the cell body is in a long fusiform shape, and the boundary is clear; the LPMSCs have poor growth state, irregular shapes and spreading appearance, fuzzy cell boundaries, disappearance of stereoscopic impression and obvious cytoplast granular sensation. Analysis of the cell morphology parameters, as shown in fig. 1b, revealed that the surface area of the LPMSCs was significantly increased compared to the EPMSCs, and the aspect ratio was decreased.
In FIG. 1c, LPMSCs stained significantly more blue cells than EPMSCs. Statistical analysis shows that the proportion of blue-stained cells (SA-beta-gal positive) in the LPMSCs is obviously higher than that of the EPMSCs, which indicates that the number of aged cells in the LPMSCs is obviously increased.
FIG. 1d shows the RT-qPCR technology for detecting aging-related factor P16INK4amRNA levels, results indicate P16 in LPMSCs relative to EPMSCsINK4aThe mRNA expression level is obviously increased. Therefore, the invention successfully establishes the in vitro replicative failure of the MSCsThe old model, EPMSCs as young cells, LPMSCs as senescent cells.
Example 2
Detection of glycometabolism level and PFKM expression of senescent MSCs
2.1 detection of glucose consumption (glucose assay kit, Nanjing was established)
EPMSCs and LPMSCs are mixed at 4 × 103Density of individual cells/well in 96-well plates, 5% CO2And a constant temperature incubator for cells at 37 ℃. When the cells grew adherent, the original culture solution was discarded, 50. mu.l of serum-free DMEM/F-12 was added to each well, and the culture supernatant was collected the next day. Centrifuging the supernatant at room temperature of 1200prm for 3min, removing the precipitate, and collecting the supernatant to another EP tube to be tested;
the specific procedures according to the kit instructions are shown in table 6:
TABLE 6 glucose consumption measurement protocol
After the liquid is uniformly mixed, the mixture is put in a water bath kettle at 37 ℃ for reaction for 5min, and then a spectrophotometer is used for detecting the absorbance value of each hole at the position of 505nm of wavelength, and the glucose content is calculated.
2.2 detection of lactic acid level (lactic acid detection kit, Nanjing was built)
Cell culture supernatant was collected and tested in the same manner as glucose consumption, according to kit instructions, see table 7:
TABLE 7 lactic acid level detection operating procedure
Mixing the liquid with ddH2O is adjusted to zero, and the absorbance value (wavelength 530nm) of each tube is detected.
2.3 detection of ATP content (ATP detection kit, Biyuntian)
And taking out cells with good culture state in a 6-hole plate, washing the cells for 3 times by using PBS (phosphate buffer solution), adding 200 mu l of lysis solution into each hole to ensure that the lysis solution is fully contacted with the cells, centrifuging at 4 ℃ for 10min after lysis at 12000rpm, and transferring the obtained supernatant to another EP (ultraviolet) tube for detecting the ATP (adenosine triphosphate) content in the cells. Diluting the ATP detection reagent diluent, preparing an ATP detection working solution with the ATP detection reagent according to a ratio of 4:1, adding 100 mu l of ATP detection working solution into each hole of a sterile 96-hole detection plate, standing for 5-10 min, and consuming background ATP. Diluting the sample and the standard substance with ATP detection lysate, adding 20 mul of the sample or the diluted standard substance into a sample hole, uniformly mixing, detecting the RLU value with a chemiluminescence apparatus, making a standard curve, and substituting the standard curve into a formula to calculate the ATP content of the sample.
2.4 detection of ECAR levels (extracellular acid Rate detection kit, Luxcel Biosciences)
Cells were aligned at 8X 104Inoculating to 96-well plate at density of one well, setting 3 multiple wells, standing at 37 deg.C and 5% CO2Culturing in a constant-temperature incubator; the next day, drug treatment was performed according to ECAR assay instructions, and the 96-well plates were placed in CO-free format2Incubating in a constant-temperature incubator at 37 ℃, and detecting on a computer for 3-4 hours after 3 hours. Standard curves were drawn and statistically analyzed.
2.5 RT-qPCR detection of PFKM mRNA expression levels in EPMSCs and LPMSCs
The real-time quantitative fluorescent PCR method for detecting the expression of PFKM in EPMSCs and LPMSCs is the same as 1.5, and the sequences of the primers are as follows:
TABLE 8 primers for real-time quantitative fluorescent PCR detection
2.6 Western Blot to detect the expression level of PFKM protein in EPMSCs and LPMSCs
(1) Extraction of Total cellular protein
Digesting when the cells are fused to 80-90%, placing in a 15ml centrifuge tube after digestion is ended, and centrifuging at 1200rpm for 5min at room temperature. Resuspend with 3ml PBS, add appropriate amount of protein lysate (PMSF: RIPA ═ 1:100 prepared in advance), lyse on ice for 15min, repeatedly blow and mix until cells are fully lysed. After lysis, cells and lysate were centrifuged at 12000rpm for 15 min; after centrifugation, the liquid on the upper part of the EP tube is slightly sucked out, so that the sediment is prevented from being sucked up, and the liquid is transferred into a new EP tube. Mu.l of protein sample was taken for concentration determination, and the remaining protein sample was quickly stored at-80 ℃ to avoid protein degradation.
(2) BCA method for determining protein concentration
Using a 96-well plate, 0. mu.l, 1. mu.l, 2. mu.l, 4. mu.l, 8. mu.l, 12. mu.l, 16. mu.l, and 20. mu.l of a 0.5mg/ml protein standard were added, and PBS was added to the plate to make up 20. mu.l. Preparing BCA working solution (the ratio of the reagent A to the reagent B is 50:1), adding 200 mu l of the working solution into each well when the working solution is used in the field, and then adding the working solution in the absence of CO2Incubate at 37 ℃ for 30 min. And (3) placing the 96-well plate on an enzyme labeling instrument for detection, setting the wavelength to be 562nm, measuring the absorbance value, and finally calculating the total protein concentration of the sample to be detected according to the standard curve.
(3) Western blotting procedure
After the concentration is measured, 20 mu g of protein sample is taken and mixed evenly, PBS is filled to 20 mu l, the tube opening is sealed by a sealing film, and the boiling is carried out for about 7 min. Preparing 12% separation gel and 5% concentration gel, performing electrophoresis on the upper layer concentration gel at 80V for 50min and the lower layer separation gel at 120V for 70 min. And (3) taking down the whole gel plate after electrophoresis, determining the position of the target protein, cutting gel with proper size, wherein the area of the gel is the minimum, then, the filter paper and the PVDF membrane are the maximum, recording the area of the PVDF membrane, activating the PVDF membrane for 15sec by using a methanol solution, and finishing activation when the membrane becomes semitransparent. The filter paper-PVDF membrane-gel-filter paper are stacked on a semi-dry membrane rotating instrument in sequence, constant voltage and current of 2.5 times membrane area are set, and membrane rotation is carried out for 45 min. Sealing the transferred PVDF membrane for 1-2 h by using milk powder, then incubating primary antibody (PFKM antibody, Proteintetech), placing the PVDF membrane in a primary antibody solution (PFKM1: 1000; beta-actin 1:2000), incubating for 2h by a shaking table, and then, refrigerating at 4 ℃ overnight. After the primary antibody incubation was completed, the membrane was washed 3 times with 1 × TBST for 10min each time; the secondary antibody (. beta. -actin antibody, Sangon Biotech) was used after dilution at 1:5000, and the secondary antibody was incubated at room temperature for 1h, after which the membrane was washed 3 times with 1 XTSST for 10min each. Working solution is prepared according to the ECL hypersensitive luminous liquid specification, and images are collected after the color development of a color development system.
As shown in FIG. 2a, the glucose consumption of the cells was reduced for LPMSCs compared to EPMSCs, indicating that the glucose consumption of senescent MSCs was significantly lower than that of young MSCs.
FIG. 2b shows that lactate levels in extracellular fluids, LPMSCs, are decreased compared to EPMSCs, and the results indicate that senescent MSCs have a diminished capacity to secrete lactate, indicating a decreased level of glycolysis in senescent cells.
As shown in fig. 2c for intracellular ATP levels, LPMSCs produced lower ATP than EPMSCs, indicating that cellular energy production gradually decreased as MSCs senesced.
FIG. 2d shows the extracellular acid secretion rate, with lower ECAR values for LPMSCs than for EPMSCs. Indicating that glycolytic levels of senescent MSCs are reduced.
FIG. 2e shows the expression level of PFKM mRNA in EPMSCs and LPMSCs detected by RT-qPCR, and PFKM mRNA in LPMSCs is lower than that in EPMSCs, indicating that PFKM mRNA expression in senescent MSCs is reduced.
FIG. 2f shows that Western Blot detects the expression level of PFKM proteins in EPMSCs and LPMSCs, and the PFKM protein expression of LPMSCs is lower than that of EPMSCs, which indicates that the PFKM protein expression is gradually reduced in the aging process of MSCs.
Example 3
Lentiviral infection of senescent MSCs to obtain improved senescent MSCs
Construction of improved aging MSCs with high expression of PFKM
A lentivirus for stably expressing PFKM is constructed by Shanghai Jikai gene medical science and technology GmbH (a GV358 vector: Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin is used as a framework vector, and a PFKM gene is constructed on the vector by a double enzyme digestion method (AgeI/AgeI enzyme digestion)). The titer of PFKM-overexpressing lentiviruses was 3E + 8. Determining the optimal multiplicity of infection, setting PFKM lentivirus infection gradient to infect the aged MSCs according to 50, 100, 150, 200 and 250, setting the MOI value with the infection efficiency of more than 80% and the minimum cytotoxicity after 48h culture as the optimal MOI value, adopting the optimal MOI value of 200 to be used in subsequent experiments, obtaining an experimental group LV-PFKM after 72h culture, and infecting the aged MSCs in the same state with the lentivirus of a control group (GV358 Vector) to obtain a negative control group LV-Vector.
RT-qPCR was used to detect the fold increase in PFKM mRNA expression in the experimental group after lentivirus infection compared to the negative control group.
The Western Blot assay detects the fold increase in PFKM protein expression in the experimental group after lentivirus infection compared to the negative control group.
FIGS. 3a and 3b show that after PFKM lentivirus overexpression is performed with different MOI values, each group of MSCs expresses green fluorescent protein, and when the MOI value is 200, the green fluorescent protein is expressed most strongly and the cytotoxicity is minimal. The RT-qPCR results are shown in FIG. 3c, compared with the negative control group, the mRNA expression level of PFKM in the overexpression group is improved by about 7 times, and the results in FIG. 3d show that the protein expression level of PFKM is improved by about 2.5 times. Thus, the slow virus with over-expression of PFKM can cause the PFKM to be stably and highly expressed after infecting aged MSCs.
Example 4
Improved carbohydrate metabolism levels of senescent MSCs and senescence assay
4.1 testing the Effect of overexpression of PFKM on the level of carbohydrate metabolism in cells
4.1.1 measurement of cell glucose consumption
As shown in FIG. 4a, glucose was up-regulated by about 1.4 times in the LV-PFKM group compared with that in the LV-Vector group. Indicating that the glucose consumption of the improved aged MSCs is obviously up-regulated.
4.1.2 detection of cellular lactate levels
As shown in FIG. 4b, cellular lactate levels in the LV-PFKM group were up-regulated by about 1.6-fold compared to the LV-Vector group. Indicating that lactate levels in the modified senescent MSCs are significantly upregulated.
4.1.3 detection of cellular ATP content
As shown in FIG. 4c, the ATP levels in the LV-PFKM group were up-regulated by about 1.4-fold compared to the LV-Vector group, indicating that the ATP levels in the improved senescent MSCs were significantly up-regulated.
4.1.4 detection of cellular ECAR levels
As shown in FIG. 4d, ECAR levels were up-regulated by about 1.5 fold in the LV-PFKM group compared to the LV-Vector group, indicating a significant up-regulation of ECAR in the improved senescent MSCs.
4.2 testing the Effect of overexpression of PFKM on cellular senescence
4.2.1 Observation of cell morphology characteristics by inverted phase contrast microscope and statistical analysis
As shown in FIG. 4e, compared with the LV-Vector group, the cells in the LV-PFKM group are in a long spindle shape, and have stronger stereoscopic impression and clearer boundary. As shown in FIG. 4f, the morphological parameters of the two groups of cells were statistically analyzed, and it was found that the cell surface area of the LV-PFKM group was down-regulated by 1.5 times and the aspect ratio was up-regulated by 1.7 times. Indicating that the morphology of the aged MSCs cells with the improved expression of PFKM is more youthful.
4.2.2 SA-beta-gal staining to detect cellular senescence
As shown in FIG. 4g, the number of blue-stained cells in the LV-PFKM group was significantly reduced and the SA- β -gal positive rate was down-regulated by about 1.5 times, as compared with the negative control group. Indicating that overexpression of PFKM improved senescence in senescent MSCs.
4.2.3 RT-qPCR detection of aging-related factor expression
As shown in FIG. 4h, RT-qPCR detected the senescence-associated factor P16INK4amRNA expression level, results show: p16 in LV-PFKM group compared with negative control groupINK4aExpression of (a) was down-regulated by 1.4-fold. Indicating that the expression of the senescence-associated factors in the improved senescent MSCs is significantly down-regulated.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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| CN116041477A (en) * | 2022-10-13 | 2023-05-02 | 呈诺再生医学科技(北京)有限公司 | Application of TDGF1 gene in preparing medicine for treating senility related diseases or reversing cell senility |
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