CN115029307A

CN115029307A - Method for delaying aging of MSCs

Info

Publication number: CN115029307A
Application number: CN202210896484.7A
Authority: CN
Inventors: 何旭; 于晓; 孙亚楠; 高星宇; 张畅; 孙慧; 石英爱; 张海英; 黄凡珂; 王一茗; 闫悦琪
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2022-07-28
Filing date: 2022-07-28
Publication date: 2022-09-09

Abstract

The invention is suitable for the technical field of senescence delaying, and provides a method for delaying senescence of MSCs, which comprises the following steps: step 1: obtaining aged MSCs; step 2: the slow virus carrying SCD2 is used for infecting aged MSCs, so that the lipid synthesis level of the aged MSCs is up-regulated, and the aging of the MSCs is delayed. The invention organically relates the aging of SCD2 and MSCs, explores the aging of stem cells from the novel perspective of stem cell lipid metabolism, finds that SCD2 can delay the aging of mesenchymal stem cells by improving the synthesis level of cell lipid, provides experimental basis for clarifying the regulation and control mechanism of the aging of stem cells, provides a new idea and a new technology for obtaining a large amount of young MSCs by in vitro culture, is beneficial to maintaining the number and functions of stem cells, is beneficial to delaying the aging of organisms and preventing and treating various aging diseases, and has important significance for the long-term healthy development of the society.

Description

Method for delaying aging of MSCs

Technical Field

The invention relates to the technical field of senescence delaying, in particular to a method for delaying senescence of MSCs.

Background

With the advancement of society and the rapid development of economy, the aging of the population will become a major socioeconomic problem all over the world, and aging is one of the most important causes of the occurrence thereof. Aging is a complex process, manifested by decreased physiological activity, low levels of energy metabolism, stress-induced loss of homeostasis, leading to increased risk of illness and death. At present, over 300 theories or hypotheses have been proposed by the scientific community to explain the occurrence, development and mechanism of aging, wherein the stem cell aging theory is one of the latest theories in the aging mechanism of organisms at present. The stem cell aging theory considers that adult stem cell aging is an important reason for the aging of tissues and organs, the aging of individuals and the occurrence of various aging diseases, and stem cells are extremely important models for researching aging mechanisms. Therefore, the aging-activated stem cells are effective measures for delaying individual aging and preventing aging-related diseases such as neurodegenerative diseases, metabolic diseases and various malignant tumors, and can furthest improve the life quality of the aged population. Aging is closely related to metabolic disorders, which are key markers of aging. Metabolic pathways related to aging, such as the mTOR and AMPK pathways, rapamycin, metformin, and exercise, etc., can directly or indirectly regulate the pathways, and have become major targets for anti-aging intervention. With age, membrane phospholipids are also more unsaturated and lipid peroxidation products increase, which may lead to cell damage. Furthermore, different types of dietary interventions may affect aging, and these interventions are often accompanied by changes in lipid homeostasis. The dietary lipid can regulate the expression of lipid metabolism related genes by influencing the activity of adult stem cells, enhance the lipid decomposition capability and contribute to the realization of healthy aging. Lipids can modulate the biological properties of stem cells by affecting changes in energy storage, plasma membrane composition, signal transduction, and gene expression. However, how changes in lipid metabolism affect stem cell senescence has not been explored to date.

Adult stem cells are undifferentiated cells existing in differentiated tissues in an adult body, and Mesenchymal Stem Cells (MSCs) are a class of adult stem cells having high-efficiency self-renewal and multi-differentiation potential. They are easy to culture in vitro, capable of contacting host cells, and are immune tolerant, and are ideal seed cells for tissue engineering and treatment of various diseases. However, during in vitro amplification culture, the lipid synthesis capacity of MSCs is reduced, and MSCs show senescence-characteristic phenotypes such as morphological changes, reduced proliferative activity, and physiological function imbalance. Therefore, sufficient numbers of MSCs cannot be obtained by in vitro expansion to meet the needs of clinical treatment. The aging of stem cells severely limits clinical applications such as autologous stem cell transplantation, tissue and organ regeneration and repair, etc. of patients.

MSCs are the basis of cellular therapy for many diseases, an earlier and more deeply studied adult stem cell, and have become the main seed cell 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 proliferation activity and the multi-directional differentiation potential of the MSCs are reduced along with the increase of the in vitro culture times, 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. Therefore, in view of the above situation, there is an urgent need to provide a method for delaying the aging of MSCs to overcome the shortcomings in the current practical application.

Disclosure of Invention

The invention aims to provide a method for delaying the aging of MSCs, and aims to solve the problems in the technical background.

The present invention is thus achieved, a method of delaying aging of MSCs, the method comprising the steps of:

step 1: obtaining aged MSCs;

step 2: the slow virus carrying SCD2 is used for infecting aged MSCs, so that the lipid synthesis level of the aged MSCs is up-regulated, and the aging of the MSCs is delayed.

As a further scheme of the invention: in step 1, the specific steps for obtaining senescent MSCs are:

step 1.1: extracting primary cells from rat bone marrow, and performing in vitro subculture by using a full bone marrow wall attaching method to obtain early-generation secondary MSCs (EPMSCs, P2-P3) and late-generation MSCs (LPMSCs, P9-P10);

step 1.2: by morphological observation (cell morphology characteristics, cell surface area and aspect ratio), senescence-associated beta-galactosidase (SA-beta-gal) staining and senescence-associated factor P16 ^INK4a To establish replicative senescence MSCs.

As a further scheme of the invention: in step 1.1, the rats are 1-2 month old male Wistar rats.

As a further scheme of the invention: in step 2, the time for infecting senescent MSCs with a lentivirus carrying SCD2 ranged from 48h to 72 h.

As a further scheme of the invention: the method is applied to the preparation of the anti-aging medicine.

As a further scheme of the invention: the method is applied to the preparation of the anti-skin-aging health care product.

As a further scheme of the invention: the use of the method in the preparation of a food additive.

Compared with the prior art, the invention has the beneficial effects that:

the invention discusses about the influence of SCD2 on MSCs lipid synthesis level regulation cell senescence, organically connects SCD2 with MSCs senescence for the first time, researches stem cell senescence from a novel perspective of stem cell lipid metabolism, finds that SCD2 can delay mesenchymal stem cell senescence by improving the cell lipid synthesis level, provides experimental basis for clarifying a regulation mechanism of stem cell senescence, also provides a new idea and a new technology for obtaining a large amount of young MSCs by in vitro culture, is beneficial to maintaining the number and functions of stem cells, is beneficial to delaying organism senescence and preventing and treating various aging diseases, and has important significance for long-term healthy development of society.

Drawings

FIG. 1a shows the cell morphology of EPMSCs and LPMSCs under the microscope in example 1 of the present invention;

FIG. 1b is a diagram showing the structure of the cell morphology parameter analysis of EPMSCs and LPMSCs in example 1 of the present invention;

FIG. 1c shows the result of SA-. beta. -gal staining in example 1 of the present invention;

FIG. 1d shows the detection of aging-related factor P16 by RT-qPCR in example 1 of the present invention ^INK4a Results for mRNA levels are shown;

FIG. 2a is a comparison graph of the LPMSCs and EPMSCs stained with alizarin red in example 2 of the present invention;

FIG. 2b is a diagram schematically showing the result of Western Blot in example 2 of the present invention;

FIG. 2c is a graph showing the comparison of red fluorescence intensity between EPMSCs and LPMSCs in example 2 of the present invention;

FIG. 2d is a diagram of RT-qPCR detection of the expression level of SCD2 mRNA in EPMSCs and LPMSCs according to example 2 of the present invention;

FIG. 2e is a diagram of Western Blot to detect the expression level of SCD2 protein in EPMSCs and LPMSCs in example 2 of the present invention;

FIG. 3a is a schematic diagram showing that after the SCD2 lentivirus is over-expressed, each group of MSCs expresses green fluorescent protein according to example 3 of the present invention;

FIG. 3b is a diagram showing the result of RT-qPCR in example 3 of the present invention;

FIG. 3c is a diagram schematically showing the result of Western Blot detection in example 3 of the present invention;

FIG. 4a is a schematic representation of SA- β -gal staining for cellular senescence in example 4 of the present invention;

FIG. 4b is the expression diagram of the aging-associated factor detected by RT-qPCR in example 4 of the present invention;

FIG. 4c is a schematic diagram of alizarin red staining for detecting osteogenic differentiation capacity of cells in example 4 of the present invention;

FIG. 4d is a schematic diagram of the Western Blot detection of osteogenic differentiation capacity of cells in example 4 of the present invention;

FIG. 4e is a graph showing the effect of Nile Red staining assay for detecting the overexpression of SCD2 on the level of lipid synthesis in cells of example 4.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Specific implementations of the present invention are described in detail below with reference to specific embodiments.

The embodiment of the invention provides a method for delaying the aging of MSCs, which comprises the following steps:

step 1: obtaining aged MSCs;

specifically, primary cells are extracted from bone marrow of 1-2 month old male Wistar rats, and early generation secondary MSCs (EPMSCs, P2-P3) and late generation secondary MSCs (LPMSCs, P9-P10) are obtained by using a full bone marrow wall attaching method and in-vitro subculture;

by morphological observation (cell morphological characteristics, cell surface area and aspect ratio), senescence-associated beta-galactosidase (SA-. beta. -gal) staining and senescence-associated factor P16 ^INK4a Establishing replicative senescence MSCs;

the lipid synthesis level and osteogenic differentiation potential of the aging MSCs are obviously reduced through triglyceride content detection, alizarin red staining and Runx2 protein expression level detection;

step 2: infecting aged MSCs with a lentivirus carrying SCD2, so that the lipid synthesis level of the aged MSCs is up-regulated, thereby delaying the aging of the MSCs;

specifically, the slow virus carrying SCD2 is used for infecting aged MSCs, and the infection time is 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 SCD2 can be obviously up-regulated;

age-related factor P16 by SA-beta-gal staining ^INK4a The indexes of expression detection, alizarin red staining, Runx2 protein expression detection, Nile red staining and the like find that SCD2 delays the aging of MSCs by up-regulating the lipid synthesis level of the aging MSCs, provides a new thought and a new technology for obtaining a large amount of young MSCs by in vitro culture, deepens the understanding of the aging potential mechanism of the MSCs, and is beneficial to the exploration of a new target point for preventing and treating aging-related diseases.

Example 1 establishment of rat MSCs model of senescence by in vitro replication

1.1 isolation of Primary cells

Taking a healthy male Wistar rat to be in a quiet and stable state, taking out a neck, killing the rat and sterilizing the rat, then quickly separating the femur, the tibia and the humerus of the rat by using an instrument, putting the rat into a 50ml centrifugal tube, soaking the rat in PBS (phosphate buffer solution) containing 1% of double antibodies, and then quickly moving the rat into a super clean bench;

removing muscles and tissues on the surface of the bone, and soaking the bone in a complete culture solution;

opening the marrow cavities at two ends of the bone by using rongeur, sucking culture solution by using a 5-10ml syringe to flush the marrow cavities, and flushing for 3-4 times until the marrow cavities become semitransparent;

putting the washed liquid on a cell filter screen for filtration, centrifuging at room temperature at the rotating speed of 1500rpm for 5min, then resuspending the cell suspension by using a 5ml pipette, and putting the cell suspension in a 6cm cell culture dish for culture;

half of the liquid change is carried out the next day, the culture is continued, and the cells are fused to about 80 percent for subculture.

1.2 construction of MSCs in vitro replication aging model

Extracting primary MSCs from bone marrow of 150-180g healthy Wistar male rats, carrying out in-vitro subculture to about P10 generation, and carrying out senescence identification on different generation cells to successfully establish a MSCs replicative senescence model;

P2-P3MSCs are early-generation MSCs (EpMSCs), and P9-P10MSCs are late-generation MSCs (LPMSCs).

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

Culturing EPMSCs and LPMSCs in a 6-well plate respectively; when the cells reach 70% fusion, washing with PBS 3 times, adding 500 μ l of fixing solution into each hole to fix the cells for 15-18min, and washing the cells with PBS again; adding 1ml of working solution (containing 10 μ l of each of staining solution A and B, 930 μ l of staining solution C, and 50 μ l of X-Gal solution) into each well after washing, sealing with sealing film, preservative film and tinfoil, and keeping out of the sun at 37 deg.C without CO ₂ The incubator is kept overnight; and observing the staining result of the positive cells under an inverted phase contrast microscope and taking a picture.

1.5 fluorescent quantitative PCR (RT-qPCR) for detecting aging-related factor mRNA expression level

(1) Extraction of cellular RNA

When the cell growth density reaches 80-90%, absorbing and removing the culture solution, washing with PBS for 2-3 times, adding 1ml Trizol into the culture dish, cracking for 15min at room temperature, repeatedly blowing until all cells are completely cracked, absorbing all the liquid into 1.5ml EP tube, centrifuging for 15min at 4 ℃, rotating at 12000rpm, and absorbing the supernatant into another EP tube; adding chloroform into Trizol of 200 mul/ml, violently shaking and mixing, forbidding vortex, standing for 15min at room temperature, centrifuging for 15min at 4 ℃ environment, rotating at 12000rpm, absorbing upper water phase into a new EP tube, adding isopropanol into Trizol of 500 mul/ml, standing for 15min at room temperature after gentle oscillation, centrifuging for 15min at 4 ℃ environment, rotating at 12000rpm, discarding supernatant, precipitating at the bottom of the tube to be RNA, adding 1ml of 75% ethanol precooled in advance, flicking the bottom of the tube to suspend the precipitate, centrifuging for 15min at 4 ℃ environment, rotating at 12000rpm, discarding supernatant, drying for about 10min at room temperature, and making the precipitate semitransparent; adding 10-20 μ l DEPC water to dissolve RNA, detecting RNA concentration and purity on computer, and optionally adding or subtracting DEPC water amount according to RNA concentration.

(2) RNA reverse transcription into cDNA (TransScript All-in-One First-Strand cDNA Synthesis Supermix for qPCR, TransGen Biotech)

According to the kit specification, the following operations are carried out:

TABLE 1 reverse transcription reaction System

Name	Volume
		SuperMix(5×)	2μl
Total RNA	500ng/RNA concentration
		DEPC Water	To volume 10μl
gDNA	0.5μl

TABLE 2 reverse transcription reaction procedure

Temperature	Time	Cycles
				40℃	15min	1
85℃	5s	1
			4℃	+∞

The synthesized cDNA can be directly subjected to qPCR 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

Name	Volume
		Forward Primer	0.4μl
cDNA	2μl
		Reverse Primer	0.5μl
Passive Reference Dye(50×)	0.4μl
		TransStart Top Green qPCR SuperMix(2×)	10μl
DEPC Water	To volume 20μl

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, as shown in figure 1a, two groups of cells are observed to have obvious morphological difference 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.

FIG. 1c shows the results of SA-. beta. -gal staining, which indicates that 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 detection of aging-associated factor P16 by RT-qPCR ^INK4a mRNA levels, results indicate P16 in LPMSCs relative to EPMSCs ^INK4a The mRNA expression level is obviously increased. Therefore, we successfully establish a MSCs replicative senescence model, and take EPMSCs as young cells and LPMSCs as senescent cells.

Example 2 detection of osteogenic differentiation potential, lipid Synthesis levels and SCD2 expression of senescent MSCs

2.1 alizarin red staining and Western Blot for detecting osteogenic differentiation capacity of cells

1ml of 0.1% gelatin is added to a 6-well plate, shaken well and incubated in CO ₂ After the incubator is used for at least 30min, gelatin is sucked off, MSCs are inoculated in a 6-hole plate, the cell density is determined according to the growth state, and 2ml of prepared complete culture solution is added into each hole and then cultured in a cell incubator; when the cell fusion reaches about 75%, abandoning the culture solution, adding osteogenesis induced differentiation culture solution into the pore plate, and changing the culture solution every 2-3 days; after induction for 14-17 days, selecting whether alizarin red staining is carried out or not according to the growth condition of cells, wherein the staining time can be shortened if the number of the cells is gradually reduced; after osteogenesis induction is finished, removing an induction liquid, washing with PBS for 2-3 times, and fixing with 4% paraformaldehyde solution at room temperature for 25-30 min; discarding the fixative, washing with PBS for 2-3 times to ensure thorough washing of the fixative, and adding 2ml alizarin red working solution into each hole for dyeing for 8-10 min; discard staining solution, wash with PBS 2-3 times, add 2ml PB per wellAnd S, observing a dyeing result under a microscope, and judging the osteogenic differentiation effect of the MSCs.

As shown in fig. 2a, alizarin red staining revealed a significant reduction in red bone matrix calcium deposition in LPMSCs compared to EPMSCs.

As shown in fig. 2b, Western Blot results further confirm that expression of the osteogenic differentiation related protein Runx2 of the LPMSCs cells is significantly reduced, indicating that osteogenic differentiation potential of senescent MSCs is reduced.

2.2 Nile Red staining for cellular lipid Synthesis levels

Dissolving nile red in DMSO to prepare 1mM stock solution, subpackaging, freezing and storing in dark place; removing the culture solution by suction, washing with PBS for 2-3 times, 3min each time, and fixing with 4% paraformaldehyde solution at room temperature for 30 min; adding 1 XNile red working solution (1mM storage solution: PBS 1:1000), and incubating at 37 deg.C in dark for 5-10 min; washing with PBS for 2-3 times, adding DAPI dye solution, incubating at 37 deg.C in dark for 5-10min, washing with PBS for 2-3 times, observing red fluorescence intensity and taking picture for recording.

As shown in fig. 2c, the red fluorescence intensity of LPMSCs was weaker compared to EPMSCs; the quantitative analysis result further proves that the triglyceride content in LPMSCs is obviously reduced, so that the triglyceride content in the aged MSCs is reduced, and the lipid synthesis capability is reduced.

2.3 RT-qPCR detection of SCD2 mRNA expression level

The real-time quantitative fluorescent PCR method for detecting the expression of SCD2 mRNA in EPMSCs and LPMSCs is the same as 1.5, and the sequences of the primers are as follows:

TABLE 6 real-time quantitative fluorescent PCR method detection primers

2.4 Western Blot detection of SCD2 protein expression level

(1) Extraction of Total cellular protein

Digesting with pancreatin when the cell growth density is over 80%, stopping digestion of the complete culture solution, blowing, sucking the liquid into a centrifugal tube, and centrifuging at room temperature for 5min at the rotation speed of 1200 rpm; adding 2ml PBS for suspension, then adding PMSF and RIPA according to the proportion respectively to ensure that the protein is fully cracked, shaking and uniformly mixing once every 10min for 3 times to ensure that the cells are fully cracked; after the cracking is completed, centrifuging at room temperature for 25min at the rotating speed of 1200rpm, and sucking the upper layer liquid into a new 1.5ml EP tube after centrifuging; the supernatant is the protein sample to be detected, and can be used for subsequent protein concentration detection or frozen storage at-80 ℃.

(2) BCA method for determining protein concentration

Adding 0, 1, 2, 4, 8, 12, 16 and 20 μ l of 0.5mg/ml protein standard substance into a 96-well plate, and adding PBS to make up to 20 μ 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 CO ₂ Incubating for 30min in an incubator at 37 ℃; 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

Taking 30 mu g of protein sample, SDS protein loading buffer solution and PBS, wherein the total amount is 10-20 mu l, sealing a sealing membrane, and boiling for 5-7 min; preparing 8-12% of separation gel and 5% of concentrated gel according to the molecular weight of the protein, and carrying out electrophoresis by selecting different voltages (80V for the upper layer of gel and 120V for the lower layer of gel); after electrophoresis is finished, preparing a material for gel according to the area size of PVDF membrane > filter paper, then activating by using methanol, and after 30 seconds, observing that the membrane becomes semitransparent, namely activating; stacking the filter paper-membrane-gel-filter paper on a semi-dry membrane converter in sequence, calculating current according to the length and width of 2.5 times of PVDF, and converting the membrane for 40-50 min; sealing with milk powder for 1-2h, incubating different primary antibodies with dilution ratios of (SCD 21: 500, beta-actin 1:2000, Runx21:1000), washing membrane with 1 × TBST at 4 deg.C overnight, and washing for three times for 30 min; the dilution ratio of the secondary antibody is 1:3000, the secondary antibody is incubated at room temperature for 1-2h, and then is washed by 1 × TBST for three times for 30 min; ECL hypersensitive luminous liquid is prepared in a ratio of 1:1 in a dark place, and the result is observed after color development is finished, and the image is stored in a TIFF format.

FIG. 2d shows that the expression level of SCD2 mRNA in EPMSCs and LPMSCs is detected by RT-qPCR, and the result shows that SCD2 mRNA in LPMSCs is lower than that in EPMSCs, indicating that the expression level of SCD2 mRNA in senescent MSCs is reduced.

FIG. 2e shows that Western Blot detects the expression level of SCD2 protein in EPMSCs and LPMSCs, and the result shows that the expression level of SCD2 protein in LPMSCs is lower than that of EPMSCs, which indicates that the expression level of SCD2 protein in aging of aged MSCs is reduced.

Example 3 Lentiviral infection of senescent MSCs to obtain modified senescent MSCs

Constructing improved aged MSCs highly expressing SCD 2:

constructing lentivirus for stably expressing SCD2 by Shanghai Jikai gene medicine science and technology GmbH, culturing for 72h to obtain experimental group LV-SCD2, and infecting aging MSCs in the same state with lentivirus of control group to obtain LV-Vector of control group; RT-qPCR is used for detecting the increased multiple of the SCD2 mRNA expression in the experimental group after the lentivirus infection compared with the control group; the Western Blot assay measures the fold increase in SCD2 protein expression in the experimental group compared to the control group following lentiviral infection.

FIG. 3a shows that after SCD2 lentivirus was overexpressed, each group of MSCs expressed green fluorescent protein.

FIG. 3b shows the RT-qPCR results, and compared with the control group, the mRNA expression level of SCD2 in the over-expression group was increased by about 3.5 times.

FIG. 3c shows the result of Western Blot detection, and the protein expression level of SCD2 in the over-expressed group is significantly higher than that in the control group, about 2 times.

The above results indicate that the lentivirus overexpressing SCD2 stably and highly expresses SCD2 after infecting senescent MSCs.

Example 4 measurement of senescence and lipid Synthesis levels of modified senescent MSCs

4.1 testing the Effect of overexpression of SCD2 on cellular senescence

4.1.1 SA-beta-gal staining to detect cellular senescence

As shown in FIG. 4a, the number of blue-stained cells in LV-SCD2 group was significantly reduced and the SA- β -gal positive rate was significantly down-regulated compared to the control group, indicating that over-expression of SCD2 improved the senescence status of senescent MSCs.

4.1.2RT-qPCR detection of aging-related factor expression

As shown in FIG. 4b, RT-qPCR detected the senescence-associated factor P16 ^INK4a Expression of mRNA; the results show that: p16 in LV-SCD2 group compared with control group ^INK4a The expression of mRNA is obviously reduced, which indicates that the expression of the senescence-associated factors in the improved senescent MSCs is obviously reduced.

4.1.3 alizarin Red staining and Western Blot to detect osteogenic differentiation capability of cells

As shown in FIG. 4c, alizarin red staining revealed significantly increased calcium deposition of the red bone matrix in LV-SCD2 group compared to LV-Vector group.

As shown in fig. 4d, Western Blot results further confirm that the expression of osteogenic differentiation related protein Runx2 of cells in LV-SCD2 group is significantly up-regulated, which indicates that the overexpression of SCD2 can promote osteogenic differentiation of MSCs, and it is presumed that SCD2 can improve osteogenic differentiation capability of MSCs by inhibiting senescence of MSCs.

4.2 Nile Red staining to examine the Effect of over-expression of SCD2 on the level of cellular lipid Synthesis

As shown in FIG. 4e, cells in LV-SCD2 group showed stronger red fluorescence compared to LV-Vector group; the quantitative analysis result further proves that the intracellular triglyceride content in the LV-SCD2 group is obviously increased, so that the over-expression of SCD2 can up-regulate the lipid synthesis capability of the aging MSCs, and the SCD2 can inhibit the aging of the MSCs by promoting the lipid synthesis.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. A method of delaying the senescence of MSCs comprising the steps of:

step 1: obtaining aged MSCs;

2. The method for delaying the senescence of MSCs according to claim 1, wherein in step 1, the step of obtaining senescent MSCs comprises:

step 1.1: extracting primary cells from rat bone marrow, and obtaining early-generation MSCs and late-generation MSCs by using a full bone marrow wall attaching method and in-vitro subculture;

step 1.2: by morphological observation, age-related beta-galactosidase staining and age-related factor P16 ^INK4a And establishing replicative senescence MSCs.

3. The method for delaying the senescence of MSCs according to claim 2, wherein in step 1.1 the rat is a male Wistar rat at 1-2 months of age.

4. The method of claim 1, wherein the time for infecting aged MSCs with SCD2 is 48-72 hours in step 2.

5. The method for delaying the senescence of MSCs according to any one of claims 1-4, wherein the method is used for the manufacture of a medicament for delaying senescence.

6. The method for delaying the aging of MSCs according to any of claims 1-4, wherein the method is used for the preparation of anti-skin aging health products.

7. The method of delaying the senescence of MSCs according to any of claims 1-4, wherein the method is used for the preparation of a food supplement.