CN110791473A

CN110791473A - Application of human sirt3 gene in stem cell induced differentiation

Info

Publication number: CN110791473A
Application number: CN201810872937.6A
Authority: CN
Inventors: 孙晗笑; 利时雨
Original assignee: Guangzhou Abduction Biotechnology Co Ltd
Current assignee: Guangzhou Abduction Biotechnology Co Ltd
Priority date: 2018-08-02
Filing date: 2018-08-02
Publication date: 2020-02-14

Abstract

The invention discloses an application of a human sirt3 gene or sirt 3-like gene or a product thereof in stem cell induced differentiation, and provides a method for promoting stem cell differentiation or mitochondrial biogenesis increase. The highly expressed sirt3 protein can remarkably enhance aerobic metabolism of stem cells, and promote the conversion of stem cell metabolism from glycolysis to oxidative phosphorylation; sirt3 improves mitochondrial biogenesis of stem cells, and facilitates induction of stem cell differentiation. After induced differentiation by sirt3, the stem cells can be developed into various tissues and used for artificial organ regeneration, such as bone tissues for 3D printing by differentiating the bone marrow mesenchymal stem cells into osteoblasts.

Description

Application of human sirt3 gene in stem cell induced differentiation

Technical Field

The invention belongs to the field of protein research and application mechanisms, and particularly relates to application of a human sirt3 gene or sirt 3-like gene or a product thereof in stem cell induced differentiation.

Background

Stem cells are primitive cells that function as the "stem" in the growth and development of an individual organism, a population of cells with the potential for self-renewal, high proliferation and multipotentiality. On one hand, the stem cells are orderly differentiated by taking time as a main axis to form different types of cells; on the other hand, the cells are proliferated, migrated, arranged and combined with other types of cells according to the overall structural blueprint strictly predetermined by the morphogenesis to form various tissues and organs until the complete individual is developed.

Stem cells can be classified into embryonic stem cells and adult stem cells according to the developmental stage. Embryonic stem cells are the basis for tissue organ development: the embryonic stage is from 15 th to 56 th day after fertilization, and the cells at the stage are embryonic stem cells which are generated by totipotent stem cells in a differentiation mode, lose totipotency, cannot develop into complete individuals, have strong signal receiving capacity and can differentiate cells with complete types and good quality in time. Adult stem cells: refers to undifferentiated cells that exist in an already differentiated tissue, such cells being capable of self-renewal and differentiation to form this type of tissue. The adult stem cells can be obtained from patients, have no histocompatibility problem, and can avoid the harm to the patients caused by long-term application of immunosuppressant during treatment. Examples of the adult stem cells found so far include hematopoietic stem cells, bone marrow mesenchymal stem cells, neural stem cells, hepatic stem cells, muscle satellite stem cells, skin surface stem cells, intestinal epithelial stem cells, retinal neural stem cells, and pancreatic stem cells.

Mesenchymal Stem Cells (MSCs) were first found in bone marrow. As early as 1867, Cohnheim, germany, experimentally speculated that non-hematopoietic stem cells were present in the bone marrow. In 1869, Goujon demonstrated bone marrow osteogenic capacity through experimentation, and further intensive studies on bone marrow non-hematopoietic stem cells (bone marrow MSCs) did not actually begin until the 70's in the 20 th century. MSCs are a class of pluripotent stem cells belonging to the mesoderm, and have the ability to differentiate into various cells such as osteocytes, chondrocytes, adipocytes, myocytes (tendon) cells, bone marrow stroma, and even liver cells and nerve cells. In recent years, due to the fact that MSCs have high in-vitro amplification, multidirectional differentiation and easiness in transfection and expression of exogenous genes, domestic and foreign researches are increasingly carried out.

MSC is usually present in bone marrow, is also found in bone, cartilage, muscle, tendon, adipose tissue, dermis, peripheral blood and umbilical cord blood, can secrete various cytokines such as SCF, Flt3, Tpo, LIF, IL-6, IL-11, TNF- α and the like, can be differentiated into bone marrow stromal cells, is closely related to stem cell homing, and has a very important position in hematopoietic reconstitution.

Due to the good characteristics of the MSCs, the MSCs can find wide application prospects through the research. In tissue engineering, the cells are used as seed cells, and after being cultured and expanded, the seed cells are combined with different biological materials to repair tissue defects such as bones, cartilages, tendons and the like. In the treatment of cell transplantation, the allogeneic MSCs are infused in an orderly manner to promote the recovery of hematopoietic function. Or inducing and differentiating into myocardial cells, nerve cells and the like directionally for cell transplantation treatment. In gene therapy, exogenous genes are transferred to make the exogenous genes effectively and durably expressed, diseases caused by lack of circulating proteins, such as hemophilia B, are treated, or growth factor genes beneficial to directed differentiation are transferred to MSCs, so that directed differentiation is promoted, and the process of in vivo tissue repair is accelerated.

Mitochondria are important organelles of eukaryotes, not only provide energy for cell growth and development through oxidative phosphorylation, but also play an important role in the aspects of amino acid biosynthesis and fatty acid and steroid metabolism, and participate in the regulation and control of cellular calcium ion homeostasis, reactive oxygen level, apoptosis and signal transduction. Mitochondria are organelles that change dynamically, and maintain the homeostasis of their network structure through continuous fusion and division, and the dynamic balance of mitochondrial fusion and division plays an important role in regulating the pluripotency and survival of stem cells. At present, it is widely believed that somatic mitochondria are mature and long, and have a large number of cristae and a high matrix density. However, stem cells have a unique ultrastructure compared to somatic mitochondria, are small in mitochondrial number, immature, and spherically distributed around the nucleus, and have a immature mitochondrial structure, a deficient cristae, and a low matrix density. As stem cells differentiate and pluripotency decreases, mitochondrial morphological structures will change. For example, it has been found that during in vitro culture differentiation of human ESCs, mitochondria become elongated networks and the mitochondrial cristae assumes a mature ultrastructure. In other researches, the copy number of the mitochondrial mtRNA of the Pluripotent Stem Cells (PSCs) is low, and the copy number of the mtRNA is increased while the mitochondria are mature after differentiation. These findings indicate that lower mitochondrial activity and an unexplained network structure are important features of PSCs.

High expression of pluripotency genes (Nanog, Oct4 and Sox2) is a main characteristic of the stem cells for maintaining the self-replication capacity, and mitochondrial function metabolism is closely related to the pluripotency of the stem cells and plays a key role in regulating and controlling the pluripotency of the cells. Studies have demonstrated that somatic cells produce energy through oxidative phosphorylation-based aerobic metabolism, while PSCs and adult stem cells rely primarily on anaerobic glycolysis to produce energy, and that glycolytic metabolism provides important co-activators and substrates for proliferation and mitochondrial biosynthesis in these stem cells (fig. 1). Although glycolysis is inefficient in energy production, the amount of Reactive Oxygen Species (ROS) produced while supplying energy is small, which plays an important role in maintaining the pluripotency of stem cells under hypoxic conditions. It is found that anaerobic stimulation of glycolysis or inhibition of mitochondrial respiration promotes maintenance of pluripotency of PSCs, whereas inhibition of glycolysis or enhancement of mitochondrial function impairs pluripotency of PSCs, and inhibits the proliferative capacity of embryonic stem cells, leading to apoptosis.

Sirtuins, a class of NAD + dependent histones, deacetylates unspecifically and deacetylates non-histone substrates in addition to histone substrates. The mammal Sirtuins family has 7 members (SIRTl-SIRT 7), and the gene location, tissue expression, cell location, enzyme activity, main action substrates and function formulas are different. The SIRT 1-SIRT 7 family members play an important regulation role in the aspects of attenuated gene silencing, cell metabolism, cell growth and apoptosis, circadian rhythm and the like.

The human sirt3 gene is located on chromosome 11, p15.5(11p15.5), and is composed of 21902 bases. The sirt3 gene encoded protein consists of 399 amino acids and has a molecular weight of about 44 kDa. The N end of the mutant contains a mitochondrion positioning signal peptide consisting of 25 amino acid residues, after being recognized and cut by peptidase in a mitochondrion matrix, a mature enzyme protein with the size of 28 kDa is formed, and the sirt3 two alternative splicing transcription variants code two different protein subtypes, namely 1-399 amino acids (full-length sirt3) and 142-399 amino acids (mature sirt 3).

Cooper et al believe that the full-length sirt3 is localized only to the mitochondria. However, with the progress of research, Iwahara et al found that after stress stimulation of cells, the full-length sirt3 was enzymatically cleaved to mature sirt 3. Due to the lack of mitochondrial localization signals, mature sirt3 is non-specifically distributed in the mitochondria or cytoplasm and even occurs in the nucleus. Sirt3 was found to have deacetylase activity, whether localized to the mitochondria, cytoplasm, or nucleus. Thus, the deacetylation function of sirt3 may be independent of its cellular location.

However, sirt3 distribution is clearly tissue specific and correlates with organ metabolic activity. The more metabolically active organs, such as liver, brown adipose tissue, heart and kidney, etc., also have higher sirt3 expression. Research shows that in a low-temperature environment, the organism increases the metabolic rate of the organism to maintain the body temperature by enhancing sirt3 expression in brown fat cells, deacetylating and activating uncoupling protein activity. Similarly, exercise, diet, energy restriction, and the like, can enhance sirt3 expression activity; and long-term high fat diet can reduce the expression. Although the specific mechanism of tissue-specific distribution of sirt3 is not clear, it is highly expressed in high-energy metabolic organs, suggesting that it is closely related to the regulation of cellular energy metabolism.

Mitochondria have the main function of energy synthesis and also have the biological activities of regulating the generation of Reactive Oxygen Species (ROS), cell metabolism (such as cholesterol, hormone synthesis and the like), apoptosis and the like. sirt3, as a major deacetylase in mitochondria, can regulate cellular energy synthesis and mitochondrial physiological activity, and also participate in the synthesis of biomolecules (such as proteins and lipids) required for regulating and maintaining cell survival. sirt3 is involved in the regulation of almost all signaling pathways associated with cellular metabolism in the body, such as ROS production and clearance, Tricarboxylic Acid Cycle (TAC), Fatty acid oxidation (FAM), ketone body production, Urea Cycle (UC) cell growth and apoptosis, etc. (fig. 2). sirt3 can participate in oxidative stress reaction, reducing intracellular ROS level; can also be used as a tumor inhibiting factor to promote apoptosis of cells. In certain breast cancer cells sirt3 expression is down-regulated. Decreased expression of sirt3 or sirt3, knockout, can affect the development of metabolic-related aging diseases such as heart disease, cancer, and the like.

Mitochondria are a very critical organelle in the intermediary metabolic process because they can convert carbon source in cells into useful cellular energy ATP, ATP synthesis in the metabolic process can be regulated by various factors such as nutritional status, phosphorylation, allosteric regulation, reactive oxygen species, and divalent cations calcium and magnesium, etc. mitochondria are also the most basic site for acetyl CoA production, providing acetyl groups for enzyme acetylation, utilization of carbon source in mitochondria is also involved in acetylation modification of protein lysine, lysine acetylation is a new post-transcriptional modification that coordinates metabolism of carbon source substances, co-regulated by acetyltransferase and deacetylase, various studies show that acetylation modification of lysine can regulate fatty acid β -oxidation, TCA cycle, oxidative phosphorylation and oxidative stress in mitochondria by targeting key enzymes, sirt3 is an important enzyme in the process of lysine acetylation modification, and can affect the development of age-related diseases such as cancer, heart disease and neurodegenerative disease.

With the progress of research on Sirtuins, sirt3 has been found to play an important role in mitochondria, especially in the middle of mitochondrial metabolism, energy metabolism, and oxidative stress. To investigate the biological role of Sirtuins in mitochondria, the Lombard et al research group knocked out Sirtuins sirt3, sirt4 and sirt5, respectively, in a mouse model. It was found that sirt3 was knocked out in mice, proteins in liver mitochondria and brown adipose tissue were highly acetylated, while the acetylation status was not altered in mice knocked out sirt4 and sirt5, these evidences show that sirt3, a major deacetylase, acts extensively on proteins in mitochondria, whereas sirt4 and sirt5 are limited in deacetylase activity, further confirming the biological role of sirt3 in mitochondria. sirt3 also deacetylates proteins that produce ROS and detoxify them, reduces the enzymatic activity of these proteins, inhibits ROS production, and thus plays an important role in mitochondrial oxidative stress.

Disclosure of Invention

The invention aims to further research sirt3 gene and determine the function of sirt3 gene in the differentiation process of stem cells, so that sirt3 gene can play a role in disease treatment or related food and medicines.

The invention provides the use of a human sirt3 gene or sirt 3-like gene or product thereof in promoting stem cell induced differentiation.

The application of the separated sirt3 gene or product in medicines for promoting stem cell differentiation means that the sirt3 gene is used as medicines or preparations for promoting stem cell induced differentiation and is used for tissue engineering, artificial organs or stem cell treatment and the like.

Compared with the prior art, experiments show that the MSCs with the sirt3 gene or product highly expressed can obviously enhance the aerobic metabolism, promote the conversion of the MSCs metabolism from glycolysis to oxidative phosphorylation, increase the mitochondrial biogenesis, and differentiate the MSCs transfected by sirt3 into osteoblasts. Our findings indicate that sirt3 gene or product can induce the directional differentiation of stem cells, and can be used for tissue engineering or artificial organs after induced differentiation, and can also be used for inducing the directional differentiation in vivo/in vitro during stem cell therapy.

Drawings

FIG. 1 is a graph of the major differences in energy metabolism of pluripotent stem cells and differentiated cells. A: the pluripotent stem cell has higher enzyme expression for regulating glycolytic metabolism, lower pyruvate dehydrogenase activity and higher uncoupling protein 2 expression, and inhibits the pyruvate from entering mitochondria, so that the pyruvate can generate ATP to provide energy mainly through glycolysis; b: the expression of the glycolytic metabolic pathway related enzyme gene of the differentiated cell is down-regulated, the expression of uncoupling protein 2 is reduced, and the pyruvate enters mitochondria energy metabolism and is converted from glycolysis to oxidative phosphorylation.

FIG. 2 is a diagram of sirt 3-related regulatory signal pathways.

FIG. 3 is a diagram of the construction of the MSCs integration expression vector pc-DNA3.1-sirt3 of example 1.

FIG. 4 is a graph showing the results of the double restriction enzyme identification of pc-DNA3.1-sirt3 in example 1, M: DNA Marker; lane 1: double enzyme digestion of pc-DNA3.1-sirt 3; lane 2: pc-DNA3.1 plasmid (negative control); lane 3: sirt3 protein.

FIG. 5 is a graph of mRNA levels of sirt3 detected by qPCR in example 2, with pc-DNA3.1 at 1 and pc-DNA3.1-sirt3 at 2.

FIG. 6 is a protein level graph of sirt3 in Western blot assay cells of example 2, where pc-DNA3.1 is 1 and pc-DNA3.1-sirt3 is 2.

Fig. 7 is a graph of mitochondrial staining for example 3, left: empty plasmid transfection group (vector); and (3) right: recombinant plasmid pc-DNA3.1-sirt 3.

FIG. 8 is a graph showing the intracellular ATP content in example 3.

FIG. 9 is a graph of the effect of sirt3 protein on oxidative phosphorylation process in example 3.

FIG. 10 is a graph of alkaline phosphatase activity assay of example 4.

FIG. 11 is a diagram of osteogenic differentiation-associated genes detected by RT-PCR in example 4.

Detailed Description

The present invention will be described in further detail with reference to examples.

Example 1 preparation of sirt3 Gene and plasmid construction

1. Experimental materials and methods

1.1 cloning of sirt3 Gene

The primers are used to amplify the sirt3 target gene of human, and the PCR product is purified and recovered. The specific operation steps are as follows:

sirt3 primer sequence: 5'-ATCGATGGGCTTGAGAGAGT-3'

R:3′-AGGTTCCATGAGCTTCAACC-5′

The PCR reaction system is as follows:

10×PCR Buffer	5.00 μl
		DNA polymerase	0.25 μl
dNTP（2.5mM）	2.00 μl
		MgCl₂	1.50 μl
UL43-for（10μM）	1.00 μl
		UL43-rev（10μM）	1.00 μl
Form panel	4.00 μl
		ddH₂O	12.75 μl
Total of	25.00 μl

Setting PCR conditions: 5 min at 94 ℃, 1 min at 55 ℃, 1 min at 72 ℃ for 30 s, 30 cycles; 10min at 72 ℃. After the PCR is finished, the PCR product is stored at 4 ℃ and separated and identified by 1% agarose gel electrophoresis.

1.2 recovery and purification of PCR products

The PCR products were verified by nucleic acid electrophoresis using 1% agarose gel. And recovering and purifying the target fragment by using the gel recovery kit. The specific operation steps are as follows:

(1) after the electrophoresis is finished, the agarose gel block containing the bright band of the DNA fragment is cut off under UV irradiation with a sterilized clean rubber cutter. When cutting the gel, the empty agar block is cut as much as possible, and the gel cutting speed is high, so that excessive DNA loss is prevented.

(2) To convert the volume of about 1. mu.l gel per 1 mg gel, 3 times the volume of Buffer W1 was added to the EP tube, and the tube was incubated in a water bath at 65 ℃ for 10min, all with shaking until the gel was completely dissolved.

(3) Then, half the volume of Buffer W2 as much as the amount of Buffer W1 was added to the EP tube, mixed well, centrifuged at 12000rpm for 30 s, the supernatant was discarded, and the operation was repeated once.

(4) Rinsing twice with washing solution, centrifuging at 12000rpm for 1 min, and discarding the supernatant.

(5) Adding 35 μ l of Eluent, standing for 5 min, centrifuging at 12000rpm for 2 min, and eluting the DNA to obtain the purified target DNA.

1.3 in vitro recombination of fragments of interest and cloning vectors

Plasmid pcDNA3.1 has low copy, multiple cloning site and other features, and has two enhancer/promoter regions, one of which is CMV IE enhancer/promoter region and is used for exogenous gene in front of the multiple cloning site.

(1) And (3) carrying out independent PCR (polymerase chain reaction) process by using upstream and downstream primers and pcDNA3.1 plasmid as a template, carrying out 1% agarose gel electrophoresis identification on a PCR product, and recovering and purifying DNA.

(2) And carrying out double enzyme digestion on the PCR product recovered from the pcDNA3.1, and then recovering the product. At the same time, the empty plasmid pcDNA3.1 was digested in duplicate.

(3) The product recovered after double digestion of Sirt3 is treated with T4 DNA ligase to obtain Sirt3 expression plasmid pcDNA3.1-Sirt 3.

1.4 preparation of competent cells

(1) The DH5 α strain frozen in the laboratory was inoculated into LB plate and cultured at 37 ℃ for 16 h.

(2) A single colony was selected and inoculated into 50 ml of LB liquid medium and cultured at 37 ℃ until the OD value became about 0.5 (range 0.4-0.6).

(3) Transferring 25 ml of the bacterial solution into a precooled 50 ml EP tube, standing on ice for 30min, cooling the culture to 0 ℃, centrifuging at 4 ℃, 4000 rpm for 10min, discarding the supernatant, and recovering the thalli.

(4) 5 ml of precooled 0.1 mol/L CaCl are added₂Resuspend each pellet and place on an ice bath for 30 min. Centrifuging at 4 deg.C and 4000 rpm for 10min, removing supernatant, and recovering thallus.

(5) 1 ml of 0.1 mol/L CaCl precooled with ice is added₂(containing 15% glycerol) resuspend the pellet.

(6) Cells were split into small portions, 100 ul/portion, frozen at-80 ℃ for use on ice.

1.5 transformation of recombinant plasmids

(1) Taking a proper amount of DH5 α competent cells, adding 9 μ l of the product after reaction, mixing uniformly, and carrying out ice bath for 30 min;

(2) placing into 42 deg.C water bath, and heat shocking for 2 min; immediately transferring to an ice water bath for cooling for 1 min;

(3) cooling, adding 400 μ l LB liquid culture medium, culturing at 37 deg.C and 180 rpm under shaking for 1 hr to activate

(4) Spreading on LB solid culture medium containing X-gal, IPT, Amp, placing in biochemical incubator at 37 deg.C for 15 min, and performing inverted culture for 16-20 h after the bacterial liquid is completely absorbed.

1.6 identification and extraction of plasmids

White colonies were picked and inoculated into 5 ml LB liquid medium (with ampicillin resistance) and cultured overnight, according to the plasmid mini-extraction kit, as follows:

(1) 2 ml of the bacterial culture was centrifuged at 12000rpm for 1 min, the supernatant was discarded, and 250. mu.l of solution 1 (containing RNaseA) was added thereto, followed by shaking to suspend the pellet thoroughly.

(2) Add solution 2 to EP tube, turn 6-8 times up and down. Adding the solution 3, turning over for 6-8 times, and mixing. Centrifuge at 12000rpm for 10min and transfer the supernatant to another EP tube.

(3) Adding the supernatant into adsorption column, standing at room temperature for 2 min, centrifuging at 12000rpm for 1 min, discarding the waste liquid, and replacing the adsorption column into the collection tube.

(4) Adding 750 μ l of column washing solution into the elution column, centrifuging for 1 min (12000 r/min), discarding the solution, pouring 250 μ l of column washing solution, centrifuging for 5 min (12000 r/min), and discarding the solution.

2. Results of the experiment

2.1 construction of plasmid pc-DNA3.1-sirt3

sirt3 plasmid (vector pc-DNA 3.1), pc-DNA3.1 plasmid was purchased from Add-gene. The relevant method of the reference, using homologous recombination principles to design sirt3 gene integration expression vectors, is shown in FIG. 3.

The query of NCBI indicates that the size of sirt3 gene of human is 21902bp, primers are utilized to construct pc-DNA3.1-sirt3 eukaryotic expression vector plasmid, PCR amplification products can be observed through electrophoresis, and two amplified specific target bands (as shown in figure 4) of about 21902bp can be observed, and the results are consistent with the expected results before experiments. Thus, the successful construction of the plasmid pc-DNA3.1-sirt3 is demonstrated.

Example transfection and expression of the sirt3 Gene

1. Experimental materials and methods

1.1 Liposome transfection

Taking 6-8 week male SD rat, taking two side femur bone marrow, preparing into single cell suspension, and inoculating the separated cells into L-DMEM culture medium containing Fetal Bovine Serum (FBS) with volume fraction of 0.1. Culturing in an incubator at 37 deg.C under 5% CO₂. The cells can be purified by differential adherence, and subculture is carried out when 90% of the cells are fused. Taking the third generation MSC_STransfection was performed with reference to screening optimized transfection conditions: cell density 80% -90% fusion, DNA concentration 6.0 μm/ml, DNA: lipofeetamine 2000(trg: trL) = 3: 4. after 6h of transfection, L-DMEM medium containing FBS (volume fraction 0.1) was replaced and incubation was continued. Cells were harvested for 48h of transfection.

1.2, Western blot detection of sirt3 gene overexpression protein level

Species of MSCs cells (8X 10 per well)⁴) In a 6-well plate, after 24h, the plasmid expressing sirt3 is transfected into MSCs cells, and meanwhile pc-DNA3.1 is used as a blank control, after 24h, the solution is changed and the cells are continuously cultured for 72h, and then the cells are lysed for Western blot detection.

2. Results of the experiment

2.1 qPCR detection of overexpression Effect of sirt3

Cell culture and transgenic primary culture approximately 2 weeks later at passage 1. And the cells of the 4 th generation have uniform morphology, and the flow cytometry detects the surface antigen of the MSCs, and the expression of CD29, CD44 and CD105 is positive, but CD34 and CD45 are not expressed. qPCR was used to detect the overexpression effect of sirt3, plasmid of sirt3 was transfected into MSCs cells, and total RNA was extracted 72h after transfection with pc-DNA3.1 as blank control. The results showed a significant increase in sirt3 plasmid transfected MSCsmRNA levels compared to the control, indicating that overexpression of the sirt3 gene was successful. As shown in fig. 5.

2.2 Western blot detection of protein levels of sirt3

The plasmid of sirt3 was transfected into MSCs cells, and at the same time, cellular protein lysate was extracted 72h after transfection with pc-DNA3.1 as a blank control for Western blot detection, which showed that the protein level of sirt3 gene in MSCs cells transfected with sirt3 plasmid was significantly increased compared to the control group, indicating that the protein level of sirt3 was also successfully overexpressed, as shown in fig. 6.

Example Effect of trisert 3 on cells

1. Experimental materials and methods

1.1 mitochondrial fluorescent staining

After the plasmid pc-DNA3.1-sirt3 transfects MSCs cells, the cells are cultured in a CO2 incubator at 37 ℃ for 4 hours, and then the culture medium without resistance and serum is changed into a normal culture medium for continuous culture. Mitochondria were stained with MitoTracker green FM (Invitrogen) for about 48h, and green fluorescence was observed with a fluorescence microscope and photographed.

1.2 measurement of intracellular ATP content

Intracellular ATP content was measured using luciferin-luciferase in reaction with an ATP bioluminescent cell assay kit (Sigma). Cells harvested at 0, 12, 24, 36, 48h respectively were suspended in KRH buffer containing 0.1mM glucose and 0.2% BSA. And incubated at 37 ℃ in a shaking water bath. Immediately after addition of the assay mixture containing luciferin and luciferase, luminescence was measured in a bioluminescent meter equipped with a syringe (Lumat LB 9507, Berthold, Bad wilddad, germany). From this, the ATP content was calculated.

1.3、Real Time PCR

The level of oxidative phosphorylation of the cells can be measured by measuring the expression levels of mRNA of the four complexes, namely NDUFB-3, SDHB, CYC-1, SURF-1. The gene sequence was obtained from NCBI and the TaKaRa company was entrusted with the design of four pairs of primers (see Table 1). Related reagents such as SsoFas EvaGreen Supermix were placed on ice according to the instructions.

TABLE 1 Gene primer sequences

Gene	Forward primer	Reverse primer
			NDUFB-3	5’-TCAGATTGCTGTCAGACATGG-3’	5’-TGGTGTCCCTTCTATCTTCCA-3’
SDHB	5’-AAATGTGGCCCCATGGTATTG-3’	5’-AGAGCCACAGATGCCTTCTCTG-3’
			CYC-1	5’-TGGCCCCTCCCATCTACAC-3’	5’-ATCCTTGGCTATCTGGGACATG-3’
SURF-1	5’-CAAACCTACGCCAAAATCCA-3’	5’-GAAATGAATGAGCCTACAGA -3’
			β-tubulin	5’-CCCAACAATGTGAAGACGG-3’	5’-GCCTCGGTGAACTCCATCT-3’

The reaction system is as follows:

SsoFas EvaGreen Supermix	5.0 μl
		10 μ M upstream primer	0.5 μl
10 μ M downstream primer	0.5 μl
		cDNA template	2.0 μl
Sterile water	2.0 μl
		In all	10.0 μl

The relative expression level of the relevant gene was determined by analyzing the results obtained with the CFX manager software using the cell group as a control group and β -tubulin as an internal control for the expression level.

2. Results of the experiment

2.1 Effect of sirt3 on MSCs cell mitochondria after transfection

The recombinant plasmid pc-DNA3.1-sirt3 and the empty plasmid pc-DNA3.1 are respectively transfected into MSCs cells, and the observation of an inverted fluorescence microscope after 48 hours shows that: the cells of the empty plasmid pc-DNA3.1 group are very weak in green (FIG. 7 left), and the recombinant plasmid pc-DNA3.1-sirt3 group shows strong positive fluorescent green (FIG. 7 right).

2.2 Effect of sirt3 on ATP in MSCs cells after transfection

We found that intracellular ATP levels significantly increased after sirt3 transfection of MSCs cells. The higher ATP level of sirt3 transfected cells was about 2-fold higher than that of WT and empty vector transfected cells at 36 h, and slightly increased at 24h after transfection, and the experimental results are shown in FIG. 8.

2.3 Effect of sirt3 protein on oxidative phosphorylation Process

To further verify whether sirt3 protein can promote oxidative phosphorylation in host cells, we examined the mRNA of three complexes of this process to preliminarily evaluate the effect of sirt3 protein on oxidative phosphorylation.

The electron transfer chain of the oxidative phosphorylation process mainly comprises three enzyme complexes (I, II and IV), and NADH dehydrogenase 1- β (NDUFB 3), succinate dehydrogenase complex β Subunit (SDHB) and Surf logic protein (SURF 1) are subunits of the complexes I, II and IV respectively.

As shown in FIG. 9, the expression levels of the three genes are increased after the sirt3 gene is transfected, and the expression level reaches the maximum value after 36 h and then is reduced; the cells transfected with no load have no significant difference compared with the wild type cells.

Example Effect of Sirt3 on cell differentiation of MSCs

1. Experimental materials and methods

1.1 osteogenic Induction of differentiation

The SD rat bone marrow mesenchymal stem cells are digested with pancreatin when being cultured to the fourth generation, and the digestion is carried out at 5X 10⁷L^-1The cells were inoculated on a medium at a density and transferred to a carbon dioxide incubator at 37 ℃ for 3 hours. After 3h, 1.0 mL of 10% fetal calf blood was added to each wellThe DMEM complete growth medium was replaced after 24h with DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin diabody, as well as dexamethasone at a concentration of 100 nM, vitamin C at 10. mu.g/mL, and sodium β -glycerophosphate at 10 mM.

1.2 detection of expression level of osteogenesis-related Gene

The levels of osteoblast-associated gene transcription factor 2(Runx2), Osteocalcin (OCN) were determined 1, 7, 14 and 21 days after osteogenic induction. The plate was freed of medium, washed 2 times with phosphate buffer, frozen in liquid nitrogen and the samples were ground to powder, total cellular RNA extracted using TaKaRa MiniBEST RNA extraction kit and cDNA synthesis using PrimeScript RT MasterMix kit. After the reverse transcription is finished, a specific osteogenesis related gene primer is added into a real-time fluorescence quantitative PCR analyzer, the expression (GAPDH) of glyceraldehyde-3-phosphate dehydrogenase is used as an internal reference gene, and 2^−ΔΔCTThe expression of the relevant gene is calculated. The primer list is shown in Table 2.

TABLE 2 RT-PCR primer Table

2. Results of the experiment

2.1 osteogenic Induction of differentiation and detection

The alkaline phosphatase activity results are shown in FIG. 10. Bone marrow mesenchymal stem cells are subjected to osteogenic induction, and the osteogenic differentiation effect is detected by measuring the activity of alkaline phosphatase. The results showed that the alkaline phosphatase activity underwent a change from rising to falling within 21 days, the alkaline phosphatase activity reached a maximum after 14 days of culture, and the alkaline phosphatase activity decreased after 21 days of culture.

2.2 detection of expression level of osteogenesis-related Gene

The results of osteogenic differentiation-related gene expression are shown in fig. 11. The bone marrow mesenchymal stem cells are subjected to osteogenesis induction, and the osteogenesis differentiation effect is detected by detecting the expression level change of osteogenesis related gene transcription factor 2(Runx2) and Osteocalcin (OCN). The expression of Runx2 underwent a tendency to rise first and then fall, reaching a maximum at 7 days and finally gradually falling. Expression of OCN also underwent a first-up and then-down trend, with expression reaching a maximum after 14 days of culture.

Runx2 regulates osteoblast differentiation of mesenchymal stem cell item, and its expression change shows that mesenchymal stem cells largely differentiate into osteoblasts in osteogenesis induction medium, and the differentiation effect is most obvious at 7 days and then gradually decreases. The OCN is a specific protein of early differentiation shown by osteogenic induced differentiation of the bone marrow mesenchymal stem cells into osteoblasts, and the expression level of the OCN shows that the OCN is increased and then decreased, which indicates that the osteogenic differentiation reaches a maximum value in 14 days, and then the cells stop growing.

In this chapter of research, cells are isolated from the marrow cavity of SD rat and cultured and identified in vitro, and experiments show that the extracted mesenchymal stem cells grow well and have osteogenic differentiation potential. After osteogenic induced differentiation, good cell biocompatibility was shown after 21 days of in vitro co-culture.

Claims

1. Use of a human sirt3 gene or sirt 3-like gene or product thereof in inducing differentiation of stem cells.

2. The use of the human sirt3 gene or sirt 3-like gene or product thereof in inducing differentiation of stem cells according to claim 1, wherein the human sirt3 gene is transfected into stem cells by transgenic technology to make the stem cells highly expressed, and the highly expressed sirt3 protein can promote differentiation of stem cells and significantly enhance aerobic metabolism of stem cells, and promote the conversion of stem cell metabolism from glycolysis to oxidative phosphorylation; the biogenesis of mitochondria is improved.

3. The use of the human sirt3 gene or sirt 3-like gene or products thereof according to claim 1 for inducing differentiation of stem cells, wherein the stem cells used are all embryonic stem cells, hematopoietic stem cells, bone marrow mesenchymal stem cells, and the like.

4. Use of the human sirt3 gene or sirt 3-like gene or products thereof in induced differentiation of stem cells according to claim 3, wherein the stem cells are induced to differentiate into various tissues and artificial organs.

5. The use of the human sirt3 gene or sirt 3-like gene or product thereof in stem cell induced differentiation according to claim 4, wherein bone marrow mesenchymal stem cells transfected with sirt3 are differentiated into osteoblasts for 3D printed bone tissue preparation.

6. The use of the human sirt3 gene or sirt 3-like gene or product thereof in inducing differentiation of stem cells according to claim 4, wherein sirt3 transfected stem cells are used for induction of in vivo/ex vivo directed differentiation at the time of stem cell therapy.