Method for inhibiting Ehhadh to promote somatic cell reprogramming to produce stem cells and application thereof

Abstract

The invention discloses a method for inhibiting Ehhadh to promote somatic cell reprogramming to produce stem cells and application of the method, and belongs to the field of molecular biology. The research finds that the Ehhadh gene can regulate and control cell fate remodeling for the first time. The invention constructs an Ehhadh gene interference fragment introduction reprogramming system, discovers that the Ehhadh gene can promote the reprogramming efficiency, establishes an OSKM + shEhhadh-iPS cell line, and verifies the pluripotency of the cell line from the aspects of pluripotency and differentiation ability. The invention also constructs an Ehhadh gene overexpression vector, and the Ehhadh gene overexpression vector is introduced into the embryonic stem cells to promote the differentiation of the embryonic stem cells. The invention provides a new angle for obtaining an efficient somatic cell reprogramming method, provides a new method for rapid differentiation of embryonic stem cells, and provides a new research direction for gene regulation cell fate remodeling in the fields of stem cell production, stem cell treatment, gene editing of large animal breeding embryos, directional differentiation of embryos, organ regeneration and the like. The method has important practical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a method for inhibiting Ehhadh to promote somatic cell reprogramming to produce stem cells and an application thereof. Background Art

[0002] Reprogramming of cell fate is widely used in medicine and animal genetic breeding and reproduction. Studying the regulation mechanism of cell fate plasticity and developmental potential is a major need for regenerative medicine and coping with an aging society. It provides a theoretical basis and methodological strategies for stem cell therapy, in vivo repair, and organ regeneration. It also provides a new direction for protecting rare animal genetic resources and breaking through the barriers of distant hybridization.

[0003] Embryonic stem cells (ESCs) are derived from early embryos and have high pluripotency and self-renewal capabilities. However, the isolation, culture and establishment of ESCs are relatively difficult, which limits their application. Induced pluripotent stem cells (iPSCs) are pluripotent stem cells that have a series of induction factors introduced into mature somatic cells and reprogrammed into pluripotent stem cells with characteristics similar to those of embryonic stem cells. They can differentiate into a variety of functional cells and can be cultured in vitro. Embryonic stem cells and induced pluripotent stem cells complement each other and have broad application prospects in many fields such as clinical treatment and drug development.

[0004] Somatic cell reprogramming is a complex and difficult process that is not only regulated by many signaling pathways, but also affected by epigenetic modifications. Takahashi and Yamanaka demonstrated that the expression of four transcription factors, Oct4, Sox2, Klf4, and c-Myc (collectively referred to as "OSKM"), reprograms the developmental potential of adult cells, enabling them to transform into various cell types. These findings have opened up the field of cell reprogramming. However, the reprogramming efficiency of the traditional OSKM four factors needs to be further improved.

[0005] Enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase (Ehhadh) is a bifunctional enzyme that exists in both mitochondria and peroxisomes and is one of the four important enzymes in the peroxisomal β-oxidation pathway. This enzyme is involved in a variety of important metabolic processes, such as AA degradation and metabolism, fatty acid metabolism, and PPAR signaling pathways. Previous studies have shown that Ehhadh is associated with the occurrence, development, and drug resistance of various tumors, such as osteosarcoma and ovarian cancer; however, the role of this gene in somatic cell reprogramming and embryonic stem cell differentiation has not yet been elucidated. Summary of the invention

[0006] In view of the above prior art, the purpose of the present invention is to provide the application of Ehhadh gene in regulating somatic cell reprogramming and embryonic stem cell differentiation. The present invention has found that Ehhadh gene can regulate cell fate remodeling, improve the reprogramming efficiency of somatic cells, and promote the rapid differentiation of embryonic stem cells.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] In a first aspect of the present invention, there is provided the use of the Ehhadh gene as a target in regulating the efficiency of somatic cell reprogramming;

[0009] The Ehhadh gene is a DNA molecule as shown in the following i) or ii):

[0010] i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1;

[0011] ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

[0012] The nucleotide sequence of the CDS region of the Ehhadh gene is shown in SEQ ID NO.1, and is as follows:

[0013]

[0014] The amino acid sequence of the protein encoded by the Ehhadh gene is shown in SEQ ID NO.1, and is as follows:

[0015] maeylrlphslamirlcnppvnaisptvitevrnglqkasldhtvraivicgandnfcagadihgfksptgltlgslvdeiqryqkpvvaaiqgvalggglelalgchyrianakarvgfpevmlgilpgargtqllprvvgvpvaldlitsgrhistdealklgildvvvksdpveea ikfaqtvigkpieprrilnkpvpslpnmdsvfaeaiakvrkqypgrlapetcvrsvqasvkhpyevaikeeaklfmylrgsgqaralqyaffaeksankwstpsgaswktasaqpvssvgvlglgtmgrgiaisfarvgipvvavesdpkqldtakkiitstlekeasksgqasakpnlr fssstkelssvdlvieavfedmnlkkkvfaelsalckpgaflctntsaldvddiasstdrpqlvigthffspahimrllevipsrysspttiatvmslskrigkigvvvgncygfvgnrmlapyynqgyflieegskpedvdgvleefgfrmgpfrvsdlagldvgwkvrkgqgltgps lppgtptrkrgntryspiadmlceagrfgqktgkgwyqydkplgrihkpdpwlseflsqyrethikqrsiskeeilerclyslineafrileegmaaspehidviylhgygwprhvggpmyyaasvglptvleklqkyyrqnpdipqlepsdylrrlvaqgspplkewqslagphsskl

[0016] In the above applications, the Ehhadh gene can be used as a target gene to downregulate the expression of the Ehhadh gene, which significantly improves the efficiency of somatic cell reprogramming compared to the traditional OSKM four-factor reprogramming. Therefore, the Ehhadh gene can be used in the fields of stem cell production, stem cell therapy, etc.

[0017] The second aspect of the present invention provides the use of a substance for down-regulating the expression of the Ehhadh gene in any one of the following (1)-(3):

[0018] (1) Improve the efficiency of somatic cell reprogramming;

[0019] (2) preparing products that improve the efficiency of somatic cell reprogramming;

[0020] (3) Produce stem cells.

[0021] In the above application, the somatic cell reprogramming is the OSKM four-factor reprogramming system.

[0022] In the above application, the substances for downregulating the expression of the Ehhadh gene include: small interfering RNA (siRNA), shRNA, long non-coding RNA (lncRNA) and circular RNA (circRNA), etc. These substances can form complexes with proteins as interfering fragments, bind to RNA, cut off RNA and degrade RNA, thereby achieving the purpose of preventing gene expression.

[0023] Preferably, the substance that downregulates the expression of the Ehhadh gene is shRNA, and its nucleotide sequence is shown in SEQ ID NO.3.

[0024] The third aspect of the present invention provides the use of the Ehhadh gene in preparing a product for promoting the differentiation of embryonic stem cells.

[0025] In the above applications, the Ehhadh gene can be used as a target gene, and by upregulating the expression of the Ehhadh gene, the differentiation of embryonic stem cells can be promoted. Therefore, the Ehhadh gene can be applied to the fields of embryo gene editing, embryo directed differentiation, organ regeneration, etc.

[0026] The fourth aspect of the present invention provides the use of an overexpression vector containing the Ehhadh gene in the preparation of a product for promoting the differentiation of embryonic stem cells.

[0027] A fifth aspect of the present invention provides a method for improving the efficiency of somatic cell reprogramming, comprising the following steps:

[0028] Introduce the Ehhadh gene interference fragment into the somatic cell reprogramming system.

[0029] Preferably, the somatic cell reprogramming system includes four transcription factors: Oct4, Sox2, Klf4 and c-Myc.

[0030] Preferably, the nucleotide sequence of the Ehhadh gene interference fragment is shown as SEQ ID NO.3.

[0031] A sixth aspect of the present invention provides a method for producing stem cells, comprising the following steps:

[0032] Substances that downregulate Ehhadh gene expression and four transcription factors, Oct4, Sox2, Klf4 and c-Myc, were transferred into somatic cells.

[0033] Preferably, the substance that downregulates the expression of the Ehhadh gene is shRNA, and its nucleotide sequence is shown in SEQ ID NO.3; the shRNA is linked to the PsicoR vector and then transferred into the somatic cells.

[0034] A seventh aspect of the present invention provides a method for promoting embryonic stem cell differentiation, comprising the following steps:

[0035] An overexpression vector containing the Ehhadh gene is introduced into embryonic stem cells to overexpress the Ehhadh gene.

[0036] Beneficial effects of the present invention:

[0037] The present invention is the first to study and discover that the Ehhadh gene can regulate cell fate remodeling. The present invention constructs an Ehhadh gene interference fragment into a reprogramming system, finds that the Ehhadh gene can promote the efficiency of reprogramming, and establishes an OSKM+shEhhadh-iPS cell line, which verifies that the cell line has stem cell characteristics in terms of pluripotency and differentiation ability. The present invention also constructs an Ehhadh gene overexpression vector, introduces it into embryonic stem cells, and finds that it promotes the differentiation of embryonic stem cells. The present invention provides a new angle for obtaining an efficient reprogramming method, provides a new method for the rapid differentiation of embryonic stem cells, and provides a new research direction for gene-regulated cell fate remodeling in the fields of stem cell production, stem cell therapy, gene editing of large animal breeding embryos, directed differentiation of embryos, organ regeneration, etc., and has important practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Knockdown of Ehhadh (shEhhadh) significantly improves the efficiency of somatic cell reprogramming (A. shEhhadh knockdown efficiency detection; B. Clonal morphology of knockdown of Ehhadh under bright field and dark field, scale bar 200μm).

[0039] Figure 2 After knockdown of Ehhadh induced somatic cell reprogramming, alkaline phosphatase activity (AP) staining was used to detect the pluripotency of iPSCs. Scale bar 1 mm.

[0040] Figure 3 After the reprogramming of somatic cells induced by knockdown of Ehhadh was completed, the proportion of Oct4+ cells was detected by flow cytometry analysis to indicate the rate of successful reprogramming into stem cells.

[0041] Figure 4 Overexpression of Ehhadh significantly inhibited the induction rate of iPSCs (A. Ehhadh was successfully overexpressed on the third day of reprogramming; B. The clone morphology of overexpressing Ehhadh under bright field and fluorescence, scale bar 200 μm).

[0042] Figure 5 After the reprogramming of somatic cells induced by overexpression of Ehhadh was completed, the pluripotency of iPSCs was detected by alkaline phosphatase activity (AP) staining. Scale bar 1 mm.

[0043] Figure 6 After the somatic cell reprogramming induced by overexpression of Ehhadh was completed, the proportion of Oct4+ cells was detected by flow cytometry analysis to indicate the rate of successful reprogramming into stem cells.

[0044] Figure 7 iPSCs clones established by knockdown of Ehhadh. Scale bar: 200 μm.

[0045] Figure 8 Further verification of the pluripotency of iPSCs clones established by knocking down Ehhadh (A. RT-qPCR detection of transcriptional expression of pluripotency genes; B. Immunofluorescence detection of expression of endogenous pluripotency proteins, scale bar 50μm).

[0046] Fig. 9 Verification of the differentiation potential of iPSCs clones established by knocking down Ehhadh (A. iPSCs clones can form embryoid bodies and undergo adherent differentiation process, scale bar 200μm; B. Differentiated embryoid bodies, pluripotent gene expression is inhibited; C. Differentiated embryoid bodies, the expression of marker genes for three-germ layer differentiation is increased; D. Differentiated embryoid bodies, the expression of marker proteins for three-germ layer differentiation is increased, scale bar 100μm).

[0047] Fig.10 Overexpression of Ehhadh promotes differentiation of ESCs (A. The transcription level of pluripotency genes is reduced; B. The transcription level of three-germ layer differentiation marker genes is increased; C. Immunofluorescence detection of the expression of pluripotency proteins and three-germ layer differentiation marker proteins, scale bar 100μm). DETAILED DESCRIPTION

[0048] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0049] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0050] The test materials used in the examples of the present invention are all conventional test materials in the field and can be purchased through commercial channels. The experimental methods without detailed conditions are carried out in accordance with conventional test methods or the operating instructions recommended by the supplier. Among them:

[0051] PsicoR-RFP and Fuw-TetOn vectors were purchased from Addgene.

[0052] FM medium is a feeder cell medium (Feeder medium), and the formula is as follows:

[0053]

[0054] All the above reagents were purchased from Gibco.

[0055] ESM medium is mouse pluripotent stem cell culture medium, and its formula is as follows:

[0056]

[0057] Recombinant Mouse LIF Protein was purchased from Millipore, 2-Mercaptoethanol was purchased from ThermoFisher, and the remaining reagents were purchased from Gibco.

[0058] Example 1: Construction of Ehhadh gene interference fragment and Ehhadh gene recombinant expression vector

[0059] 1. Construction of Ehhadh gene interference fragment:

[0060] According to the mRNA sequence of Mus musculus (house mouse) Ehhadh (GenBank: NM_023737.3) published by NCBI, shRNA that interferes with the expression of the Ehhadh gene was designed. The nucleotide sequence is shown in SEQ ID NO.3, which is as follows:

[0061] shEhhadh: 5′-TTTCGAGTGTGGATTTAGT-3′.

[0062] The Ehhadh gene interference fragment sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The PsicoR vector was double-digested with Hpa I and Xho I, and the interference fragment of the Ehhadh gene was annealed and connected to the PsicoR vector after restriction digestion. The plasmid was extracted according to the plasmid extraction kit of Kangwei Century Company. The recombinant plasmid was sequenced and verified to be correct by Sangon Biotech (Shanghai) Co., Ltd., and the Ehhadh gene interference fragment (shEhhadh) was constructed.

[0063] 2. Construction of Ehhadh gene recombinant expression vector:

[0064] According to the mRNA sequence of Mus musculus (house mouse) Ehhadh (GenBank: NM_023737.3) published by NCBI, Hpa I and Xho I were selected as restriction sites, and primers were designed to amplify the CDS region of the Ehhadh gene. The Fuw-TetOn vector was double-digested with Hpa I and XhoI, and the CDS region of the Ehhadh gene was connected to the Fuw-TetOn vector after restriction digestion. The plasmid was extracted according to the plasmid extraction kit of Kangwei Century Company. The recombinant plasmid was synthesized and verified to be correct by sequencing by Sangon Biotech (Shanghai) Co., Ltd., and the Ehhadh gene recombinant expression vector (mEhhadh) was constructed.

[0065] Example 2: Effect of the Ehhadh gene on the efficiency of mouse somatic cell reprogramming

[0066] 1. Test method:

[0067] (1) Transfection and preparation of viral fluid

[0068] When HEK293T (human embryonic kidney cells) grow to 60% to 70% of the bottom of a 10 cm culture dish, start transfection of the plasmid; replace the preheated fresh culture medium 1 hour before transfection, and then put it in a CO2 incubator, and then prepare the transfection working solution; the mass ratio of target gene / negative control plasmid: lentiviral packaging element psPAX2 (Pol / Gag) plasmid: pMD2_G (VSV-G) plasmid is 5:3:2. After preparation, dilute it with physiological saline according to the usage ratio in the instructions (see Weige for details). Vigofect reagent manual of Las Biotechnology (Beijing) Co., Ltd.); add 392 μL of physiological saline to 8 μL of transfection reagent VigoFect to make the total volume reach 400 μL, let it stand at room temperature for 5 minutes, then add the transfection reagent dilution solution dropwise to the plasmid solution prepared above), mix gently, and let it stand at room temperature for 15 minutes to obtain the transfection working solution; add the working solution dropwise to the above culture dish, mix it by cross method, and put it in the incubator; after 8 to 10 hours, replace it with fresh FM culture medium, and then put it in the incubator to continue culturing.

[0069] When the target gene plasmid was shEhhadh, the negative control plasmid was PsicoR empty vector; when the target gene plasmid was mEhhadh, the negative control plasmid was Fuw-TetOn empty vector. The treatments of transfecting the target gene plasmid and the negative control plasmid simultaneously transferred four transcription factors, Oct4, Sox2, Klf4 and c-Myc.

[0070] 48 to 72 hours after transfection, the cell culture medium was collected and filtered through a 0.45 μM filter membrane, and then the culture medium was collected to obtain the virus solution for infection.

[0071] (2) Mouse somatic cell reprogramming process

[0072] Take out OR-MEF cells (mouse fibroblasts) from liquid nitrogen for recovery. First, put the frozen cells into a 37°C water bath. Add FM culture medium three times the volume of the frozen cell liquid, slowly add the thawed cell suspension into the culture medium, and centrifuge at 1,000rpm for 5 minutes. After centrifugation, carefully aspirate the supernatant, resuspend with an appropriate amount of FM culture medium, add to a 35mm cell culture dish that has been pre-filled with culture medium, and place in a CO2 incubator. After about 12 to 24 hours, when the cells are fully grown, subculture and plate the OR-MEF cells.

[0073] 14-16 h before collecting the virus solution, MEF cells were cultured at 2.0×10 4 Cells were seeded per well in a 12-well plate.

[0074] Collecting virus solution and infection: discard the original culture medium, add the filtered fresh virus solution to each well of the 12-well plate, and add 200 μL of the target gene virus solution to each well; after 10-12 hours, change the medium, aspirate the virus solution, add ESM containing 1000x doxycycline (Dox) (i.e., add 50 μl Dox to 50 ml ESM culture medium) to start induction, which is recorded as the first day of reprogramming;

[0075] Afterwards, in the early stage of reprogramming, the medium was replaced with ESM containing 1000x doxycycline (Dox) every other day. In the middle and late stages of reprogramming, the amount of cells increased, and the time and amount of medium replacement were changed according to the situation. Dox was withdrawn after a large number of Oct4-GFP green fluorescent clones appeared, and subsequent experiments were carried out three days after the clones became stable.

[0076] (3) AP staining to detect somatic cell reprogramming efficiency

[0077] After the reprogramming induction is completed, select the iPS clone wells in good condition and wash them with DPBS three times; then add 4% paraformaldehyde to fix the cells and place them in a 4°C refrigerator overnight; after fixation, discard the fixative and wash them with DPBS three times; prepare the staining solution (see the instructions of the Pluripotent Stem Cell Alkaline Phosphatase Colorimetric Kit of Biotech Co., Ltd. for details), slowly add the staining solution along the wall of the dish to the culture dish to cover the cells, protect the reaction from light and observe the clone color at any time, and quickly absorb the staining solution when the cells change color; after discarding the staining solution, wash them with DPBS three times, take pictures or scan the AP staining results to retain the results, count the number of AP-positive clones, and perform corresponding analysis.

[0078] (4) Flow cytometry to detect somatic cell reprogramming efficiency

[0079] After reprogramming induction is completed, remove the original culture medium and wash with DPBS 1 to 2 times; add 300 μL of trypsin and digest in a cell culture incubator at 37°C for 5 minutes, observe under a microscope, and digest until the cells are loose and detached from the bottom of the dish; neutralize with an equal amount of FM culture medium to terminate the digestion, then blow and observe under a microscope until they become single cells; centrifuge at 1,000 rpm for 5 minutes; add 1 mL of DPBS to resuspend, and perform on-machine analysis and detection according to the BD LSRFortessa flow cytometry operation manual.

[0080] 2. Test results:

[0081] We collected samples of cells on the third day after the introduction of shEhhadh for reprogramming, and the results of real-time fluorescence quantitative PCR showed that the expression level of Ehhadh was significantly lower than that of the empty control ( Figure 1 A), indicating that the target gene was successfully knocked down; we conducted three batches of reprogramming experiments, and we found that the number of Oct4-GFP+ clones produced by knocking down Ehhadh increased significantly compared with the control ( Figure 1 B). The results of AP staining test showed that the number of AP+ clones produced by knocking down Ehhadh increased significantly compared with the control ( Figure 2 ). The results of flow cytometry experiments also showed that compared with the empty control, after knocking down Ehhadh, Oct4-GFP + The proportion of cells increased significantly ( Figure 3 ), based on this, we can conclude that knocking down Ehhadh improves the induction efficiency of mouse somatic cell reprogramming.

[0082] After the introduction of mEhhadh, we collected cell samples on the third day of reprogramming and used the MEF cell samples introduced with the Fuw-TetOn empty vector as a control. The results showed that the expression level of the target gene Ehhadh was 4 times that of the control, indicating that the target gene was successfully overexpressed ( Figure 4A). The clone morphology during the reprogramming process was recorded, and the number of Oct4-GFP+ clones produced by overexpressing Ehhadh was significantly reduced compared with the control ( Figure 4 B). Through AP staining experiments, we found that compared with the control, the number of AP+ clones produced by overexpression of Ehhadh was significantly reduced ( Figure 5 ). Flow cytometry revealed that compared with the empty control, the proportion of Oct4-GFP+ cells was significantly reduced after overexpression of Ehhadh ( Figure 6 ).

[0083] Example 3: Establishment of OSKM+shEhhadh cell line and detection of differentiation ability

[0084] 1. Test method:

[0085] The somatic cell reprogramming of Example 2 was induced to be completed in about 15 days, and Dox was removed. After 2 to 3 days, the clones were stabilized and grown to a sufficient size, and single clones were picked. 12 to 24 hours before picking clones, a 96-well plate was prepared, 100 μL FM culture medium was added to each well, and feeder (mouse feeder layer cells) was seeded. After the growth was stable, single clones were picked. Note that before picking single clones, the medium was changed to ESM medium with double antibodies (penicillin / streptomycin, 500 μL each was added to 50 ml of medium);

[0086] Prepare a new 96-well plate and add 30 μL of trypsin to each well in advance; observe under a fluorescence microscope to find a clone with a full morphology and strong green fluorescence. There should be no other clones around the clone. Use a small white gun tip to suck out the selected single clone under a stereo microscope in the clean bench and put it into a 96-well plate with trypsin. Place it under a fluorescence microscope to observe whether there are green fluorescent clones and mark them; Digest it in a cell culture incubator at 37°C for 5 minutes. After the clones are loose, add an equal amount of ESM with double antibodies and blow it, and transfer all the suspensions to a 96-well plate with a feeder, and place it in an incubator for culture; After the cells adhere to the wall for 10 to 12 hours, replace it with fresh preheated ESM culture medium with double antibodies (penicillin / streptomycin, add 500 μl each to 50 ml culture medium);

[0087] After that, fresh ESM medium was replaced every day until the clones grew to an appropriate size, and then the cells were passaged at appropriate proportions and gradually transferred to 6-well and 12-well plates; cell lines with good morphology and growth status were selected for freezing and subsequent cell line verification tests.

[0088] (1) Fluorescence quantitative PCR detection of pluripotent gene expression levels:

[0089] After the iPS cell line has grown well, the original culture medium is aspirated, and the cells are washed once with DPBS. 500 μL of trypsin is added to digest in a 37°C incubator for about 5 minutes. After observing that the clones are loose under the microscope, an equal amount of culture medium is added to form a cell suspension by blowing. Centrifuge at 1,000 rpm for 5 minutes and discard the supernatant. Add 2 mL of ESM to resuspend, transfer to a new 60 mm culture dish, and place in an incubator for 25 to 35 minutes to allow the feeder cells to adhere to the wall. Carefully aspirate the cell suspension and add it to a new centrifuge tube, centrifuge at 1,000 rpm for 5 minutes, discard the supernatant, and retain the cell pellet. At the same time, ESCs (mouse embryonic stem cells) are collected as positive controls, and MEFs (mouse fibroblasts) are collected as negative controls. Total RNA of the above cells is extracted according to the RNAprep Pure Cell / Bacteria Kit method and its concentration is detected by NanoDrop. Total RNA is stored at -80°C.

[0090] Reverse transcription was performed using the FastKing gDNA DispellingRT SuperMix kit. The reverse transcription system was 20 μL: Total RNA 2 μg, 5×FastKing-RT SuperMix 4Μ, and RNase-Free ddH2O was added to 20 μL. Reaction conditions: 42°C for 15 minutes, 95°C for 3 minutes, and terminated at 4°C. The cDNA samples were frozen in a -80°C refrigerator for later use.

[0091] The real-time fluorescence quantitative PCR primers of pluripotency genes are shown in Table 1 , and the expression changes of the three germ layer related genes and pluripotency genes were detected.

[0092] Table 1: RT-qPCR primer sequences for pluripotency genes

[0093]

[0094] (2) Immunofluorescence staining to detect pluripotent protein expression levels

[0095] Prepare the punching solution: take 10 μL of TritonX-100, dilute it with 10 mL of PBS to a working concentration of 0.1%, and store it at room temperature; Blocking solution: use an electronic balance to weigh 0.3 g of BSA powder and add it to 10 mL of PBS to make a blocking solution with a working concentration of 3%, and store it at -20°C for later use;

[0096] The specific steps of the experiment are as follows: the cells were cultured on a cell slide in a 24-well plate. After the iPS cell line clone density and size grew to a suitable level, the ESM was removed and the cells were washed once with DPBS. A 4% paraformaldehyde solution was slowly added along the wall of the dish and fixed overnight at 4°C. The fixative was removed and the cells were washed three times with DPBS, each time for 5 minutes. The prepared 0.1% TritonX-100 solution was added and the cells were punched at room temperature for 15 minutes. The punching solution was removed and the cells were washed twice with DPBS, each time for 5 minutes. A 3% BSA solution was added and the cells were blocked at room temperature at 4°C overnight. The primary antibody was blocked with an appropriate amount of 3% The cells were diluted with BSA solution and incubated at room temperature for 90 minutes. After the reaction, the primary antibody was discarded and the culture dish was washed 3 times with DPBS, each time for 5 minutes. The corresponding secondary antibody was diluted with blocking solution in an appropriate proportion and reacted at room temperature for 1 hour. After the reaction, the secondary antibody was discarded and the cells were washed 2 times with DPBS, each time for 5 minutes. The diluted nuclear staining solution DPAI was added and allowed to stand at room temperature for 5 to 10 minutes. The DPAI was removed and the culture dish was washed once with DPBS. New DPBS was added, the culture dish was wrapped with tin foil and stored at 4°C away from light. The samples were scanned and photographed using a laser confocal microscope.

[0097] (3) Embryoid body (EB) differentiation of cell lines to detect their differentiation ability

[0098] iPS clones with good growth status were digested into single cells with trypsin, neutralized with 2 times FM medium, and centrifuged at 1,000 rpm for 5 minutes; the supernatant was discarded, and the feeder was removed by differential adhesion method; ESM medium without LIF (leukemia inhibitory factor) was used to gently pipette and count the cells, and the cell density was adjusted to 5×10 4 / mL; prepare a 10cm cell culture dish in advance, add 6mL DPBS in the dish in advance; arrange the cell suspension evenly on the lid of the 10cm cell culture dish at 20μL / drop (the cell density is 2000-3000 / 20μL at this time), quickly flip the lid over and cover the culture dish with DPBS, and culture it in a 37℃5% CO2 incubator; after culturing for 48h, EB balls can be observed to form in the hanging drops, collect the EB balls, let it stand for 5 minutes, and discard the supernatant; put the EB balls in a six-well plate treated with 0.1% Gelatin, change the medium every other day, and culture in a 37℃ incubator for 3 days (i.e., let the EB balls adhere to the wall and differentiate), wait for the cells to differentiate spontaneously, and use a microscope to take pictures and record the entire differentiation process; the real-time fluorescence quantitative PCR primers of the three germ layer marker genes are shown in Table 2, and the differentiated cells are detected by fluorescence quantitative PCR and immunofluorescence to detect the expression of pluripotent genes and three germ layer marker genes and proteins of embryoid bodies.

[0099] Table 2: Primer sequences of three germ layer marker genes of embryoid body

[0100]

[0101] 2. Test results:

[0102] (1) Establishment of OSKM+shEhhadh cell line

[0103] After reprogramming induction, Oct4-GFP+ monoclones with clear edges, bright green fluorescence, smooth cell surface and tight cell connections were selected for cell line establishment, and OSKM+shEhhadh-iPS cell lines were expanded and cryopreserved ( Figure 7 ).

[0104] (2) OSKM+shEhhadh-iPS cell line expresses pluripotent genes

[0105] The qualified clones screened out were tested for pluripotency expression, and pluripotency was tested at both the protein level and the RNA level. Fluorescence quantitative detection showed that the expression levels of pluripotency genes Nanog, Oct4, Sox2, Rex1, and Utf1 were significantly higher than those of the negative control MEFs, and comparable to those of the positive control (R1 ES) ( Figure 8 A). Immunofluorescence staining revealed that the shEhhadh-iPS cell line expressed pluripotency marker proteins NANOG and SSEA1, showing red fluorescence as shown in the figure ( Figure 8 B), indicating that the cell lines tested express endogenous pluripotency proteins.

[0106] (3) OSKM+shEhhadh-iPS cell line has differentiation potential

[0107] EB balls transplanted into six-well plates began to differentiate spontaneously at the edge of the embryoid bodies after 24 hours of differentiation. After 72 hours, the cell differentiation became more obvious, most of the cells had spontaneously differentiated, and the green fluorescence displayed by Oct4-GFP+ was significantly weakened ( Fig. 9 A), and then the samples were collected after all the cells were differentiated. Real-time fluorescence quantitative PCR was performed to detect the pluripotency genes. The expression level of pluripotency genes in the differentiated shEhhadh-iPS cell line was significantly lower than that in the undifferentiated ( Fig. 9 B). Then, the expression of three germ layer differentiation marker genes was detected at both RNA and protein levels. Their mRNA expression levels were significantly higher than those of the shEhhadh-iPS cell line ( Fig. 9 C), immunofluorescence staining revealed that the shEhhadh-iPS cell line expressed three germ layer differentiation marker proteins, showing red fluorescence as shown in the figure ( Fig. 9 D), indicating that the detected cells express three germ layer differentiation marker proteins. These indicate that the EB spheres generated by the established shEhhadh-iPS cell line have significantly reduced pluripotency levels after differentiation, and have the ability to randomly differentiate into three germ layers.

[0108] Example 4: Effect of Ehhadh gene on differentiation of mouse embryonic stem cells

[0109] 1. Test method:

[0110] The ESCs differentiation system was established by using the differentiation of the mouse R1 embryonic stem cell line; when the cell clusters grew to a suitable size, the original culture medium was removed, washed once with DPBS, and digested in a 37°C incubator for about 5 minutes with trypsin; after observing the clones loose under the microscope, an equal amount of culture medium was added to form a cell suspension; centrifuged at 1,000rpm for 5 minutes, and the supernatant was discarded; 2mL ESM was added to resuspend, transferred to a new culture dish, and placed in an incubator for 25 to 35 minutes to allow the feeder cells to adhere to the wall; the cell suspension was carefully removed and added to a new centrifuge tube, centrifuged at 1,000rpm for 5 minutes, the supernatant was discarded, and the cell pellet was retained; ESM culture medium without LIF (leukemia inhibitory factor) was gently blown, and the cells were seeded in a plate coated with 0.1% gelatin at a ratio of 1:5. After 72 hours of passage, we can observe from the microscope that the edges of the cell clusters have clearly begun to differentiate, the shape has changed, and the irregular edges indicate that ESCs have differentiated normally and can be used for subsequent experiments. R1 ES was passaged, feeder and LIF were removed, and cultured in a 37°C, 5% CO2 cell culture incubator for 12 hours, and then the empty plasmid and Ehhadh overexpression plasmid (mEhhadh) were introduced into the cells at the same time. Samples were collected 48 hours after transfection, and then we detected the expression of genes at both RNA and protein levels. The real-time fluorescence quantitative PCR primers for pluripotent genes are shown in Table 1, and the real-time fluorescence quantitative PCR primers for three germ layer marker genes are shown in Table 2.

[0111] 2. Test results:

[0112] The results of real-time fluorescence quantitative PCR showed that, compared with the empty plasmid, the expression of pluripotent genes Nanog and Sox2 was significantly downregulated by overexpression of Ehhadh, and the expression of three germ layer key genes Sox17, Eomes, and Cdx2 was significantly upregulated ( Fig.10 A, B). The protein expression level is consistent with that of mRNA. As shown in the figure, after overexpression of Ehhadh, the fluorescence intensity of pluripotent protein detected by immunofluorescence was significantly higher than that of the control group, while the fluorescence intensity of the three germ layer marker proteins was the opposite ( Fig.10 C). This indicates that overexpression of Ehhadh promotes ESCs differentiation.

[0113] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Application of Ehhadh gene as a target in regulating the efficiency of somatic cell reprogramming; The Ehhadh gene is a DNA molecule as shown in the following i) or ii): i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

2. Use of a substance for down-regulating Ehhadh gene expression in any of the following (1)-(3): (1) Improve the efficiency of somatic cell reprogramming; (2) preparing products that improve the efficiency of somatic cell reprogramming; (3) Produce stem cells.

3. The use according to claim 2, characterized in that: The substance for down-regulating the expression of the Ehhadh gene is shRNA, and its nucleotide sequence is shown in SEQ ID NO.

3.

4. A method for improving the efficiency of somatic cell reprogramming, characterized in that: The following steps are involved: Introduce the Ehhadh gene interference fragment into the somatic cell reprogramming system.

5. The method according to claim 4, characterized in that The nucleotide sequence of the Ehhadh gene interference fragment is shown in SEQ ID NO.

3.

6. A method for producing stem cells, characterized in that: The following steps are involved: Substances that downregulate Ehhadh gene expression and four transcription factors, Oct4, Sox2, Klf4 and c-Myc, were transferred into somatic cells.

7. The production method according to claim 6, characterized in that: The substance for down-regulating the expression of the Ehhadh gene is shRNA, and its nucleotide sequence is shown in SEQ ID NO.3; the shRNA is linked to the PsicoR vector and then transferred into the somatic cells.

8. Application of Ehhadh gene in the preparation of products promoting embryonic stem cell differentiation.

9. Use of an overexpression vector containing the Ehhadh gene in the preparation of a product for promoting embryonic stem cell differentiation.

10. A method for promoting embryonic stem cell differentiation, characterized in that: The following steps are involved: An overexpression vector containing the Ehhadh gene is introduced into embryonic stem cells to overexpress the Ehhadh gene.

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