CN112143705B

CN112143705B - Double-gene modified stem cell and application thereof

Info

Publication number: CN112143705B
Application number: CN202011012627.0A
Authority: CN
Inventors: 段海峰; 薛冰华; 于婷婷; 解晶; 庞如梦; 刘丽华
Original assignee: Beijing Huaao Xuande Biomedical Technology Co ltd
Current assignee: Beijing Huaao Xuande Biomedical Technology Co ltd
Priority date: 2020-09-23
Filing date: 2020-09-23
Publication date: 2022-09-06
Anticipated expiration: 2040-09-23
Also published as: CN112143705A

Abstract

The invention discloses a double-gene modified stem cell, a preparation method and application thereof, and also provides a pharmaceutical composition comprising the stem cell. Wherein the stem cell comprises a first exogenous nucleic acid and a second exogenous nucleic acid in the same expression vector, the first exogenous nucleic acid encoding a nucleotide sequence comprising a first protein selected from adiponectin, a variant of adiponectin, or a first fusion protein comprising the adiponectin or a variant of adiponectin; the second foreign nucleic acid encodes a nucleotide sequence comprising a second protein selected from the group consisting of sphingosine kinase1, a sphingosine kinase1 variant, or a second fusion protein comprising said sphingosine kinase1 or sphingosine kinase1 variant. The double-gene modified stem cell provided by the invention shows the synergistic effect of the adipiectin gene and the mutated SPHK1 gene, and can obviously reduce the blood sugar and the blood fat of a tested animal and reduce the weight of the tested animal.

Description

Double-gene modified stem cell and application thereof

Technical Field

The invention belongs to the technical field of cell therapy, particularly relates to a stem cell for treating diabetes, and particularly relates to a stem cell jointly modified by Adiponectin (Adiponectin) and mutated sphingosine kinase 1(sphingosine kinase1, SPHK1) genes and application thereof.

Background

Obesity and type 2 diabetes (T2DM) are among the major public health problems that afflict modern society. Obesity and its associated insulin resistance are key factors in the onset of type 2 diabetes, and 80-90% of type 2 diabetic patients are investigated to be overweight or obese. Therefore, effective control of blood glucose and body weight has always been the subject of focus in the research field. According to statistics, the number of the attack groups of type 2 diabetes mellitus in the world reaches 4 hundred million, and accounts for 90% -95% of all diabetes mellitus patients. Hyposecretion, decreased activity or defective function of endogenous hormones, cytokines or enzymes is closely related to the onset of metabolic diseases, such as insulin resistance and relative hyposecretion, which are central causes of diabetes. Therefore, these key molecules for regulating the metabolic balance of glycolipids are also important targets for the development of drugs for treating metabolic syndromes such as diabetes.

Adiponectin (Adiponectin), an endogenous biologically active polypeptide or protein secreted by adipocytes, acts as an insulin-sensitizing hormone and improves insulin resistance and arteriosclerosis in mice. The research on human bodies finds that the adiponectin level can indicate the development of II type diabetes and coronary heart disease, and shows the potential of resisting diabetes, atherosclerosis and inflammation in clinical tests. Adiponectin, as an insulin hypersensitizing hormone, can increase and promote fatty acid oxidation and sugar absorption of skeletal muscle cells, obviously enhances the gluconeogenesis inhibition effect of insulin, inhibits glycogenesis of liver, and is an important regulating factor in a regulation network of lipid metabolism and blood sugar homeostasis of an organism.

In experimental models of atherosclerosis, plasma adiponectin levels are inversely related to triglycerides and low density lipoproteins and positively related to high density lipoproteins. The adiponectin is used for treating, so that the content of triglyceride and low-density lipoprotein in blood can be obviously reduced, the content of high-density lipoprotein can be increased, and atherosclerotic lesion can be relieved. There is increasing evidence that adiponectin and its artificial synthetases are of value in the treatment of type 2 diabetes and the metabolic syndrome associated with insulin resistance. Recent studies have reported that rapid in vivo administration of truncated adiponectin reduces postprandial plasma free fatty acids in mice on a high-fat diet, and if this protein is administered slowly, it causes the mice to lose weight continuously without affecting their food intake. Scherer and his assistant also demonstrated that rapid injection of recombinant adiponectin would completely ameliorate hyperglycemia in several animal models of diabetes, including ob/ob mice, non-obese diabetic mice, and streptozotocin-treated mice.

Sphingosine kinase 1(SphK1) is a newly discovered family of lipid kinases that has been evolutionarily conserved in humans, mice, yeast and plants, an enzyme that is a key enzyme in the sphingolipid metabolic pathway, catalyzes the formation of sphingosine-1-phosphate (S1P) from sphingosine, and is a "rheostat" that regulates the synthesis of ceramide and sphingosine-1-phosphate (S1P).

After being combined with a receptor, the S1P can regulate cell processes such as cell growth, apoptosis, differentiation, hematopoiesis and the like. The SphK1/S1P signaling pathway is involved in a variety of biological processes and disease processes, including tumorigenesis and diabetes. In addition, diabetic mice showed reduced blood glucose and blood lipid levels compared to control mice after injection of adenovirus carrying the human SphK1 gene. At present, SphK1 is mainly researched on the basis of the effect of SphK1 in cells, drugs developed by taking the protein as a target point mainly use antibodies or antagonists thereof, and the protein is not directly prepared into drugs for treatment; another method is gene therapy using a virus or the like as a vector to introduce the SPHK1 gene into cells, for example, gene therapy using adenovirus as a vector, but this method is liable to cause drug resistance in vivo, and long-term administration is required for the treatment of metabolic diseases such as diabetes, and therefore, the use of this method is limited.

Therefore, there is a need to develop a novel method for fundamentally treating glycolipid metabolic diseases.

Disclosure of Invention

In order to overcome the above problems, the present inventors have conducted intensive studies and, as a result, found that: the mesenchymal stem cells simultaneously modified by the Adiponectin gene and the mutated SPHK1 gene can obviously reduce the blood sugar and the blood fat of tested animals and reduce the weight, can be safely applied to human subjects without causing immunogenic reaction, and provides an important research basis for the treatment of metabolic diseases, thereby completing the invention.

Specifically, the present invention aims to provide the following:

in a first aspect of the invention, there is provided a double-genetically modified stem cell comprising a first exogenous nucleic acid comprising a nucleotide sequence encoding a first protein and a second exogenous nucleic acid comprising a nucleotide sequence encoding a second protein, wherein,

the first protein is selected from adiponectin, an adiponectin variant, or a first fusion protein comprising the adiponectin or the adiponectin variant,

the second protein is selected from the group consisting of sphingosine kinase1, a sphingosine kinase1 variant, or a second fusion protein comprising the sphingosine kinase1 or sphingosine kinase1 variant.

Wherein the first protein has the amino acid sequence shown in SEQ ID NO: 1;

the second protein has the amino acid sequence shown in SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.

Wherein the cell further comprises an expression vector, the first exogenous nucleic acid and the second exogenous nucleic acid are contained in the same or different expression vectors,

preferably in the same expression vector.

Wherein the double-gene modified stem cell is obtained by introducing a first exogenous nucleic acid and a second exogenous nucleic acid into a mesenchymal stem cell.

In a second aspect, a method for preparing a double-gene modified stem cell is provided, preferably for preparing the above-mentioned stem cell, wherein the preparation method comprises the steps of linking a first exogenous nucleic acid and a second exogenous nucleic acid, and introducing into a mesenchymal stem cell.

Wherein, the preparation method comprises the following steps:

step 1, obtaining a first exogenous nucleic acid and a second exogenous nucleic acid, and connecting the first exogenous nucleic acid and the second exogenous nucleic acid with a vector plasmid after enzyme digestion to obtain a recombinant plasmid;

step 2, transfecting the recombinant plasmid and the packaging plasmid to obtain a recombinant lentiviral vector containing a first exogenous nucleic acid and a second exogenous nucleic acid;

and 3, transfecting the obtained recombinant lentiviral vector to mesenchymal stem cells to obtain the double-gene modified stem cells.

In a third aspect, a double-gene modified stem cell prepared by the method is provided.

In a fourth aspect, an application of the stem cell or the double-gene modified stem cell prepared by the method in preparing a medicament for treating metabolic diseases is provided.

Wherein the metabolic disease is selected from obesity, type I diabetes, type II diabetes, dyslipidemia, insulin resistance, hyperglycemia, metabolic syndrome, atherosclerosis, coronary heart disease, and secondary complications thereof.

In a fifth aspect, a pharmaceutical composition is provided, wherein the composition comprises the stem cell or the double-gene modified stem cell prepared by the method.

The invention has the advantages that:

(1) the double-gene modified stem cell provided by the invention can simultaneously express Adiponectin and sphingosine kinase1, shows the synergistic effect of the adipoetin gene and the mutated SPHK1 gene, can obviously reduce the blood sugar and blood fat of a tested animal, and can reduce the weight;

(2) the stem cell modified by double genes provided by the invention not only maintains the functions of mesenchymal stem cells, but also can well express adiponectin and sphingosine kinase1 protein, can be safely applied to human subjects, and does not cause immunogenic reaction;

(3) the double-gene modified stem cell provided by the invention has the biological activities of reducing blood sugar, blood fat and the like which are obviously improved, can be effectively used for preparing a medicament for treating metabolic diseases, and has great clinical value.

Drawings

FIG. 1 illustrates a plasmid map of pCDH-AQS gene according to a preferred embodiment of the present invention; FIG. 2 is a graph showing the results of detecting the expression levels of Adiponectin and QSPHK1 by Realtime-PCR in Experimental example 1; FIG. 3 is a graph showing the results of the expression levels of adipoectin and QSPHK1 measured by ELISA in Experimental example 1; FIG. 4 is a graph showing the results of examining the influence of each sample on the expression level of sterol metabolism gene SREBP1c mRNA in Experimental example 2; FIG. 5 shows the results of examination of the influence of each sample on the levels of sterol metabolism genes SREBP1 and SREBP2 protein in Experimental example 2; FIG. 6 shows the results of examining the influence of each sample on the expression levels of lipid metabolism-controlling genes p-AMPK and AMPK in Experimental example 3; FIG. 7 is a graph of the random weight change trend in diabetic model mice, with arrows representing the time points of drug and cell administration; FIG. 8 is a graph showing the trend of fasting glucose changes in diabetic mice, with arrows representing the time points of drug and cell administration; FIG. 9 is a figure showing a comparison of body types of diabetes model mice in different experimental groups 28 days after cell treatment; FIG. 10 is a graph of HE staining of liver and abdominal subcutaneous fat in different experimental groups of diabetic model mice 28 days after cell treatment; FIG. 11 is a histogram of the blood lipid levels in serum of diabetes model mice of different experimental groups 28 days after cell treatment.

Detailed Description

The present invention will be described in further detail below with reference to preferred embodiments and examples. The features and advantages of the present invention will become more apparent from the description.

The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

The inventor finds that stem cells simultaneously expressing Adiponectin and sphingosine kinase1 have biological activities of remarkably improving blood sugar and blood fat reduction and the like through research and repeated exploration on Adiponectin and sphingosine kinase1 in glycolipid metabolic balance. In consideration of the defects of protein medicines or gene therapy in the prior art, the inventor modifies stem cells by using an Adiponectin (Adiponectin) gene and a sphingosine kinase1 gene (SPHK1 gene) from the perspective of gene modification, so that the remarkable synergistic effect of the two genes can be shown, the immunogenic reaction can be avoided, and the clinical value is great.

Thus, in a first aspect of the invention there is provided a double-genetically modified stem cell comprising a first exogenous nucleic acid comprising a nucleotide sequence encoding a first protein and a second exogenous nucleic acid comprising a nucleotide sequence encoding a second protein, wherein,

According to a preferred embodiment of the invention, the adiponectin variant has one or more amino acid substitution, insertion or deletion compared to the sequence from which it is derived, or has at least 80% or more, preferably 95% or more, such as 99% sequence identity and retains adiponectin activity;

the sphingosine kinase1 variant has one or more amino acid substitutions, insertions or deletions compared to the sequence from which it is derived, or has at least 80% or more, preferably at least 95% or more, such as 99% sequence identity, and retains the activity of sphingosine kinase 1.

Wherein the adiponectin variant retains adiponectin activity, i.e., retains its ability to increase fatty acid oxidation and sugar absorption in skeletal muscle cells, potentiate gluconeogenesis inhibition by insulin, lower blood triglyceride and low density lipoprotein levels;

the sphingosine kinase1 variant retains the activity of sphingosine kinase1, i.e. retains its ability to catalyze the production of S1P from sphingosine, a metabolite of ceramide.

In a further preferred embodiment, the first protein is adiponectin or a first fusion protein comprising adiponectin;

the second protein is sphingosine kinase1 or a second fusion protein comprising sphingosine kinase 1.

In a still further preferred embodiment, the first protein has the amino acid sequence as set forth in SEQ ID NO:1 (c) or a pharmaceutically acceptable salt thereof,

According to a preferred embodiment of the invention, the first foreign nucleic acid encodes a first protein and the second foreign nucleic acid encodes a second protein,

preferably, the first foreign nucleic acid is an Adiponectin gene (Adiponectin gene) and the second foreign nucleic acid is a mutated SPHK1 gene (designated as QSPHK1 gene).

The inventor analyzes the protein sequence of sphingosine kinase1 and finds that the N-terminal of the sphingosine kinase1 contains a conserved GKGK structural domain which appears in a plurality of protein sequences which are modified by acetylation, wherein K is the site of the modification by acetylation, and the acetylation of sphingosine kinase1 occurs on K of the GK structural domain. After being acetylated and modified, the SPHK1 has enhanced stability, two Ks (lysines) in the GKGK structural domain are respectively mutated into arginine (R) or glutamine (Q), the ubiquitination modification of SPHK1 is blocked, the degradation by proteasomes is avoided, and the activity of the SPHK1 is enhanced.

Based on this, in the invention, the SPHK1 gene is preferably mutated, so that K in the GKGK structural domain of the encoded sphingosine kinase1 is mutated into R or Q, thereby improving the stability and action activity of the protein.

Among them, mutation can be carried out by a method commonly used in the art, for example, mutation of the base encoding K in the gkgkgkgkk domain in SPHK1 gene by using a point mutation assay technique.

In a further preferred embodiment, the first exogenous nucleic acid has the sequence as set forth in SEQ ID NO:3 or the sequence shown in SEQ ID NO:3, preferably a nucleotide sequence having the sequence shown in SEQ ID NO:3, and (b) is a sequence shown in the specification;

the second exogenous nucleic acid has the sequence shown in SEQ ID NO:4 or SEQ ID NO:4, preferably a nucleotide sequence having the sequence shown in SEQ ID NO: 4.

For example: the nucleotide sequence of the first exogenous nucleic acid is set forth in SEQ ID NO:3, and the nucleotide sequence of the second exogenous nucleic acid is shown as SEQ ID NO:4, respectively.

According to a preferred embodiment of the present invention, the double-gene modified stem cell further comprises an expression vector, the first exogenous nucleic acid and the second exogenous nucleic acid are contained in the same or different expression vectors,

preferably in the same expression vector.

In a further preferred embodiment, the first exogenous nucleic acid and the second exogenous nucleic acid are linked by a sequence encoding a self-cleaving peptide,

the sequence encoding the self-cleaving peptide is linked between the 3 'end of the first exogenous nucleic acid and the 5' end of the second exogenous nucleic acid.

In the present invention, the self-cleaving peptide includes, but is not limited to, 2A peptide derived from Foot and Mouth Disease Virus (FMDV), Equine Rhinitis A Virus (ERAV) or brown wing moth virus, preferably 2A peptide derived from brown wing moth virus.

In a still further preferred embodiment, the 2A peptide comprises the amino acid sequence shown as SEQ ID NO. 5, preferably the amino acid sequence of the 2A peptide is shown as SEQ ID NO. 5.

Preferably, the sequence encoding the self-cleaving peptide comprises the nucleotide sequence shown in SEQ ID NO. 6, preferably the sequence encoding the self-cleaving peptide is shown in SEQ ID NO. 6.

According to a preferred embodiment of the present invention, the ligated first and second exogenous nucleic acids comprise the nucleotide sequences shown in SEQ ID NO. 7,

preferably, the nucleotide sequences of the first exogenous nucleic acid and the second exogenous nucleic acid after connection are shown as SEQ ID NO. 7.

In a further preferred embodiment, the linked first and second foreign nucleic acids encode a protein having the amino acid sequence shown in SEQ ID NO 8,

preferably, the ligated first and second exogenous nucleic acids encode a protein having the amino acid sequence shown in SEQ ID NO. 8.

According to a preferred embodiment of the invention, the stem cells are mesenchymal stem cells, derived from adipose tissue, umbilical cord, bone marrow or cord blood, preferably derived from umbilical cord.

Wherein the Mesenchymal Stem Cell (MSC) is a pluripotent stem cell having all the commonality of stem cells, i.e., self-renewal and multipotentiality. In addition, it has immunoregulation function, and can inhibit T cell proliferation and immune reaction thereof by intercellular interaction and cytokine production, thereby playing the role of immune reconstitution; meanwhile, the mesenchymal stem cells have convenient sources, are easy to separate, culture, amplify and purify, still have the characteristics of the stem cells after multiple times of passage amplification, and have no immunological rejection.

The umbilical cord mesenchymal stem cells are derived from in vitro umbilical cord tissues, are convenient to obtain, wide in source and free from the debate and limitation of ethics, meanwhile, the obtained materials are non-invasive to a donor and are not influenced by the age factor of the donor, the in vitro separation culture is simple, the amplification is rapid, the immunogenicity is low, the tumorigenicity is avoided, and the umbilical cord mesenchymal stem cells can be used as a cell subcarrier for gene therapy. Under the specific induction condition in vivo or in vitro, the umbilical cord mesenchymal stem cells can be differentiated into various tissue cells, still have multidirectional differentiation potential after continuous subculture and cryopreservation, and are ideal seed cells for cell transplantation treatment.

Therefore, umbilical cord-derived mesenchymal stem cells are preferably used in the present invention.

In a further preferred embodiment, the double-genetically modified stem cell is obtained by introducing a first exogenous nucleic acid and a second exogenous nucleic acid into a mesenchymal stem cell.

The stem cell modified by the double genes shows the synergistic effect of the adipiectin gene and the mutated SPHK1 gene, can obviously reduce the blood sugar and the blood fat of a tested animal and reduce the weight, and can be safely applied to a human subject without causing immunogenic reaction.

In a second aspect of the present invention, there is provided a method for preparing a double-gene-modified stem cell, the method comprising the steps of linking a first exogenous nucleic acid and a second exogenous nucleic acid, and introducing into a mesenchymal stem cell.

According to a preferred embodiment of the present invention, the preparation method comprises the steps of:

and 3, transfecting the obtained recombinant lentiviral vector to a mesenchymal stem cell to obtain the double-gene modified stem cell.

The preparation of the double-gene modified stem cells is further described below:

step 1, obtaining a first exogenous nucleic acid and a second exogenous nucleic acid, and connecting the first exogenous nucleic acid and the second exogenous nucleic acid with a vector plasmid after enzyme digestion to obtain a recombinant plasmid.

Wherein the first exogenous nucleic acid is preferably an adipinctin gene having the nucleotide sequence as shown in SEQ ID NO: 3; the second exogenous nucleic acid is preferably a mutated SPHK1 gene having the amino acid sequence as set forth in SEQ ID NO: 4.

The first exogenous nucleic acid and the second exogenous nucleic acid are preferably connected through a T2A sequence, and the T2A sequence is shown as SEQ ID NO. 6.

In the invention, the adipinctin-T2A fragment is obtained by PCR amplification, then the QSPHK1 fragment is obtained, and the fragment is recovered by agarose gel electrophoresis.

According to a preferred embodiment of the present invention, the primer pair for amplifying the Adiponectin-T2A fragment is EF1-BamHI-Adiponectin-F and T2A-SPHK1-R, and has the nucleotide sequences shown in SEQ ID NO 9 and SEQ ID NO 10, respectively;

the primer pair for amplifying the QSPHK1 fragment is T2A-SPHK1-F and SPHK1-SalI-WPRE-R, and the nucleotide sequences are respectively shown as SEQ ID NO. 11 and SEQ ID NO. 12.

In the invention, a vector plasmid is subjected to double enzyme digestion by a method commonly used in the prior art, then is subjected to recombination connection with an amplified adipinectin-T2A fragment and a QSPHK1 fragment, is transformed into an escherichia coli DH-5 alpha competent cell, a positive single colony after transformation is selected for PCR identification, a correct colony is sequenced, and a colony with correct sequencing is subjected to plasmid extraction to obtain a recombinant clone plasmid, wherein the pCDH-AQS plasmid is shown in figure 1, the nucleotide sequence of adipinectin-QSPHK 1 is shown in SEQ ID NO. 7, and the coded amino acid sequence is shown in SEQ ID NO. 8.

Wherein the vector plasmid is a recombinant lentiviral vector plasmid pCDH-EF1, and enzymes adopted by double enzyme digestion are preferably BamHI and SalI.

And 2, transfecting the recombinant plasmid and the packaging plasmid to a lentivirus packaging cell to obtain the recombinant lentivirus vector containing the first exogenous nucleic acid and the second exogenous nucleic acid.

Wherein, step 2 comprises the following substeps:

step 2-1, culturing the packaging cells.

In the invention, the packaging cells are preferably 293T cells, the 293T cells are activated and then resuspended, the cells are inoculated into a culture dish for culture, and digestion is carried out when the confluency of the cells reaches over 90%; after termination of the digestion, the cells are centrifuged, resuspended and the cells are seeded in each coated culture dish for packaging the virus, preferably 1X 10 cells per dish (150mm) ⁷ ～1.5×10 ⁷ Each cell, preferably 1.2X 10, is inoculated per culture dish ⁷ And (4) cells.

The medium of the 293T cells is 10% FBS + DMEM medium.

Step 2-2, transfection of packaging cells.

Specifically, the packaging plasmid is mixed with the recombinant cloning plasmid (pCDH-AQS) obtained in step 1, and after incubation, transfection reagents are added and incubated at room temperature to form a DNA-lipofection complex.

In the present invention, the packaging plasmid can be a lentivirus packaging Helper plasmid commonly used in the prior art, such as Helper1 plasmid, Helper2 plasmid and Helper3 plasmid, which are purchased from Addgene, and the mass ratio of pCDH-AQS plasmid, Helper1 plasmid, Helper2 plasmid and Helper3 plasmid is 2:1:1:1 is preferred.

The transfection reagent may be a reagent commonly used in the art, for example, a Lipofectamin 2000 (available from Invitrogen) solution, and the transfection reagent is added dropwise to the plasmid mixing system after mixing with DMEM medium.

And (3) uniformly mixing the DNA-liposome transfection compound with the packaging cells, culturing, periodically changing the liquid in the culture process, collecting supernatant liquid after changing the liquid, and storing the supernatant liquid at 4 ℃.

According to a preferred embodiment of the invention, the collected supernatant is concentrated and subjected to a virus titer determination,

the concentration is preferably performed by centrifuging the supernatant at low temperature, discarding the precipitate, and concentrating the supernatant on a Millipore protein ultrafiltration column (100 KD).

Wherein the virus titer determination is preferably carried out according to the method commonly used in the prior art, and the virus is diluted to 1 × 10 according to the determination result ⁸ TU/ml and stored at-80 ℃.

Through the steps, a lentivirus vector carrying the adipinectin and QSPHK1 can be obtained and is marked as Lenti-AQS.

Preferably, the mesenchymal stem cells are isolated by using a climbing umbilical cord tissue block method.

Specifically, the method comprises the following steps: the isolated umbilical cord from normal labor is placed in PBS buffer containing 200U/mL penicillin and 200U/mL streptomycin, and in order to ensure the activity of the umbilical cord tissue, the fresh umbilical cord is separated within 6 h. Flushing residual hematocele in umbilical vein and umbilical artery with 20mL syringe, and cutting umbilical cord tissue into pieces of 1mm by tissue scissors ³ Filtering the small umbilical cord tissue blocks with a 200-mesh filter screen, collecting umbilical cord tissue blocks on the 200-mesh filter screen, and removing the small umbilical cord tissue blocks to obtain umbilical cord tissue blocks with the diameter of 1-1.5 mm. Collecting tissue blocks with diameter of 1-1.5mm, directly inoculating into culture flask, and directly placing in 5% CO ₂ 37 ℃ incubatorAnd standing for 1-2 h. After the tissue block adheres firmly, adding alpha-MEM culture solution containing 10% fetal calf serum, and placing in 5% CO ₂ Continuously culturing in a 37 ℃ culture box, and after five days, the proliferation of the umbilical cord tissue mesenchymal stem cells is fully paved by about 80 percent in a culture bottle; the cells obtained were primary cells after digestion with 0.25% trypsin (0.01% EDTA). MSC is isolated and cultured by an umbilical cord tissue block climbing method, a small amount of cells climb out around the umbilical cord tissue after 72 hours, the cells are free from the tissue after about 7 days and gradually form clone, and the prepared mesenchymal stem cells are frozen and stored.

Step 3 comprises the following substeps:

and 3-1, culturing and inoculating the stem cells.

Resuscitating and culturing the pre-frozen stem cells, digesting the cells by 0.05 percent of trypsin after the resuspension cells are full, terminating the digestion by a culture medium containing serum, and centrifuging and resuspending the cells after the digestion is terminated. The cells are then seeded onto culture dishes, preferably 2X 10 cells per dish (150mm) ⁶ ～2.5×10 ⁶ Individual cells, the media was changed the next day after inoculation.

And 3-2, adding a lentivirus vector Lenti-AQS, and continuously culturing.

Polybrene (Polybrene) was added to the inoculated cells, and a corresponding volume of lentiviral vector was added according to the MOI value of the cells and the virus titer, and the cells were cultured.

Wherein, the added lentiviral vector volume is (MOI × cell number)/viral titer. The MOI refers to the ratio of virus to cell number during transduction, and in the invention, the MOI is preferably 10-50, such as 40.

At 37 deg.C, 5% CO ₂ Culturing for 6-8h at saturated humidity, removing virus-containing culture medium, replacing with serum-free culture medium, and culturing at 37 deg.C and 5% CO2 at saturated humidity for 2-3 days.

And 3-3, carrying out subculture on the cells.

In the present invention, after the genetically modified cells were confluent, the cells were digested with 0.05% trypsin, the digestion was stopped with serum-containing medium, the cell suspension was centrifuged at 800rpm for 5min, and the cells obtained by centrifugation were used for bloodless cultureResuspending in clear medium, subculturing at 1:6 ratio, and culturing in serum-free medium at 37 deg.C and 5% CO ₂ Culturing for 3 days until the cells grow full to obtain the stem cells with double gene modification, which are marked as MSC-AQS.

In a third aspect of the present invention, the double-gene modified stem cell prepared by the above method is provided.

In a fourth aspect of the present invention, there is provided a use of the double-gene modified stem cell of the first aspect or the stem cell prepared by the method of the second aspect in the preparation of a medicament for treating metabolic diseases.

Preferably, the metabolic disease is selected from the group consisting of obesity, type I diabetes, type II diabetes, dyslipidemia, insulin resistance, hyperglycemia, metabolic syndrome, atherosclerosis, coronary heart disease, and secondary complications thereof.

More preferably, the metabolic disease is selected from obesity, type II diabetes, dyslipidemia, hyperglycemia, and secondary complications of the above.

For example, the metabolic disorder is selected from obesity, type II diabetes, dyslipidemia, and hyperglycemia.

In a fifth aspect of the invention, there is provided a pharmaceutical composition comprising the double-gene modified stem cell of the first aspect or the stem cell prepared by the method of the second aspect.

The dosage form of the pharmaceutical composition can be any form known in the medical field, and is preferably tablets, pills, suspensions, emulsions, solutions, injections, gels, capsules, powders, granules or suppositories, and is more preferably an injection.

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient, preferably comprising a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution (e.g., a balanced salt solution or physiological saline), dispersion, suspension or emulsion.

More preferably, the pharmaceutical composition may be implanted in the form of a suspension, gel, colloid, slurry, or mixture.

In the present invention, the pharmaceutical composition is prepared according to the principles of cellular drugs prescribed in the prior art, for example, according to Hematopoietic Stem Cell Therapy (Hematopoietic Stem Cell Therapy), e.g., d.ball, j.list & p.law, churchlill Livingstone, 2000.

According to a preferred embodiment of the present invention, the pharmaceutical composition may be administered to a subject by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection or oral administration,

the subject is a mammal, e.g., a human.

Examples

The present invention is further described below by way of specific examples, which are merely exemplary and do not limit the scope of the present invention in any way.

Example 1 mesenchymal Stem cell obtaining

The method for separating the mesenchymal stem cells by adopting the umbilical cord tissue block climbing method comprises the following specific steps:

the isolated umbilical cord from normal labor is placed in PBS buffer containing 200U/mL penicillin and 200U/mL streptomycin, and in order to ensure the activity of the umbilical cord tissue, the fresh umbilical cord is separated within 6 h. Flushing residual hematocele in umbilical vein and umbilical artery with 20mL syringe, and cutting umbilical cord tissue into pieces of 1mm by tissue scissors ³ Filtering the small umbilical cord tissue blocks with a 200-mesh filter screen, collecting umbilical cord tissue blocks on the 200-mesh filter screen, and removing the small umbilical cord tissue blocks to obtain umbilical cord tissue blocks with the diameter of 1-1.5 mm. Collecting tissue blocks with diameter of 1-1.5mm, directly inoculating the tissue blocks into culture flask, and directly placing in 5% CO ₂ And standing for 1-2h in an incubator at 37 ℃. After the tissue blocks adhered firmly, the culture medium of alpha-MEM (purchased from Gibco) containing 10% fetal bovine serum was added and placed in 5% CO ₂ Continuously culturing in a 37 ℃ culture box, and after five days, the proliferation of the umbilical cord tissue mesenchymal stem cells is fully paved by about 80 percent in a culture bottle; the cells obtained were primary cells after digestion with 0.25% trypsin (0.01% EDTA). Culturing MSC by block-climbing method, allowing a small amount of cells to climb out around the umbilical cord tissue after 72h, and allowing the cells to free out of the tissue after about 7 days and gradually culturingForming clone, and freezing and storing the prepared mesenchymal stem cells.

Example 2 cloning of Adiponectin and QSPHK1 genes and vector construction

DNA molecules encoding Adiponectin and QSPHK1 were synthesized from the whole gene of Zhongmeitai and Biotechnology (Beijing) Co., Ltd, wherein the Adiponectin (amino acid sequence SEQ ID NO:1, coding sequence SEQ ID NO:3) and QSPHK1 (amino acid sequence SEQ ID NO:2, coding sequence SEQ ID NO:4) were linked by a T2A sequence (amino acid sequence SEQ ID NO:5, coding sequence SEQ ID NO: 6).

The Adiponectin-T2A fragment (851bp) was amplified with primer EF1-BamHI-Adiponectin-F (nucleotide sequence: SEQ ID NO:9) and primer T2A-SPHK1-R (nucleotide sequence: SEQ ID NO:12), the QSPHK1 fragment (1278bp) was amplified with primer T2A-SPHK1-F (nucleotide sequence: SEQ ID NO:11) and primer SPHK1-SalI-WPRE-R (nucleotide sequence: SEQ ID NO:10), and the amplified products were analyzed by agarose gel electrophoresis.

The recombinant lentiviral vector plasmid pCDH-EF1 (purchased from Addge) was digested simultaneously with BamHI and SalI, the recovered vector was digested with 2X mix recombinase (purchased from CloneSmarter) and ligated with the Adiponectin-T2A fragment and QSPHK1 fragment to transform DH 5. alpha. competent cells, 100. mu.L of the resulting suspension was spread on LB plates containing ampicillin resistance, and cultured overnight at 37 ℃. Selecting a single clone to carry out colony PCR, sending a positive clone to a sample for sequencing, storing the clone with correct sequencing result, extracting a plasmid, and naming the plasmid as pCDH-AQS, wherein the nucleotide sequence is shown as SEQ ID NO. 7, and the amino acid sequence is shown as SEQ ID NO. 8.

Example 3 preparation of Lentiviral vectors carrying Adiponectin Gene and QSPHK1 Gene

1 frozen 293T cell (purchased from Addgene) was taken out of liquid nitrogen, quickly placed in a 37 ℃ water bath until ice disappeared, added dropwise to a 15ml centrifuge tube containing 5ml of pre-warmed medium, centrifuged at 1200rpm for 3min, the supernatant was discarded, the cells were re-suspended in 293T medium (10% FBS + DMEM) and seeded into a 150mm petri dish, 37 ℃ and 5% CO ₂ And (5) culturing at saturated humidity. When the confluence degree of cells reaches above 90%, discarding the old culture medium, adding 5ml of ethanolAnd (3) slightly shaking the bacteria PBS solution, washing the cells, removing the PBS solution, adding 2ml of 0.25% Trypsin-EDTA digestive juice, and digesting for 1-2min until the cells are completely digested. Adding serum-containing medium to stop digestion, centrifuging the cell suspension at 1200rpm for 3min, resuspending the centrifuged cells in the medium, inoculating 1.2X 10 cells per 150mm dish ⁷ Cells were used for packaging lentiviruses at 37 ℃ with 5% CO ₂ Saturated humidity culture, 20ml medium/dish.

2h before transfection, the 293T cell culture medium was replaced with 18ml of DMEM medium, 1ml of pre-warmed DMEM medium was added to A sterile centrifuge tubes, and then pCDH-AQS plasmid, Helper1 plasmid, Helper2 plasmid and Helper 3(pCDH-AQS: pHelper1: pHelper 2: Helper 3: 2:1:1:1, total 54. mu.g, Helper1, Helper2 and Helper3 plasmids were lentiviral-packaged Helper plasmids, purchased from Addgene) prepared above were added and mixed well. 1ml of pre-warmed DMEM medium was added to the B sterile centrifuge tube, followed by 108. mu.l of Lipofectamin 2000 (from Invitrogen) solution and mixed well. Tubes A and B were incubated at room temperature for 5 minutes. The liquid in tube B was added drop-wise to tube A, mixed well and incubated at room temperature for 20min to form a DNA-lipofectin complex.

Transferring the DNA-liposome mixture to 293T cells with liquid replaced in advance, mixing, and performing 5% CO at 37 deg.C ₂ And (5) culturing at saturated humidity. After 6-8h of incubation, the medium containing the transfection mixture was aspirated, and 20ml of preheated DMEM medium containing 5% FBS was added to each dish of cells, at 37 deg.C and 5% CO ₂ And (5) culturing at saturated humidity. After the liquid change, at 24h and 48h, respectively, the supernatant was collected and temporarily stored at 4 ℃, and 20ml of fresh medium was changed. Centrifuging the collected liquid at 4 deg.C and 3500rpm for 15min, discarding the precipitate, and concentrating the supernatant with Millipore protein ultrafiltration column (100KD) to obtain lentiviral vector (Lenti-AQS) carrying Adiponectin and QSPHK 1; simultaneously performing virus titer determination, and diluting the virus to 1 × 10 according to the determination result ⁸ TU/ml, the virus after subpackaging was stored at-80 ℃.

Example 4 Dual Gene modification of mesenchymal Stem cells

Resuscitating pre-frozen P3 generation mesenchymal stem cells to one150mm dish, 20ml serum-free medium 37 deg.C, 5% CO ₂ And (5) culturing in saturated humidity. After the revived cells were confluent, the cells were digested with 0.05% trypsin, the digestion was stopped with serum-containing medium, the cell suspension was centrifuged at 800rpm for 5min, and the centrifuged cells were resuspended in MSC serum-free medium (purchased from Bioind).

Inoculating 2-2.5X 10 cells per 150mm culture dish ⁶ Cells, the medium from which the cells were aspirated the next day after inoculation was discarded, replaced with serum-free α -MEM medium, 20ml of medium/dish, 16 μ l of Polybrene (purchased from Sigma) was added, and the Lenti-AQS lentivirus obtained in 2 (titer 1X 10) was added simultaneously with the multiplicity of infection of 40MOIs (Amersham pharmacia Biotech) ⁸ U/ml)，37℃、5％CO ₂ Culturing for 6-8h under saturated humidity. After 6 to 8 hours, the virus-containing α -MEM medium was discarded and replaced with a serum-free medium, and the culture was continued at 37 ℃ and 5% CO2 saturated humidity for 2 to 3 days.

After the cells after gene editing grow to full, digesting the cells by 0.05 percent trypsin, stopping digestion by a culture medium containing serum, centrifuging the cell suspension at 800rpm for 5min, resuspending the centrifuged cells by a serum-free culture medium, carrying out passage according to the passage ratio of 1:6, and carrying out passage by the serum-free culture medium at 37 ℃ and 5 percent CO ₂ The culture was carried out for 3 days. The mesenchymal stem cell modified by double genes is named as MSC-AQS.

Comparative example

Comparative example 1

The mesenchymal stem cells which are monogenetically modified by the adipoectin are prepared according to the method of the embodiment 1 to the embodiment 4 and are marked as MSC-A.

Comparative example 2

QSPHK1 single-gene modified mesenchymal stem cells, designated as MSC-QS, were prepared according to the methods described in examples 1-4.

Comparative example 3

The sample in this comparative example is mesenchymal stem cells modified with empty vector and is denoted as MSC.

Examples of the experiments

Experimental example 1 verification of biological function

(1) Identification of cell phenotype:

selecting P6 generation unmodified mesenchymal stem cells and double-gene modified mesenchymal stem cells (MSC-AQS), digesting with 0.05% pancreatin, washing twice with PBS, labeling cells with mouse anti-human CD14-PerCp-Cy5.5, CD19-PE, CD34-PE, CD45-PE or CD45-FITC, HLA-DR-PE, CD73-PE, CD90-PE and CD105-PE (Becton, Dickinson and Company), labeling each sample to be tested with 1 × 10 ⁶ And incubating the cells for 30min at room temperature in a dark place, washing the cells twice by PBS, fixing the cells by 2% paraformaldehyde, and detecting the cells by a flow cytometer.

The results show that the surface marker of the mesenchymal stem cells (MSC-AQS) after lentivirus infection by the Lenti-AQS still maintains the characteristics of mesenchymal stem cells, i.e. CD73, CD90 and CD105 are positive (> 90%), CD14, CD19, CD34, CD45 and HLA-DR are negative (< 1%).

(2) Adipogenic and osteogenic differentiation induction:

selecting P6 generation unmodified mesenchymal stem cells and double-gene modified mesenchymal stem cells (MSC-AQS), digesting with 0.05% pancreatin at 2 × 10 ⁵ Cell density per well was plated in 12-well plates and changed to adipogenic induction medium and osteogenic induction medium (purchased from BI corporation) on day 2, then changed every 2 days, adipogenic induction was stained with oil red O after 18 days, and osteogenic induction was stained with alizarin red-S after 23 days.

Wherein, the dyeing process of oil red O: the medium was aspirated and washed once with DPBS (1 ml/well). Fixing: DPBS was aspirated off and 10% formalin (4% formaldehyde; 1ml/well) was added; fixing for 30-60 minutes at room temperature; formalin was aspirated and washed with 60% isopropanol for 2-3 minutes (1 ml/well); removing isopropanol by suction, and adding an oiled-O staining working solution (1 ml/well); standing for 10-30 minutes at room temperature; washing with distilled water to remove excess dye.

Alizarin red-S staining process: the medium was aspirated and washed once with DPBS (1 ml/well); fixing: absorbing DPBS, adding 1ml of 70% EtOH into each hole, and fixing for 30-60 minutes at room temperature; EtOH was aspirated off and washed 3 times with distilled water (1 ml/well); removing distilled water by suction, and adding 1ml of 2% ARS staining working solution into each hole; standing for 30-60 minutes at room temperature; the staining solution was aspirated off and washed 4 times with 1ml of distilled water (1 ml/well); 1ml of distilled water was added to each well to avoid drying the cells.

The staining result shows that the MSC cells (MSC-AQS) after Lenti-AQS lentivirus infection still have adipogenic and osteogenic differentiation capability.

(3) Detecting the expression conditions of adippectin and QSPHK 1:

taking the mesenchymal stem cells (marked as MSC) modified by the empty vector in the comparative example 3, the mesenchymal stem cells (marked as MSC) modified by the single gene in the comparative examples 1 and 2, and the cell lysate of the mesenchymal stem cells (MSC-AQS) modified by the double genes, extracting RNA, carrying out RT-qPCR detection after reverse transcription, taking the mesenchymal stem cells without any gene modification as a common control (marked as a control group), and carrying out fluorescent real-time quantitative kit (according to Takara) (

Premix Ex Taq ^TM ) The results of the measurements are shown in FIG. 2.

The cell culture supernatants of MSC and MSC-AQS were collected and assayed for secreted protein concentration according to the ELISA kit instructions, with the results shown in FIG. 3.

As can be seen from fig. 2 and 3, adipoectin and QSPHK1 were present in high amounts in the supernatant of the MSC cells (MSC-AQS) that were double genetically modified with adipoectin and QSPHK 1.

Experimental example 2 Effect on sterol metabolism Gene expression level

(1) mRNA expression level:

the cells (MSC-QS, MSC-A, MSC-QS and MSC) of example 1 and comparative examples 1-3 were cultured in 100mm dishes, and when the confluency of the cells reached 70% -80%, the original MSC serum-free medium was discarded, 10ml of alpha-MEM medium was added, and the mixture was incubated at 37 ℃ and 5% CO ₂ The cultivation was continued for 48h at saturated humidity. Collecting culture supernatants of the four cells, and storing at 4 deg.C for use, wherein the cells need to be placed in a refrigerator at-80 deg.C for long-term storage. Mesenchymal stem cells without any genetic modification were used as a normal control (labeled as control group).

1 frozen HePG2 cells (human hepatoma cells, purchased from ATCC) were removed from liquid nitrogen and placed in a flashAdding into 37 deg.C water bath until ice blocks disappear, adding dropwise into 15ml centrifuge tube containing 5ml pre-heated medium, centrifuging at 1200rpm for 3min, discarding supernatant, re-suspending cells with HePG2 medium (10% FBS + DMEM) and inoculating into 100mm culture dish, and culturing at 37 deg.C with 5% CO ₂ And (5) culturing in saturated humidity. And the two to three days are passed once, and the passage ratio is 1: 6. After the cells are full, discarding the old culture medium, adding 2ml of sterilized PBS solution, slightly shaking, washing the cells, discarding the PBS solution, adding 2ml of 0.25% Trypsin-EDTA digestive juice, and digesting for 2-3min until the cells are completely digested. Adding serum-containing medium to terminate digestion, centrifuging the cell suspension at 1200rpm for 3min, resuspending the centrifuged cells in the medium, and inoculating 1 × 10 cells per well of 6-well plate ⁶ -2×10 ⁶ Adding 2ml of culture medium into each cell, and performing cell culture at 37 deg.C and 5% CO ₂ And (4) performing saturated humidity culture, and performing functional verification when the cell density reaches 70-80%.

Removing HePG2 culture medium from original 6-well plate, adding 2ml culture supernatant of pre-collected MSC, MSC-A, MSC-QS and MSC-AQS, culturing at 37 deg.C with 5% CO ₂ The culture was carried out at saturated humidity for 24 hours. After digesting the cells and terminating centrifugation, collecting the cells and extracting RNA by TRIzol method, after measuring the RNA concentration, inverting 500ng of total RNA to cDNA using reverse transcription kit (All-in-One cDNA Synthesis SuperMix).

RT-qPCR detection, fluorescent real-time quantification kit according to Takara: (

Premix Ex Taq ^TM ) And (5) carrying out the description. Wherein, the detection primer of RT-qPCR comprises SREBP1C-F, SREBP 1C-R, beta-Actin-F and beta-Actin-R, and the nucleotide sequences are respectively shown as SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16. The results of the detection are shown in FIG. 4.

As can be seen from FIG. 4, the culture supernatant of the MSC cells (MSC-AQS) modified by the double genes of Adiponectin and QSPHK1 can obviously inhibit the mRNA expression level of SREBP1c, and the inhibition activity of the culture supernatant is obviously higher than that of the culture supernatants of MSC-A and MSC-QS. It can be seen that the adipoectin and QSPHK1 double-gene modified MSC cells exhibited significant synergy in regulating lipid metabolism.

(2) Protein level

The expression levels of sterol metabolism genes SREBP1 and SREBP2 (protein expression levels of SREBP1 and SREBP2 in fig. 5) were measured by Western Blot using mesenchymal stem cells without any gene modification as common controls (labeled as a control group) for the cells (MSC-QS, MSC-A, MSC-QS, and MSC) of example 1 and comparative examples 1 to 3, and the results are shown in fig. 5.

As can be seen from FIG. 5, the MSC-AQS group can significantly reduce the expression of cholesterol synthesis genes SREBP1 and SREBP2, thereby inhibiting the synthesis of cholesterol.

Experimental example 3 Effect on the expression levels of lipid metabolism-controlling genes p-AMPK and AMPK

The results of Western Blot analysis of the expression of the protein levels of lipid metabolism-controlling genes p-AMPK and AMPK in the cells (MSC-QS, MSC-A, MSC-QS and MSC) of example 1 and comparative examples 1-3 using the mesenchymal stem cells without any genetic modification as common controls (labeled as control group) are shown in FIG. 6.

As can be seen from FIG. 6, the MSC-AQS group can significantly increase the phosphorylation level of lipid metabolism regulatory gene AMPK, thereby enhancing the degradation of lipid.

Experimental example 4 evaluation of in vivo biological Activity

(1) Experimental groups

25 male C57BL/6J mice induced by a high-fat high-sugar diet (mice purchased from sbeft biotechnology limited) were selected for 17-18 weeks. The experiment is divided into: control group (saline), MSC group (cells), MSC-a group (cells), MSC-AQS group (cells), and MSC-Q group (cells), for a total of 5 groups, each group consisting of 5 mice, with the mice without any treatment being the wild group.

(2) Treatment regimens

The administration time is as follows: the cell group required injection of cells every 7 days for a total of 3 cells.

The dose for each group:

control group (saline group): each mouse was injected with 100. mu.l of physiological saline each time, and was intraperitoneally injected.

Cell group: the injection amount per mouse is 1 × 10 ⁶ Cells/100 ul, once every 7 days, were injected intraperitoneally.

(3) Detecting the index

Measurement of fasting glucose and random body weight: fasting blood glucose was measured 6 hours after the mice were starved, and random body weights were measured regularly weekly after the mice were returned to normal diet. And (5) drawing a weight change curve according to the average blood sugar and the weight of the mouse.

Detecting serum biochemical indexes: after 28 days of treatment, blood is taken from the eyeballs of the mice, serum is separated by centrifugation at 3000rpm for 10min, and the samples are sent to Beijing and Doubbo medical laboratory Co., Ltd for detection of Triglyceride (TG), total cholesterol (TG), High Density Lipoprotein (HDL) and Low Density Lipoprotein (LDL).

Hematoxylin-eosin (HE) staining of animal tissues: after 28 days of treatment, the mice were sacrificed after neck amputation, a median abdominal incision was made, the abdomen was fully exposed, the liver and abdominal subcutaneous fat of the mice were removed, fixed with 4% paraformaldehyde in a volume 10 times that of the abdomen for 24 hours, and paraffin embedding and hematoxylin-eosin (HE) staining were performed.

(4) The result of the detection

Model mice were treated with 3 cells and fasting blood glucose and body weight of the mice were measured once a week from the first treatment, and two weeks of data observation continued after the treatment was stopped. The average body weight and blood glucose profiles of the mice are shown in fig. 7 and 8, respectively.

The results show that after three times of treatment, the mice of the MSC-AQS cell treatment group have obviously reduced random body weight (P <0.05) compared with the mice of the control group, and are obviously superior to the mice of the MSC-A and MSC-QS groups; meanwhile, compared with the control group mice, the mice in the MSC-AQS cell treatment group have extremely obvious difference of fasting blood glucose (P <0.01), and have obvious treatment effect in the third cell treatment, and are obviously superior to the MSC-A and MSC-QS groups.

After 3 times of treatment, the body type of mice in the MSC-AQS cell treatment group is also obviously smaller than that of control mice (figure 9), the subcutaneous fat content of the abdomen is obviously reduced, HE (high-grade hematoxylin) staining of a liver histopathology section shows that the liver of the Con mice has severe steatosis, the liver tissue is filled with fat cells, the liver cells are necrotized in large scale, and the steatosis of the liver is obviously improved and the fibrosis degree is reduced after the MSC-AQS cell treatment. Abdominal subcutaneous fat HE staining (lower part of fig. 10) showed that the Con group mice were over-expanded in adipocytes and more irregular in nuclei; following MSC-AQS treatment, the adipocyte volume was significantly reduced.

After 3 times of treatment, biochemical detection of mouse serum shows that substances such as TC (total cholesterol), LDL (low density lipoprotein), HDL (high density lipoprotein) and the like in the mouse serum are remarkably reduced after MSC-AQS treatment, and are superior to MSC-A and MSC-QS groups (figure 11).

Therefore, the MSC-AQS can obviously reduce the weight and the blood sugar of a mouse, simultaneously can reduce the blood fat content, relieve the occurrence of fatty liver and improve the abnormal blood fat metabolism, has better curative effect than MSC-A and MSC-QS groups, and has obvious synergistic effect. Therefore, the double-gene modified mesenchymal stem cells of the present invention are particularly useful for treating metabolic disorders.

The invention has been described in detail with reference to specific embodiments and illustrative examples, but the description is not intended to be construed in a limiting sense. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its embodiments without departing from the spirit and scope of the present invention, which fall within the scope of the present invention.

SEQUENCE LISTING

<110> Beijing Shuangyin Biotechnology Co., Ltd

<120> double-gene modified stem cell and application thereof

<130> 2020

<160> 16

<170> PatentIn version 3.5

<210> 1

<211> 244

<212> PRT

<213> Adiponectin amino acid sequence (Artificial sequence)

<400> 1

Met Leu Leu Leu Gly Ala Val Leu Leu Leu Leu Ala Leu Pro Gly His

1 5 10 15

Asp Gln Glu Thr Thr Thr Gln Gly Pro Gly Val Leu Leu Pro Leu Pro

20 25 30

Lys Gly Ala Cys Thr Gly Trp Met Ala Gly Ile Pro Gly His Pro Gly

35 40 45

His Asn Gly Ala Pro Gly Arg Asp Gly Arg Asp Gly Thr Pro Gly Glu

50 55 60

Lys Gly Glu Lys Gly Asp Pro Gly Leu Ile Gly Pro Lys Gly Asp Ile

65 70 75 80

Gly Glu Thr Gly Val Pro Gly Ala Glu Gly Pro Arg Gly Phe Pro Gly

85 90 95

Ile Gln Gly Arg Lys Gly Glu Pro Gly Glu Gly Ala Tyr Val Tyr Arg

100 105 110

Ser Ala Phe Ser Val Gly Leu Glu Thr Tyr Val Thr Ile Pro Asn Met

115 120 125

Pro Ile Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn His Tyr Asp

130 135 140

Gly Ser Thr Gly Lys Phe His Cys Asn Ile Pro Gly Leu Tyr Tyr Phe

145 150 155 160

Ala Tyr His Ile Thr Val Tyr Met Lys Asp Val Lys Val Ser Leu Phe

165 170 175

Lys Lys Asp Lys Ala Met Leu Phe Thr Tyr Asp Gln Tyr Gln Glu Asn

180 185 190

Asn Val Asp Gln Ala Ser Gly Ser Val Leu Leu His Leu Glu Val Gly

195 200 205

Asp Gln Val Trp Leu Gln Val Tyr Gly Glu Gly Glu Arg Asn Gly Leu

210 215 220

Tyr Ala Asp Asn Asp Asn Asp Ser Thr Phe Thr Gly Phe Leu Leu Tyr

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His Asp Thr Asn

<210> 2

<211> 408

<212> PRT

<213> QSPHK1 amino acid sequence (Artificial sequence)

<400> 2

Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Leu Ala Gly Leu Cys

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Cys Leu Val Pro Val Ser Leu Ala Met Asp Pro Ala Gly Gly Pro Arg

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Gly Val Leu Pro Arg Pro Cys Arg Val Leu Val Leu Leu Asn Pro Arg

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Gly Gly Gln Gly Gln Ala Leu Gln Leu Phe Arg Ser His Val Gln Pro

50 55 60

Leu Leu Ala Glu Ala Glu Ile Ser Phe Thr Leu Met Leu Thr Glu Arg

65 70 75 80

Arg Asn His Ala Arg Glu Leu Val Arg Ser Glu Glu Leu Gly Arg Trp

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Asp Ala Leu Val Val Met Ser Gly Asp Gly Leu Met His Glu Val Val

100 105 110

Asn Gly Leu Met Glu Arg Pro Asp Trp Glu Thr Ala Ile Gln Lys Pro

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Leu Cys Ser Leu Pro Ala Gly Ser Gly Asn Ala Leu Ala Ala Ser Leu

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Asn His Tyr Ala Gly Tyr Glu Gln Val Thr Asn Glu Asp Leu Leu Thr

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Asn Cys Thr Leu Leu Leu Cys Arg Arg Leu Leu Ser Pro Met Asn Leu

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Leu Ser Leu His Thr Ala Ser Gly Leu Arg Leu Phe Ser Val Leu Ser

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Leu Ala Trp Gly Phe Ile Ala Asp Val Asp Leu Glu Ser Glu Lys Tyr

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Arg Arg Leu Gly Glu Met Arg Phe Thr Leu Gly Thr Phe Leu Arg Leu

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Ala Ala Leu Arg Thr Tyr Arg Gly Arg Leu Ala Tyr Leu Pro Val Gly

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Arg Val Gly Ser Lys Thr Pro Ala Ser Pro Val Val Val Gln Gln Gly

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Pro Val Asp Ala His Leu Val Pro Leu Glu Glu Pro Val Pro Ser His

260 265 270

Trp Thr Val Val Pro Asp Glu Asp Phe Val Leu Val Leu Ala Leu Leu

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His Ser His Leu Gly Ser Glu Met Phe Ala Ala Pro Met Gly Arg Cys

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Ala Ala Gly Val Met His Leu Phe Tyr Val Arg Ala Gly Val Ser Arg

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Ala Met Leu Leu Arg Leu Phe Leu Ala Met Glu Lys Gly Arg His Met

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Glu Tyr Glu Cys Pro Tyr Leu Val Tyr Val Pro Val Val Ala Phe Arg

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Leu Glu Pro Lys Asp Gly Lys Gly Val Phe Ala Val Asp Gly Glu Leu

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Met Val Ser Glu Ala Val Gln Gly Gln Val His Pro Asn Tyr Phe Trp

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<210> 3

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<213> Adiponectin nucleotide sequence (Artificial sequence)

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atgctgttgc tgggagctgt tctactgcta ttagctctgc ccggtcatga ccaggaaacc 60

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gtgctcctgc atctggaggt gggcgaccaa gtctggctcc aggtgtatgg ggaaggagag 660

cgtaatggac tctatgctga taatgacaat gactccacct tcacaggctt tcttctctac 720

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<213> QSPHK1 nucleotide sequence (Artificial sequence)

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gtgctggtgc tgctgaaccc gcgcggcggc cagggccagg ccttgcagct cttccggagt 180

cacgtgcagc cccttttggc tgaggctgaa atctccttca cgctgatgct cactgagcgg 240

cggaaccacg cgcgggagct ggtgcggtcg gaggagctgg gccgctggga cgctctggtg 300

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aactgcacgc tattgctgtg ccgccggctg ctgtcaccca tgaacctgct gtctctgcac 540

acggcttcgg ggctgcgcct cttctctgtg ctcagcctgg cctggggctt cattgctgat 600

gtggacctag agagtgagaa gtatcggcgt ctgggggaga tgcgcttcac tctgggcacc 660

ttcctgcgtc tggcagccct gcgcacctac cgcggccgac tggcttacct ccctgtagga 720

agagtgggtt ccaagacacc tgcctccccc gttgtggtcc agcagggccc ggtagatgca 780

caccttgtgc cactggagga gccagtgccc tctcactgga cagtggtgcc cgacgaggac 840

tttgtgctag tcctggcact gctgcactcg cacctgggca gtgagatgtt tgctgcaccc 900

atgggccgct gtgcagctgg cgtcatgcat ctgttctacg tgcgggcggg agtgtctcgt 960

gccatgctgc tgcgcctctt cctggccatg gagaagggca ggcatatgga gtatgaatgc 1020

ccctacttgg tatatgtgcc cgtggtcgcc ttccgcttgg agcccaagga tgggaaaggt 1080

gtgtttgcag tggatgggga attgatggtt agcgaggccg tgcagggcca ggtgcaccca 1140

aactacttct ggatggtcag cggttgcgtg gagcccccgc ccagctggaa gccccagcag 1200

atgccaccgc cagaagagcc cttatga 1227

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Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

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<210> 7

<211> 2013

<212> DNA

<213> Adiponectin-QSPHK1 nucleotide sequence (Artificial sequence)

<400> 7

atgctgttgc tgggagctgt tctactgcta ttagctctgc ccggtcatga ccaggaaacc 60

acgactcaag ggcccggagt cctgcttccc ctgcccaagg gggcctgcac aggttggatg 120

gcgggcatcc cagggcatcc gggccataat ggggccccag gccgtgatgg cagagatggc 180

acccctggtg agaagggtga gaaaggagat ccaggtctta ttggtcctaa gggagacatc 240

ggtgaaaccg gagtacccgg ggctgaaggt ccccgaggct ttccgggaat ccaaggcagg 300

aaaggagaac ctggagaagg tgcctatgta taccgctcag cattcagtgt gggattggag 360

acttacgtta ctatccccaa catgcccatt cgctttacca agatcttcta caatcagcaa 420

aaccactatg atggctccac tggtaaattc cactgcaaca ttcctgggct gtactacttt 480

gcctaccaca tcacagtcta tatgaaggat gtgaaggtca gcctcttcaa gaaggacaag 540

gctatgctct tcacctatga tcagtaccag gaaaataatg tggaccaggc ctccggctct 600

gtgctcctgc atctggaggt gggcgaccaa gtctggctcc aggtgtatgg ggaaggagag 660

cgtaatggac tctatgctga taatgacaat gactccacct tcacaggctt tcttctctac 720

catgacacca acgagggcag aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct 780

ggacctatgc cgtcttctgt ctcgtggggc atcctcctgc tggcaggcct gtgctgcctg 840

gtccctgtct ccctggctat ggacccagcg ggcggccccc ggggcgtgct cccgcggccc 900

tgccgcgtgc tggtgctgct gaacccgcgc ggcggccagg gccaggcctt gcagctcttc 960

cggagtcacg tgcagcccct tttggctgag gctgaaatct ccttcacgct gatgctcact 1020

gagcggcgga accacgcgcg ggagctggtg cggtcggagg agctgggccg ctgggacgct 1080

ctggtggtca tgtctggaga cgggctgatg cacgaggtgg tgaacgggct catggagcgg 1140

cctgactggg agaccgccat ccagaagccc ctgtgtagcc tcccagcagg ctctggcaac 1200

gcgctggcag cttccttgaa ccattatgct ggctatgagc aggtcaccaa tgaagacctc 1260

ctgaccaact gcacgctatt gctgtgccgc cggctgctgt cacccatgaa cctgctgtct 1320

ctgcacacgg cttcggggct gcgcctcttc tctgtgctca gcctggcctg gggcttcatt 1380

gctgatgtgg acctagagag tgagaagtat cggcgtctgg gggagatgcg cttcactctg 1440

ggcaccttcc tgcgtctggc agccctgcgc acctaccgcg gccgactggc ttacctccct 1500

gtaggaagag tgggttccaa gacacctgcc tcccccgttg tggtccagca gggcccggta 1560

gatgcacacc ttgtgccact ggaggagcca gtgccctctc actggacagt ggtgcccgac 1620

gaggactttg tgctagtcct ggcactgctg cactcgcacc tgggcagtga gatgtttgct 1680

gcacccatgg gccgctgtgc agctggcgtc atgcatctgt tctacgtgcg ggcgggagtg 1740

tctcgtgcca tgctgctgcg cctcttcctg gccatggaga agggcaggca tatggagtat 1800

gaatgcccct acttggtata tgtgcccgtg gtcgccttcc gcttggagcc caaggatggg 1860

aaaggtgtgt ttgcagtgga tggggaattg atggttagcg aggccgtgca gggccaggtg 1920

cacccaaact acttctggat ggtcagcggt tgcgtggagc ccccgcccag ctggaagccc 1980

cagcagatgc caccgccaga agagccctta tga 2013

<210> 8

<211> 670

<212> PRT

<213> Adiponectin-QSPHK1 amino acid sequence (Artificial sequence)

<400> 8

Met Leu Leu Leu Gly Ala Val Leu Leu Leu Leu Ala Leu Pro Gly His

1 5 10 15

Asp Gln Glu Thr Thr Thr Gln Gly Pro Gly Val Leu Leu Pro Leu Pro

20 25 30

Lys Gly Ala Cys Thr Gly Trp Met Ala Gly Ile Pro Gly His Pro Gly

35 40 45

His Asn Gly Ala Pro Gly Arg Asp Gly Arg Asp Gly Thr Pro Gly Glu

50 55 60

Lys Gly Glu Lys Gly Asp Pro Gly Leu Ile Gly Pro Lys Gly Asp Ile

65 70 75 80

Gly Glu Thr Gly Val Pro Gly Ala Glu Gly Pro Arg Gly Phe Pro Gly

85 90 95

Ile Gln Gly Arg Lys Gly Glu Pro Gly Glu Gly Ala Tyr Val Tyr Arg

100 105 110

Ser Ala Phe Ser Val Gly Leu Glu Thr Tyr Val Thr Ile Pro Asn Met

115 120 125

Pro Ile Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn His Tyr Asp

130 135 140

Gly Ser Thr Gly Lys Phe His Cys Asn Ile Pro Gly Leu Tyr Tyr Phe

145 150 155 160

Ala Tyr His Ile Thr Val Tyr Met Lys Asp Val Lys Val Ser Leu Phe

165 170 175

Lys Lys Asp Lys Ala Met Leu Phe Thr Tyr Asp Gln Tyr Gln Glu Asn

180 185 190

Asn Val Asp Gln Ala Ser Gly Ser Val Leu Leu His Leu Glu Val Gly

195 200 205

Asp Gln Val Trp Leu Gln Val Tyr Gly Glu Gly Glu Arg Asn Gly Leu

210 215 220

Tyr Ala Asp Asn Asp Asn Asp Ser Thr Phe Thr Gly Phe Leu Leu Tyr

225 230 235 240

His Asp Thr Asn Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val

245 250 255

Glu Glu Asn Pro Gly Pro Met Pro Ser Ser Val Ser Trp Gly Ile Leu

260 265 270

Leu Leu Ala Gly Leu Cys Cys Leu Val Pro Val Ser Leu Ala Met Asp

275 280 285

Pro Ala Gly Gly Pro Arg Gly Val Leu Pro Arg Pro Cys Arg Val Leu

290 295 300

Val Leu Leu Asn Pro Arg Gly Gly Gln Gly Gln Ala Leu Gln Leu Phe

305 310 315 320

Arg Ser His Val Gln Pro Leu Leu Ala Glu Ala Glu Ile Ser Phe Thr

325 330 335

Leu Met Leu Thr Glu Arg Arg Asn His Ala Arg Glu Leu Val Arg Ser

340 345 350

Glu Glu Leu Gly Arg Trp Asp Ala Leu Val Val Met Ser Gly Asp Gly

355 360 365

Leu Met His Glu Val Val Asn Gly Leu Met Glu Arg Pro Asp Trp Glu

370 375 380

Thr Ala Ile Gln Lys Pro Leu Cys Ser Leu Pro Ala Gly Ser Gly Asn

385 390 395 400

Ala Leu Ala Ala Ser Leu Asn His Tyr Ala Gly Tyr Glu Gln Val Thr

405 410 415

Asn Glu Asp Leu Leu Thr Asn Cys Thr Leu Leu Leu Cys Arg Arg Leu

420 425 430

Leu Ser Pro Met Asn Leu Leu Ser Leu His Thr Ala Ser Gly Leu Arg

435 440 445

Leu Phe Ser Val Leu Ser Leu Ala Trp Gly Phe Ile Ala Asp Val Asp

450 455 460

Leu Glu Ser Glu Lys Tyr Arg Arg Leu Gly Glu Met Arg Phe Thr Leu

465 470 475 480

Gly Thr Phe Leu Arg Leu Ala Ala Leu Arg Thr Tyr Arg Gly Arg Leu

485 490 495

Ala Tyr Leu Pro Val Gly Arg Val Gly Ser Lys Thr Pro Ala Ser Pro

500 505 510

Val Val Val Gln Gln Gly Pro Val Asp Ala His Leu Val Pro Leu Glu

515 520 525

Glu Pro Val Pro Ser His Trp Thr Val Val Pro Asp Glu Asp Phe Val

530 535 540

Leu Val Leu Ala Leu Leu His Ser His Leu Gly Ser Glu Met Phe Ala

545 550 555 560

Ala Pro Met Gly Arg Cys Ala Ala Gly Val Met His Leu Phe Tyr Val

565 570 575

Arg Ala Gly Val Ser Arg Ala Met Leu Leu Arg Leu Phe Leu Ala Met

580 585 590

Glu Lys Gly Arg His Met Glu Tyr Glu Cys Pro Tyr Leu Val Tyr Val

595 600 605

Pro Val Val Ala Phe Arg Leu Glu Pro Lys Asp Gly Lys Gly Val Phe

610 615 620

Ala Val Asp Gly Glu Leu Met Val Ser Glu Ala Val Gln Gly Gln Val

625 630 635 640

His Pro Asn Tyr Phe Trp Met Val Ser Gly Cys Val Glu Pro Pro Pro

645 650 655

Ser Trp Lys Pro Gln Gln Met Pro Pro Pro Glu Glu Pro Leu

660 665 670

<210> 9

<211> 50

<212> DNA

<213> primer EF1-BamHI-Adiponectin-F (Artificial sequence)

<400> 9

tccatttcag gtgtcgtgag gaattggatc cgccgccacc atgctgttgc 50

<210> 10

<211> 53

<212> DNA

<213> primer SPHK1-SalI-WPRE-R (Artificial sequence)

<400> 10

ttgtaatcca gaggttgatt gtcgactcat aagggctctt ctggcggtgg cat 53

<210> 11

<211> 50

<212> DNA

<213> primer T2A-SPHK1-F (Artificial sequence)

<400> 11

tgacgtcgag gagaatcctg gacctatgcc gtcttctgtc tcgtggggca 50

<210> 12

<211> 50

<212> DNA

<213> primer T2A-SPHK1-R (Artificial sequence)

<400> 12

tgccccacga gacagaagac ggcataggtc caggattctc ctcgacgtca 50

<210> 13

<211> 20

<212> DNA

<213> primer SREBP1C-F (Artificial sequence)

<400> 13

cactgtgacc tcgcagatcc 20

<210> 14

<211> 20

<212> DNA

<213> primer SEBP1C-R (Artificial sequence)

<400> 14

ataggcagct tctccgcatc 20

<210> 15

<211> 18

<212> DNA

<213> primer beta-Actin-F (Artificial sequence)

<400> 15

cctggcaccc agcacaat 18

<210> 16

<211> 18

<212> DNA

<213> primer beta-Actin-R (Artificial sequence)

<400> 16

gggccggact cgtcatac 18

Claims

1. A method for preparing a double-gene modified stem cell, which comprises the following steps:

step 1, obtaining a first exogenous nucleic acid and a second exogenous nucleic acid, carrying out enzyme digestion, and connecting with a vector plasmid pCDH-EF1 to obtain a recombinant plasmid pCDH-AQS, wherein the structure of the recombinant plasmid pCDH-AQS is shown in figure 1;

the first exogenous nucleic acid is an adiponectin gene, and the nucleotide sequence of the first exogenous nucleic acid is shown as SEQ ID NO:3 is shown in the specification;

the second exogenous nucleic acid is mutated sphingosine kinase1 gene, the mutated sphingosine kinase1 gene is obtained by mutating two Ks in a GKGK structural domain of the mutated sphingosine kinase1 gene into arginine or glutamine respectively, and the nucleotide sequence of the second exogenous nucleic acid is shown as SEQ ID NO:4 is shown in the specification;

the first exogenous nucleic acid and the second exogenous nucleic acid are contained in the same expression vector;

the first exogenous nucleic acid and the second exogenous nucleic acid are connected through a sequence for coding self-cutting peptide, and the sequence for coding self-cutting peptide is shown as SEQ ID NO. 6;

the nucleotide sequences of the first exogenous nucleic acid and the second exogenous nucleic acid after connection are shown as SEQ ID NO. 7; the first exogenous nucleic acid and the second exogenous nucleic acid after connection encode proteins with amino acid sequences shown as SEQ ID NO. 8;

step 2, transfecting the recombinant plasmid and the packaging plasmid with a lentivirus packaging cell to obtain a recombinant lentivirus vector containing first exogenous nucleic acid and second exogenous nucleic acid;

step 2 comprises the following substeps:

step 2-1, culturing packaging cells; the packaging cell is a 293T cell;

step 2-2, transfecting a packaging cell;

mixing the packaging plasmid with the recombinant plasmid pCDH-AQS obtained in the step 1, adding a transfection reagent after incubation, and incubating at room temperature to form a DNA-liposome transfection complex; uniformly mixing the DNA-liposome transfection compound with the packaging cells, and culturing to obtain a recombinant lentiviral vector;

the packaging plasmids are Helper1 plasmids, Helper2 plasmids and Helper3 plasmids, and the mass ratio of the recombinant plasmids to the Helper1 plasmids to the Helper2 plasmids to the Helper3 plasmids is 2:1:1: 1;

step 3, transfecting the obtained recombinant lentiviral vector to mesenchymal stem cells to obtain double-gene modified stem cells;

the mesenchymal stem cells are derived from umbilical cord.

2. Use of the double-genetically-modified stem cell prepared by the method of claim 1 in the preparation of a medicament for treating a metabolic disorder selected from the group consisting of obesity, type II diabetes, dyslipidemia, insulin resistance, and atherosclerosis.