CN118291386A - A stem cell expressing FGF1 protein and FGF21 protein and its use - Google Patents

Abstract

The present invention relates to the technical field of biomedicine, and more specifically, it relates to a stem cell expressing FGF1 protein and FGF21 protein and uses thereof. The main points of the technical solution are: the stem cell expressing FGF1 protein and FGF21 protein of the present invention has an amino acid sequence as shown in SEQ ID NO:8, and has significant advantages; the modified stem cells of the present invention exhibit significant synergistic effects, can significantly reduce blood sugar and blood lipids of test animals, reduce body weight, and can be safely administered to human subjects without inducing immunogenic reactions; the modified stem cells of the present invention can be used to treat metabolic diseases and have great clinical value.

Description

A stem cell expressing FGF1 protein and FGF21 protein and its use

Technical Field

The present invention relates to the technical field of biomedicine, and more specifically, to a stem cell expressing FGF1 protein and FGF21 protein and a use thereof.

Background technique

Metabolic diseases represented by diabetes are closely related to some endogenous molecules involved in metabolic regulation. There are three main types of endogenous molecules that have been discovered: one is hormones, including insulin, glucagon, GLP1, glucocorticoids, etc.; the second is cell growth factors with hormone-like functions, including FGF1, FGF19, FGF21 and FGF23, etc.; the third is enzymes involved in metabolic regulation or cell signal transduction, including SPK1, PI3K, HSL, etc.

Insufficient secretion, reduced activity or functional defects of endogenous hormones, cytokines or enzymes are closely related to the occurrence of metabolic diseases. For example, insulin resistance and relative insufficient secretion are the core causes of diabetes. Therefore, these key molecules for regulating the balance of glucose and lipid metabolism are also the focus of drug development for the treatment of metabolic syndromes such as diabetes. FGF1 protein and FGF21 protein have hormone-like effects and play an important role in regulating glucose and lipid metabolism.

Fibroblast growth factor (FGF) is an important type of tissue growth factor, including 22 family members. They have hormone-like effects and play an important role in the regulation of glucose and lipid metabolism. Among them, FGF1 binds to tyrosine kinase receptors through heparin sulfate proteoglycans and is expressed in the embryonic pancreas to play a corresponding role. It can be applied to wounds or ulcers, neuropathy, diabetes, etc., and can have a good therapeutic effect. FGF1 is involved in embryonic development, wound repair, etc., and can promote tissue repair and wound healing in patients with trauma, ulcers, etc.; FGF1 can act on nerve cells, protect neurons from the influence of neurodegenerative diseases, and has a certain therapeutic effect on diseases such as optic atrophy and sensorine deafness; FGF1 can inhibit the hypothalamus-pituitary-adrenal axis, reduce the synthesis of acetyl coenzyme A in the liver, and then increase insulin sensitivity. It can play a hypoglycemic role in people with high blood sugar levels and diabetes. Among them, FGF21 is mainly synthesized by the liver, acts on adipose tissue through endocrine pathways, and regulates the metabolism of glucose and lipids. As a lipid metabolism regulatory molecule, FGF21 has been developed as a drug for the treatment of metabolic diseases, as an insulin sensitizer, improving metabolism in patients with obesity and type 2 diabetes, reducing hepatic steatosis and fibrosis in patients with non-alcoholic steatohepatitis, etc. It is widely used in the prevention and rehabilitation of metabolic diseases such as liver fat and lipid metabolism, as well as cardiovascular diseases.

Therefore, there is an urgent need for a new, reliable product and method that can fundamentally treat metabolic diseases.

Summary of the invention

In view of the deficiencies in the prior art, the object of the present invention is to provide a stem cell expressing FGF1 protein and FGF21 protein and uses thereof.

The above technical objectives of the present invention are achieved through the following technical solutions: A modified stem cell, comprising:

(1) FGF1 protein, which is selected from: FGF1 protein or its variant;

(2) FGF21 protein, which is selected from: FGF21 protein or its variant;

Wherein, the FGF1 variant has at least 90% sequence identity with wild-type FGF1 and has FGF1 activity;

The FGF21 variant has at least 90% sequence identity with wild-type FGF21 and has FGF21 activity.

In one embodiment, the FGF1 has an amino acid sequence as shown in SEQ ID NO: 1;

The FGF21 has an amino acid sequence as shown in SEQ ID NO:2.

In one embodiment, the FGF1 variant has an amino acid sequence as shown in SEQ ID NO: 3;

The FGF21 variant has the amino acid sequence shown in SEQ ID NO:4.

In one embodiment, the stem cells express: FGF1 protein having an amino acid sequence as shown in SEQ ID NO: 3;

The FGF21 protein has the amino acid sequence shown in SEQ ID NO:4.

In one embodiment, it also includes:

(1) an exogenous nucleic acid expressing FGF1 protein, which comprises a nucleotide sequence encoding the FGF1 protein;

(2) An exogenous nucleic acid expressing FGF21 protein, which comprises a nucleotide sequence encoding the FGF21 protein.

In one embodiment, the exogenous nucleic acid expressing FGF1 protein is linked to the exogenous nucleic acid expressing FGF21 protein via a nucleotide sequence encoding a self-cleaving peptide;

The self-cleaving peptide has an amino acid sequence as shown in SEQ ID NO:5.

The above-mentioned method for preparing modified stem cells comprises the following steps:

Isolation of mesenchymal stem cells;

Constructing lentivirus carrying FGF1-FGF21 genes;

The lentivirus infects the mesenchymal stem cells to obtain FGF1-FGF21-stem cells that stably express FGF1 and FGF21.

In one embodiment, the mesenchymal stem cells are derived from one of the umbilical cord or the placenta.

A pharmaceutical composition comprising any of the above stem cells;

The pharmaceutical composition is an injection.

A use of a modified stem cell and a pharmaceutical composition as described above for preparing a medicament for treating a metabolic disorder in a subject, wherein the metabolic disorder is selected from obesity, type II diabetes, dyslipidemia, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, atherosclerosis, coronary heart disease, and hypertension;

The subject is a human.

The above-mentioned stem cells expressing FGF1 protein and FGF21 protein and their use have the following beneficial effects:

Firstly, the stem cells expressing FGF1 protein and FGF21 protein of the present invention have the amino acid sequence shown in SEQ ID NO: 8, which has significant advantages.

Secondly, the modified stem cells of the present invention exhibit a significant synergistic effect, can significantly reduce blood sugar and blood lipids and reduce body weight in test animals, and can be safely administered to human subjects without inducing immunogenic reactions.

Thirdly, the modified stem cells of the present invention can be used to treat metabolic diseases and have great clinical value.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Flow cytometry of stem cells after transformation;

Figure 2 is a fasting blood glucose change trend chart of volunteers (sample A, sample B, sample C, sample D, sample E);

Figure 3 is a graph showing the changing trend of low-density cholesterol in volunteers (sample A, sample B, sample C, sample D, sample E);

Figure 4 is a graph showing the trend of changes in serum triglycerides of volunteers (sample A, sample B, sample C, sample D, sample E).

Sequence information

Information on the partial sequences involved in the present invention is provided in Table 1 below.

Table 1: Description of sequences

SEQ ID NO describe 1 Wild-type FGF1 amino acid sequence 2 Wild-type FGF21 amino acid sequence 3 FGF1 variant amino acid sequences 4 FGF21 variant amino acid sequences 5 Self-cleaving peptide (2A) amino acid sequence 6 Self-cleaving peptide (2A) nucleic acid sequence 7 Signal peptide 8 FGF1-FGF21 amino acid sequences

Detailed ways

The present invention is described in detail below in conjunction with the accompanying drawings and examples. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art and commercially available common instruments and reagents, and references can be made to the Molecular Cloning Experiment Guide (4th Edition) (Science Press) and the relevant experimental guidelines of CFDA and the manufacturer's instructions of the corresponding instruments and reagents.

A modified stem cell comprising:

(1) FGF1 protein, which is selected from: FGF1 protein or its variant;

(2) FGF21 protein, which is selected from: FGF21 protein or its variant;

Wherein, the FGF1 variant has at least 90% sequence identity with wild-type FGF1 and has FGF1 activity, and FGF1 has an amino acid sequence as shown in SEQ ID NO: 1;

The FGF21 variant has at least 90% sequence identity with wild-type FGF21 and has FGF21 activity. FGF21 has the amino acid sequence shown in SEQ ID NO:2.

Further, the FGF1 variant has the amino acid sequence shown in SEQ ID NO:3;

The FGF21 variant has the amino acid sequence shown in SEQ ID NO:4.

At the same time, the stem cells express: FGF1 protein having an amino acid sequence as shown in SEQ ID NO:3;

The FGF21 protein has the amino acid sequence shown in SEQ ID NO:4.

Among them, stem cells also include:

(1) an exogenous nucleic acid expressing FGF protein, which comprises a nucleotide sequence encoding FGF1 protein;

(2) An exogenous nucleic acid expressing FGF21 protein, which comprises a nucleotide sequence encoding FGF21 protein.

Further, the exogenous nucleic acid expressing the FGF1 protein is linked to the exogenous nucleic acid expressing the FGF21 protein via a nucleotide sequence encoding a self-cleaving peptide;

Preferably, the self-cleaving peptide is a 2A peptide;

The self-cleaving peptide is the 2A peptide of the beta-tetrasomal virus (TaV) of the Acanthopanax punctatus;

The self-cleaving peptide has the amino acid sequence shown in SEQ ID NO:5.

Example 1

The preparation of stem cells expressing FGF1 and FGF21 comprises the following steps:

Mesenchymal stem cells are isolated from the umbilical cord or placenta;

In this example, mesenchymal stem cells were isolated from umbilical cord;

Specifically, take 6 50ml centrifuge tubes, add 20ml of normal saline containing 1% double antibody to 3 tubes, and add 20ml of normal saline without double antibody to the other 3 tubes; wash the umbilical cord: take out the umbilical cord into a 10cm culture dish, and cut it into several small pieces, and put them into normal saline containing double antibodies and then normal saline without double antibodies in sequence for thorough washing; take the washed umbilical cord and put it into a new 10cm culture dish, remove the artery and vein, tear off the Wharton's jelly and cut it into pieces; add normal saline without double antibodies to 45ml. 500g, 5min centrifugal washing twice; divide the shredded Wharton's jelly equally into T175 culture bottles added with 25ml complete culture medium; place the culture bottle flat so that the tissue blocks are as evenly distributed as possible on the entire bottom surface, and place the culture bottle in an incubator with saturated humidity of 37°C and 5% CO2 for cultivation; after 5-7 days, discard 13ml of culture medium in the culture bottle and add 13ml of new culture medium; place the culture bottle flat so that the tissue blocks are as evenly distributed as possible on the entire bottom surface, and place the culture bottle in an incubator with saturated humidity of 37°C and 5% CO2 for cultivation; discard the culture medium and tissue blocks in the culture bottle, add physiological saline to wash twice; add 5ml of trypsin, let it stand for 1min, and observe under a microscope until most cells become round. Add 10ml culture medium to stop digestion; aspirate into a 50ml centrifuge tube, centrifuge at 500g for 5min; discard supernatant, add culture medium to count; inoculate at 5000 cells/cm2, add new culture medium, and mark as P1; place the culture bottle flat so that the cells are as evenly distributed as possible on the entire bottom surface, and place the culture bottle in an incubator with saturated humidity at 37℃ and 5% CO2 for culture; discard the culture medium in the culture bottle, add physiological saline to wash twice; add 5mL pancreatin, let stand for 1min, and observe under a microscope until most cells become round. Add 10ml culture medium to stop digestion; aspirate into a 50ml centrifuge tube, centrifuge at 500g for 5min; discard supernatant, add culture medium to count; inoculate at 5000 cells/cm2, and add new culture medium. Mark the corresponding name generation; place the culture bottle flat so that the cells are as evenly distributed as possible on the entire bottom surface, and place the culture bottle in an incubator with saturated humidity at 37℃ and 5% CO2 for culture.

Constructing lentivirus carrying FGF1-FGF21 genes;

Specifically, take out a frozen 293 stem cell from liquid nitrogen and quickly put it in a 37°C water bath until the ice disappears, add dropwise to a 15ml centrifuge tube containing 5ml preheated culture medium, centrifuge at 1200rpm for 3min, discard the supernatant, resuspend the cells with 293T culture medium (10% FBS+DMEM) and inoculate them into a T75 culture flask, and culture at 37°C and 5% CO ₂ saturation. When the cell confluence reaches more than 90%, discard the old culture medium, add 5ml sterile PBS solution, shake gently, wash the cells and discard the PBS solution, add 2ml 0.25% trypsin-EDTA digestion solution, digest for 1-2min until the cells are completely digested. Add serum-containing culture medium to terminate digestion, centrifuge the cell suspension at 1200rpm for 3min, resuspend the centrifuged cells with culture medium, inoculate 1.2×10 ⁷ cells per T75 culture flask for packaging lentivirus, culture at 37°C and 5% CO ₂ saturation, 20ml culture medium/dish.

2h before transfection, replace the 293 stem cell culture medium with 17ml DMEM culture medium, add 1ml preheated DMEM culture medium to sterile centrifuge tube A, then add the pCDH-FG plasmid, pHelper1 plasmid and pHelper2 plasmid prepared in 1.2 above (pCDH-FG: pHelper1: pHelper2 = 1:1:1, a total of 54μg, pHelper1 and pHelper2 plasmids are auxiliary plasmids for lentiviral packaging), and mix well. Add 1ml preheated DMEM culture medium to sterile centrifuge tube B, then add 108μl Lipofectamin2000 solution and mix well. Incubate tubes A and B at room temperature for 5 minutes. Add the liquid in tube B to tube A dropwise, mix well, and incubate at room temperature for 20min to form a DNA-liposome transfection complex.

The DNA-liposome mixture was transferred to the 293 stem cells that had been replaced with the medium in advance, mixed, and cultured at 37°C and 5% CO ₂ saturation. After 6-8 hours of culture, the culture medium containing the transfection mixture was discarded, and 20 ml of preheated DMEM culture medium containing 5% FBS was added to each dish of cells, and cultured at 37°C and 5% CO ₂ saturation. After 24h and 48h after the medium was replaced, the supernatant was collected and temporarily stored at 4°C, and 20 ml of fresh culture medium was replaced. The collected liquid was centrifuged at 4°C and 3500rpm for 15min, the precipitate was discarded, and the supernatant was concentrated with an ultrafiltration column (10KD) to obtain a lentiviral vector (Lenti-FGF1/FGF21) carrying FGF1/FGF21; at the same time, the virus titer was determined, and the virus was diluted to 1×109TU/ml according to the determination results, and the aliquoted virus was stored at -80°C.

Mesenchymal stem cells were infected with lentivirus to obtain FGF1-FGF21-stem cells that stably expressed FGF1 and FGF21;

The specific operations are as follows:

The cultured stem cells were added into a T75 culture flask, and cultured in 20 ml serum-free medium at 37°C and 5% CO ₂ saturation.

Each T75 culture flask was inoculated with 2-2.5×106 cells. The next day after inoculation, the culture medium of the cells was removed and replaced with serum-free culture medium. 20 ml culture medium/flask, 16 μl Polybrene was added, and the previously obtained Lenti-FGF1/FGF21 lentivirus (titer of 1×109TU/ml) was added at a multiplicity of infection of 30 MOIs. The cells were cultured at 37°C and 5% CO ₂ saturation for 6-8 hours. After 6-8 hours, the a-MEM culture medium containing the virus was discarded and replaced with serum-free culture medium. The cells were cultured at 37°C and 5% CO ₂ saturation for 2-3 days. The cells were subcultured at a subculture ratio of 1:3, and the serum-free culture medium was cultured at 37°C and 5% CO ₂ for 12 days. The stem cells modified with the FGF1/FGF21 gene were named FGF-MSC.

Example 2

Evaluation of biological activity of FGF1/FGF21 modified stem cells in vivo

experimental design

According to the clinical conditions of the patients, five patients who had suffered from type II diabetes for many years were selected as volunteers. All patients took metformin to control blood sugar and were numbered as sample A, sample B, sample C, sample D, and sample E respectively.

The initial status is as follows: Sample A: LDL cholesterol: 6.98mmol/L; triglycerides: 4.37mmol/L; fasting blood glucose: 11.5mmol/L. Sample B: LDL cholesterol: 7.48mmol/L; triglycerides: 6.22mmol/L; fasting blood glucose: 13.3mmol/L. Sample C: LDL cholesterol: 6.05mmol/L; triglycerides: 5.15mmol/L; fasting blood glucose: 12.6mmol/L. Sample D: LDL cholesterol: 5.86mmol/L; triglycerides: 4.07mmol/L; fasting blood glucose: 10.3mmol/L. Sample E: LDL cholesterol: 6.58mmol/L; triglycerides: 3.75mmol/L; fasting blood glucose: 9.6mmol/L.

The FGF-MSCs prepared in advance were injected intravenously once every seven days, and all injections were required to be given in the morning. A total of four cell infusions were given, on the 1st day, the 7th day, the 14th day, and the 21st day.

Detection Indicator

All patients had blood drawn on day 1, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84 and 90 for measurement of fasting blood glucose, low-density lipoprotein cholesterol and triglycerides. Change curves were drawn based on the patients' average blood glucose, triglycerides and low-density lipoprotein cholesterol.

Experimental Results

The volunteers were treated with cells for 4 times, and their fasting blood glucose, low-density cholesterol, and triglycerides were measured every 7 days from the first treatment, and the observation was continued until 90 days after the treatment was stopped. The fasting blood glucose change curve, low-density cholesterol change curve, and triglyceride change curve of the volunteers are shown in Figure 2, Figure 3, and Figure 4, respectively. The results showed that after 90 days of FGF-stem cell treatment, the fasting blood glucose, low-density cholesterol, and triglycerides of the volunteers were significantly reduced.

In summary, FGF-stem cells can significantly reduce fasting blood sugar and low-density cholesterol in volunteers, and can also reduce triglyceride content, alleviate fatty liver, improve dyslipidemia, and repair pancreatic islet cell function, which has a significant synergistic effect. Therefore, the modified stem cells of the present invention are particularly suitable for treating metabolic diseases.

The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims (10)

1. A modified stem cell comprising:

(1) FGF1 protein selected from the group consisting of: FGF1 protein or a variant thereof;

(2) FGF21 protein selected from the group consisting of: FGF21 protein or a variant thereof;

Wherein the FGF1 variant has at least 90% or more sequence identity as compared to wild-type FGF1 and has FGF1 activity;

the FGF21 variant has at least 90% sequence identity or more as compared to wild-type FGF21 and has FGF21 activity.

2. A modified stem cell according to claim 1, wherein: the FGF1 has an amino acid sequence shown as SEQ ID NO. 1;

FGF21 has an amino acid sequence as shown in SEQ ID NO. 2.

3. A modified stem cell according to claim 1, wherein: the FGF1 variant has an amino acid sequence shown as SEQ ID NO. 3;

the FGF21 variant has an amino acid sequence as shown in SEQ ID NO. 4.

4. A modified stem cell according to claim 1, wherein the stem cell expresses: FGF1 protein having an amino acid sequence as shown in SEQ ID NO. 3;

FGF21 protein having the amino acid sequence as shown in SEQ ID NO. 4.

5. A modified stem cell according to claim 1, further comprising:

(1) An exogenous nucleic acid that expresses an FGF protein comprising a nucleotide sequence encoding said FGF1 protein;

(2) An exogenous nucleic acid that expresses an FGF21 protein comprising a nucleotide sequence encoding said FGF21 protein.

6. A modified stem cell according to claim 1, wherein: the exogenous nucleic acid for expressing the FGF1 protein is connected with the exogenous nucleic acid for expressing the FGF21 protein through a nucleotide sequence for encoding self-cutting peptide;

The self-cleaving peptide has an amino acid sequence as shown in SEQ ID NO. 5.

7. A method of preparing a modified stem cell according to any one of claims 1 to 6, comprising the steps of:

Isolating mesenchymal stem cells;

constructing a slow virus carrying FGF1-FGF21 genes;

the lentivirus infects the mesenchymal stem cells to obtain FGF1-FGF 21-stem cells stably expressing FGF1 and FGF 21.

8. The method of claim 7, wherein the method comprises: the mesenchymal stem cells are derived from one of an umbilical cord or a placenta.

9. A pharmaceutical composition characterized by: comprising any one of the stem cells of claims 1-6;

The pharmaceutical composition is an injection.

10. Use of a modified stem cell according to claims 1-6 and a pharmaceutical composition according to claim 9 for the preparation of a medicament for treating a metabolic disorder in a subject, characterized in that: the metabolic disorder is selected from obesity, type II diabetes, dyslipidemia, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, atherosclerosis, coronary heart disease, hypertension;

The subject is a human.