Use of umbilical cord mesenchymal stem cell-derived exosomes in preparation of insulin sensitizers

Abstract

The application provides application of umbilical cord mesenchymal stem cell-derived exosomes in preparation of an insulin sensitizer, and the sensitization effect is realized by overexpression of Galectin-1. The umbilical cord mesenchymal stem cell-derived exosomes overexpressing Galectin-1 are prepared through the following steps: S1, human umbilical cord mesenchymal stem cells are obtained by separation and culture; S2, human umbilical cord mesenchymal stem cells are transfected with a lentivirus loaded with a Galectin-1 gene CDS sequence plasmid, and human umbilical cord mesenchymal stem cells overexpressing Galectin-1 molecules are obtained through screening; and S3, after the human umbilical cord mesenchymal stem cells overexpressing Galectin-1 are cultured in a culture medium, culture supernatant is collected, and the umbilical cord mesenchymal stem cell-derived exosomes overexpressing Galectin-1 are obtained after centrifugation.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of exosomes derived from umbilical cord mesenchymal stem cells in the preparation of insulin sensitizers. Background Technology

[0002] Insulin resistance refers to a significant decrease in the efficiency of insulin in promoting glucose uptake and utilization. Obesity is a major contributing factor to insulin resistance. With rapid economic development and changes in lifestyle, the overweight and obesity rates among Chinese residents aged 18 and above are 34.3% and 16.4%, respectively, resulting in more than half of adults facing a crisis of reduced insulin sensitivity. Insulin resistance accelerates the development of metabolic syndrome, cardiovascular disease, and polycystic ovary syndrome, and in particular, it can lead to type 2 diabetes. The latest epidemiological survey of diabetes in China shows that among adults over 20 years old, 9.7% have diabetes (approximately 92.4 million), and another 15.5% (approximately 148 million) are in the prediabetes stage. These data indicate that my country has become the world's largest country in terms of diabetes prevalence.

[0003] Insulin resistance is a chronic subclinical inflammatory process, and suppressing inflammation has become a novel approach to treating it. Human umbilical cord mesenchymal stem cells (UC-MSCs) are a type of stem cell with multiple differentiation potential and immunomodulatory effects, currently used in the treatment of various diseases such as diabetes and autoimmune diseases. However, their short survival time in patients, weak biological activity, and difficulty in reaching target tissues due to lung aggregation significantly weaken their therapeutic efficacy. The therapeutic activity of UC-MSCs is highly dependent on their paracrine properties, especially exosomes (Exos). Exosomes are lipid bilayer cystic vesicles, 50-150 nm in diameter, containing various substances including lipid molecules, proteins, and nucleic acids (including DNA, RNA, and various non-coding RNAs). They play an indispensable role in the early diagnosis, clinical treatment, and prognosis of diseases and have begun to replace UC-MSCs in therapeutic applications. Galectin-1 (LGALS1) is one of the main components of mesenchymal stem cell exosomes and an important mediator for the immunosuppressive function of exosomes. It may be a breakthrough in treating insulin resistance through anti-inflammatory pathways.

[0004] Currently, the availability of insulin sensitizers in clinical practice is limited, and they often have significant side effects (such as edema, weight gain, hypoglycemia, and increased risk of heart failure). Therefore, this invention provides the application of exosomes derived from umbilical cord mesenchymal stem cells in the preparation of insulin sensitizers. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing the application of exosomes derived from umbilical cord mesenchymal stem cells containing high doses of Galectin-1 overexpression in the preparation of insulin sensitizers.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The application of exosomes derived from umbilical cord mesenchymal stem cells in the preparation of insulin sensitizers shows that the sensitizing effect is further enhanced by overexpression of Galectin-1 on the basis of the exosome's own sensitizing effect.

[0008] To optimize the above technical solution, the specific measures also include:

[0009] Furthermore, exosomes derived from umbilical cord mesenchymal stem cells overexpressing Galectin-1 were prepared via the following steps:

[0010] S1. Human umbilical cord mesenchymal hepatocytes were isolated and cultured.

[0011] S2. Human umbilical cord mesenchymal stem cells were transfected with lentivirus carrying a plasmid containing the Galectin-1 gene CDS sequence, and human umbilical cord mesenchymal stem cells overexpressing Galectin-1 were obtained by screening.

[0012] S3. After culturing human umbilical cord mesenchymal stem cells overexpressing Galectin-1 in a culture medium, the culture supernatant was collected, centrifuged, and exosomes derived from human umbilical cord mesenchymal stem cells overexpressing Galectin-1 were obtained.

[0013] Furthermore, the CDS sequence is the nucleotide sequence of SEQ ID NO.1.

[0014] Furthermore, the culture medium is serum-free DMEM / F12 medium.

[0015] Furthermore, the incubation period is 36–48 hours.

[0016] Furthermore, the centrifugation method is a combination of ultracentrifugation and density gradient centrifugation.

[0017] The beneficial effects of this invention are:

[0018] Mesenchymal stem cells are widely available, easy to obtain and isolate, and can be expanded on a large scale. Galectin-1 has a strong anti-inflammatory effect, which can inhibit acute inflammation, induce T cell death, promote macrophage reprogramming, and downregulate the synthesis of pro-inflammatory cytokines. This is different from the functions of Galectin-2 and Galectin-3, which also belong to the galactolectin family, in inducing increased inflammation and inhibiting insulin sensitivity. This suggests that overexpression of the Galectin-1 gene in umbilical cord mesenchymal stem cells and increasing the content of Galectin-1 in exosomes can enhance the immunosuppressive capacity of exosomes and make exosomes have a more potent insulin sensitizing effect.

[0019] This invention utilizes a lentivirus carrying a Galectin-1 gene CDS sequence plasmid to efficiently transfect human umbilical cord mesenchymal stem cells, stably inducing cells to overexpress Galectin-1. The human umbilical cord mesenchymal stem cells overexpressing Galectin-1 are cultured in a culture medium, the supernatant is collected, and exosomes derived from human umbilical cord mesenchymal stem cells overexpressing Galectin-1 (Galectin-1-Exos) are obtained after ultracentrifugation combined with density gradient centrifugation.

[0020] Transmission electron microscopy, nanoparticle tracking analysis, and Western blotting were used to identify abundant exosomes secreted by Galectin-1-Exos. Comparative experiments showed that Galectin-1-Exos treatment, compared with Exos treatment, could further effectively reduce serum insulin levels in type 2 diabetic mice and significantly improve insulin tolerance and systemic insulin sensitivity. In addition, Galectin-1-Exos treatment significantly increased the levels of phosphorylated AKT (p-AKT) and phosphorylated insulin receptor (p-IR) in the liver, white adipose tissue, and skeletal muscle of type 2 diabetic mice, indicating that the activity of insulin signaling pathways in these tissues was significantly enhanced, which indirectly proves that overexpression of Galectin-1-Exos improves the insulin sensitivity of exosomes.

[0021] The Galectin-1-Exos prepared by this invention possesses the immunosuppressive properties of mesenchymal stem cells, and also has the advantages of easy storage (it can be stored for a long time in an ultra-low temperature freezer at -80℃ and maintain its biological activity), systemic circulation and crossing the blood-brain barrier, and no immune rejection. It can overcome the defects of stem cells such as short lifespan, weak biological activity, and difficulty in reaching target tissues due to lung aggregation. Attached Figure Description

[0022] Figure 1 Schematic diagram of Galectin-1 expression level in exosomes derived from umbilical cord mesenchymal stem cells detected by Western blotting, **p<0.01;

[0023] Figure 2 A schematic diagram of exosomes derived from umbilical cord mesenchymal stem cells that overexpress Galectin-1, identified using transmission electron microscopy. Scale bar = 100 nm.

[0024] Figure 3 A schematic diagram of the detection of exosomes derived from umbilical cord mesenchymal stem cells overexpressing Galectin-1 using a nanoparticle tracker;

[0025] Figure 4 This image shows the detection of exosome marker proteins derived from umbilical cord mesenchymal stem cells that overexpress Galectin-1. TSG101 and CD63 are exosome positive marker proteins, while GRP94 is an exosome negative marker protein.

[0026] Figure 5 Treatment of exosomes derived from umbilical cord mesenchymal stem cells overexpressing Galectin-1 reduced blood glucose in type 2 diabetic mice (p<0.05, **p<0.01, ***p<0.001).

[0027] Figure 6 A schematic diagram illustrating how treatment with exosomes derived from umbilical cord mesenchymal stem cells overexpressing Galectin-1 improves glucose tolerance in type 2 diabetic mice; the type 2 diabetic group compared to the treatment group... # p<0.05, ### p<0.0012; compared with the Galectin-1-Exos treatment group, *p<0.05, **p<0.01, ***p<0.001;

[0028] Figure 7 Schematic diagram showing how treatment with exosomes derived from umbilical cord mesenchymal stem cells overexpressing Galectin-1 reduced serum insulin levels in type 2 diabetic mice. *p<0.05, **p<0.01, ***p<0.001;

[0029] Figure 8 A schematic diagram illustrating how treatment with exosomes derived from umbilical cord mesenchymal stem cells overexpressing Galectin-1 improves insulin resistance in type 2 diabetic mice; the type 2 diabetic group compared to the treatment group... # p<0.05, ## p<0.01, ### p<0.001; *p<0.05 compared with the Galectin-1-Exos treatment group;

[0030] Figure 9Schematic diagram illustrating how treatment with exosomes derived from umbilical cord mesenchymal stem cells overexpressing Galectin-1 enhances the activity of the hepatic insulin signaling pathway in type 2 diabetic mice. *p<0.05, **p<0.01;

[0031] Figure 10 Schematic diagram illustrating how treatment with exosomes derived from umbilical cord mesenchymal stem cells overexpressing Galectin-1 enhances the activity of the white adipose tissue insulin signaling pathway in type 2 diabetic mice. *p<0.05, **p<0.01, ***p<0.001;

[0032] Figure 11 A schematic diagram illustrating how treatment with exosomes derived from umbilical cord mesenchymal stem cells overexpressing Galectin-1 enhances the activity of the insulin signaling pathway in skeletal muscle of type 2 diabetic mice. *p<0.05, **p<0.01. Detailed Implementation

[0033] Example 1: Isolation and purification of exosomes derived from umbilical cord mesenchymal stem cells overexpressing Galectin-1

[0034] 1.1 Isolation and culture of human umbilical cord mesenchymal stem cells

[0035] With consent, fresh umbilical cords were harvested from healthy mothers after normal delivery. The cords were rinsed with PBS to remove cord blood, cut into approximately 1mm x 1mm pieces, and placed in DMEM / F12 (Gibco 31331093) medium containing 10% FBS (Gibco 10099141). The medium was incubated at 37°C in a 5% CO2 incubator. During culture, non-adhesive cells were removed by PBS washing, and the medium was changed every 3 days. When well-developed fibroblast-like cell colonies appeared after 10 days, they were digested with 0.25% trypsin and passaged into new culture flasks for further expansion. When the cells reached 80–85% confluence, they were digested with 0.25% trypsin and passaged. Human umbilical cord mesenchymal stem cells from passages 3–6 were used in experiments. Tests showed that these stem cells possess excellent proliferation and differentiation potential, capable of differentiating into osteoblasts, adipocytes, chondrocytes, etc. Flow cytometry analysis showed that HLA-DR, CD34, and CD45 were negative (<5%); CD90, CD105, and CD73 were positive (>90%).

[0036] 1.2 Construction of umbilical cord mesenchymal stem cells overexpressing Galectin-1 (Galectin-1-Exos)

[0037] The CDS sequence (SEQ ID NO.1) of the Galectin-1 gene was obtained from the NCBI database. An overexpression plasmid for Galectin-1 was obtained by inserting the CDS sequence (from Genscript Biotech Co., Ltd.) into the vector plasmid pLVX-HA-IRES-2A-EGFP. The Galectin-1 overexpression plasmid, along with two lentiviral packaging plasmids, pCMV-VSV-G and pCMV-dR8.91, was transfected into 293T cells using Lipofectamine 3000 (Invitrogen L3000150). The lentiviral supernatant was collected within 72 hours post-transfection. The lentivirus was then concentrated using a lentivirus concentration kit (Jiangyuan Biotechnology JY03012). Concentrated lentiviruses were used to infect umbilical cord mesenchymal stem cells with polybrene (8 μg / mL). After 72 hours of infection, cells overexpressing Galectin-1 were screened with puromycin (2 μg / mL), and the exosomal Galectin-1 content was detected by Western blotting to verify the overexpression effect.

[0038] 1.3 Isolation and purification of exosomes

[0039] Ordinary umbilical cord mesenchymal stem cells (UCMSCs) and UCMSCs overexpressing Galectin-1 were cultured in serum-free DMEM / F12 for 48 hours. The culture supernatant was collected and ultracentrifuged. First, centrifuged at 2000g for 30 minutes, and the supernatant was transferred to a new centrifuge tube. Then, it was centrifuged again at 13000g for 30 minutes, and the supernatant was filtered through a 0.22μm filter (Millipore SLGP033RB). The filtrate was transferred to a high-speed centrifuge tube and centrifuged at 100000g for 70 minutes. The supernatant was carefully discarded, and the precipitate was washed with PBS. The precipitate was then centrifuged again at 100000g for 70 minutes, and the supernatant was discarded. The supernatant was then used to clean the precipitate using OptiPrep. TM A density gradient medium (STEMCELL 07820) was used to prepare 10%, 20%, and 30% iodixanol solutions, which were then spread into centrifuge tubes. Exosomes obtained by ultracentrifugation were centrifuged in this density gradient medium at 350,000 g for 60 min. The fractions containing exosomes were collected to obtain high-purity exosomes, which were then stored at -80°C. The total protein concentration in the isolated exosomes was determined using the BCA method (ThermoFisher 23225).

[0040] 1.4 Western blot analysis for Galectin-1 content

[0041] Exosomes derived from ordinary umbilical cord mesenchymal stem cells and exosomes derived from umbilical cord mesenchymal stem cells overexpressing Galectin-1 (Galectin-1-Exos) were lysed thoroughly in lysis buffer (ThermoFisher 89901) containing a mixture of protease and phosphatase inhibitors (ThermoFisher 78440). The supernatant was collected by high-speed centrifugation to obtain protein samples, and protein concentration was determined. Four protein samples were mixed with one loading buffer (ThermoFisher 39000) and boiled at 100°C for 5 minutes. Next, equal volumes of protein from the two exosome sources were subjected to polyacrylamide gel electrophoresis. The proteins were then transferred to a PVDF membrane (Millipore IPVH00010), blocked with 5% skim milk, and incubated overnight at 4°C with diluted primary antibody. The membrane was washed three times with TBST and incubated at room temperature for 1 hour with diluted horseradish peroxidase (HRP)-labeled secondary antibody. The blot membrane was rinsed three times with TBST, and ECL developer (ThermoFisher 32106) was added to the membrane. Development was then performed using a gel imaging system (Tanon 4600SF). Quantitative analysis was finally performed using ImageJ software. Primary antibodies used included Rabbit anti-Galectin-1 antibody (1:1000, Proteintech 11858-1-AP) and Mouse anti-GAPDH antibody (1:8000, Proteintech 60004-1-Ig).

[0042] like Figure 1 As shown, the exosomes derived from umbilical cord mesenchymal stem cells overexpressing Galectin-1 (Galectin-1-Exos) showed a significantly increased Galectin-1 content compared to exosomes derived from ordinary umbilical cord mesenchymal stem cells (Exos), indicating that umbilical cord mesenchymal stem cells overexpressing Galectin-1 were successfully constructed.

[0043] Example 2: Identification of exosomes derived from umbilical cord mesenchymal stem cells overexpressing Galectin-1

[0044] 2.1 Transmission electron microscopy observation and nanoparticle tracking analysis (NTA)

[0045] Take 10 μL of Galectin-1-Exos sample, drop it onto a copper grid, allow it to absorb for 1 minute, then blot off excess liquid with filter paper. Next, drop 10 μL of 2% phosphotungstic acid onto the copper grid, stain for 2 minutes, and blot off excess liquid with filter paper. Allow the copper grid to air dry at room temperature, then perform electron microscopy (Hitachi HT-7700) at 100 kV for imaging.

[0046] like Figure 2 As shown, transmission electron microscopy observations indicate that Galectin-1-Exos exhibits a bilayered, circular cup-shaped structure.

[0047] Particle size analysis: After the instrument performance test with standard samples was passed, 30 μL of Galectin-1-Exos sample was taken and particle size analysis was performed using the Nanosight LM10 system (Malvern instruments, USA).

[0048] like Figure 3 As shown, nanoparticle tracking analysis results indicate that the prepared Galectin-1-Exos has an average diameter of 100.3 nm and a concentration of 5.89 × 10⁻⁶. 7 Particles / ml.

[0049] 2.2 Western blot analysis for the expression of exosome marker proteins

[0050] First, Galectin-1-Exos protein was extracted and subjected to polyacrylamide gel electrophoresis. The protein was then transferred to a PVDF membrane, blocked, incubated with primary antibody, washed with TBST, and then incubated with horseradish peroxidase (HRP)-labeled secondary antibody. After TBST washing and ECL development, the protein was imaged using a gel imaging system. The primary antibodies used included Mouse anti-CD63 antibody (1:1000, Proteintech 67605-1-Ig), Mouse anti-TSG101 antibody (1:1000, Proteintech 67381-1-Ig), and Rabbit anti-Grp94 antibody (1:1000, Cell Signaling Technology 0292S).

[0051] like Figure 4 As shown, Western blot analysis revealed that the isolated vesicles expressed the Galectin-1-Exos-specific protein markers TSG101 and CD63, but did not express the endoplasmic reticulum protein marker GRP94. These results indicate that umbilical cord mesenchymal stem cells overexpressing Galectin-1 secrete abundant exosomes.

[0052] Example 3: Application of exosomes derived from umbilical cord mesenchymal stem cells overexpressing Galectin-1 in the preparation of insulin sensitizers.

[0053] 3.1 Construction of type 2 diabetic mice and administration of Galectin-1-Exos

[0054] Four-week-old SPF-grade male C57BL / 6 mice (Speford (Beijing) Biotechnology Co., Ltd.) were placed in a 12-hour light / 12-hour dark environment with free access to food and water. After one week of acclimatization, the mice were divided into four groups: a control group, a type 2 diabetes group, a group treated with exosomes derived from ordinary umbilical cord mesenchymal stem cells (Exos treatment group), and a group treated with exosomes derived from umbilical cord mesenchymal stem cells overexpressing Galectin-1 (Galectin-1-Exos treatment group). Mice in the type 2 diabetes group and the two exosome treatment groups were fed a high-fat diet (60 kcal% high-fat diet, ResearchDiets D12492) for 25 weeks, while mice in the control group were given a normal diet. After 25 weeks, mice in the two exosome treatment groups were injected via tail vein with the corresponding exosomes at a dose of 10 mg / kg (exosome protein / mouse body weight), once a week for 4 weeks. The control group and the type 2 diabetes group were injected with an equal volume of PBS. Seven days after the last injection, fasting blood glucose, insulin, and insulin sensitivity in mice were measured.

[0055] 3.2 Detection of fasting blood glucose and glucose tolerance in mice

[0056] After fasting for 12 hours, blood was collected from the tails of four groups of mice to determine the glucose concentration, which was taken as the blood glucose at 0 minutes, i.e., fasting blood glucose. Subsequently, a glucose solution (1g / kg) was injected intraperitoneally, and blood was collected from the tails at 30, 60, and 120 minutes after the glucose load, and blood glucose was measured using a rapid blood glucose meter.

[0057] Compared with the type 2 diabetes group, the fasting blood glucose levels of mice in the Exos treatment group and the Galectin-1-Exos treatment group were significantly reduced. Figure 5 ), glucose tolerance level significantly increased ( Figure 6 Among them, the improvement was more significant in mice treated with Galectin-1-Exos.

[0058] 3.3 Detection of fasting insulin levels and insulin tolerance in mice

[0059] After fasting for 12 hours, whole blood was collected from mice in four groups, and serum was obtained by centrifugation. Serum insulin levels were then detected using an insulin ELISA kit (Crystal Chem 90080). After fasting for 6 hours, blood was collected from the tails of the mice in all four groups to determine glucose concentration as the blood glucose level at 0 minutes. Subsequently, recombinant insulin (0.75 U / kg) was injected intraperitoneally, and blood was collected from the tails at 30, 60, and 120 minutes for rapid blood glucose measurement (insulin tolerance test).

[0060] The results showed that Exos treatment and Galectin-1-Exos treatment effectively reduced serum insulin levels in type 2 diabetic mice. Figure 7 It significantly improved insulin tolerance in type 2 diabetic mice. Figure 8 Among them, Galectin-1-Exos treatment showed a more significant effect. These results indicate that both Exos treatment and Galectin-1-Exos treatment can effectively increase systemic insulin sensitivity in type 2 diabetic mice, with Galectin-1-Exos exhibiting a more significant effect.

[0061] Example 4: Application of exosomes derived from umbilical cord mesenchymal stem cells overexpressing Galectin-1 in enhancing the activity of insulin signaling pathways in the liver, muscle, and adipose tissue.

[0062] After fasting for 8 hours, mice in the four groups of mice in Example 3 were intraperitoneally injected with recombinant insulin (0.75 U / kg). Ten minutes later, liver, white adipose tissue, and skeletal muscle were collected and placed in a lysis buffer containing a mixture of protease and phosphatase inhibitors, and homogenized thoroughly in a homogenizer. The supernatant was collected by high-speed centrifugation to obtain protein samples, and the protein concentration was determined. An equal amount of protein from each experimental group was subjected to polyacrylamide gel electrophoresis, and the protein was transferred to a PVDF membrane. The membrane was blocked, incubated with primary antibody, washed with TBST, and then incubated with horseradish peroxidase (HRP)-labeled secondary antibody, washed with TBST, developed with ECL contrast agent, and imaged using a gel imaging system. The primary antibodies used include Rabbit anti-phospho-Akt antibody (1:1000, Cell Signaling Technology 4060S), Rabbit anti-Akt antibody (1:1000, Cell Signaling Technology 9272S), Rabbit anti-phospho-IRβ antibody (1:1000, Cell Signaling Technology 3024S), and Rabbit anti-IRβ antibody (1:1000, Cell Signaling Technology 3025S).

[0063] The results showed that Exos treatment and Galectin-1-Exos treatment significantly improved liver function in type 2 diabetic mice. Figure 9 ), white adipose tissue ( Figure 10 ) and skeletal muscle ( Figure 11 The levels of phosphorylated AKT (p-AKT) and phosphorylated insulin receptor (p-IR) in these tissues were measured, indicating that the activity of the insulin signaling pathway was significantly enhanced in these tissues. Among them, Galectin-1-Exos showed the most significant effect.

[0064] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should be considered within the scope of protection of the present invention.

Claims

1. Use of umbilical cord mesenchymal stem cell-derived exosomes overexpressing Galectin-1 in the preparation of a drug for treating type 2 diabetes, characterized in that, Exosomes derived from umbilical cord mesenchymal stem cells overexpressing Galectin-1 were prepared via the following steps: S1. Human umbilical cord mesenchymal stem cells were isolated and cultured. S2. Human umbilical cord mesenchymal stem cells were transfected with a lentivirus carrying a plasmid containing the Galectin-1 gene CDS sequence, and human umbilical cord mesenchymal stem cells overexpressing Galectin-1 were obtained by screening; the CDS sequence is the nucleotide sequence of SEQ ID NO.1; S3. After culturing human umbilical cord mesenchymal stem cells overexpressing Galectin-1 in a culture medium, the culture supernatant was collected, centrifuged, and exosomes derived from human umbilical cord mesenchymal stem cells overexpressing Galectin-1 were obtained.

2. Use according to claim 1, characterized in that, The culture medium is serum-free DMEM / F12 medium.

3. Use according to claim 1, characterized in that, The incubation period is 36-48 hours.

4. Use according to claim 1, characterized in that, The centrifugation method was a combination of ultracentrifugation and density gradient centrifugation.

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