Method for improving hypoxia damage resistance of islet cells

Abstract

The invention discloses a method for improving hypoxia damage resistance of islet cells, and belongs to the technical field of biology. According to the invention, islet cells in a packaging bracket and a culture medium containing a mesenchymal stem cell exosome are incubated together, and the mesenchymal stem cell exosome is loaded with a miRNA mimics composition and a miRNA inhibitor; the miRNA mimic composition is prepared from one or more of miR-539-3p mimic, miR-21-5p mimic, miR-27a-3p mimic and miR-155-5p mimic, and the miRNA mimic composition is prepared from one or more of miR-539-3p mimic, miR-21-5p mimic, miR-27a-3p mimic and miR-155-5p mimic; the miRNA inhibitor is a miR-485-3p inhibitor, and the miRNA inhibitor is a miR-485-3p inhibitor. According to the present invention, the exosome is adopted as the natural shuttle vector, the miRNA mimics and / or the inhibitor are / is loaded into the islet cells, the hypoxia damage resistance gene expression of the islet cells is subjected to targeted regulation, the survival of the islet cells under the hypoxia condition is promoted, and the activity of the islet cells under the hypoxia condition can be effectively improved by using the method provided by the present invention.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for improving the anti-hypoxic injury of pancreatic islet cells. Background Art

[0002] Diabetes affects the health of hundreds of millions of people globally and brings a huge economic burden to the world. Islet transplantation is an effective treatment for type 1 diabetes and brittle diabetes, which can significantly improve the blood glucose level of patients and even enable patients to completely get rid of insulin. However, how to maintain islet function after transplantation is a huge challenge faced by this technology. Hypoxia is an important factor in islet cell apoptosis and loss of function. In the pancreas, islet cells account for only 2% of pancreatic cells, but consume more than 15% of the oxygen supply. Hypoxia can lead to a large number of apoptotic deaths of islet cells in the encapsulation scaffold, limiting the application of islet encapsulation technology in clinical islet transplantation. Therefore, improving the hypoxia tolerance of islet cells in the encapsulation scaffold, thereby enhancing the activity of islet cells, will help the islet encapsulation technology to enter the clinical stage and improve the effect of islet transplantation.

[0003] To solve the problem of islet hypoxia in the encapsulation scaffold, some researchers have developed implantable oxygen delivery containers using silicone to deliver oxygen in the air to the hypoxic graft site through diffusion; there are also oxygen-generating biomaterials, such as biphasic calcium phosphate scaffolds coated with an O 2 generating system to improve the survival rate of transplanted islets; some scholars have also used alginate gel to heat-stabilize microalgae or thermophilic cyanobacteria, and use the photosynthesis of these organisms to provide O 2 . Although these oxygen-producing materials can maintain islet function to a certain extent under hypoxic conditions, they will gradually lose their oxygen-producing function as the experiment progresses. Therefore, there is an urgent need for a method to improve the anti-hypoxic injury of islet cells. Summary of the Invention

[0004] The present invention makes improvements in view of the above problems existing in the prior art, that is, the technical problem to be solved by the present invention is to provide a method that can improve the activity of islet cells in the encapsulation scaffold, effectively improve the activity of islet cells under hypoxic conditions, help the islet encapsulation technology to enter the clinical stage, and improve the effect of islet cell transplantation.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows: A method for improving the anti-hypoxic injury of pancreatic islet cells, comprising the steps of: co-incubating pancreatic islet cells in an encapsulation scaffold with a culture medium containing mesenchymal stem cell exosomes, wherein the mesenchymal stem cell exosomes are loaded with a miRNA mimics composition and / or a miRNA inhibitor; the miRNA mimics composition comprises one or any combination of miR-539-3p mimic, miR-21-5p mimic, miR-27a-3p mimic and miR-155-5p mimic; the miRNA inhibitor is miR-485-3p inhibitor.

[0006] Specifically, it includes the following steps: (1) Isolate and obtain mesenchymal stem cell exosomes; (2) Co-incubate the exosomes with miRNA mimics and miRNA inhibitor at 4 °C for 4-12 hours; (3) Add the exosomes loaded with miRNA mimics and / or miRNA inhibitor to the biocompatible scaffold loaded with pancreatic islet cells and co-incubate.

[0007] Further, in step (1), the concentration of mesenchymal stem cell exosomes is 1×10 9 ~1×10 11 particles / mL.

[0008] Further, the conditions for the co-incubation in step (3) are culturing in a hypoxic incubator at 37 °C, with a CO2 concentration of 5% and an O2 concentration of 5% for 48 h.

[0009] The nucleotide sequence of the above miR-539-3p mimic is as shown in SEQ ID NO.1, the nucleotide sequence of miR-21-5p mimic is as shown in SEQ ID NO.2, the nucleotide sequence of miR-27a-3p mimic is as shown in SEQ ID NO.3, and the nucleotide sequence of miR-155-5p mimic is as shown in SEQ ID NO.4.

[0010] The concentration of the above miR-539-3p mimic is 1-100 nM; the concentration of miR-155-5p mimic is 1-100 nM; the concentration of miR-21-5p mimic is 1-100 nM; the concentration of miR-27a-3p mimic is 1-100 nM.

[0011] The nucleotide sequence of the above miR-485-3p inhibitor is as shown in SEQ ID NO.5.

[0012] The concentration of the above miR-485-3p inhibitor is 10-200 nM.

[0013] The remarkable advantages of the present invention: The present invention uses exosomes as natural shuttle vectors to load miRNA mimics and / or inhibitors into pancreatic islet cells, target and regulate the gene expression of pancreatic islet cells against hypoxic injury, promote the survival of pancreatic islet cells under hypoxic conditions, and effectively improve the activity of pancreatic islet cells under hypoxic conditions through the method of the present invention. Description of the Drawings

[0014] Figure 1 Exosomes obtained by the present invention (electron microscopy image).

[0015] Figure 2 Expression results of miRNAs related to exosomes loaded with miRNA mimics.

[0016] Figure 3 Activity of pancreatic islet cells cultured in this example and activity of pancreatic islet cells in the control group under hypoxic conditions (fluorescent staining).

[0017] Figure 4 Apoptosis of pancreatic islet cells cultured in this example and apoptosis of pancreatic islet cells in the control group under hypoxic conditions (flow cytometry analysis graph / result analysis).

[0018] Figure 5 Number of pancreatic islet cells cultured in this example under hypoxic conditions.

[0019] Figure 6 Expression of genes related to promoting insulin secretion of pancreatic islet cells in this example under hypoxic conditions.

[0020] Figure 7 Inhibition of gene expression of damaged pancreatic islets in this example under hypoxic conditions.

[0021] Figure 8 Results of exosomes loaded with miRNAs entering pancreatic islet cells in this example (fluorescent labeling).

[0022] Figure 9 Comparison of the promotion of pancreatic islet cell activity by exosomes loaded with miRNAs and inhibitors alone and exosomes loaded in combination under hypoxic conditions (CCK-8).

[0023] Figure 10 Comparison of the reduction of pancreatic islet cell apoptosis by exosomes loaded with miRNAs and inhibitors alone and exosomes loaded in combination under hypoxic conditions in this example.

[0024] Figure 11 For Figure 10 apoptosis data statistics. Specific implementation manners

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0026] The test materials used in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels.

[0027] Example 1: Exosomes loaded with miRNA mimics promote the activity of islet cells under hypoxia 1) Isolate and collect mesenchymal stem cell exosomes [References: Chen Junqiu, Chen Jin, Huang Lianghu, et al. Study on the promotion of angiogenesis of mouse islet endothelial cells by mesenchymal cell exosomes [J]. Chinese Journal of Cells and Stem Cells (Electronic Edition), 2018, 8(4): 243-248.] and [CHEN J, CHEN J, CHENG Y, et al. Mesenchymal stem cell-derived exosomes protect beta cells against hypoxia-induced apoptosis via miR-21 by alleviating ER stress and inhibiting p38 MAPK phosphorylation [J]. Stem cell research & therapy, 2020, 11(1): 97.], and adjust the concentration of mesenchymal stem cell exosomes to: 1×10 10 particles / mL; 2) Add 50 nM of miR-539-3p mimic, 50 nM of miR-155-5p mimic, 50 nM of miR-27a-3p mimic, and 100 nM of miR-485-3p inhibitor, and incubate at 4°C for 6 hours; 3) Collect the exosomes in step 2) and perform PCR to detect the expression levels of miR-539-3p, miR-155-5p, and miR-27a-3p; 4) Load 2×10 islet cells into the biological scaffold 4cells, and 2 ml of basal medium RPMI 1640 (hypoxic control group) and exosomes loaded with miRNA mimics and inhibitors (intervention group of this method) were added respectively; placed at 37 °C, CO 2 concentration was 5%; O 2 cultured in a hypoxic incubator with an O concentration of 5% for 48 h; 5) AOPI fluorescence staining was used to detect the activity of islet cells; 6) Cells were collected, and the apoptosis of islet cells was detected and analyzed using an Annexin-V / PI apoptosis analysis kit; 7) Total RNA of islet cells was extracted, and qPCR was used to detect and analyze the expression levels of insulin secretion genes GLUT2, IRS-2, NKX6.1, PDX1; and the damage islet gene UCP-2.

[0028] The electron micrograph of the extracted exosomes is as Figure 1 shown; after loading miRNA mimic, the corresponding miRNA expression level in exosomes increased significantly ( Figure 2 ); exosomes loaded with miRNA mimic significantly improved the activity of islet cells ( Figure 3 ); reduced the apoptosis of islet cells ( Figure 4 ); the number of cultured islet cells increased significantly ( Figure 5 ); the apoptosis rate of islet cells decreased significantly ( Figure 6 ). The expression of insulin secretion genes increased significantly, and the expression of islet damage gene UCP-2 could be significantly inhibited ( Figure 7 ).

[0029] Example 2: Exosomes loaded with miRNA mimcs and inhibitors can shuttle into islet cells 1) Mesenchymal stem cell exosomes were isolated and collected, and the concentration was adjusted to: 1×10 9 particles / mL; 2) Add 50 nM of miR-539-3p mimic, 50 nM of miR-155-5p mimic, 50 nM of miR-27a-3p mimic, 100 nM of miR-485-3p inhibitor, and incubate at 4 °C for 6 hours; 3) Add 5 μL of fluorescent dye CFSE to label the exosomes (green fluorescence); 4) Load 2×10 4 islet cells in the biological scaffold, and add 2 ml of basal medium RPMI 1640 containing exosomes loaded with miRNA; placed at 37 °C, CO 2 concentration was 5%; O 2Cultivate for 24 h and 48 h in a hypoxia incubator with a concentration of 5%.

[0030] 5) After staining the cell nuclei with 5 μL of DAPI, observe under a fluorescence microscope.

[0031] The results are as Figure 8 shown. At 24 h, exosomes loaded with miRNA mimics and inhibitors can shuttle into islet cells and gradually increase over time ( Figure 8 ).

[0032] Example 3: Comparison of the effects of exosomes loaded with a single miRNA mimic or inhibitor and exosomes loaded in combination on the activity of islet cells under hypoxia 1) Isolate and collect mesenchymal stem cell exosomes, and adjust the concentration of mesenchymal stem cell exosomes with basal medium RPMI 1640 to: 1×10 10 particles / mL; 2) Add to the mimics + inhibitor group (50 nM miR-21-5p mimic + 50 nM miR-539-3p mimic + 50 nM miR-155-5p mimic + 50 nM miR-27a-3p mimic + 100 nM miR-485-3p inhibitor), combined mimics group (50 nM miR-21-5p mimic + 50 nM miR-539-3p mimic + 50 nM miR-155-5p mimic + 50 nM miR-27a-3p mimic), miR-21-5p group (50 nM miR-21-5p mimic), miR-539-3p group (50 nM miR-539-3p mimic), miR-27a-3p group (50 nM miR-27a-3p mimic), miR-485-3p inhibitor group (100 nM miR-485-3p inhibitor) respectively, and incubate at 4°C for 6 hours; 3) Load 2×10 4 islet cells into the biological scaffold, and add 2 ml of basal medium RPMI 1640 (hypoxia control group), 2 ml of basal medium RPMI 1640 (normoxia control group) and exosomes loaded with miRNA mimics and inhibitors (intervention group + hypoxia by this method, the specific group settings are shown in step 2); Place under hypoxic conditions at 37°C, CO 2 concentration is 5%; O 2Cultured in a hypoxic incubator with a concentration of 5% for 48 h; placed under normoxic conditions at 37 °C, CO 2 with a concentration of 5%; O 2 Cultured in an incubator with a concentration of 21% for 48 h.

[0033] 5) The activity of islet cells was determined by the CCK-8 method; 6) Cells were collected, and the apoptosis of islet cells was detected and analyzed using an Anexin-V / PI apoptosis analysis kit; The results were as Figure 9 described. Under hypoxic conditions, compared with the activity of islet cells (100%) under normal oxygen concentration, exosomes loaded with miRNA mimics or inhibitors significantly increased the activity of islet cells. The exosomes loaded with a combination of multiple miRNA mimics and miRNA inhibitors had the most obvious effect, and the exosomes loaded with miRNA mimics or miRNA inhibitors alone also increased the activity of islet cells. The activity of islet cells in the hypoxic control group was 68.16% ± 4.61%; the group loaded with exosomes of combined miRNA mimics and miRNA inhibitors (97.29% ± 6.96%); the group loaded with combined mimics (96.90% ± 7.68%); the group loaded with a single miR-21-5p mimic (88.78% ± 4.15%); the group loaded with a single miR-539-3p mimic (89.46% ± 5.84%); the group loaded with a single miR-27a-3p mimic (77.50% ± 5.38%); the group loaded with a single miR155-5p mimic (75.95% ± 3.88%); the group loaded with a single miR-485-3p inhibitor (76.42% ± 4.27%).

[0034] Under hypoxic conditions, the apoptosis of islet cells increased significantly (16.91% ± 1.88%), and exosomes loaded with miRNA mimics or inhibitors significantly reduced the apoptosis of islet cells ( Figure 10 ). The group loaded with exosomes of combined mimics and inhibitors (7.90% ± 0.72%); the group loaded with combined mimics (9.22% ± 1.37%); the group loaded with a single miR-21-5p mimic (9.40% ± 1.57%); the group loaded with a single miR-539-3p mimic (10.07% ± 2.32%); the group loaded with a single miR-27a-3p mimic (14.69% ± 1.57%); the group loaded with a single miR155-5p mimic (14.71% ± 1.75%); the group loaded with a single miR-485-3p inhibitor (14.93% ± 1.48%), seeFigure 11 。

[0035] Those skilled in the art can easily understand that the above are only preferred examples of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for improving the resistance of pancreatic islet cells to hypoxic damage, characterized in that: The pancreatic islet cells in the encapsulated scaffold are co-incubated with a culture medium containing mesenchymal stem cell exosomes, wherein the mesenchymal stem cell exosomes are loaded with a miRNA mimics composition and / or a miRNA inhibitor; the miRNA mimics composition includes one or any several of miR-539-3p mimic, miR-21-5pmimic, miR-27a-3p mimic and miR-155-5p mimic; and the miRNA inhibitor is a miR-485-3p inhibitor.

2. The method according to claim 1, characterized in that: The specific steps include: (1) Isolation and acquisition of mesenchymal stem cell exosomes; (2) Incubate the exosomes with miRNA mimics and miRNA inhibitor at 4°C for 4 to 12 hours; (3) Exosomes loaded with miRNA mimics and / or miRNA inhibitor are added to the bioscaffold loaded with pancreatic islet cells and incubated together.

3. The method for improving the resistance of pancreatic islet cells to hypoxic damage according to claim 2, characterized in that: In step (1), the concentration of mesenchymal stem cell exosomes was 1×10 9 ~1×10 11 particles / mL.

4. The method for improving the resistance of pancreatic islet cells to hypoxia damage according to claim 2, characterized in that: The incubation conditions in step (3) are culturing in a hypoxic incubator at 37° C., 5% CO 2 concentration, and 5% O 2 concentration for 48 hours.

5. The method for improving the resistance of pancreatic islet cells to hypoxia damage according to claim 1, characterized in that: The nucleotide sequence of the miR-539-3p mimic is shown in SEQ ID NO.1, the nucleotide sequence of the miR-21-5p mimic is shown in SEQ ID NO.2, the nucleotide sequence of the miR-27a-3p mimic is shown in SEQ ID NO.3, and the nucleotide sequence of the miR-155-5p mimic is shown in SEQ ID NO.

4.

6. The method for improving the resistance of pancreatic islet cells to hypoxia damage according to claim 1, characterized in that: The concentration of the miR-539-3p mimic is 1~100 nM; the concentration of the miR-155-5p mimic is 1~100 nM; the concentration of the miR-21-5p mimic is 1~100 nM; and the concentration of the miR-27a-3p mimic is 1~100 nM.

7. The method for improving the resistance of pancreatic islet cells to hypoxia damage according to claim 1, characterized in that: The nucleotide sequence of the miR-485-3p inhibitor is shown in SEQ ID NO.

5.

8. The method for improving the resistance of pancreatic islet cells to hypoxic damage according to claim 1, characterized in that: The concentration of the miR-485-3p inhibitor is 10~200 nM.

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