Preparation for promoting in-vitro proliferation of pancreatic stem cells and application thereof

Abstract

The invention relates to a preparation for promoting in-vitro proliferation of pancreatic stem cells and application of the preparation. A nucleotide sequence of a miR-802 inhibitor comprises a sequence as shown in SEQ ID No. 1. According to the composition containing the miR-802 inhibitor, the in-vitro proliferation capacity of pancreatic stem cells is remarkably improved, and the capacity of pancreatic duct cells for forming pancreatic stem cell colonies is remarkably improved. Experiments prove that the miR-802 inhibitor has the functions of promoting dry gene expression and inhibiting endocrine differentiation gene expression in pancreatic stem cells cultured in vitro.

Description

Technical Field

[0001] The present invention relates to the field of cell technology, and in particular to a preparation for promoting in vitro proliferation of pancreatic stem cells and application thereof. Background Art

[0002] Pancreatic stem cells are a type of cell with the potential for self-renewal and multidirectional differentiation. They play an important role in pancreatic development, regeneration, and disease treatment. Pancreatic stem cells are mainly found in the pancreatic ducts and islets of the pancreas. They are adult stem cells with the ability to self-renew and differentiate into all lineages of pancreatic cells. They are currently considered to be important candidate seed cells for cell replacement therapy for diabetes.

[0003] In the treatment of diabetes, the application of pancreatic stem cells is particularly eye-catching. Since insufficient insulin secretion caused by dysfunction of pancreatic islet cells is one of the main characteristics of diabetes, obtaining functional pancreatic β cells through inducing differentiation of pancreatic stem cells has become an important strategy for treating diabetes. At present, human pluripotent stem cells have been successfully induced to differentiate into pancreatic β cells, and their effect on improving diabetic symptoms has been verified in animal models. In addition, there are studies using pancreatic stem cells to prepare pancreatic organoids, which contain all cell types of the pancreas and can respond quickly to sugar stimulation and secrete insulin. These pancreatic organoids can not only be used as in vitro models to study the proliferation and differentiation of pancreatic islet cells and their interaction with the microenvironment, but can also be used as transplant materials for the treatment of diabetes.

[0004] However, although pancreatic stem cells have shown great potential in the treatment of diabetes, their clinical application still faces many challenges, such as the low efficiency of pancreatic stem cell expansion in vitro and the inability to maintain their stem cell properties during in vitro expansion, which reduces their differentiation effect. Therefore, how to efficiently isolate and expand pancreatic stem cells is an urgent problem to be solved. Summary of the invention

[0005] To solve the above technical problems, the present invention provides a preparation for promoting the proliferation of pancreatic stem cells in vitro and its application. The present invention finds that a composition containing a miR-802 inhibitor can effectively promote the proliferation and self-renewal ability of pancreatic stem cells in vitro.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides use of a miR-802 inhibitor in the preparation of a preparation for promoting the proliferation of pancreatic stem cells.

[0008] SEQ ID No.1:

[0009] AAGGATGAATCTTTGTTACTGA.

[0010] In the present invention, it is found that miR-802 inhibitors can significantly promote the proliferation and self-renewal ability of pancreatic stem cells in vitro, and can increase the expression of pancreatic stem cell stemness genes, which can further develop preparations that promote pancreatic stem cell proliferation and methods for in vitro amplification of pancreatic stem cells.

[0011] In a second aspect, the present invention provides a preparation for promoting the proliferation of pancreatic stem cells in vitro, the preparation comprising the miR-802 inhibitor as claimed in claim 1;

[0012] Preferably, the miR-802 inhibitor is expressed via an expression vector;

[0013] Preferably, the expression vector comprises a lentiviral expression vector;

[0014] Preferably, the lentiviral vector comprises any one of pEZX-AM03, GV159, pLKO.1 or pmiRZip.

[0015] In a third aspect, the present invention provides a composition for promoting the proliferation of pancreatic stem cells in vitro, the composition comprising DMEM / F12 culture medium, matrigel, methylcellulose, EGF protein, nicotinamide, an adjuvant and the inhibitor described in the first aspect or the second aspect.

[0016] In the present invention, the composition is based on DMEM / F12 culture medium and added with a variety of reagents that promote the proliferation of pancreatic stem cells in vitro. DMEM / F12 culture medium is used to provide components such as water, essential amino acids, inorganic salts and sugars required for the growth of stem cells. Matrigel is used to provide matrix proteins required for the growth of stem cells in a three-dimensional environment. Methylcellulose is used to provide a supporting skeleton for the growth of stem cells in a three-dimensional environment. EGF protein is used to promote the proliferation of pancreatic stem cells. And the adjuvant is used to promote the maintenance of the stemness of stem cells.

[0017] Preferably, the volume concentration of the matrix gel in the composition for promoting the proliferation of pancreatic stem cells in vitro is 3-8%, for example, 3%, 4%, 5%, 6%, 7% or 8%.

[0018] Preferably, the volume concentration of methylcellulose in the composition for promoting the proliferation of pancreatic stem cells in vitro is 0.5-2%, for example, it can be 0.5%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8% or 2%.

[0019] Preferably, the viscosity of methylcellulose in the composition for promoting the proliferation of pancreatic stem cells in vitro is 1000-2000, for example, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000.

[0020] Preferably, the concentration of EGF protein in the composition for promoting the proliferation of pancreatic stem cells in vitro is 10-30 ng / mL, for example, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL or 30 ng / mL.

[0021] Preferably, the concentration of nicotinamide in the composition for promoting the proliferation of pancreatic stem cells in vitro is 5-15mmol / L, for example, it can be 5mmol / L, 6mmol / L, 7mmol / L, 8mmol / L, 9mmol / L, 10mmol / L, 11mmol / L, 12mmol / L, 13mmol / L, 14mmol / L or 15mmol / L.

[0022] Preferably, the volume concentration of the inhibitor in the composition for promoting the proliferation of pancreatic stem cells in vitro is 0.5-5%, for example, it can be 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%, etc.

[0023] Preferably, the auxiliary agent comprises any one of Noggin protein, VEGF protein or B27 cell culture additive or a combination of at least two thereof.

[0024] Preferably, the concentration of the auxiliary agent in the composition for promoting the proliferation of pancreatic stem cells in vitro is 5-15 ng / mL, for example, it can be 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL or 15 ng / mL, etc.

[0025] In a fourth aspect, the present invention provides a method for in vitro expansion of pancreatic stem cells, the method comprising: inhibiting the expression of miR-802 in pancreatic stem cells using the miR-802 inhibitor described in the first aspect or the second aspect.

[0026] Preferably, the method specifically comprises: placing pancreatic total cells in the composition for promoting the proliferation of pancreatic stem cells in vitro for culturing

[0027] Preferably, the volume ratio of the composition to the total pancreatic cells is 500 μL:(5000-20000). The (5000-20000) may be, for example, 5000, 6000, 8000, 10000, 12000, 14000, 16000, 18000 or 20000.

[0028] Preferably, the culture temperature is 35-40°C and the culture time is 10-18 days. The 35-40°C may be, for example, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C, etc. The 10-18 days may be, for example, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days or 18 days, etc.

[0029] Preferably, the method for preparing the total pancreatic cells comprises cutting the pancreas into pieces and adding digestive fluid to digest the pancreas;

[0030] Preferably, the digestive solution comprises 0.05-0.4% type II collagenase, for example, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35% or 0.4%, etc.

[0031] Preferably, the preparation method of the composition comprises adding matrigel, methylcellulose, EGF protein, nicotinamide, adjuvants and the miR-802 inhibitor of the first aspect or the second aspect to DMEM / F12 culture medium and stirring the mixture.

[0032] In a fourth aspect, the present invention provides a use of the inhibitor for promoting the in vitro proliferation of pancreatic stem cells described in the first aspect, the composition for promoting the in vitro proliferation of pancreatic stem cells described in the second aspect, or the method for amplifying pancreatic stem cells described in the third aspect in the preparation of pancreatic stem cells.

[0033] Compared with the prior art, the present invention has at least the following beneficial effects:

[0034] The present invention provides a composition, which includes a miR-802 inhibitor, which significantly improves the proliferation ability of pancreatic stem cells in vitro, and significantly increases the ability of pancreatic duct cells to form pancreatic stem cell colonies. Experimental verification shows that the miR-802 inhibitor has the function of promoting stemness gene expression and inhibiting endocrine differentiation gene expression in pancreatic stem cells cultured in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Figure 1 is a light microscopic image of primary pancreatic stem cells, wherein Figure A is a light microscopic image of a blank control group, Figure B is a light microscopic image of miR-802 overexpression, Figure C is a light microscopic image of Example 1, Figure D is a light microscopic image of Example 2, and Figure E is a light microscopic image of Example 3.

[0036] Figure 2 Graphs A and B are bar graphs showing the proliferation capacity of primary pancreatic stem cells, wherein Graph A is a bar graph showing colony formation rate, Graph B is a bar graph showing colony diameter, and Graph C is a bar graph showing cell number.

[0037] Figure 3This is a light microscopic image of pancreatic stem cells after 5 generations.

[0038] Figure 4 It is a bar chart analysis of the proliferation ability of subcultured pancreatic stem cells, wherein Figure A is a bar chart analysis of the colony formation rate after subculture, and Figure B is a bar chart analysis of the number of cells after subculture.

[0039] Figure 5 This is the immunofluorescence result of the expression of stemness genes and proliferation marker genes of pancreatic stem cells.

[0040] Figure 6 Figure 1 is a graph showing the detection results of stemness gene expression of pancreatic stem cells, wherein Figure A is a graph showing the detection results of Ki67 positive cells, and Figure B is a graph showing the detection results of Ki67 expression levels.

[0041] Figure 7 Figure 1 is the analysis diagram of flow cytometry detection results. Figure A is the flow cytometry detection result diagram, and Figure B is the CD133 + Histogram of cell ratios.

[0042] Figure 8 This is a graph showing the analysis results of stemness gene and endocrine differentiation gene expression. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0044] Example 1

[0045] In this example, pancreatic stem cells were expanded in vitro

[0046] (1) Preparation of inhibitors

[0047] HEK293T cells were seeded in 10 cm culture dishes at a density of 70% confluence. HEK293T cells were cultured in a medium without antibiotics. 10 μg of the lentiviral vector plasmid pEZX-AM03-miR802 constructed with SEQ ID No.1 was mixed with 7.5 μg of psPAX2 packaging plasmid and 2.5 μg of pMD2.G envelope plasmid. The plasmid mixture and 60 μg of polyethyleneimine were diluted in 500 μL of serum-free culture medium to form a plasmid mixture solution and a polyethyleneimine solution. The plasmid mixture solution and the polyethyleneimine solution were mixed, allowed to stand at room temperature for 15 minutes, and then added dropwise to the cell culture dish and gently shaken to mix. Fresh culture medium was replaced after 8 hours. After 48 hours, the culture supernatant was collected, centrifuged at 500g for 5 minutes to remove cell debris, and then filtered with a 0.45 μm filter membrane. The filtered supernatant was centrifuged at 20,000g for 2 hours at 4°C, and the supernatant was discarded to obtain the lentiviral precipitate. Resuspend the pellet in 100 μL of phosphate buffer to obtain the inhibitor.

[0048] (2) Preparation of composition

[0049] A composition was prepared on ice, and matrigel, 1500 viscosity methylcellulose, EGF protein, nicotinamide, inhibitor and Noggin protein were added to the DMEM / F12 culture medium mother solution, the volume concentration of matrigel was 5%, the volume concentration of 1500 viscosity methylcellulose was 1%, the concentration of EGF protein was 20 ng / mL, the concentration of nicotinamide was 10 mmol / L, the concentration of Noggin protein was 10 ng / mL, and the volume concentration of inhibitor was 1%.

[0050] (3) In vitro expansion of pancreatic stem cells

[0051] Place the pancreas in a cold dish, remove the tissue, and cut into 1 mm pieces. 3 Tissue blocks were digested with a digestion solution containing 0.2% (v / v) type II collagenase to obtain total pancreatic cells.

[0052] Use a pre-cooled pipette tip to add the cell suspension to the three-dimensional semi-solid culture medium. After shaking evenly, use a syringe to inject into a low-adhesion 24-well plate, 500 μL of the composition culture medium per well, and put 10,000 total pancreatic cells. Place in a 37°C, 5% CO2 constant temperature incubator for 14 days.

[0053] Example 2

[0054] In this example, pancreatic stem cells were expanded in vitro

[0055] (1) Preparation of inhibitors

[0056] HEK293T cells were seeded in 10 cm culture dishes at a density of 60% confluence. HEK293T cells were cultured in a medium without antibiotics. 10 μg of the lentiviral vector plasmid pEZX-AM03-miR802 constructed with SEQ ID No.1 was mixed with 7.5 μg of psPAX2 packaging plasmid and 2.5 μg of pMD2.G envelope plasmid. The plasmid mixture and 60 μg of polyethyleneimine were diluted in 500 μL of serum-free culture medium to form a plasmid mixture solution and a polyethyleneimine solution. The plasmid mixture solution and the polyethyleneimine solution were mixed, allowed to stand at room temperature for 15 minutes, and then added dropwise to the cell culture dish and gently shaken to mix. Fresh culture medium was replaced after 8 hours. After 48 hours, the culture supernatant was collected, centrifuged at 500g for 5 minutes to remove cell debris, and then filtered with a 0.45 μm filter membrane. The filtered supernatant was centrifuged at 20000g for 2 hours at 4°C, and the supernatant was discarded to obtain the lentiviral precipitate. Resuspend the pellet in 100 μL of phosphate buffer to obtain the inhibitor.

[0057] (2) Preparation of composition

[0058] A composition was prepared on ice, and matrigel, 1500 viscosity methylcellulose, EGF protein, nicotinamide, inhibitor and Noggin protein were added to the DMEM / F12 culture medium mother solution, the volume concentration of matrigel was 3%, the volume concentration of 2000 viscosity methylcellulose was 0.5%, the concentration of EGF protein was 10 ng / mL, the concentration of nicotinamide was 15 mmol / L, the concentration of Noggin protein was 5 ng / mL, and the volume concentration of inhibitor was 2%.

[0059] (3) In vitro expansion of pancreatic stem cells

[0060] Place the pancreas in a cold dish, remove the tissue, and cut into 1 mm pieces. 3 Tissue blocks were digested with a digestion solution containing 0.4% (v / v) type II collagenase to obtain total pancreatic cells.

[0061] Use a pre-cooled pipette tip to add the cell suspension to the three-dimensional semi-solid culture medium. After shaking evenly, inject it into a low-adhesion 24-well plate with a syringe, 500 μL of the composition culture medium per well, and put 10,000 total pancreatic cells. Place in a 35°C, 5% CO2 constant temperature incubator for 14 days.

[0062] Example 3

[0063] In this example, pancreatic stem cells were expanded in vitro

[0064] (1) Preparation of inhibitors

[0065] HEK293T cells were seeded in 10 cm culture dishes at a density of 70% confluence. HEK293T cells were cultured in a medium without antibiotics. 10 μg of the lentiviral vector plasmid pEZX-AM03-miR802 constructed with SEQ ID No.1 was mixed with 7.5 μg of psPAX2 packaging plasmid and 2.5 μg of pMD2.G envelope plasmid. The plasmid mixture and 60 μg of polyethyleneimine were diluted in 500 μL of serum-free culture medium to form a plasmid mixture solution and a polyethyleneimine solution. The plasmid mixture solution and the polyethyleneimine solution were mixed, allowed to stand at room temperature for 15 minutes, and then added dropwise to the cell culture dish and gently shaken to mix. Fresh culture medium was replaced after 8 hours. After 48 hours, the culture supernatant was collected, centrifuged at 500g for 5 minutes to remove cell debris, and then filtered with a 0.45 μm filter membrane. The filtered supernatant was centrifuged at 20,000g for 2 hours at 4°C, and the supernatant was discarded to obtain the lentiviral precipitate. Resuspend the pellet in 100 μL of phosphate buffer to obtain the inhibitor.

[0066] (2) Preparation of composition

[0067] A composition was prepared on ice, and matrigel, 1500 viscosity methylcellulose, EGF protein, nicotinamide, inhibitor and Noggin protein were added to the DMEM / F12 culture medium mother solution, the volume concentration of matrigel was 8%, the volume concentration of 1000 viscosity methylcellulose was 2%, the concentration of EGF protein was 30 ng / mL, the concentration of nicotinamide was 5 mmol / L, the concentration of Noggin protein was 15 ng / mL, and the volume concentration of inhibitor was 5%.

[0068] (3) In vitro expansion of pancreatic stem cells

[0069] Place the pancreas in a cold dish, remove the tissue, and cut into 1 mm pieces. 3 Tissue blocks were digested with a digestion solution containing 0.05% (v / v) type II collagenase to obtain total pancreatic cells.

[0070] Use a pre-cooled pipette tip to add the cell suspension to the three-dimensional semi-solid culture medium. After shaking evenly, use a syringe to inject into a low-adhesion 24-well plate, 500 μL of the composition culture medium per well, and put 10,000 pancreatic total cells. Place in a 40°C, 5% CO2 constant temperature incubator for 14 days.

[0071] Test Example 1

[0072] This test example detects the colony-forming ability of pancreatic stem cells after expansion

[0073] Primary mouse pancreatic single cells overexpressing miR-802 (miR-802 overexpression group) and primary mouse pancreatic single cells loaded with blank vector (control group) were inoculated with 10,000 cells in each culture well and cultured for 10 days. Figure 1 A in the middle is the control group. It can be seen that in the absence of inhibitors, pancreatic single cells formed typical cystic hollow pancreatic stem cell colonies. Figure 1 Figure B in the middle is the miR-802 overexpression group. It can be seen that when miR-802 is overexpressed, the number of pancreatic stem cell colonies formed decreases. Figure 1 Figures C, D and F in the middle are pancreatic stem cells of Example 1, Example 2 and Example 3 respectively. It can be seen that the colony formation ratio and the size of the colony are improved.

[0074] The Example 1, the control group and the miR-802 overexpression group were compared. Figure 2 As shown in Figures A, B and C, the colony formation rate, colony diameter and total cell amount of pancreatic stem cells after expansion in Example 1 were significantly improved. This proves that after inhibiting miR-802, the ability of mouse pancreatic ductal cells to form pancreatic stem cell colonies increased significantly.

[0075] Test Example 2

[0076] This test case detects the proliferation ability of pancreatic stem cells after passage

[0077] The primary mouse pancreatic single cells of the control group, miR-802 overexpression group and Example 1 were digested into single cells and then subcultured. After five consecutive subcultures, Figure 3 It can be seen that the control group and the miR-802 overexpression group formed colonies with smaller diameters, most of which were solid, while the miR-802 inhibitor group of Example 1 still formed cystic hollow colonies with relatively large diameters and stable morphology. The formation rate and total number of stem cell colonies were observed after 10 consecutive passages, and the results were analyzed by Figure 4 As shown in Figure A, the colony formation rate of the miR-802 inhibitor group increased significantly with the increase in the number of passages, while the colony formation rates of the control group and the miR-802 overexpression group did not change much. Figure 4 As can be seen from Figure B, the number of cells also increased significantly in the miR-802 inhibitor group, and the number of cells in the miR-802 overexpression group decreased, indicating that inhibition of miR-802 promoted the proliferation of pancreatic stem cells.

[0078] Test Example 3

[0079] This test case detects the stemness gene of pancreatic stem cells

[0080] Immunofluorescence was used to detect the expression of the stemness marker Pdx1 and cell proliferation marker Ki67 in the control group, miR-802 overexpression group and primary mouse pancreatic stem cell of Example 1. Figure 5 It can be seen that pancreatic stem cell stemness marker Pdx1 and cell proliferation marker Ki67 can be detected in pancreatic stem cell colonies, and miR-802 inhibitor can increase the expression intensity of Pdx1 and Ki67. Figure 6 Figure A is a statistical analysis of the number of positive cells. It can be seen that the miR-802 inhibitor significantly increased the proportion of Ki67 positive cells. Figure 6 As shown in Figure B, similar changes in Ki67 expression levels were observed by fluorescent quantitative PCR detection of Ki67 mRNA. The results suggest that miR-802 inhibitors significantly promote the proliferation of pancreatic stem cells in vitro.

[0081] Test Example 4

[0082] This test case was performed by flow cytometry

[0083] Flow cytometry was used to detect pancreatic stem cell colonies cultured for 14 days. Figure 7 As shown in Figures A and B, it was found that miR-802 inhibitor significantly promoted the proportion of pancreatic stem cell marker CD133 positive cells. Figure 8 As shown in the figure, the results of fluorescence quantitative PCR showed that miR-802 inhibitor significantly promoted the expression of Pdx1 and CD133, and inhibited the expression of Ins1, Ins2, Ngn3 and NeuroD1. The results suggest that miR-802 inhibitor has the function of promoting the expression of stemness genes and inhibiting the expression of endocrine differentiation genes in pancreatic stem cells cultured in vitro.

[0084] In summary, the composition containing miR-802 inhibitor provided by the present invention significantly improves the proliferation ability of pancreatic stem cells in vitro, and the ability of pancreatic ductal cells to form pancreatic stem cell colonies is significantly increased. Experimental verification shows that miR-802 inhibitor has the function of promoting stemness gene expression and inhibiting endocrine differentiation gene expression in pancreatic stem cells cultured in vitro.

[0085] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. Use of a miR-802 inhibitor in the preparation of a preparation for promoting the proliferation of pancreatic stem cells, characterized in that: The nucleic acid sequence of the miR-802 inhibitor includes the sequence shown in SEQ ID No.

1.

2. A preparation for promoting the proliferation of pancreatic stem cells in vitro, characterized in that: The formulation comprises the miR-802 inhibitor described in claim 1; Preferably, the miR-802 inhibitor is expressed via an expression vector; Preferably, the expression vector comprises a lentiviral expression vector; Preferably, the lentiviral vector comprises any one of pEZX-AM03, GV159, pLKO.1 or pmiRZip.

3. A composition for promoting the proliferation of pancreatic stem cells in vitro, characterized in that: The composition comprises DMEM / F12 culture medium, matrix gel, methylcellulose, EGF protein, nicotinamide, an adjuvant and the miR-802 inhibitor according to claim 1 or 2.

4. The composition for promoting the proliferation of pancreatic stem cells in vitro according to claim 3, characterized in that: The volume concentration of the matrix gel is 3-8%; Preferably, the volume concentration of methylcellulose in the composition for promoting the proliferation of pancreatic stem cells in vitro is 0.5-2%; Preferably, the viscosity of the methylcellulose in the composition for promoting the proliferation of pancreatic stem cells in vitro is 1000-2000; Preferably, the concentration of EGF protein in the composition for promoting the proliferation of pancreatic stem cells in vitro is 10-30 ng / mL; Preferably, the concentration of nicotinamide in the composition for promoting the proliferation of pancreatic stem cells in vitro is 5-15 mmol / L; Preferably, the volume concentration of the inhibitor in the composition for promoting the proliferation of pancreatic stem cells in vitro is 0.5-5%.

5. The composition for promoting the proliferation of pancreatic stem cells in vitro according to claim 3 or 4, characterized in that: The auxiliary agent includes any one of Noggin protein, VEGF protein or B27 serum-free additive or a combination of at least two thereof; Preferably, the concentration of the auxiliary agent in the composition for promoting the proliferation of pancreatic stem cells in vitro is 5-15 ng / mL.

6. Use of miR-802 inhibitor in promoting the proliferation of pancreatic stem cells, characterized in that: The nucleic acid sequence of the miR-802 inhibitor includes the sequence shown in SEQ ID No.

1.

7. A method for in vitro expansion of pancreatic stem cells, characterized in that: The method comprises: using the miR-802 inhibitor described in claim 1 or 2 to inhibit the expression of miR-802 in pancreatic stem cells.

8. The method for in vitro expansion of pancreatic stem cells according to claim 7, characterized in that: The method specifically comprises: placing pancreatic total cells into the composition for promoting the in vitro proliferation of pancreatic stem cells according to any one of claims 3 to 5 for culturing; Preferably, the volume ratio of the composition to the total pancreatic cells is 500 μL: (5000-20000); Preferably, the culture temperature is 35-40°C and the culture time is 10-18 days; Preferably, the method for preparing the total pancreatic cells comprises cutting the pancreas into pieces and adding digestive fluid to digest the pancreas; Preferably, the digestion solution comprises 0.05-0.4% type II collagenase.

9. The method for expanding pancreatic stem cells according to any one of claims 6 to 8, characterized in that: The preparation method of the composition comprises adding matrix gel, methylcellulose, EGF protein, nicotinamide, an adjuvant and the miR-802 inhibitor according to claim 1 or 2 into a DMEM / F12 culture medium and stirring the mixture.

10. Use of the miR-802 inhibitor according to claim 1 or 2, the composition for promoting the proliferation of pancreatic stem cells in vitro according to any one of claims 3 to 5, or the method for amplifying pancreatic stem cells according to any one of claims 7 to 9 in preparing pancreatic stem cells.

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