Nano liver stem cell peptide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of cell culture, in particular to nano liver stem cell peptide as well as a preparation method and application thereof. The preparation method of the nano liver stem cell peptide comprises the following steps: (1) mixing induced pluripotent stem cells with a first induced differentiation culture medium, and culturing for 3-5 days to obtain an intermediate cell 1; (2) inoculating the intermediate cell 1 into a second induced differentiation culture medium, and culturing for 3-5 days to obtain an intermediate cell 2; and (3) inoculating the intermediate cell 2 into a third induced differentiation culture medium, and culturing for 3-5 days to obtain the liver stem cell, and (4) after the liver stem cells are subjected to ultrafiltration, taking substances of which the molecular weight cutoff is less than or equal to 50kDa, centrifuging, taking precipitates, and drying to obtain the liver stem cell peptide. The liver stem cell peptide prepared by the method has the particle size of 1-20nm, can target the liver, has a good effect in treatment of liver injury, and does not generate drug resistance.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and particularly relates to a nano hepatic stem cell peptide, a preparation method thereof, and an application thereof. Background Art

[0002] Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cells that can be directly generated from somatic cells. Since they can reproduce infinitely and generate all other cell types in the body (such as neurons, heart, pancreas, etc.), they represent a single cell source that can be used to replace cells lost due to injury or diseases. Thus, iPSCs can induce the generation of a class of cells with targeted therapy for injury or diseases.

[0003] Liver injury refers to the damage and dysfunction of the liver caused by various reasons. Liver injury can lead to the regeneration disorder and necrosis of hepatocytes, and then cause liver cirrhosis; it can lead to the disorder of lipid metabolism in the patient's body, and then increase the blood viscosity, causing atherosclerosis; it can directly lead to the disorder of lipid metabolism and induce diabetes, etc. The drugs for treating and preventing liver injury in the prior art have poor efficacy and can also cause adverse reactions. Therefore, a drug that can target the liver and has excellent efficacy is needed to meet the deficiencies of the prior art.

[0004] Whether iPSCs can be used to induce hepatic stem cells to target the liver for treating and preventing liver injury has not been reported in the prior art. Based on this, the present invention is proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a hepatic stem cell peptide that can target the liver and effectively treat liver injury, and particularly relates to a nano hepatic stem cell peptide, a preparation method thereof, and an application thereof.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a method for inducing pluripotent stem cells to differentiate into hepatic stem cell peptides, comprising the following steps:

[0008] (1) Mixing induced pluripotent stem cells with a first induced differentiation medium and culturing for 3 - 5 days to obtain intermediate cell 1;

[0009] (2) Inoculating intermediate cell 1 into a second induced differentiation medium and culturing for 3 - 5 days to obtain intermediate cell 2;

[0010] (3) Inoculating intermediate cell 2 into a third induced differentiation medium and culturing for 3 - 5 days to obtain hepatic stem cells;

[0011] (4) After ultrafiltrating the hepatic stem cells, taking the substance with a cut-off molecular weight ≤ 50 kDa, centrifuging, taking the precipitate, and drying to obtain hepatic stem cell peptides.

[0012] Preferably, the first induced differentiation medium is based on DMEM / F12 medium and further includes components with the following concentrations:

[0013] Hepatocyte growth factor 8 - 12 ng / mL, epidermal growth factor 5 - 10 ng / mL, activin A 6 - 15 ng / mL, phloridzin 10 - 20 μg / mL, vitamin C 10 - 20 μg / mL.

[0014] Preferably, the second induced differentiation medium is based on DMEM / F12 medium and further includes components with the following concentrations:

[0015] Hepatocyte growth factor 8 - 12 ng / mL, epidermal growth factor 5 - 10 ng / mL, creatine phosphate 20 - 50 ng / mL, prostaglandin 30 - 50 ng / mL.

[0016] Preferably, the third induced differentiation medium is based on DMEM / F12 medium and further includes components with the following concentrations:

[0017] Hepatocyte growth factor 10 - 20 ng / mL, epidermal growth factor 3 - 6 ng / mL, valproic acid 0.05 - 0.1 mM, glutamine 0.1 - 0.3 mM.

[0018] Preferably, the culture temperature in steps (1) - (3) is independently 20 - 30 °C, and the oxygen concentration for culture is independently 3 - 10%.

[0019] Preferably, the pore size of the filter membrane during ultrafiltration in step (4) is 2 - 20 nm.

[0020] Preferably, the rotation speed of centrifugation in step (4) is 20000 - 30000 rpm, and the centrifugation time is 8 - 10 min.

[0021] The present invention also provides a hepatic stem cell peptide prepared by the described method, and the particle size of the hepatic stem cell peptide is 1 - 20 nm.

[0022] The present invention also provides the application of the described hepatic stem cell peptide in the preparation of a nano - drug for treating and / or preventing liver injury.

[0023] The present invention also provides the application of the hepatic stem cell peptide prepared by the described method in the preparation of a nano - drug for treating and / or preventing liver injury.

[0024] The present invention provides a nano liver stem cell peptide, a preparation method thereof and an application. The induction method of the present invention includes the following steps: (1) Mixing induced pluripotent stem cells with a first induced differentiation medium and culturing for 3 - 5 days to obtain intermediate cell 1; (2) Inoculating intermediate cell 1 into a second induced differentiation medium and culturing for 3 - 5 days to obtain intermediate cell 2; (3) Inoculating intermediate cell 2 into a third induced differentiation medium and culturing for 3 - 5 days to obtain liver stem cells; (4) After ultrafiltrating the liver stem cells, taking the substance with a cut-off molecular weight ≤ 50 kDa, centrifuging, taking the precipitate and drying to obtain the liver stem cell peptide. The liver stem cell peptide prepared by the method of the present invention has a particle size of 1 - 20 nm, can target the liver, has good efficacy in treating liver injury and will not produce drug resistance. It provides a basis for the preparation of highly effective drugs for treating liver injury. Description of the Drawings

[0025] Figure 1 Shows the situation of alanine aminotransferase secreted by induced pluripotent stem cells cultured by different methods at different time points;

[0026] Figure 2 Shows the situation of aspartate aminotransferase secreted by induced pluripotent stem cells cultured by different methods at different time points;

[0027] Figure 3 Shows the situation of blood urea nitrogen secreted by induced pluripotent stem cells cultured by different methods at different time points. Detailed Embodiments

[0028] The present invention provides a method for the directed differentiation of induced pluripotent stem cells into liver stem cell peptides, including the following steps:

[0029] (1) Mixing induced pluripotent stem cells with a first induced differentiation medium and culturing for 3 - 5 days to obtain intermediate cell 1; (2) Inoculating intermediate cell 1 into a second induced differentiation medium and culturing for 3 - 5 days to obtain intermediate cell 2; (3) Inoculating intermediate cell 2 into a third induced differentiation medium and culturing for 3 - 5 days to obtain liver stem cells; (4) After ultrafiltrating the liver stem cells, taking the substance with a cut-off molecular weight ≤ 50 kDa, centrifuging, taking the precipitate and drying to obtain the liver stem cell peptide.

[0030] In the present invention, the first induced differentiation medium is based on DMEM / F12 medium and further includes the following components at the following concentrations:

[0031] Hepatocyte growth factor 8 - 12 ng / mL, preferably 10 ng / mL;

[0032] Epidermal growth factor 5 - 10 ng / mL, preferably 8 ng / mL;

[0033] Activin A is 6 - 15 ng / mL, preferably 10 ng / mL;

[0034] Phloridzin is 10 - 20 μg / mL, preferably 15 μg / mL;

[0035] Vitamin C is 10 - 20 μg / mL, preferably 15 μg / mL.

[0036] In the present invention, the second induced differentiation medium is based on DMEM / F12 medium and further includes components with the following concentrations:

[0037] Hepatocyte growth factor is 8 - 12 ng / mL, preferably 10 ng / mL;

[0038] Epidermal growth factor is 5 - 10 ng / mL, preferably 8 ng / mL;

[0039] Creatine phosphate is 20 - 50 ng / mL, preferably 35 ng / mL;

[0040] Prostaglandin is 30 - 50 ng / mL, preferably 40 ng / mL.

[0041] In the present invention, the third induced differentiation medium is based on DMEM / F12 medium and further includes components with the following concentrations:

[0042] Hepatocyte growth factor is 10 - 20 ng / mL, preferably 15 ng / mL;

[0043] Epidermal growth factor is 3 - 6 ng / mL, preferably 4.5 ng / mL;

[0044] Valproic acid is 0.05 - 0.1 mM, preferably 0.08 mM;

[0045] Glutamine is 0.1 - 0.3 mM, preferably 0.2 mM.

[0046] In the present invention, the culture temperature in steps (1) - (3) is independently 20 - 30 °C, preferably 25 °C, and the oxygen concentration for culture is independently 3 - 10%, preferably 5%.

[0047] In the present invention, the pore size of the filter membrane during ultrafiltration in step (4) is 2 - 20 nm, and the pressure during ultrafiltration is 0.1 - 0.5 Mpa, more preferably 0.2 Mpa.

[0048] In the present invention, the rotation speed during centrifugation in step (4) is 20000 - 30000 rpm, preferably 25000 rpm, and the centrifugation time is 8 - 10 min, preferably 9 min.

[0049] The present invention also provides a hepatic stem cell peptide prepared by the described method. The particle size of the hepatic stem cell peptide is 1 - 20 nm, preferably 5 - 10 nm, and more preferably 8 nm.

[0050] The present invention also provides the use of the described hepatic stem cell peptide in the preparation of a nano - drug for treating and / or preventing liver injury.

[0051] The present invention also provides the use of the hepatic stem cell peptide prepared by the described method in the preparation of a nano - drug for treating and / or preventing liver injury.

[0052] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0053] The DMEM / F12 medium was purchased from Wuhan Punosai Life Science Co., Ltd.

[0054] The induced pluripotent stem cells of the present invention are DYR0100 human induced pluripotent stem cells, purchased from Shanghai Yaji Biotechnology Co., Ltd.

[0055] Example 1

[0056] Preparation method of the first induction medium: Based on the DMEM / F12 medium, the following components are further added at the following concentrations: hepatocyte growth factor 10 ng / mL, epidermal growth factor 6 ng / mL, activin A 10 ng / mL, phloridzin 15 μg / mL, vitamin C 15 μg / mL.

[0057] Preparation method of the second induction medium: Based on the DMEM / F12 medium, the following components are further added at the following concentrations: hepatocyte growth factor 10 ng / mL, epidermal growth factor 6 ng / mL, creatine phosphate 30 ng / mL, prostaglandin 40 ng / mL.

[0058] Preparation method of the third induction medium: Based on the DMEM / F12 medium, the following components are further added at the following concentrations: hepatocyte growth factor 15 ng / mL, epidermal growth factor 5 ng / mL, valproic acid 0.1 mM, glutamine 0.2 mM.

[0059] The human induced pluripotent stem cells were seeded at 3×10 4Cells / holes were inoculated into cell culture dishes containing the first induction medium and cultured for 5 days at a temperature of 25°C and an oxygen concentration of 5% to obtain intermediate cells 1, with the culture medium being changed once a day. After resuspending intermediate cells 1 with DMEM / F12 medium, they were inoculated into cell culture dishes containing the second induction medium and cultured for 4 days at a temperature of 25°C and an oxygen concentration of 8%, with the culture medium being changed once a day, to obtain intermediate cells 2; after resuspending intermediate cells 2 with DMEM / F12 medium, they were inoculated into cell culture dishes containing the third induction medium and cultured for 4 days at a temperature of 25°C and an oxygen concentration of 10%, with the culture medium being changed once a day, to obtain hepatic stem cells. The hepatic stem cells were ultrafiltered in an ultrafiltration tube. During ultrafiltration, the pore size of the ultrafiltration membrane was 10 nm, and the ultrafiltration pressure was 0.3 MPa. Substances with a molecular weight cut-off ≤50 kDa were taken, placed in a centrifuge with a rotational speed of 25000 rpm, centrifuged for 8 minutes, and the precipitate was taken and freeze-dried to obtain hepatic stem cell peptides.

[0060] Example 2

[0061] Preparation method of the first induction medium: Based on DMEM / F12 medium, the following components were further added at the following concentrations: hepatocyte growth factor 8 ng / mL, epidermal growth factor 10 ng / mL, activin A 10 ng / mL, phloridzin 10 μg / mL, vitamin C 20 μg / mL.

[0062] Preparation method of the second induction medium: Based on DMEM / F12 medium, the following components were further added at the following concentrations: hepatocyte growth factor 8 ng / mL, epidermal growth factor 10 ng / mL, creatine phosphate 25 ng / mL, prostaglandin 45 ng / mL.

[0063] Preparation method of the third induction medium: Based on DMEM / F12 medium, the following components were further added at the following concentrations: hepatocyte growth factor 10 ng / mL, epidermal growth factor 6 ng / mL, valproic acid 0.08 mM, glutamine 0.3 mM.

[0064] Human induced pluripotent stem cells were taken at 4×10 4Cells / holes were inoculated into cell culture dishes containing the first induction medium and cultured for 4 days at a temperature of 25°C and an oxygen concentration of 3% to obtain intermediate cells 1, with the culture medium being changed once a day. After resuspending intermediate cells 1 with DMEM / F12 medium, they were inoculated into cell culture dishes containing the second induction medium and cultured for 5 days at a temperature of 25°C and an oxygen concentration of 6%, with the culture medium being changed once a day, to obtain intermediate cells 2. After resuspending intermediate cells 2 with DMEM / F12 medium, they were inoculated into cell culture dishes containing the third induction medium and cultured for 5 days at a temperature of 25°C and an oxygen concentration of 9%, with the culture medium being changed once a day, to obtain hepatic stem cells. The hepatic stem cells were ultrafiltered in an ultrafiltration tube. During ultrafiltration, the pore size of the ultrafiltration membrane was 10 nm and the ultrafiltration pressure was 0.3 MPa. Substances with a molecular weight cut-off ≤50 kDa were taken, placed in a centrifuge with a rotational speed of 25000 rpm, centrifuged for 8 minutes, and the precipitate was taken and freeze-dried to obtain hepatic stem cell peptides.

[0065] Example 3

[0066] Preparation method of the first induction medium: Based on DMEM / F12 medium, the following components were added at the following concentrations: hepatocyte growth factor 12 ng / mL, epidermal growth factor 5 ng / mL, activin A 8 ng / mL, phloridzin 20 μg / mL, vitamin C 10 μg / mL.

[0067] Preparation method of the second induction medium: Based on DMEM / F12 medium, the following components were added at the following concentrations: hepatocyte growth factor 12 ng / mL, epidermal growth factor 5 ng / mL, creatine phosphate 30 ng / mL, prostaglandin 30 ng / mL.

[0068] Preparation method of the third induction medium: Based on DMEM / F12 medium, the following components were added at the following concentrations: hepatocyte growth factor 20 ng / mL, epidermal growth factor 3 ng / mL, valproic acid 0.08 mM, glutamine 0.1 mM.

[0069] Human induced pluripotent stem cells were taken at 4×10 4Cells / holes were inoculated into cell culture dishes containing the first induction medium and cultured for 4 days at a temperature of 25°C and an oxygen concentration of 4% to obtain intermediate cells 1, with the culture medium changed once a day. After resuspending intermediate cells 1 with DMEM / F12 medium, they were inoculated into cell culture dishes containing the second induction medium and cultured for 5 days at a temperature of 25°C and an oxygen concentration of 8%, with the culture medium changed once a day, to obtain intermediate cells 2; after resuspending intermediate cells 2 with DMEM / F12 medium, they were inoculated into cell culture dishes containing the third induction medium and cultured for 5 days at a temperature of 25°C and an oxygen concentration of 10%, with the culture medium changed once a day, to obtain hepatic stem cells. The hepatic stem cells were ultrafiltered in an ultrafiltration tube. During ultrafiltration, the pore size of the ultrafiltration membrane was 10 nm, and the ultrafiltration pressure was 0.3 MPa. Substances with a molecular weight cut-off ≤50 kDa were taken, placed in a centrifuge with a rotation speed of 25000 rpm, centrifuged for 8 minutes, and the precipitate was taken and freeze-dried to obtain hepatic stem cell peptides.

[0070] Comparative Example 1

[0071] Comparative Example 1 was set up according to the scheme of Example 1. The difference from Example 1 was that phloretin in Example 1 was replaced with salidroside at the same concentration in the first induction medium.

[0072] Comparative Example 2

[0073] Comparative Example 1 was set up according to the scheme of Example 1. The difference from Example 1 was that prostaglandin was not added to the second induction medium.

[0074] Comparative Example 3

[0075] Comparative Example 1 was set up according to the scheme of Example 1. The difference from Example 1 was that glutamine was not added to the third induction medium.

[0076] Experimental Example 1

[0077] Cells cultured in Example 1 and Comparative Examples 1 to 3 until the 5th, 9th, and 13th days were taken, and the supernatant was taken to detect the secretion of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and urea nitrogen.

[0078] Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and urea nitrogen are stem cell-specific biochemical indicators used to detect whether human induced pluripotent stem cells are transformed into hepatic stem cells. The results are as Figures 1 - 3 shown.

[0079] The results showed that the contents of ALT, AST, and urea nitrogen secreted by human induced pluripotent stem cells induced by Example 1 of the present invention were higher than those of other groups, indicating that the method of Example 1 of the present invention can transform human induced pluripotent stem cells into hepatic stem cells.

[0080] Experimental Example 2

[0081] The particle size of the hepatic stem cell peptides prepared in Example 1 and Comparative Examples 1-3 was detected by light scattering method.

[0082] The detection results showed that the particle size of the hepatic stem cell peptides prepared in Example 1 and Comparative Examples 1-3 was 10±2 nm.

[0083] Experimental Example 3

[0084] Twenty-four healthy NIH mice with an average body weight of 20 g were randomly divided into 6 groups: a normal control group, a model control group, an Example 1 group, a Comparative Example 1 group, a Comparative Example 2 group, and a Comparative Example 3 group, with 4 mice in each group. The mice in the normal control group and the model control group were intragastrically administered 1 mL of normal saline every day. The mice in the Example 1 group were intragastrically administered 1 mg of the hepatic stem cell peptide of Example 1 every day. The mice in the Comparative Example 1 group were intragastrically administered 1 mg of the hepatic stem cell peptide of Comparative Example 1 every day. The mice in the Comparative Example 2 group were intragastrically administered 1 mg of the hepatic stem cell peptide of Comparative Example 2 every day. The mice in the Comparative Example 3 group were intragastrically administered 1 mg of the hepatic stem cell peptide of Comparative Example 3 every day, and intragastric administration was continued for 7 d. On the 3rd and 7th days of intragastric administration, except for the mice in the normal control group, the mice in the other groups were subcutaneously injected with 0.1 mL of 2% CCl4. 24 h after the last injection of CCl4 and then intragastric administration of the drug, blood was taken from the orbital cavities of the mice in each group, and the contents of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of each group of mice were measured by the Reitman method. The results are shown in Table 1. The contents of interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) in the serum of each group of mice were measured by the King method. The results are shown in Table 2.

[0085] Table 1 Effects of hepatic stem cell peptides prepared by different methods on ALT and AST in mouse serum

[0086] Group ALT (U / L) AST (U / L) Normal control group 264 253 Model control group 864 748 Example 1 group 376 368 Comparative example 1 group 589 567 Comparative example 2 group 567 548 Comparative example 3 group 613 602

[0087] As can be seen from Table 1, the hepatic stem cell peptide cultured in Example 1 of the present invention has better effects than other groups.

[0088] Table 2 Effects of hepatic stem cell peptides prepared by different methods on the contents of inflammatory factors in mouse serum

[0089] Group IL-6 (pg / mL) TNF-α (pg / mL) Normal control group 207 186 Model control group 316 352 Example 1 group 247 234 Comparative example 1 group 289 304 Comparative example 2 group 296 326 Comparative example 3 group 277 299

[0090] As can be seen from Table 2, the levels of IL-6 and TNF-α in the model control group mice were significantly increased, and the levels of IL-6 and TNF-α in the Example 1 mice were significantly lower than those in other groups, indicating that the hepatic stem cell peptide prepared in Example 1 of the present invention can effectively alleviate CCl4-induced liver injury.

[0091] As can be seen from the above embodiments, the present invention provides a nano liver stem cell peptide, its preparation method and application. The preparation method of the present invention comprises the following steps: (1) Mixing induced pluripotent stem cells with a first induced differentiation medium and culturing for 3-5 days to obtain intermediate cell 1; (2) Inoculating intermediate cell 1 into a second induced differentiation medium and culturing for 3-5 days to obtain intermediate cell 2; (3) Inoculating intermediate cell 2 into a third induced differentiation medium and culturing for 3-5 days to obtain liver stem cells; (4) After ultrafiltering the liver stem cells, taking the substance with a molecular weight cut-off ≤50 kDa, centrifuging, taking the precipitate, and drying to obtain liver stem cell peptide. The liver stem cell peptide prepared by the method of the present invention has a particle size of 1-20 nm, can target the liver, has good efficacy in treating liver injury, and does not produce drug resistance.

[0092] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for inducing pluripotent stem cells to differentiate into hepatic stem cell peptides, characterized in that, It includes the following steps: (1) Mix induced pluripotent stem cells with the first induced differentiation medium and culture for 3 - 5 days to obtain intermediate cell 1; (2) Inoculate intermediate cell 1 into the second induced differentiation medium and culture for 3 - 5 days to obtain intermediate cell 2; (3) Inoculate intermediate cell 2 into the third induced differentiation medium and culture for 3 - 5 days to obtain hepatic stem cells; (4) After ultrafiltration of the hepatic stem cells obtained above, take the substances with a molecular weight cut-off ≤ 50 kDa, centrifuge, take the precipitate, and dry to obtain hepatic stem cell peptides.

2. The method according to claim 1, wherein The first induced differentiation medium is based on DMEM / F12 medium and further includes components with the following concentrations: Hepatocyte growth factor 8 - 12 ng / mL, epidermal growth factor 5 - 10 ng / mL, activin A 6 - 15 ng / mL, phloridzin 10 - 20 μg / mL, vitamin C 10 - 20 μg / mL.

3. The method according to claim 1, wherein The second induced differentiation medium is based on DMEM / F12 medium and further includes components with the following concentrations: Hepatocyte growth factor 8 - 12 ng / mL, epidermal growth factor 5 - 10 ng / mL, creatine phosphate 20 - 50 ng / mL, prostaglandin 30 - 50 ng / mL.

4. The method according to claim 1, wherein The third induced differentiation medium is based on DMEM / F12 medium and further includes components with the following concentrations: Hepatocyte growth factor 10 - 20 ng / mL, epidermal growth factor 3 - 6 ng / mL, valproic acid 0.05 - 0.1 mM, glutamine 0.1 - 0.3 mM.

5. The method according to any one of claims 1 to 4, characterized in that The culture temperature in steps (1) - (3) is independently 20 - 30 °C, and the oxygen concentration during culture is independently 3 - 10%.

6. The method according to claim 1, characterized in that The pore size of the filter membrane during ultrafiltration in step (4) is 2 - 20 nm.

7. The method according to claim 1, characterized in that The rotation speed of centrifugation in step (4) is 20000 - 30000 rpm, and the centrifugation time is 8 - 10 min.

8. The hepatic stem cell peptide prepared by the method according to any one of claims 1 to 7, characterized in that, The particle size of the hepatic stem cell peptides is 1 - 20 nm.

9. Use of the hepatic stem cell peptides according to claim 8 in the preparation of nano-drugs for treating and / or preventing liver injury.

10. Use of the hepatic stem cell peptides prepared by the method according to any one of claims 1 - 7 in the preparation of nano-drugs for treating and / or preventing liver injury.

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