A pluripotent stem cell culture solution containing a maternal factor

Abstract

The present application relates to the biomedical field, and particularly relates to a kind of omnipotent stem cell culture medium containing maternal factors.The present application first uses maternal factors and pomegranate seed active peptide extract jointly to prepare omnipotent stem cell culture medium, the maternal factors in the culture medium are selected from TPRX1, TPRXL, miR-302 and IL1 alpha combination, verified analysis, the above-mentioned joint existence synergies, can effectively improve the expression of omnipotent stem cell in Oct3 / 4 gene and cell cluster number, further promote the induction capacity of somatic cell to omnipotent stem cell, so that the production of large quantities of omnipotent stem cell can be induced, meanwhile, the present application uses omnipotent stem cell culture medium as fibroblast proliferation-promoting culture medium, can effectively promote fibroblast proliferation, and is beneficial to the application in wound healing.

Description

TECHNICAL FIELD

[0001] The present application relates to the biomedical field, in particular to a kind of omnipotent stem cell culture solution containing maternal factors. BACKGROUND

[0002] When animal fertilization, sperm is mainly released to the egg in male pronucleus, the zygote formed by female, male pronucleus fusion into zygote nucleus, but the genome of fertilized egg is not transcribed in the first few mitosis, reasonable logical speculation is that its early development completely depends on the RNA and protein stored in egg juice, i.e maternal factors.

[0003] The latest research uses four-factor expression cassette (Hsf1, Zar1, Padi6 and Npm2) to directly convert mouse or human somatic cells into 2C-like totipotent stem cells (MFi2CLTSCs) in vitro, express 2C markers such as MERVL and Zscan4, and have the ability to form blastocysts and chimeric embryos. This strategy avoids the tumor risk of iPSC, the limitation of SCNT egg source, and the insufficient specificity of small molecules of EPSC, and provides an efficient and controllable new way for regenerative medicine, endangered species protection and egg-free totipotent cell preparation. However, how to effectively improve the induction efficiency of totipotent stem cells using maternal factors is an urgent problem to be solved. SUMMARY

[0004] In view of the technical problems existing in the prior art, the present application provides a totipotent stem cell culture solution containing maternal factors. The present application first uses maternal factors in combination with pomegranate seed active peptide extract to prepare a totipotent stem cell culture solution. The maternal factors in the culture solution are selected from the combination of TPRX1, TPRXL, miR-302 and IL1α. Through verification analysis, the above-mentioned combination has a synergistic effect, which can effectively improve the expression of Oct3 / 4 gene in totipotent stem cells and the number of cell clusters, further improve the induction ability of somatic cells to totipotent stem cells, so that a large number of totipotent stem cells can be induced, and at the same time, the totipotent stem cell culture solution can be used as a fibroblast proliferation-promoting culture solution, which can effectively promote the proliferation of fibroblasts and is beneficial to the application in wound healing.

[0005] Specifically, the present application first provides a totipotent stem cell culture solution containing maternal factors, comprising the following components: 10% (v / v) FBS, 1-3 mM TPRX1, 1-3 mM TPRXL, 0.5-1.5 mM miR-302, 1-2 mM IL1α, 0.05 mM β-mercaptoethanol, 0.1-3 mM pomegranate seed active peptide extract, 100 u / L penicillin, 100 u / L streptomycin, and the balance of RPMI-1640 culture solution. The above-mentioned substances are prepared into a totipotent stem cell culture solution by a conventional preparation method. When used, the above-mentioned culture solution is preheated at 37°C and then used.

[0006] Preferably, the pomegranate seed active peptide extract is an active polypeptide shown in SEQ ID NO. 1.

[0007] Preferably, the separation method of the pomegranate seed active peptide extract comprises the following steps: after the pomegranate seeds are physically crushed, distilled water is added in a mass ratio of 1: (10-20), heating treatment is performed, the temperature is maintained at 50-60°C for 3-5 h, then papain is added in a mass ratio of 100: (5-9) for enzymolysis for 6-8 h, then an equal amount of papain is continuously added to the enzymolysis solution for secondary enzymolysis treatment for 12-18 h, after the enzymolysis is completed, ultrafiltration is performed, and the active peptide in the enzymolysis solution is separated by HPLC-MS, and is obtained after identification.

[0008] Preferably, the totipotent stem cell culture solution containing maternal factors comprises the following components: 10% (v / v) FBS, 1 mM TPRX1, 1 mM TPRXL, 0.5 mM miR-302, 1 mM IL1α, 0.05 mM β-mercaptoethanol, 0.1 mM pomegranate seed active peptide extract, 100 u / L penicillin, 100 u / L streptomycin, and the balance of RPMI-1640 culture solution. The above-mentioned substances are prepared into a totipotent stem cell culture solution by a conventional preparation method. When used, the above-mentioned culture solution is preheated at 37°C and then used.

[0009] Preferably, the totipotent stem cell culture solution containing maternal factors comprises the following components: 10% (v / v) FBS, 2 mM TPRX1, 2 mM TPRXL, 1.0 mM miR-302, 1.5 mM IL1α, 0.1 mM β-mercaptoethanol, 0.2 mM pomegranate seed active peptide extract, 100 u / L penicillin, 100 u / L streptomycin, and the balance of RPMI-1640 culture solution. The above-mentioned substances are prepared into a totipotent stem cell culture solution by a conventional preparation method. When used, the above-mentioned culture solution is preheated at 37°C and then used.

[0010] Preferably, the all-purpose stem cell culture solution containing maternal factors comprises the following components: 10% (v / v) FBS, 3 mM TPRX1, 3 mM TPRXL, 1.5 mM miR-302, 2.0 mM IL1α, 0.15 mM β-mercaptoethanol, 0.3 mM pomegranate seed active peptide extract, 100 u / L penicillin, 100 u / L streptomycin, and the balance of RPMI-1640 culture solution. The above-mentioned substances are prepared into an all-purpose stem cell culture solution by a conventional preparation method. When used, the above-mentioned culture solution is preheated at 37°C and then used.

[0011] Further, the present application also provides the use of maternal factors in the preparation of an all-purpose stem cell culture solution, wherein the maternal factors are selected from one or more of TPRX1, TPRXL, miR-302 and IL1α.

[0012] Further, the present application also provides the use of pomegranate seed active peptide extract in the preparation of an all-purpose stem cell culture solution, wherein the pomegranate seed active peptide extract is the active polypeptide shown in SEQ ID NO. 1.

[0013] Further, the present application also provides the use of maternal factors in combination with pomegranate seed active peptide extract in the preparation of an all-purpose stem cell culture solution, wherein the maternal factors are selected from one or more of TPRX1, TPRXL, miR-302 and IL1α; and the pomegranate seed active peptide extract is the active polypeptide shown in SEQ ID NO. 1.

[0014] The advantages of the present application are as follows: the present application first uses maternal factors in combination with pomegranate seed active peptide extract to prepare an all-purpose stem cell culture solution, wherein the maternal factors are selected from a combination of TPRX1, TPRXL, miR-302 and IL1α; through verification analysis, the above-mentioned combination has a synergistic effect, can effectively improve the expression of the Oct3 / 4 gene in the all-purpose stem cells and the cell colony number, further improves the induction ability of somatic cells to all-purpose stem cells, so that a large number of all-purpose stem cells can be induced, and at the same time, the all-purpose stem cell culture solution of the present application can be used as a fibroblast proliferation-promoting culture solution, can effectively promote the proliferation of fibroblasts, and is beneficial to the application in wound healing. DETAILED DESCRIPTION

[0015] The present application will be further described in detail below in combination with specific examples, so that those skilled in the art can more clearly understand the present application.

[0016] The following examples are used to illustrate the present application and are not used to limit the scope of the present application. Based on the specific examples in the present application, all other examples obtained by those skilled in the art without creative efforts are within the protection scope of the present application.

[0017] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.

[0018] All components of the inducing agent of the present invention are commercially available products: RPMI-1640, FBS, β-mercaptoethanol, TPRX1, TPRXL, miR-302 and IL1α, penicillin and streptomycin are all commercially available. Example 1

[0019] A pluripotent stem cell culture medium containing maternal factors comprises the following components: 10% (v / v) FBS, 1 mM TPRX1, 1 mM TPRXL, 0.5 mM miR-302, 1 mM IL1α, 0.05 mM β-mercaptoethanol, 0.1 mM pomegranate seed active peptide extract, 100 u / L penicillin, 100 u / L streptomycin, and the balance being RPMI-1640 culture medium. The above substances are prepared into a pluripotent stem cell culture medium according to conventional preparation methods. Before use, the culture medium should be preheated to 37°C.

[0020] The pomegranate seed active peptide extract is the active polypeptide shown in SEQ ID NO.1. The separation method of this pomegranate seed active peptide extract includes the following steps: After physically pulverizing pomegranate seeds, distilled water is added at a mass ratio of pulverized material to distilled water of 1:10. The mixture is then heated at 50°C for 3 hours. Bromelain (enzyme parameter 10,000 u / g) is added at a mass ratio of hydrolysate to bromelain of 100:5 for enzymatic hydrolysis for 6 hours. Subsequently, papain (enzyme parameter 10,000 u / g) is added to the hydrolysate in an equal amount to bromelain for a second enzymatic hydrolysis treatment of 12 hours. After hydrolysis, ultrafiltration is performed, and the active peptides in the hydrolysate are separated using HPLC-MS. The pomegranate seed active peptide extract shown in SEQ ID NO.1 is obtained and used for future reference. Example 2

[0021] A pluripotent stem cell culture medium containing maternal factors comprises the following components: 10% (v / v) FBS, 2 mM TPRX1, 2 mM TPRXL, 1.0 mM miR-302, 1.5 mM IL1α, 0.1 mM β-mercaptoethanol, 0.2 mM pomegranate seed active peptide extract, 100 u / L penicillin, 100 u / L streptomycin, and the balance being RPMI-1640 culture medium. The above substances are prepared into a pluripotent stem cell culture medium according to conventional preparation methods. Before use, the culture medium should be preheated to 37°C. The pomegranate seed active peptide extract is the pomegranate seed active peptide extract prepared in Example 1 as shown in SEQ ID NO. 1. Example 3

[0022] A totipotent stem cell culture solution containing maternal factors, comprising the following components: 10% (v / v) FBS, 3 mM TPRX1, 3 mM TPRXL, 1.5 mM miR-302, 2.0 mM IL1a, 0.15 mM β-mercaptoethanol, 0.3 mM pomegranate seed active peptide extract, 100 u / L penicillin, 100 u / L streptomycin, and the balance of RPMI-1640 culture solution. The above-mentioned substances are prepared into a totipotent stem cell culture solution by a conventional preparation method. When used, the above-mentioned culture solution is preheated at 37°C and then used. The pomegranate seed active peptide extract is the pomegranate seed active peptide extract shown as SEQ ID NO. 1 prepared in Example 1. Comparative Example 1

[0023] A totipotent stem cell culture solution, comprising the following components: 10% (v / v) FBS, 3.5 mM TPRX1, 0.05 mM β-mercaptoethanol, 100 u / L penicillin, 100 u / L streptomycin, and the balance of RPMI-1640 culture solution. The above-mentioned substances are prepared into a totipotent stem cell culture solution by a conventional preparation method. When used, the above-mentioned culture solution is preheated at 37°C and then used. Comparative Example 2

[0024] Compared with Comparative Example 1, 3.5 mM TPRXL is used instead of 3.5 mM TPRX1. Comparative Example 3

[0025] Compared with Comparative Example 1, 3.5 mM miR-302 is used instead of 3.5 mM TPRX1. Comparative Example 4

[0026] Compared with Comparative Example 1, 3.5 mM IL1a is used instead of 3.5 mM TPRX1. Comparative Example 5

[0027] Compared with Comparative Example 1, it further contains 0.1 mM pomegranate seed active peptide extract. Example 4

[0028] A totipotent stem cell culture method, comprising the following steps

[0029] 1) Take 1 x 10 4 cell human neonatal dermal fibroblasts (Catalog # 2310, purchased from Zhijiao Xinzhou Biotechnology);

[0030] 2) The human neonatal dermal fibroblasts were inoculated into the omnipotent stem cell culture solution of the different embodiments and the comparative examples, respectively, and continuously cultured for 7 days, and the fresh culture medium was replaced every 1 day;

[0031] 3) The cells after the culture of step 2) were collected by centrifugation, and the proportion of Oct3 / 4 positive cells was detected by immunohistochemical method, and the cell colony number was counted by alkaline phosphatase staining.

[0032] The blank group was RPMI-1640 culture solution. The statistical results are shown in Table 1 below:

[0033] Table 1: Test results

[0034] Blank group Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Oct3 / 4 positive ratio (%) 0 12 15 20 4 4 5 2 8 Cell colony number 0 120 136 152 42 45 56 25 98

[0035] From the above results, it can be confirmed that the maternal factors TPRX1, TPRXL, miR-302 and IL1α can effectively improve the expression of the Oct3 / 4 gene in stem cells and the cell colony number, and the combination of the pomegranate seed active peptide extract can further improve the induction ability of somatic cells to omnipotent stem cells, thereby inducing the generation of omnipotent stem cells. Example 5

[0036] Effect of the omnipotent stem cell culture solution containing maternal factors on the proliferation of fibroblasts: The effect of the omnipotent stem cell culture solution containing maternal factors on the proliferation of fibroblasts HFF-1 was detected by MTT cell proliferation detection kit. The specific detection steps can be routinely adjusted according to the kit instructions.

[0037] The blank group was RPMI-1640 culture solution, and the experimental groups 1-3 corresponded to the omnipotent stem cell culture solutions containing the maternal factors prepared by the embodiments 1-3, respectively, and the control groups 1-5 corresponded to the omnipotent stem cell culture solutions containing the maternal factors of the comparative examples 1-5, respectively. The cell dosage in the experimental groups and the control groups was 1×10 3 .

[0038] Table 2. Analysis of the proliferation of fibroblasts

[0039] Group OD value Blank group 0.175±0.012 Experimental group 1 0.543±0.025 Experimental group 2 0.596±0.014 Experimental group 3 0.658±0.031 Control group 1 0.198±0.012 Control group 2 0.209±0.032 Control group 3 0.212±0.021 Control group 4 0.246±0.014 Control group 5 0.319±0.011

[0040] The detection results are shown in Table 2: The omnipotent stem cell culture solution containing maternal factors obtained by any one of the experimental groups 1-3 of the present application can efficiently promote the proliferation of fibroblasts, and the proliferation-promoting effect is significantly better than that of the control groups 1-5 (P<0.05). It is further confirmed that the maternal factors combined with the pomegranate seed active peptide extract can be used as a fibroblast proliferation-promoting culture solution, which can effectively promote wound healing.

[0041] It is necessary to point out here that the above embodiments are only for further illustrating and describing the technical solutions of the present application, and are not for further limiting the technical solutions of the present application. The method of the present application is only a preferred embodiment, and is not used for limiting the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A pluripotent stem cell culture medium containing maternal factors, characterized in that, The product comprises the following components: 10% v / v FBS, 1-3mM TPRX1, 1-3mM TPRXL, 0.5-1.5mM miR-302, 1-2mM IL1α, 0.05-0.15mM β-mercaptoethanol, 0.1-0.3mM pomegranate seed active peptide extract, 100u / L penicillin, 100u / L streptomycin, and the balance of RPMI-1640 culture medium. The pomegranate seed active peptide extract is the active polypeptide shown in SEQ ID NO.

1.

2. The pluripotent stem cell culture medium as described in claim 1, characterized in that, The pluripotent stem cell culture medium containing maternal factors comprises the following components: 10% v / v FBS, 1mM TPRX1, 1mM TPRXL, 0.5mM miR-302, 1mM IL1α, 0.05mM β-mercaptoethanol. 0.1 mM pomegranate seed active peptide extract, 100 u / L penicillin, 100 u / L streptomycin, and the remainder of RPMI-1640 culture medium.

3. The pluripotent stem cell culture medium as described in claim 1, characterized in that, The pluripotent stem cell culture medium containing maternal factors comprises the following components: 10% v / v FBS, 2mM TPRX1, 2mM TPRXL, 1.0mM miR-302, 1.5mM IL1α, 0.1mM β-mercaptoethanol, 0.2mM pomegranate seed active peptide extract, 100u / L penicillin, 100u / L streptomycin, and the remainder of RPMI-1640 culture medium.

4. The pluripotent stem cell culture medium as described in claim 1, characterized in that, The pluripotent stem cell culture medium containing maternal factors comprises the following components: 10% v / v FBS, 3mM TPRX1, 3mM TPRXL, 1.5mM miR-302, 2.0mM IL1α, 0.15mM β-mercaptoethanol, 0.3mM pomegranate seed active peptide extract, 100u / L penicillin, 100u / L streptomycin, and the remainder of RPMI-1640 culture medium.

5. Use of the pluripotent stem cell culture medium according to any one of claims 1-4 in the preparation of fetal fibroblast culture medium.