阅读说明：本技术 一种脐带间充质干细胞因子冻干粉的制备方法 (Preparation method of umbilical cord mesenchymal stem cell factor freeze-dried powder ) 是由 乐龙 尹鸿萍 刘永军 杨美家 徐红星 于 2019-10-21 设计创作，主要内容包括：本发明提供一种脐带间充质干细胞因子冻干粉及其制备方法,收集脐带间充质干细胞条件培养基,同时采用甘露醇作为冻干粉保护剂,通过冷冻干燥工艺进行冻干,制备得到的脐带间充质干细胞因子冻干粉有利于长期稳定保存,且具有良好的抗衰老功效。(The invention provides umbilical cord mesenchymal stem cell factor freeze-dried powder and a preparation method thereof.)

1. A preparation method of umbilical cord mesenchymal stem cell factor freeze-dried powder is characterized by comprising the following steps:

(1) taking Wharton's jelly on the umbilical cord, and performing isolated culture to obtain umbilical cord mesenchymal stem cells, which are marked as P0 generation;

(2) carrying out passage for 3-5 times, namely, replacing a serum-free blank culture medium after P3-P5 generation;

(3) continuously culturing for 24h ~ 96 h;

(4) collecting cell supernatant, centrifuging, dialyzing, collecting trapped fluid, and lyophilizing to obtain lyophilized powder.

2. The method for preparing lyophilized powder of umbilical cord mesenchymal stem cell factor according to claim 1, wherein the MSC culture medium is used for passage in step (2).

3. The method for preparing lyophilized powder of umbilical cord mesenchymal stem cell factor according to claim 1, wherein the centrifugation condition in step (4) is to centrifuge the supernatant at 800 ~ 2000rpm/min for 5 ~ 15min at 2 ~ 6 ℃ and take the supernatant.

4. The method for preparing the umbilical cord mesenchymal stem cell factor freeze-dried powder according to claim 1, wherein the dialysis in the step (4) comprises placing the supernatant in a dialysis bag with 3-50 KD, placing the dialysis bag in PBS solution, dialyzing for 24 ~ 48h at 0 ~ 6 ℃ and collecting dialysate.

5. The method for preparing lyophilized powder of umbilical cord mesenchymal stem cell factor according to claim 1, wherein the lyophilization conditions in step (4) are adding a lyophilization protectant, and lyophilizing.

6. The method for preparing lyophilized powder of umbilical cord mesenchymal stem cell factor according to claim 5, wherein the lyophilized protectant in step (4) is 0 ~ 15g of mannitol per 100mL of umbilical cord mesenchymal stem cell factor.

7. The method for preparing lyophilized powder of umbilical cord mesenchymal stem cell factor according to claim 6, wherein the process of lyophilizing in step (4) is as follows:

A. the sample is added into a 1 ~ 10 ml penicillin bottle, and a 1 ~ 5 ml/bottle

B. Reducing the temperature of the cold hydrazine to-80 ~ -60 ℃, and reducing the temperature of the box body to-60 ~ -40 ℃;

C. placing the penicillin bottle semi-pressing plug in a vacuum freeze dryer, and keeping freezing for 1 ~ 6 h;

D. reducing the pressure to 10 ~ 50 Pa;

E. heating to-25 ~ -17 deg.C at a speed of 0.5 ~ 5 deg.C/min, and maintaining for 10- ~ 20 h;

F. reducing the pressure to 0 ~ 30 Pa;

G. heating to 0 ~ 25 ℃ at the speed of 0.5 ~ 5 ℃/min, maintaining for 1 ~ 10 h, and finishing freeze-drying;

H. and (5) pressing a piston in the penicillin bottle, and taking out.

8. The application of the umbilical cord mesenchymal stem cell factor freeze-dried powder in the preparation of medicines for whitening skin, resisting aging and losing hair is characterized in that the umbilical cord mesenchymal stem cell factor freeze-dried powder is prepared according to any one of the methods in claims 1 to 7.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a preparation method of umbilical cord mesenchymal stem cell factor freeze-dried powder.

Background

Mesenchymal Stem Cells (MSCs) secrete a variety of cytokines during stable growth. TGF-beta (transforming growth factor-beta), VEGF (vascular endothelial growth factor), FGF, HGF (liver growth factor), SOD (superoxide dismutase), IL-6 (interleukin-6), EGF (epidermal growth factor), collagen (COL I, COL III), Fibronectin (FN), PDGF (platelet-derived factor) and the like secreted by MSC can effectively prevent oxidative damage, and have significant effects in the aspects of skin aging resistance, wrinkle resistance, skin whitening, wound healing and the like. TGF-beta 1 secreted by MSC plays a role in whitening by inhibiting TYR (TYR) activity, reducing expression of tyrosinase related protein, inhibiting synthesis of melanin and other processes. By collecting the supernatant (MSC-CM) after treating the cultured stem cells, a large amount of cytokines can be obtained.

The MSC-CM in liquid state has short storage time at room temperature, and is not favorable for transportation. The freeze-drying operation is carried out on the freeze-dried powder, and the prepared freeze-dried powder can keep the activity of the cell factors and is convenient to store and transport.

Disclosure of Invention

The invention aims to provide a preparation method of umbilical cord mesenchymal stem cell factor freeze-dried powder, which can quickly prepare umbilical cord mesenchymal stem cell factor freeze-dried powder which can keep activity for a long time and has good anti-aging effect. In order to achieve the purpose, the invention provides the following technical scheme:

a preparation method of umbilical cord mesenchymal stem cell factor, optionally, comprises the following steps:

(1) taking Wharton's jelly on the umbilical cord, and performing isolated culture to obtain umbilical cord mesenchymal stem cells, which are marked as P0 generation;

(2) carrying out passage for 3-5 times, namely, replacing a serum-free blank culture medium after P3-P5 generation;

(3) continuously culturing for 24-96 h;

(4) collecting cell supernatant, centrifuging, dialyzing, collecting trapped fluid to obtain umbilical cord mesenchymal stem cell factor, and lyophilizing.

And (3) MSC culture medium for passage in the step (2).

The centrifugation in the step (4) is as follows: centrifuging the supernatant at the temperature of 2-6 ℃ at the rpm of 800-2000/min for 5-15 min, and taking the supernatant.

The dialysis in the step (4) is as follows: and (3) putting the supernatant into a dialysis bag with 3-50 KD, putting the dialysis bag into a PBS solution, dialyzing for 24-48 h at the temperature of 0-6 ℃, and collecting dialysate.

The freeze-drying protective agent is as follows: and adding 0-15 g of mannitol into every 100mL of the umbilical cord mesenchymal stem cell factor.

In some of these embodiments, optionally, the lyophilization process is: (1) adding the sample into a 5ml penicillin bottle, reducing the temperature of the cold hydrazine to-80 ℃ per 1.5ml penicillin bottle (2), and reducing the temperature of a box body to-40 ℃; (3) placing the penicillin bottle semi-pressing plug in a vacuum freeze dryer, and keeping freezing for 4 hours; (4) reducing the pressure to 25 Pa; (5) heating to-20 deg.C at a speed of 0.5 deg.C/min, and maintaining for 16.5 h; (6) reducing the pressure to 5 Pa; (7) heating to 25 deg.C at a speed of 0.5 deg.C/min, maintaining for 6.5h, and lyophilizing; (8) and (5) pressing a piston in the penicillin bottle, and taking out.

The invention also provides umbilical cord mesenchymal stem cell factor freeze-dried powder, which has the following specific technical scheme:

alternatively, it is prepared by the preparation method as described above.

The invention also provides application of the umbilical cord mesenchymal stem cell factor freeze-dried powder, and the specific technical scheme is as follows:

the umbilical cord mesenchymal stem cell factor freeze-dried powder is applied to skin whitening, anti-aging and alopecia prevention.

Based on the technical scheme, the invention has the following beneficial effects:

the umbilical cord mesenchymal stem cell factor is prepared by collecting the umbilical cord mesenchymal stem cell conditioned medium. Wherein, the umbilical cord mesenchymal stem cells are selected to avoid social ethical disputes, and have the advantages of rapid proliferation and low immunological rejection. The umbilical cord mesenchymal stem cell factor freeze-dried powder is prepared from the stem cell factor prepared by culturing the umbilical cord mesenchymal stem cells by the method and a suitable freeze-dried powder protective agent, is favorable for long-term stable storage of the stem cell factor, and has an anti-aging effect.

Drawings

FIG. 1 is a graph of the effect of different concentrations of lyoprotectant on the morphology of lyophilized samples;

FIG. 2 is a DSC of lyophilized samples (MSC-CM + 4% mannitol);

FIG. 3 is a graph showing the effect of each sample group on the moisture content in the skin of mice;

FIG. 4 is a graph of the effect of each sample group on hydroxyproline content in mouse skin;

FIG. 5 is a graph showing the effect of various sample groups on SOD activity in mouse skin;

FIG. 6 is a graph of HE staining of mouse skin;

FIG. 7 is a graph of the effect of each sample on the thickness of the dermis of the skin of a mouse;

FIG. 8 is a graph of MASSON staining of mouse skin;

FIG. 9 is a graph showing the effect of each sample on the collagen content of mouse skin.

Detailed Description

The invention provides a preparation method of umbilical cord mesenchymal stem cell factor freeze-dried powder, and in order to make the purpose, technical scheme and effect of the invention clearer, the invention is further described in detail with specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention. The various reagents commonly used in the examples are all commercially available products

The invention will now be described more fully with reference to the following specific examples: