阅读说明：本技术 一种诱导脐带血中的timp2蛋白酶与细胞裂解液的修复再生方法 (Repair and regeneration method for inducing TIMP2 protease and cell lysate in umbilical cord blood ) 是由 孙长林 胡海涛 于 2021-08-23 设计创作，主要内容包括：本发明公开了一种诱导脐带血中的TIMP2蛋白酶与细胞裂解液的修复再生方法,通过获取细胞裂解液,并将获得的细胞裂解液与诱导脐带血中的TIMP2蛋白酶、NP-40裂解液混合复配,可很好地用于人体组织损伤修复再生,可很好地修复老化损伤的组织和细胞、提升真皮组织和细胞的再生能力；可以修复受损的组织和细胞；可以激活老化组织和细胞的再生能力；效果显著；且技术路线成熟,原料来源广泛、取材容易,成本较低,便于大规模工业化生产。(The invention discloses a method for repairing and regenerating TIMP2 protease in induced umbilical cord blood and cell lysate, which can be well used for repairing and regenerating human tissue damage, well repairing aged and damaged tissues and cells and improving the regeneration capacity of dermal tissues and cells by obtaining the cell lysate and mixing and compounding the obtained cell lysate with TIMP2 protease and NP-40 lysate in the induced umbilical cord blood; damaged tissues and cells can be repaired; can activate regeneration ability of aged tissue and cell; the effect is remarkable; and the technical route is mature, the raw materials are wide in source, the materials are easy to obtain, the cost is lower, and the large-scale industrial production is facilitated.)

1. A method for inducing repair and regeneration of TIMP2 protease and cell lysate in umbilical cord blood, which is characterized by comprising the following steps:

(1) collecting and obtaining animal cells under aseptic conditions;

(2) subjecting the animal cells obtained in step (1) to subculture;

(3) adjusting the cell concentration of the passage cells obtained in the step (2) to 2-4X 10⁶And (3) dissolving the mixture in physiological saline, wherein the ratio of the volume (ml) of the physiological saline to the mass (g) of the animal cells in the step (2) is 7: 4, freezing in a refrigerator at the temperature of-85 ℃ until the cell is completely frozen, then heating at the temperature of 38 ℃ to finish the freeze-thaw operation, centrifuging to obtain cell suspension X1 and precipitate X1, and collecting cell suspension X1 to obtain a primary lysate;

(4) adding physiological saline containing animal cell lysate with volume concentration of 0.15% to the precipitate X1 obtained in step (3), wherein the ratio of the volume (ml) of the physiological saline to the mass (g) of the animal cells in step (2) is 7: 4, carrying out ultrasonic treatment under the ultrasonic condition with the amplitude of 25% and the temperature of 5 ℃, centrifuging to obtain cell suspension X2 and sediment X2, collecting cell suspension X2, obtaining deep lysate, and discarding the sediment B;

(5) respectively dissolving and mixing the cell suspension X1 obtained in the step (3) and the cell suspension X2 obtained in the step (4) by using physiological saline, wherein the ratio of the volume (ml) of the physiological saline to the mass (g) of the animal cells in the step (2) is 1: 1, obtaining cell lysate;

(6) mixing the cell lysate obtained in the step (5) with TIMP2 protease and NP-40 lysate in the induced umbilical cord blood to obtain mixed lysate;

(7) and (4) using the final mixed lysate obtained in the step (6) for repairing and regenerating human tissue damage.

2. The method for inducing repair and regeneration of TIMP2 protease and cell lysate in umbilical cord blood according to claim 1, wherein in step (1), the animal cells are autologous cells.

3. The method for inducing repair and regeneration of TIMP2 protease and cell lysate in umbilical cord blood according to claim 1, wherein in the step (2), the animal cells obtained in the step (1) are subcultured to P3 generation cells.

4. The method for inducing repair and regeneration of TIMP2 protease and cell lysate in umbilical cord blood as claimed in claim 1, wherein the freezing and thawing operation in the step (3) is repeated 3-5 times.

5. The method for inducing repair and regeneration of TIMP2 protease and cell lysate in umbilical cord blood as claimed in claim 1, wherein the heating time of the freeze-thaw operation in the step (3) is 18-24 min.

6. The method for inducing repair and regeneration of TIMP2 protease and cell lysate in umbilical cord blood as claimed in claim 1, wherein the time of the sonication in the step (3) is 8-12 min.

7. The method for inducing repair and regeneration of TIMP2 protease and cell lysate in umbilical cord blood according to claim 1, wherein in step (6), the concentration of TIMP2 protease in the umbilical cord blood in the mixed lysate is 0.004-0.008 μ g/mL.

8. The method for inducing repair and regeneration of TIMP2 protease and cell lysate in umbilical cord blood as claimed in claim 1, wherein in step (6), the volume percentage concentration of NP-40 lysate in the mixed lysate is 2-6%.

Technical Field

The invention relates to the technical field of cell engineering, in particular to a method for repairing and regenerating TIMP2 protease and cell lysate in induced umbilical cord blood.

Background

With the gradual improvement of the living standard of people, people are not difficult to eat full and warm, and people are pursuing higher-level life due to the abundance of life.

Cells are the basic constitutional units of our human body, and the reason for human body aging is the aging of cells in the body. With the age, the proliferation and differentiation capacity of cells in the human body is weakened, the newly born cells are not sufficiently supplemented, and the aged cells cannot be replaced in time. The aging of cells causes the reduction and decay of the functions of various physiological systems of the human body, thereby causing various diseases and finally causing death.

The concept of repair includes a premise and three elements. The precondition is that the cell or tissue must be "depleted", which means the physiological aging and apoptosis of the tissue cells, and the "loss" means pathological damage. The three elements are:

firstly, the organism absorbs and removes necrosis, debris, foreign matters, pathogeny and the like in a wearing area through immune and inflammatory reaction;

if the lost parenchymal cells have regeneration capacity and proper conditions, repair and recovery are carried out through regeneration of the same kind of parenchymal cells which are nearby and remained, and the repair can completely recover the structures and functions of the original cells and tissues, so the repair is called regenerative repair or complete repair;

in pathological conditions, if parenchymal cells cannot regenerate or only partially regenerate, the tissue defect is completely or partially repaired and filled with regenerated fibrous connective tissue (granulation tissue) rich in small blood vessels, and scars are formed, which are called scar repair or incomplete repair because the tissue can only be restored to the integrity and cannot be completely restored to the original structure and function.

However, the current repair and regeneration methods have the following problems:

1. the medicament (cell lysate) which is used for repairing and regenerating the human tissue damage and has good effect is lacked;

2. poor effects of repairing aged and damaged tissues and cells and improving the regeneration capacity of dermal tissues and cells;

3. the technology is immature and the cost is high.

Based on the situation, the invention provides a method for repairing and regenerating the TIMP2 protease and the cell lysate in the induced umbilical cord blood, which can effectively solve the problems.

Disclosure of Invention

The invention aims to provide a method for inducing repair and regeneration of TIMP2 protease and cell lysate in umbilical cord blood. According to the method for repairing and regenerating the TIMP2 protease in the induced umbilical cord blood and the cell lysate, the cell lysate is obtained, and the obtained cell lysate is mixed with the TIMP2 protease and the NP-40 lysate in the induced umbilical cord blood, so that the method can be well used for repairing and regenerating human tissue damage, well repairing aged and damaged tissues and cells and improving the regeneration capacity of dermal tissues and cells; damaged tissues and cells can be repaired; can activate regeneration ability of aged tissue and cell; the effect is remarkable; and the technical route is mature, the raw materials are wide in source, the materials are easy to obtain, the cost is lower, and the large-scale industrial production is facilitated.

In order to solve the technical problems, the technical scheme provided by the invention is as follows:

a method for inducing repair and regeneration of TIMP2 protease and cell lysate in umbilical cord blood, comprising the following steps:

(1) collecting and obtaining animal cells under aseptic conditions;

(2) subjecting the animal cells obtained in step (1) to subculture;

(3) adjusting the cell concentration of the passage cells obtained in the step (2) to 2-4X 106, dissolving the passage cells in physiological saline, wherein the ratio of the volume (ml) of the physiological saline to the mass (g) of the animal cells in the step (2) is 7: 4, freezing in a refrigerator at the temperature of-85 ℃ until the cell is completely frozen, then heating at the temperature of 38 ℃ to finish the freeze-thaw operation, centrifuging to obtain cell suspension X1 and precipitate X1, and collecting cell suspension X1 to obtain a primary lysate;

(4) adding physiological saline containing animal cell lysate with volume concentration of 0.15% to the precipitate X1 obtained in step (3), wherein the ratio of the volume (ml) of the physiological saline to the mass (g) of the animal cells in step (2) is 7: 4, carrying out ultrasonic treatment under the ultrasonic condition with the amplitude of 25% and the temperature of 5 ℃, centrifuging to obtain cell suspension X2 and sediment X2, collecting cell suspension X2, obtaining deep lysate, and discarding the sediment B;

(5) respectively dissolving and mixing the cell suspension X1 obtained in the step (3) and the cell suspension X2 obtained in the step (4) by using physiological saline, wherein the ratio of the volume (ml) of the physiological saline to the mass (g) of the animal cells in the step (2) is 1: 1, obtaining cell lysate;

(6) mixing the cell lysate obtained in the step (5) with TIMP2 protease and NP-40 lysate in the induced umbilical cord blood to obtain mixed lysate;

(7) and (4) using the final mixed lysate obtained in the step (6) for repairing and regenerating human tissue damage.

According to the method for repairing and regenerating the TIMP2 protease in the induced umbilical cord blood and the cell lysate, the cell lysate is obtained, and the obtained cell lysate is mixed with the TIMP2 protease and the NP-40 lysate in the induced umbilical cord blood, so that the method can be well used for repairing and regenerating human tissue damage, well repairing aged and damaged tissues and cells and improving the regeneration capacity of dermal tissues and cells; damaged tissues and cells can be repaired; can activate regeneration ability of aged tissue and cell; the effect is remarkable; and the technical route is mature, the raw materials are wide in source, the materials are easy to obtain, the cost is lower, and the large-scale industrial production is facilitated.

Preferably, in step (1), the animal cells are autologous cells.

Preferably, in the step (2), the animal cells obtained in the step (1) are subcultured to P3 generation cells.

Preferably, the freezing and thawing operation in the step (3) is repeated 3-5 times.

Preferably, the heating time of the freezing and thawing operation in the step (3) is 18-24 min.

Preferably, the time of the ultrasonic treatment in the step (3) is 8-12 min.

Preferably, in step (6), the concentration of TIMP2 protease in the induced cord blood in the mixed lysate is 0.004-0.008 μ g/mL.

Preferably, in the step (6), the volume percentage concentration of the NP-40 lysate in the mixed lysate is 2-6%.

Compared with the prior art, the invention has the following advantages and beneficial effects:

according to the method for repairing and regenerating the TIMP2 protease in the induced umbilical cord blood and the cell lysate, the cell lysate is obtained, and the obtained cell lysate is mixed with the TIMP2 protease and the NP-40 lysate in the induced umbilical cord blood, so that the method can be well used for repairing and regenerating human tissue damage, well repairing aged and damaged tissues and cells and improving the regeneration capacity of dermal tissues and cells; damaged tissues and cells can be repaired; can activate regeneration ability of aged tissue and cell; the effect is remarkable; and the technical route is mature, the raw materials are wide in source, the materials are easy to obtain, the cost is lower, and the large-scale industrial production is facilitated.

Detailed Description

In order that those skilled in the art will better understand the technical solutions of the present invention, the following description of the preferred embodiments of the present invention is provided in connection with specific examples, which should not be construed as limiting the present patent.

Example 1:

a method for inducing repair and regeneration of TIMP2 protease and cell lysate in umbilical cord blood, comprising the following steps:

(1) collecting and obtaining animal cells under aseptic conditions;

(2) subjecting the animal cells obtained in step (1) to subculture;

(3) adjusting the cell concentration of the passage cells obtained in the step (2) to 2-4X 106, dissolving the passage cells in physiological saline, wherein the ratio of the volume (ml) of the physiological saline to the mass (g) of the animal cells in the step (2) is 7: 4, freezing in a refrigerator at the temperature of-85 ℃ until the cell is completely frozen, then heating at the temperature of 38 ℃ to finish the freeze-thaw operation, centrifuging to obtain cell suspension X1 and precipitate X1, and collecting cell suspension X1 to obtain a primary lysate;

(4) adding physiological saline containing animal cell lysate with volume concentration of 0.15% to the precipitate X1 obtained in step (3), wherein the ratio of the volume (ml) of the physiological saline to the mass (g) of the animal cells in step (2) is 7: 4, carrying out ultrasonic treatment under the ultrasonic condition with the amplitude of 25% and the temperature of 5 ℃, centrifuging to obtain cell suspension X2 and sediment X2, collecting cell suspension X2, obtaining deep lysate, and discarding the sediment B;

(5) respectively dissolving and mixing the cell suspension X1 obtained in the step (3) and the cell suspension X2 obtained in the step (4) by using physiological saline, wherein the ratio of the volume (ml) of the physiological saline to the mass (g) of the animal cells in the step (2) is 1: 1, obtaining cell lysate;

(6) mixing the cell lysate obtained in the step (5) with TIMP2 protease and NP-40 lysate in the induced umbilical cord blood to obtain mixed lysate;

(7) and (4) using the final mixed lysate obtained in the step (6) for repairing and regenerating human tissue damage.

Preferably, in step (1), the animal cells are autologous cells.

Preferably, in the step (2), the animal cells obtained in the step (1) are subcultured to P3 generation cells.

Preferably, the freezing and thawing operation in the step (3) is repeated 3-5 times.

Preferably, the heating time of the freezing and thawing operation in the step (3) is 18-24 min.

Preferably, the time of the ultrasonic treatment in the step (3) is 8-12 min.

Preferably, in step (6), the concentration of TIMP2 protease in the induced cord blood in the mixed lysate is 0.004-0.008 μ g/mL.

Preferably, in the step (6), the volume percentage concentration of the NP-40 lysate in the mixed lysate is 2-6%.

Example 2:

a method for inducing repair and regeneration of TIMP2 protease and cell lysate in umbilical cord blood, comprising the following steps:

(1) collecting and obtaining animal cells under aseptic conditions;

(2) subjecting the animal cells obtained in step (1) to subculture;

(3) adjusting the cell concentration of the passage cells obtained in the step (2) to be 2X 106, dissolving the passage cells in physiological saline, wherein the ratio of the volume (ml) of the physiological saline to the mass (g) of the animal cells in the step (2) is 7: 4, freezing in a refrigerator at the temperature of-85 ℃ until the cell is completely frozen, then heating at the temperature of 38 ℃ to finish the freeze-thaw operation, centrifuging to obtain cell suspension X1 and precipitate X1, and collecting cell suspension X1 to obtain a primary lysate;

(4) adding physiological saline containing animal cell lysate with volume concentration of 0.15% to the precipitate X1 obtained in step (3), wherein the ratio of the volume (ml) of the physiological saline to the mass (g) of the animal cells in step (2) is 7: 4, carrying out ultrasonic treatment under the ultrasonic condition with the amplitude of 25% and the temperature of 5 ℃, centrifuging to obtain cell suspension X2 and sediment X2, collecting cell suspension X2, obtaining deep lysate, and discarding the sediment B;

(5) respectively dissolving and mixing the cell suspension X1 obtained in the step (3) and the cell suspension X2 obtained in the step (4) by using physiological saline, wherein the ratio of the volume (ml) of the physiological saline to the mass (g) of the animal cells in the step (2) is 1: 1, obtaining cell lysate;

(6) mixing the cell lysate obtained in the step (5) with TIMP2 protease and NP-40 lysate in the induced umbilical cord blood to obtain mixed lysate;

(7) and (4) using the final mixed lysate obtained in the step (6) for repairing and regenerating human tissue damage.

In the step (1), the animal cells are autologous cells.

In the step (2), the animal cells obtained in the step (1) are subcultured to P3 generation cells.

Repeating the freezing and thawing operation in the step (3) for 3 times.

The heating time of the freezing and thawing operation in the step (3) is 18 min.

The ultrasonic treatment time in the step (3) is 8 min.

In the step (6), the concentration of the TIMP2 protease in the induced umbilical cord blood in the mixed lysate is 0.004 mu g/mL.

In the step (6), the volume percentage concentration of NP-40 lysate in the mixed lysate is 2%.

Example 3:

a method for inducing repair and regeneration of TIMP2 protease and cell lysate in umbilical cord blood, comprising the following steps:

(1) collecting and obtaining animal cells under aseptic conditions;

(2) subjecting the animal cells obtained in step (1) to subculture;

(3) adjusting the cell concentration of the passage cells obtained in the step (2) to be 4X 106, dissolving the passage cells in physiological saline, wherein the ratio of the volume (ml) of the physiological saline to the mass (g) of the animal cells in the step (2) is 7: 4, freezing in a refrigerator at the temperature of-85 ℃ until the cell is completely frozen, then heating at the temperature of 38 ℃ to finish the freeze-thaw operation, centrifuging to obtain cell suspension X1 and precipitate X1, and collecting cell suspension X1 to obtain a primary lysate;

(4) adding physiological saline containing animal cell lysate with volume concentration of 0.15% to the precipitate X1 obtained in step (3), wherein the ratio of the volume (ml) of the physiological saline to the mass (g) of the animal cells in step (2) is 7: 4, carrying out ultrasonic treatment under the ultrasonic condition with the amplitude of 25% and the temperature of 5 ℃, centrifuging to obtain cell suspension X2 and sediment X2, collecting cell suspension X2, obtaining deep lysate, and discarding the sediment B;

(5) respectively dissolving and mixing the cell suspension X1 obtained in the step (3) and the cell suspension X2 obtained in the step (4) by using physiological saline, wherein the ratio of the volume (ml) of the physiological saline to the mass (g) of the animal cells in the step (2) is 1: 1, obtaining cell lysate;

(6) mixing the cell lysate obtained in the step (5) with TIMP2 protease and NP-40 lysate in the induced umbilical cord blood to obtain mixed lysate;

(7) and (4) using the final mixed lysate obtained in the step (6) for repairing and regenerating human tissue damage.

In the step (1), the animal cells are autologous cells.

In the step (2), the animal cells obtained in the step (1) are subcultured to P3 generation cells.

Repeating the freezing and thawing operation in the step (3) for 5 times.

The heating time of the freezing and thawing operation in the step (3) is 24 min.

The ultrasonic treatment time in the step (3) is 12 min.

In the step (6), the concentration of the TIMP2 protease in the induced cord blood in the mixed lysate is 0.008 mu g/mL.

In the step (6), the volume percentage concentration of the NP-40 lysate in the mixed lysate is 6%.

Example 4:

a method for inducing repair and regeneration of TIMP2 protease and cell lysate in umbilical cord blood, comprising the following steps:

(1) collecting and obtaining animal cells under aseptic conditions;

(2) subjecting the animal cells obtained in step (1) to subculture;

(3) adjusting the cell concentration of the passage cells obtained in the step (2) to be 3X 106, dissolving the passage cells in physiological saline, wherein the ratio of the volume (ml) of the physiological saline to the mass (g) of the animal cells in the step (2) is 7: 4, freezing in a refrigerator at the temperature of-85 ℃ until the cell is completely frozen, then heating at the temperature of 38 ℃ to finish the freeze-thaw operation, centrifuging to obtain cell suspension X1 and precipitate X1, and collecting cell suspension X1 to obtain a primary lysate;

(4) adding physiological saline containing animal cell lysate with volume concentration of 0.15% to the precipitate X1 obtained in step (3), wherein the ratio of the volume (ml) of the physiological saline to the mass (g) of the animal cells in step (2) is 7: 4, carrying out ultrasonic treatment under the ultrasonic condition with the amplitude of 25% and the temperature of 5 ℃, centrifuging to obtain cell suspension X2 and sediment X2, collecting cell suspension X2, obtaining deep lysate, and discarding the sediment B;

(5) respectively dissolving and mixing the cell suspension X1 obtained in the step (3) and the cell suspension X2 obtained in the step (4) by using physiological saline, wherein the ratio of the volume (ml) of the physiological saline to the mass (g) of the animal cells in the step (2) is 1: 1, obtaining cell lysate;

(6) mixing the cell lysate obtained in the step (5) with TIMP2 protease and NP-40 lysate in the induced umbilical cord blood to obtain mixed lysate;

(7) and (4) using the final mixed lysate obtained in the step (6) for repairing and regenerating human tissue damage.

In the step (1), the animal cells are autologous cells.

In the step (2), the animal cells obtained in the step (1) are subcultured to P3 generation cells.

Repeating the freezing and thawing operation in the step (3) for 4 times.

The heating time of the freezing and thawing operation in the step (3) is 20 min.

The ultrasonic treatment time in the step (3) is 11 min.

In the step (6), the concentration of the TIMP2 protease in the induced cord blood in the mixed lysate is 0.006 mu g/mL.

In the step (6), the volume percentage concentration of the NP-40 lysate in the mixed lysate is 4%.

According to the method for repairing and regenerating the TIMP2 protease in the induced umbilical cord blood and the cell lysate, the cell lysate is obtained, and the obtained cell lysate is mixed with the TIMP2 protease and the NP-40 lysate in the induced umbilical cord blood, so that the method can be well used for repairing and regenerating human tissue damage, well repairing aged and damaged tissues and cells and improving the regeneration capacity of dermal tissues and cells; damaged tissues and cells can be repaired; can activate regeneration ability of aged tissue and cell; the effect is remarkable; and the technical route is mature, the raw materials are wide in source, the materials are easy to obtain, the cost is lower, and the large-scale industrial production is facilitated.

The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.