CN111996163A - Skin anti-aging method based on mesenchymal stem cell disrupted cytoplasm - Google Patents
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Abstract
The invention discloses a skin anti-aging method based on mesenchymal stem cell broken cytoplasm, and particularly relates to the field of application of mesenchymal stem cell anti-aging; a skin anti-aging method based on mesenchymal stem cell disrupted cytoplasm comprises disruption apparatus, disruption method, and mesenchymal stem cell culture method; breaking the cell membrane and the cell wall of the mesenchymal stem cell by applying a shearing force on the mesenchymal stem cell, so that the plasma between the cell membrane and the cell nucleus flows out; the mesenchymal stem cells are placed in a container and stirred by a stirring rod to realize the crushing of the mesenchymal stem cells; the extracted plasma inclusion is placed in a culture medium to accelerate the differentiation speed and activity of the mesenchymal stem cells; the success rate of transplantation is improved and hematopoietic reconstruction is accelerated by inputting hematopoietic stem cells and mesenchymal stem cells into a human body together.
Description
Technical Field
The invention belongs to the field of anti-aging application of mesenchymal stem cells, and particularly relates to a skin anti-aging method based on mesenchymal stem cell broken cytoplasm.
Background
With the continuous development of the current science and technology, stem cell research is more and more intensive, and the stem cell research promotes the development of regenerative medicine, which is a medical revolution following drug treatment and surgical treatment; the variety of human stem cells is more, wherein the mesenchymal stem cells have obvious effect on skin aging resistance; mesenchymal stem cells are a class of pluripotent stem cells belonging to the mesoderm, and are mainly present in connective tissues and organ mesenchymes, with the most abundant content in bone marrow tissues, and are collectively referred to as bone marrow mesenchymal stem cells because bone marrow is the main source thereof. The mesenchymal stem cells have the following characteristics: the single-cell hybrid membrane has strong proliferation capacity and multidirectional differentiation potential, and has the capacity of differentiating into various cells such as muscle cells, liver cells, osteoblasts, fat cells, cartilage cells, stroma cells and the like under a suitable in vivo or in vitro environment. And secondly, the medicine has an immunoregulation function, and plays a role in immune reconstitution by inhibiting the proliferation of T cells and immune reaction thereof through intercellular interaction and cytokine production. And thirdly, the stem cell has the characteristics of convenient source, easy separation, culture, amplification and purification, stem cell characteristics after multiple times of passage amplification and no immunological rejection. Fourthly, the face is fuzzy, the surface antigen is not obvious, the rejection of the xenotransplantation is light, and the mating requirement is not strict. Due to the immunological characteristics of the mesenchymal stem cells, the mesenchymal stem cells have wide clinical application prospect in the aspect of treating hematopathy. The structure and function of the tissue and organ can be reconstructed by autotransplantation, and immunological rejection can be avoided.
The cell disruption technique is a technique of breaking cell membranes and cell walls by external force to release cell contents including target product components. When a certain protein is separated and purified by cell disruption, the protein is firstly released from tissues or cells and is kept in the original natural state without losing activity. Appropriate methods are used to disrupt the tissue and cells. The cell is difficult to be broken in different organisms or tissues of different parts of the same organism, the used method is different, for example, cell membranes of animal organs are fragile and easy to be broken, and plants and microorganisms have firmer cell walls consisting of cellulose and hemicellulose. Common cell disruption methods include mechanical disruption, chemical disruption, and ultrasonic disruption.
Disclosure of Invention
Aiming at the defects of the prior art, the purpose of the disclosure is to provide a skin anti-aging method based on mesenchymal stem cell broken cytoplasm, and solve the problem of insufficient differentiation speed of mesenchymal stem cell culture in the prior art.
The purpose of the disclosure can be realized by the following technical scheme: a skin anti-aging method based on mesenchymal stem cell disruption plasma comprises a mesenchymal stem cell disruption method, wherein the mesenchymal stem cell disruption method comprises the following steps: by applying extrusion force and shearing force on the cell membrane of the mesenchymal stem cell, the plasma is broken along the direction of the shearing force.
Further, the apparatus for completing the mesenchymal stem cell disruption method comprises: a stir bar and a container;
further, the lower end part of the stirring rod is provided with an outer sphere, the lower end part of the container is provided with an inner sphere, and the outer sphere is matched with the inner sphere.
Further, the culture method of the mesenchymal stem cells comprises the following steps:
s1: preparing a gelatin-coated culture plate;
s2: quickly immersing the freezing tube taken out from the liquid nitrogen into warm water at 37 ℃, and quickly shaking to quickly thaw the tube within 1-2 min;
s3: centrifuging: dropwise adding the serum mesenchymal stem cells into a 15mL centrifuge tube containing a stem cell culture medium, and centrifuging at 1000rpm for 3min, wherein the culture medium comprises plasma obtained by crushing the serum mesenchymal stem cells;
s4: resuspending: after centrifugation, the supernatant is discarded, 1mL of human pluripotent stem cell culture medium is added to blow and suck stem cell sediment, and the stem cell sediment is uniformly mixed;
s5: inoculation: after the blowing and sucking are uniform, discarding the balanced instant gelatin, adding the stem cell suspension which is uniformly blown into the coated 6-hole plate, and supplementing a culture system with 2mL per hole;
s6: the 6-well plate was gently shaken by a horizontal cross to distribute the cells evenly. Culturing in a constant temperature incubator at 37 ℃ and 5% CO2, and observing the cell adherence condition on the 2 nd day;
s7: liquid changing: and changing the liquid every 24h from the time of recovery.
Further, the mesenchymal stem cells cultured in the culture dish are infused into the human skin tissue together with the hematopoietic stem cells.
Further, the method for manufacturing the gelatin-coated culture plate comprises the following steps:
diluting the gelatin coating solution to a solution with a proper concentration according to experiment needs;
coating for at least 5min when gelatin coating solution is used for cell culture;
after coating, removing gelatin coating liquid by suction, drying the culture vessel until the culture vessel is completely dried by visual observation, and blowing air in a fume hood for at least 10 minutes to complete drying.
The beneficial effect of this disclosure: accelerating the differentiation speed of the mesenchymal stem cells.
Drawings
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present disclosure, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
FIG. 1 is a schematic illustration of cell disruption according to embodiments of the disclosure;
FIG. 2 is a schematic view of a cell disruption apparatus according to an embodiment of the present disclosure.
Detailed Description
The technical solutions in the embodiments of the present disclosure will be clearly and completely described below with reference to the drawings in the embodiments of the present disclosure, and it is obvious that the described embodiments are only a part of the embodiments of the present disclosure, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments disclosed herein without making any creative effort, shall fall within the protection scope of the present disclosure.
A mesenchymal stem cell-based crushing and plasma-coating skin anti-aging method comprises a mesenchymal stem cell crushing method;
cell disruption refers to a technique of releasing cell contents including target product components by destroying cell membranes and cell walls by external force, and is the basis of separating and purifying non-secretory biochemical substances synthesized in cells.
As shown in fig. 1, in the present embodiment, the mesenchymal stem cell 3 is crushed by using a compression shearing method, and by applying a shearing force 4 to the cell membrane and the cell wall of the mesenchymal stem cell 3, the cell membrane and the cell wall are crushed along the direction of the shearing force, and the plasma 5 located between the cell membrane and the cell nucleus flows out, i.e., the crushing and the plasma extraction of the mesenchymal stem cell are completed.
As shown in fig. 2, the instrument for breaking cell membranes and cell walls of mesenchymal stem cells in this embodiment includes a stirring rod 1 and a container 2, wherein the upper end of the stirring rod 1 is provided with a handle 11 for facilitating stirring of mesenchymal stem cells, the lower end of the stirring rod 1 is provided with an outer sphere 12, the bottom of the container 2 is provided with an inner sphere 21, and the outer sphere 12 arranged on the stirring rod 1 is matched with the inner sphere 21 in the container 2;
when will be to 3 cell membranes of mesenchymal stem cells and cell wall breakage, place mesenchymal stem cells on sphere 21 in the bottom of container 2 now, then stretch into container 2 with stirring rod 1, until the interior sphere 21 contact of the outer spheroid 12 that sets up on the stirring rod 1 and container 2 bottom, stir the handle 11 that stirring rod 1 upper end set up this moment, exert an extrusion force towards outside spheroid 12 simultaneously to stirring rod 1, the mesenchymal stem cells that are located on sphere 21 in container 2 bottom receive extrusion force and shearing force 4's effect and take place the breakage, make the package thick liquid 5 outflow that is located between mesenchymal stem cell membrane and the cell nucleus, and then the completion is to package thick liquid 5's extraction.
In some disclosures, the plasma-encapsulated 5 located between the cell membrane and the cell nucleus is caused to flow out by disrupting the cell membrane and the cell wall of the mesenchymal stem cell 3 by a chemical method such as an enzymatic lysis method, and this extraction method of the plasma-encapsulated 5 is not exclusive as long as the extraction of the plasma-encapsulated 5 can be achieved without disrupting the organelles in the plasma-encapsulated 5.
The culture method of the mesenchymal stem cells 3 comprises the following steps:
1. coating the culture plate with matrigel;
2. quickly immersing the freezing tube taken out from the liquid nitrogen into warm water at 37 ℃, and quickly shaking to quickly thaw the tube within 1-2 min;
3. centrifuging: dropwise adding the suspension into a 15mL centrifuge tube containing a stem cell culture medium, centrifuging at 1000rpm for 3min, wherein the culture medium comprises nutrients such as serum and the like and also comprises plasma 5 obtained by crushing mesenchymal stem cells 3;
4. resuspending: after centrifugation, the supernatant is discarded and 1mL of human pluripotent stem cell culture medium is added to blow and suck the stem cell sediment and mix evenly
5. Inoculation: after the even blowing and sucking, the balanced instant gelatin is discarded, the stem cell suspension which is evenly blown and beaten is added into the coated 6-hole plate, and the 2mL culture system per hole is supplemented
6. Gently shaking the 6-well plate horizontally to uniformly distribute the cells, culturing in a constant-temperature incubator at 37 ℃ and 5% CO2, and observing the cell adherence condition on day 2;
7. liquid changing: and changing the liquid every 24h from the time of recovery.
In addition, the method for manufacturing the gelatin-coated culture plate comprises the following steps:
1. according to the experiment, the gelatin coating solution is diluted to a solution with a proper concentration, different cells are coated, the gelatin coating time and concentration are different, even the selection of the dilution solution is different, and the gelatin coating solution can be properly coated by referring to related documents; the gelatin coating liquid is easy to form jelly under the condition of low temperature and is used after being dissolved;
2. when the gelatin coating solution is used for cell culture, coating is carried out for at least 5min, coating needs to be carried out for 1-2h in some experiments, and coating needs to be carried out overnight in some cases;
3. after coating, the gelatin coating solution is sucked off, and the culture vessel is dried until the culture vessel is completely dry by visual observation. The drying is finished by blowing air in a fume hood for at least 10 minutes, for some experiments, the drying needs 2 hours or more, and the subsequent cell adhesion is more favorably when the drying time is longer;
the plasma inclusion 5 extracted from the mesenchymal stem cells 3 is placed in a culture medium, and the mesenchymal stem cells 3 accelerate the division of the mesenchymal stem cells 3 by drawing the protein in the plasma inclusion 5 in the culture medium, so that the divided daughter cells have higher activity.
The mesenchymal stem cells 3 obtained by culture and the hematopoietic stem cells are input into human skin tissues together, the mesenchymal stem cells 3 are combined with the hematopoietic stem cells for application, the success rate of transplantation can be improved, hematopoietic reconstruction is accelerated, and the aim of resisting aging of the human skin tissues is fulfilled by utilizing the rapid updating capability and the multidirectional differentiation capability of the mesenchymal stem cells 3.
The working principle is as follows:
breaking the cell membrane and the cell wall of the mesenchymal stem cell 3 by applying a shear force 4 on the mesenchymal stem cell 3, thereby allowing an inclusion 5 located between the cell membrane and the cell nucleus to flow out; the mesenchymal stem cells 3 are placed in the container 2 and stirred by the stirring rod 11 to realize the crushing of the mesenchymal stem cells 3; accelerating the differentiation speed and activity of the mesenchymal stem cells 3 by placing the extracted plasma 5 in a culture medium; the success rate of transplantation is improved and hematopoietic reconstruction is accelerated by inputting hematopoietic stem cells and mesenchymal stem cells 3 into human skin tissues together.
The foregoing illustrates and describes the general principles, principal features, and advantages of the present disclosure. It will be understood by those skilled in the art that the present disclosure is not limited to the embodiments described above, which are presented solely for purposes of illustrating the principles of the disclosure, and that various changes and modifications may be made to the disclosure without departing from the spirit and scope of the disclosure, which is intended to be covered by the claims.
Claims (6)
1. A skin anti-aging method based on mesenchymal stem cell disruption plasma comprises a mesenchymal stem cell (3) disruption method, and is characterized in that the mesenchymal stem cell (3) disruption method comprises the following steps: by exerting extrusion force and shearing force (4) on the cell membrane of the mesenchymal stem cell (3), the plasma inclusion (5) is broken along the direction of the shearing force (4).
2. The method for fighting skin aging based on mesenchymal stem cell disruption plasma according to claim 1, characterized in that the apparatus for carrying out the method for disrupting mesenchymal stem cells (3) comprises: a stirring rod (1) and a container (2);
the lower end part of the stirring rod (1) is provided with an outer sphere (12), the lower end part of the container (2) is provided with an inner sphere (21), and the outer sphere (12) is matched with the inner sphere (21).
3. The method for fighting skin aging based on mesenchymal stem cell disruption plasma according to claim 1, wherein the method for culturing the mesenchymal stem cells (3) comprises:
s1: preparing a gelatin-coated culture plate;
s2: quickly immersing the freezing tube taken out from the liquid nitrogen into warm water at 37 ℃, and quickly shaking to quickly thaw the tube within 1-2 min;
s3: centrifuging: dropwise adding the mixture into a 15mL centrifuge tube containing a stem cell culture medium, and centrifuging at 1000rpm for 3 min;
s4: resuspending: after centrifugation, the supernatant is discarded, 1mL of human pluripotent stem cell culture medium is added to blow and suck stem cell sediment, and the stem cell sediment is uniformly mixed;
s5: inoculation: after the blowing and sucking are uniform, discarding the balanced instant gelatin, adding the stem cell suspension which is uniformly blown into the coated 6-hole plate, and supplementing a culture system with 2mL per hole;
s6: the 6-well plate was gently shaken by a horizontal cross to distribute the cells evenly. Culturing in a constant temperature incubator at 37 ℃ and 5% CO2, and observing the cell adherence condition on the 2 nd day;
s7: liquid changing: and changing the liquid every 24h from the time of recovery.
4. The method for resisting skin aging based on mesenchymal stem cell disruption plasma according to claim 3, wherein the mesenchymal stem cells (3) cultured by the culturing method are infused into human skin tissue together with hematopoietic stem cells.
5. A method for fighting skin aging based on mesenchymal stem cell disruption inclusion pulp according to claim 3, characterized in that the inclusion pulp (5) is included in the culture medium.
6. The method for resisting skin aging based on mesenchymal stem cell disruption and plasma coating of claim 3, wherein the method for manufacturing the gelatin-coated culture plate comprises:
diluting the gelatin coating solution to a solution with a proper concentration according to experiment needs;
coating for at least 5min when gelatin coating solution is used for cell culture;
after coating, removing gelatin coating liquid by suction, drying the culture vessel until the culture vessel is completely dried by visual observation, and blowing air in a fume hood for at least 10 minutes to complete drying.
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| CN112574868A (en) * | 2020-12-15 | 2021-03-30 | 山东省农业科学院畜牧兽医研究所 | A tissue homogenizer for primary muscle cell culture of domestic animal |
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