CN113412833B - Cryopreservation protective agent for ultralow-temperature damage of mesenchymal stem cells - Google Patents
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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Abstract
The invention provides a cryopreservation protective agent for ultralow temperature damage of mesenchymal stem cells, belonging to the technical field of mesenchymal stem cells. The cryopreservation protective agent for ultralow temperature damage of the mesenchymal stem cells is a culture medium containing Ranacyclin B3 polypeptide, wherein the amino acid sequence of the polypeptide is Ala-Ala-Leu-Lys-Gly-Cys-Trp-Thr-Lys-Ser-Ile-Pro-Pro-Lys-Pro-Cys-Ser-Gly-Lys-Arg. By using the polypeptide to pretreat the cord blood mesenchymal stem cells, the activity of the cells after cryopreservation, and the proliferation and adipogenic differentiation capacity of the cells can be effectively improved.
Description
Technical Field
The invention belongs to the technical field of mesenchymal stem cells, and particularly relates to a cryopreservation protective agent for ultralow-temperature damage of mesenchymal stem cells.
Background
Mesenchymal stem cells are a seed cell with high self-renewal and multipotentiality. Under specific conditions, the mesenchymal stem cells can be induced and differentiated into various tissue cells such as skeletal muscle cells, cartilage cells, fat cells, osteoblasts or cardiac muscle cells, and the like, so that the mesenchymal stem cells can be widely applied to treatment of various diseases and tissue engineering research. Mesenchymal stem cells have a wide variety of sources, and can be classified into adipose mesenchymal stem cells, muscle mesenchymal stem cells, peripheral blood mesenchymal stem cells, umbilical blood mesenchymal stem cells, bone marrow mesenchymal stem cells, and the like according to their sources. Among them, the umbilical blood mesenchymal stem cells have the advantages of wide sources, low immunogenicity, convenient material acquisition and the like, so that the umbilical blood mesenchymal stem cells are widely applied.
Cryopreservation techniques make it possible for cells, living tissues and even living organisms to survive for long periods at low temperatures. The low-temperature preservation of the cord blood mesenchymal stem cells is beneficial to storing a large amount of cord blood mesenchymal stem cells, thereby being convenient for experimenters and medical personnel to use. However, long-term cryopreservation of the cord blood mesenchymal stem cells can cause reduction of cell activity, and can affect the proliferation and differentiation capacity of the cord blood mesenchymal stem cells to a certain extent. Therefore, how to effectively improve the activity of the umbilical cord blood mesenchymal stem cells after cryopreservation recovery is a problem which needs to be solved urgently at present.
Disclosure of Invention
The invention aims to provide a cryopreservation protective agent for ultralow temperature damage of mesenchymal stem cells.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a cryopreservation protective agent for ultralow temperature damage of mesenchymal stem cells, which is a culture medium containing Ranacyclin B3 polypeptide.
Preferably, the Ranacyclin B3 polypeptide has a sequence as follows:
Ala-Ala-Leu-Lys-Gly-Cys-Trp-Thr-Lys-Ser-Ile-Pro-Pro-Lys-Pro-Cys-Ser-Gly-Lys-Arg。
preferably, the concentration of the Ranacyclin B3 polypeptide in the protective agent is more than 50 μmol/L.
Preferably, the mesenchymal stem cell is cord blood mesenchymal stem cell.
Preferably, the medium is DMEM/F12 medium.
In addition, the invention provides an application of Ranacyclin B3 polypeptide in preparation of cryopreservation protective agent for mesenchymal stem cell ultralow temperature injury, wherein the Ranacyclin B3 polypeptide has a sequence as follows:
Ala-Ala-Leu-Lys-Gly-Cys-Trp-Thr-Lys-Ser-Ile-Pro-Pro-Lys-Pro-Cys-Ser-Gly-Lys-Arg。
in addition, the invention provides application of the Ranacyclin B3 polypeptide in preparation of a promoter for activity of mesenchymal stem cells in cryopreserved umbilical cord blood.
In addition, the invention provides application of the Ranacyclin B3 polypeptide in preparation of a proliferation promoter of mesenchymal stem cells in cryopreserved umbilical cord blood.
In addition, the invention provides application of the Ranacyclin B3 polypeptide in preparation of a frozen umbilical cord blood mesenchymal stem cell adipogenic differentiation promoter.
Preferably, the Ranacyclin B3 polypeptide has a sequence as follows:
Ala-Ala-Leu-Lys-Gly-Cys-Trp-Thr-Lys-Ser-Ile-Pro-Pro-Lys-Pro-Cys-Ser-Gly-Lys-Arg。
the invention has the beneficial effects that:
according to the invention, researches show that the cord blood mesenchymal stem cells are pretreated by using a culture medium containing Ranacyclin B3 polypeptide, so that the activity of the cells after cryopreservation, and the proliferation and adipogenic differentiation capacity of the cells can be effectively provided.
Drawings
FIG. 1-survival of groups of cells cryopreserved at 80 ℃;
FIG. 2 the survival rate of each group of cells frozen in liquid nitrogen;
FIG. 3 shows the difference in cell proliferation capacity between the control group and the 100. mu. mol/L Ranacyclin B3 polypeptide-pretreated group after freezing with liquid nitrogen;
FIG. 4 shows the difference in adipogenic differentiation capacity between cells in the control group and cells in the 100. mu. mol/L Ranacyclin B3 polypeptide-pretreated group after freezing with liquid nitrogen;
FIG. 5 shows the difference in expression of adipogenic differentiation-associated proteins between cells in the control group and cells in the 100. mu. mol/L Ranacyclin B3 polypeptide-pretreated group after freezing with liquid nitrogen.
Detailed Description
In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments. It is to be understood that these examples are not limiting of the invention and that variations of the invention, now known or further developed, are considered to fall within the scope of the invention described herein and claimed below.
Example 1
Detecting the influence of Ranacyclin B3 polypeptide pretreatment on the cryopreservation activity of umbilical cord blood mesenchymal stem cells
1. Cell processing
(1) Grouping experiments: control group (using DMEM/F12 medium to treat umbilical cord blood mesenchymal stem cells for 2 h), experimental group 1 (using DMEM/F12 medium containing 25. mu. mol/L Ranacyclin B3 polypeptide to treat umbilical cord blood mesenchymal stem cells for 2 h), experimental group 2 (using DMEM/F12 medium containing 50. mu. mol/L Ranacyclin B3 polypeptide to treat umbilical cord blood mesenchymal stem cells for 2 h), and experimental group 3 (using DMEM/F12 medium containing 100. mu. mol/L Ranacyclin B3 polypeptide to treat umbilical cord blood mesenchymal stem cells for 2 h);
(2) the frozen stock solution is prepared according to 10% DMSO, 50% FBS and 40% DMEM/F12 culture medium;
(3) collecting the cells treated in the step (1), adding the frozen stock solution, and subpackaging into frozen stock tubes, wherein the cell density in each tube is 5 multiplied by 106/ml；
(4) Storing at 4 deg.C for 10min, transferring into-80 deg.C refrigerator, placing part of cells in-80 deg.C refrigerator for 1 month, and storing part of cells in liquid nitrogen overnight for 12 months.
2. Detecting viability of cells
(1) Taking out the frozen cells, putting the cells into a water bath kettle at 37 ℃ for rapid thawing and resuscitation, and centrifuging to remove supernatant;
(2) the cells were resuspended in DMEM/F12 medium to a cell density of 5X 105And/ml, placing the sample under a common optical microscope after staining with the taloprene blue, randomly selecting 6 visual fields in each group, calculating the number of cells stained with the taloprene blue, and calculating the survival rate of the cells according to the following formula:
cell survival = (total number of cells-number of Taiwan phenol blue stained cells)/total number of counted cells × 100%
The results of the experiments obtained are shown in table 1, table 2 and fig. 1, fig. 2:
TABLE 1-80 ℃ survival rate of cryopreserved cells
TABLE 2 survival rates of frozen cells in liquid nitrogen
From the results, the survival rate of the umbilical cord blood mesenchymal stem cells after cryopreservation is obviously higher than that of an untreated group after the umbilical cord blood mesenchymal stem cells are treated by the polypeptide solution containing 50 and 100 mu mol/L of Ranacyclin B3, and the difference meets the statistical requirement. The method proves that the activity of the umbilical cord blood mesenchymal stem cells frozen and stored can be improved by using the Ranacyclin B3 polypeptide solution to treat the umbilical cord blood mesenchymal stem cells.
Example 2
Detecting the influence of the Ranacyclin B3 polypeptide pretreatment on the proliferation activity of umbilical cord blood mesenchymal stem cells
(1) Taking out the cells of the control group and the experimental group 3 which are frozen in liquid nitrogen for 12 months, putting the cells into a water bath kettle at 37 ℃ for rapid thawing and recovery, and centrifuging to remove the supernatant;
(2) inoculating cells into a 6-well plate, removing floating dead cells after the cells adhere to the wall, adding pancreatin to digest the cells and adjusting the cell concentration of each group;
(3) mixing 100ul of 1 × 105The cells were seeded in a 96-well plate, and after 3 days, 10ul of CCK-8 was added to detect the OD of the cells.
The experimental results are shown in fig. 3, the mean value of the OD values of the control group is 0.599, and the standard deviation is 0.032; the mean value of OD value of the experimental group 3 is 0.658, the standard deviation is 0.025, and P is less than 0.05, and from the above results, it can be seen that the proliferation capacity of the umbilical cord blood mesenchymal stem cells can be improved by using the Ranacyclin B3 polypeptide to pretreat the umbilical cord blood mesenchymal stem cells.
Example 3
Detecting the influence of Ranacyclin B3 polypeptide pretreatment on the adipogenic differentiation capacity of mesenchymal stem cells of cryopreserved umbilical cord blood
(1) Taking out the cells of the control group and the experimental group 3 which are frozen in liquid nitrogen for 12 months, putting the cells into a water bath kettle at 37 ℃ for rapid thawing and recovery, and centrifuging to remove the supernatant;
(2) inoculating cells into a 6-well culture plate, culturing the adipogenic induction culture medium I for 2 days when the cell fusion degree reaches 90%, then replacing the adipogenic induction culture medium I with an adipogenic induction culture medium II, continuously culturing until 8 days, and replacing the culture medium every 2 days;
(3) after the culture is finished, removing the culture medium, washing the cells for 3 times by using PBS, and adding 10% formaldehyde to fix the cells for 40 min;
(4) after removing formaldehyde, adding 500ul of oil red O staining solution, and staining for 30min at room temperature;
(5) after removing the oil red O staining solution, the cells were washed with PBS, and then photographed under an inverted microscope.
The experimental result is shown in fig. 4, and it can be seen from the figure that the number of lipid droplets in the experimental group 3 is significantly greater than that in the control group, which indicates that the lipidogenic differentiation capacity of the cryopreserved umbilical cord blood mesenchymal stem cells can be effectively improved after the pretreatment with the Ranacyclin B3 polypeptide.
Example 4
(1) Taking out the cells of the control group and the experimental group 3 which are frozen in liquid nitrogen for 12 months, putting the cells into a water bath kettle at 37 ℃ for rapid thawing and recovery, and centrifuging to remove the supernatant;
(2) inoculating cells into a 6-well culture plate, adding a adipogenic induction culture medium I for culture when the cell fusion degree reaches 90%, changing the adipogenic induction culture medium I into a adipogenic induction culture medium II after culturing for 2 days, continuing culturing until 8 days, and changing the culture medium once every 2 days;
(3) after the culture is finished, removing the culture medium, washing the cells once by using precooled PBS buffer solution, adding 200ul RIPA lysate into each hole, blowing for several seconds by using a pipette gun, centrifuging for 15min at 12000g, and collecting the supernatant;
(4) detecting the protein concentration by using a BCA kit, adding a 5 xSDS-loading buffer protein loading buffer solution, and heating at 100 ℃ for 5min to fully denature the protein;
(5) preparing electrophoresis glue and electrophoresis buffer solution, installing an electrophoresis frame, and carrying out protein sampling;
(6) after the sample loading is finished, covering the electrophoresis tank, inserting a power line plug into an electrophoresis jack of the electrophoresis apparatus, and performing electrophoresis for about 20min under 90V electrophoresis until a bromophenol blue strip reaches a boundary of concentrated gel and separation gel;
(7) adjusting the voltage to 120V until the bromophenol blue band runs to the bottom of the gel, ending electrophoresis, and taking out the electrophoresis gel;
(8) cutting a PVDF membrane and filter paper with proper sizes, soaking the filter paper in an electrotransfer solution for standby, activating the PVDF membrane in a methanol solution, and then putting the activated PVDF membrane in the electrotransfer solution;
(9) installing an electric rotating clamp according to the sandwich model, and adjusting the parameters of the electric rotating instrument to 250mA for 90 min;
(10) after the electro-conversion is finished, taking the membrane out of the electro-conversion tank, rinsing with TBST, placing in a sealing solution, and shaking and sealing on a decoloring shaking table for 1h at room temperature;
(11) dilution of the antibodies with blocking solution was performed according to the AP2, PPAR γ and β -actin antibody instructions, followed by overnight incubation in a refrigerator at 4 ℃;
(12) after incubation is finished, washing the TBST membrane washing solution on a decolorizing shaker for 3 times at room temperature, 10min each time, then diluting the secondary antibody with a sealing solution according to the proportion recommended by the specification, and incubating for 2h at room temperature;
(13) after the incubation is finished, washing for 3 times with TBST membrane washing liquid at room temperature by a decoloration shaking bed, and 10min for each time;
(14) mixing a Luminol reagent and a hydrogen peroxide reagent in the ECL kit in equal volume to prepare HRP substrate working solution, placing the HRP substrate working solution at room temperature in a dark place for 10min, dropwise adding the HRP substrate working solution to a PVDF membrane when the HRP working solution is recovered to the room temperature, incubating for 5min, and placing the HRP substrate working solution in a chemiluminescence imager for exposure.
The experimental results are shown in fig. 5, and it can be seen from the figure that the expression levels of the adipogenic differentiation-related proteins AP2 and PPAR γ in the experimental group 3 are higher than those in the control group, and the results show that the expression of the adipogenic differentiation-related proteins AP2 and PPAR γ protein in the cryopreserved umbilical cord blood mesenchymal stem cells can be effectively improved after the pretreatment with the Ranacyclin B3 polypeptide.
This summary merely illustrates some embodiments which are claimed, wherein one or more of the features recited in the claims can be combined with any one or more of the embodiments, and such combined embodiments are also within the scope of the present disclosure as if they were specifically recited in the disclosure.
Claims (5)
1. A cryopreservation protective agent for ultralow temperature damage of mesenchymal stem cells is characterized in that the protective agent is a culture medium containing Ranacyclin B3 polypeptide;
the Ranacyclin B3 polypeptide has a sequence as follows:
Ala-Ala-Leu-Lys-Gly-Cys-Trp-Thr-Lys-Ser-Ile-Pro-Pro-Lys-Pro-Cys-Ser-Gly-Lys-Arg；
the concentration of the Ranacyclin B3 polypeptide in the protective agent is 50 mu mol/L;
the mesenchymal stem cells refer to umbilical cord blood mesenchymal stem cells;
the culture medium is DMEM/F12 culture medium.
2. The application of the Ranacyclin B3 polypeptide in preparing cryopreservation protective agent for mesenchymal stem cell ultralow temperature injury is characterized in that the Ranacyclin B3 polypeptide has a sequence as follows:
Ala-Ala-Leu-Lys-Gly-Cys-Trp-Thr-Lys-Ser-Ile-Pro-Pro-Lys-Pro-Cys-Ser-Gly-Lys-Arg。
the application of the Ranacyclin B3 polypeptide in preparing the promoter for the activity of the mesenchymal stem cells of the frozen umbilical cord blood, wherein the Ranacyclin B3 polypeptide has a sequence as follows:
Ala-Ala-Leu-Lys-Gly-Cys-Trp-Thr-Lys-Ser-Ile-Pro-Pro-Lys-Pro-Cys-Ser-Gly-Lys-Arg。
the application of the Ranacyclin B3 polypeptide in preparing the proliferation promoter of the mesenchymal stem cells of the frozen umbilical cord blood, wherein the Ranacyclin B3 polypeptide has a sequence as follows:
Ala-Ala-Leu-Lys-Gly-Cys-Trp-Thr-Lys-Ser-Ile-Pro-Pro-Lys-Pro-Cys-Ser-Gly-Lys-Arg。
5. the application of the Ranacyclin B3 polypeptide in preparing a frozen umbilical cord blood mesenchymal stem cell adipogenic differentiation promoter is disclosed, wherein the Ranacyclin B3 polypeptide has a sequence as follows:
Ala-Ala-Leu-Lys-Gly-Cys-Trp-Thr-Lys-Ser-Ile-Pro-Pro-Lys-Pro-Cys-Ser-Gly-Lys-Arg。
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