阅读说明：本技术 一种促进细胞生长的生物制剂 (Biological agent for promoting cell growth ) 是由 王善粉 孙昌星 于 2021-10-15 设计创作，主要内容包括：本发明属于细胞生物学技术领域,尤其涉及一种促进细胞生长的生物制剂。本发明所提供的促进细胞生长的生物制剂为提高脐带间充质干细胞生长的生物制剂,该生物制剂由短毛藻多糖组成,本发明所提供的生物制剂可以有效的促进脐带间充质干细胞的增殖,因此,可将本发明所提供的生物制剂用于促进脐带间充质干细胞的快速生长。(The invention belongs to the technical field of cell biology, and particularly relates to a biological agent for promoting cell growth. The biological preparation for promoting the cell growth is a biological preparation for improving the growth of umbilical cord mesenchymal stem cells, and consists of the short-haired algae polysaccharide.)

1. The biological preparation for promoting the growth of umbilical cord mesenchymal stem cells is characterized by being short-haired algae polysaccharide prepared by the following preparation method:

(1) cleaning the short-haired algae raw material with clear water, drying the short-haired algae raw material in a drying box to constant weight, and crushing the short-haired algae raw material into short-haired algae powder;

(2) adding 20 times of distilled water, extracting at 90 deg.C for 3 hr, centrifuging at 8500g for 30min, and collecting supernatant to obtain short-hair algae extract;

(3) rotary evaporating the extractive solution of short-haired algae to 1/4;

(4) adding 3 times of anhydrous ethanol, standing for 24 hr, centrifuging for 20min at 5000g, and freeze drying the precipitate to obtain crude polysaccharide of Dunaliella;

(5) adding distilled water to dissolve the crude polysaccharide of the short-haired algae to obtain a crude polysaccharide dissolving solution of the short-haired algae;

(6) passing the crude polysaccharide solution through a 50kDa ultrafiltration membrane, and freeze-drying the trapped fluid to obtain the polysaccharide of the short-hair algae;

the concentration of the short-hair algae polysaccharide is more than or equal to 10 mg/ml.

2. The application of the short-haired algae polysaccharide in preparing the biological preparation for promoting the proliferation of the umbilical cord mesenchymal stem cells is characterized in that the short-haired algae polysaccharide is prepared by the following preparation method:

(1) cleaning the short-haired algae raw material with clear water, drying the short-haired algae raw material in a drying box to constant weight, and crushing the short-haired algae raw material into short-haired algae powder;

(2) adding 20 times of distilled water, extracting at 90 deg.C for 3 hr, centrifuging at 8500g for 30min, and collecting supernatant to obtain short-hair algae extract;

(3) rotary evaporating the extractive solution of short-haired algae to 1/4;

(4) adding 3 times of anhydrous ethanol, standing for 24 hr, centrifuging for 20min at 5000g, and freeze drying the precipitate to obtain crude polysaccharide of Dunaliella;

(5) adding distilled water to dissolve the crude polysaccharide of the short-haired algae to obtain a crude polysaccharide dissolving solution of the short-haired algae;

(6) and (3) passing the crude polysaccharide dissolving solution through a 50kDa ultrafiltration membrane, and freeze-drying the trapped fluid to obtain the short-hair algae polysaccharide.

3. The use according to claim 2, wherein the use comprises the protein expression of the SCF, a protein for promoting the proliferation of the SCF in the umbilical cord mesenchymal stem cells, by using the brevium polysaccharide.

4. The use according to claim 2, wherein the use comprises the sargassum brevifolia polysaccharide inhibiting protein expression of cord mesenchymal stem cell antiproliferative protein p 21.

5. The application of the short-hair algae polysaccharide in preparing the protein expression promoter of the umbilical cord mesenchymal stem cell proliferation promoting protein SCF is characterized in that the short-hair algae polysaccharide is prepared by the following preparation method:

(1) cleaning the short-haired algae raw material with clear water, drying the short-haired algae raw material in a drying box to constant weight, and crushing the short-haired algae raw material into short-haired algae powder;

(2) adding 20 times of distilled water, extracting at 90 deg.C for 3 hr, centrifuging at 8500g for 30min, and collecting supernatant to obtain short-hair algae extract;

(3) rotary evaporating the extractive solution of short-haired algae to 1/4;

(4) adding 3 times of anhydrous ethanol, standing for 24 hr, centrifuging for 20min at 5000g, and freeze drying the precipitate to obtain crude polysaccharide of Dunaliella;

(5) adding distilled water to dissolve the crude polysaccharide of the short-haired algae to obtain a crude polysaccharide dissolving solution of the short-haired algae;

(6) and (3) passing the crude polysaccharide dissolving solution through a 50kDa ultrafiltration membrane, and freeze-drying the trapped fluid to obtain the short-hair algae polysaccharide.

6. The application of the short-haired algae polysaccharide in preparing the protein expression inhibitor of the proliferation inhibiting protein p21 of the umbilical cord mesenchymal stem cells is characterized in that the short-haired algae polysaccharide is prepared by the following preparation method:

(1) cleaning the short-haired algae raw material with clear water, drying the short-haired algae raw material in a drying box to constant weight, and crushing the short-haired algae raw material into short-haired algae powder;

(2) adding 20 times of distilled water, extracting at 90 deg.C for 3 hr, centrifuging at 8500g for 30min, and collecting supernatant to obtain short-hair algae extract;

(3) rotary evaporating the extractive solution of short-haired algae to 1/4;

(4) adding 3 times of anhydrous ethanol, standing for 24 hr, centrifuging for 20min at 5000g, and freeze drying the precipitate to obtain crude polysaccharide of Dunaliella;

(5) adding distilled water to dissolve the crude polysaccharide of the short-haired algae to obtain a crude polysaccharide dissolving solution of the short-haired algae;

(6) and (3) passing the crude polysaccharide dissolving solution through a 50kDa ultrafiltration membrane, and freeze-drying the trapped fluid to obtain the short-hair algae polysaccharide.

Technical Field

The invention belongs to the technical field of cell biology, and particularly relates to a biological agent for promoting cell growth.

Background

The mesenchymal stem cell is an adult stem cell with multidirectional differentiation potential and self-replication capacity. Because the mesenchymal stem cells have the characteristics of transplantability and low immunogen, the mesenchymal stem cells have wide application prospects in autoimmune diseases and various alternative treatment methods. Mesenchymal stem cells are widely present in cord blood, adipose tissue, peripheral blood, dental pulp, bone marrow, and the like. Wherein, the mesenchymal stem cells in the umbilical cord are umbilical cord mesenchymal stem cells. Under specific induction conditions, the umbilical cord mesenchymal stem cells can be induced into epidermal cells, osteoblasts, vascular endothelial cells, cardiac muscle cells and the like. Therefore, umbilical cord mesenchymal stem cells have been widely used in stem cell therapy. However, when the umbilical cord mesenchymal stem cells are cultured in vitro, the proliferation speed is relatively slow, so that the in vitro proliferation capacity of the umbilical cord mesenchymal stem cells is effectively promoted, and the method has important significance for clinical application of the umbilical cord mesenchymal stem cells.

Disclosure of Invention

The invention aims to provide a biological preparation for promoting the growth of umbilical cord mesenchymal stem cells, thereby realizing the rapid propagation of the cells.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention provides a biological preparation for promoting the growth of umbilical cord mesenchymal stem cells, which is a short-haired algae polysaccharide prepared by the following preparation method:

(1) cleaning the short-haired algae raw material with clear water, drying the short-haired algae raw material in a drying box to constant weight, and crushing the short-haired algae raw material into short-haired algae powder;

(2) adding 20 times of distilled water, extracting at 90 deg.C for 3 hr, centrifuging at 8500g for 30min, and collecting supernatant to obtain short-hair algae extract;

(3) rotary evaporating the extractive solution of short-haired algae to 1/4;

(4) adding 3 times of anhydrous ethanol, standing for 24 hr, centrifuging for 20min at 5000g, and freeze drying the precipitate to obtain crude polysaccharide of Dunaliella;

(5) adding distilled water to dissolve the crude polysaccharide of the short-haired algae to obtain a crude polysaccharide dissolving solution of the short-haired algae;

(6) passing the crude polysaccharide solution through a 50kDa ultrafiltration membrane, and freeze-drying the trapped fluid to obtain the polysaccharide of the short-hair algae;

the concentration of the short-hair algae polysaccharide is more than or equal to 10 mg/ml.

Secondly, the invention provides an application of the short-hair algae polysaccharide in preparing a biological preparation for promoting the proliferation of umbilical cord mesenchymal stem cells, wherein the short-hair algae polysaccharide is prepared by the following preparation method:

(1) cleaning the short-haired algae raw material with clear water, drying the short-haired algae raw material in a drying box to constant weight, and crushing the short-haired algae raw material into short-haired algae powder;

(2) adding 20 times of distilled water, extracting at 90 deg.C for 3 hr, centrifuging at 8500g for 30min, and collecting supernatant to obtain short-hair algae extract;

(3) rotary evaporating the extractive solution of short-haired algae to 1/4;

(4) adding 3 times of anhydrous ethanol, standing for 24 hr, centrifuging for 20min at 5000g, and freeze drying the precipitate to obtain crude polysaccharide of Dunaliella;

(5) adding distilled water to dissolve the crude polysaccharide of the short-haired algae to obtain a crude polysaccharide dissolving solution of the short-haired algae;

(6) and (3) passing the crude polysaccharide dissolving solution through a 50kDa ultrafiltration membrane, and freeze-drying the trapped fluid to obtain the short-hair algae polysaccharide.

Preferably, the application comprises the protein expression of the SCF (short-hair algae polysaccharide) promoting umbilical cord mesenchymal stem cell proliferation promoting protein.

Preferably, the application comprises the inhibition of protein expression of proliferation-suppressing protein p21 of umbilical cord mesenchymal stem cells by the short-haired algae polysaccharide.

Secondly, the invention provides an application of the short-hair algae polysaccharide in preparing a protein expression promoter of the umbilical cord mesenchymal stem cell proliferation promoting protein SCF, wherein the short-hair algae polysaccharide is prepared by the following preparation method:

(1) cleaning the short-haired algae raw material with clear water, drying the short-haired algae raw material in a drying box to constant weight, and crushing the short-haired algae raw material into short-haired algae powder;

(2) adding 20 times of distilled water, extracting at 90 deg.C for 3 hr, centrifuging at 8500g for 30min, and collecting supernatant to obtain short-hair algae extract;

(3) rotary evaporating the extractive solution of short-haired algae to 1/4;

(4) adding 3 times of anhydrous ethanol, standing for 24 hr, centrifuging for 20min at 5000g, and freeze drying the precipitate to obtain crude polysaccharide of Dunaliella;

(5) adding distilled water to dissolve the crude polysaccharide of the short-haired algae to obtain a crude polysaccharide dissolving solution of the short-haired algae;

(6) and (3) passing the crude polysaccharide dissolving solution through a 50kDa ultrafiltration membrane, and freeze-drying the trapped fluid to obtain the short-hair algae polysaccharide.

Finally, the invention provides an application of the short-hair algae polysaccharide in preparing a protein expression inhibitor of proliferation inhibiting protein p21 of umbilical cord mesenchymal stem cells, wherein the short-hair algae polysaccharide is prepared by the following preparation method:

(1) cleaning the short-haired algae raw material with clear water, drying the short-haired algae raw material in a drying box to constant weight, and crushing the short-haired algae raw material into short-haired algae powder;

(2) adding 20 times of distilled water, extracting at 90 deg.C for 3 hr, centrifuging at 8500g for 30min, and collecting supernatant to obtain short-hair algae extract;

(3) rotary evaporating the extractive solution of short-haired algae to 1/4;

(4) adding 3 times of anhydrous ethanol, standing for 24 hr, centrifuging for 20min at 5000g, and freeze drying the precipitate to obtain crude polysaccharide of Dunaliella;

(5) adding distilled water to dissolve the crude polysaccharide of the short-haired algae to obtain a crude polysaccharide dissolving solution of the short-haired algae;

(6) and (3) passing the crude polysaccharide dissolving solution through a 50kDa ultrafiltration membrane, and freeze-drying the trapped fluid to obtain the short-hair algae polysaccharide.

The invention has the beneficial effects that:

the invention provides a brevifibrum polysaccharide which can effectively promote the proliferation of umbilical cord mesenchymal stem cells, can effectively promote the protein expression of SCF (short-chain cell proliferation promoting protein) of the umbilical cord mesenchymal stem cells and can effectively reduce the protein expression of p21 (proliferation inhibiting protein) of the umbilical cord mesenchymal stem cells, so that the breviella polysaccharide can be used for preparing a biological preparation for promoting the growth of the umbilical cord mesenchymal stem cells.

Drawings

FIG. 1 CCK-8 shows the results of the test, where P is < 0.05, P is < 0.01, and P is < 0.001;

FIG. 2 detection results of EDU experiments;

FIG. 3 shows the detection results of Western Blot experiment.

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.

Example 1

(1) Cleaning 100g of the short-haired algae raw material with clear water, drying the raw material in a drying box to constant weight, and crushing the dried raw material into short-haired algae powder;

(2) adding 2000ml distilled water, extracting at 90 deg.C for 3 hr, centrifuging at 8500g for 30min, and collecting supernatant to obtain short-hair algae extract;

(3) carrying out rotary evaporation on the short-haired algae extracting solution to 500ml of the original volume;

(4) adding 1500ml of anhydrous ethanol, standing for 24h, centrifuging for 20min at 5000g, and freeze drying the precipitate to obtain crude polysaccharide of the Sphacelaria brevifolia;

(5) adding 100ml distilled water to dissolve 5g crude polysaccharide of the short-haired algae to obtain crude polysaccharide dissolving solution of the short-haired algae;

(6) and (3) passing the crude polysaccharide dissolving solution through a 50kDa ultrafiltration membrane, and freeze-drying the trapped fluid to obtain the short-hair algae polysaccharide.

Example 2

CCK-8 experiment is utilized to detect the regulation and control of the brevifibrum polysaccharide on the proliferation of umbilical cord mesenchymal stem cells

(1) Using DMEM medium to configure the short-haired algae polysaccharide into the concentrations of 1mg/ml, 10mg/ml, 20mg/ml, 50mg/ml and 100 mg/ml;

(2) inoculating umbilical cord mesenchymal stem cells of generation P3 into a 96-well plate, wherein each well has 4000 per 100ul cells, after the cells are attached to the wall, the control group is not added with the brevifibrum polysaccharide, and the experimental groups are respectively added with the brevifibrum polysaccharide with the concentrations of 1mg/ml, 10mg/ml, 20mg/ml, 50mg/ml and 100 mg/ml;

(3) the 96-well plate was placed in a cell incubator and after 48 hours of incubation, the OD value was measured using CCK-8.

The results obtained from the experiment are shown in fig. 1, wherein the OD value of the control group is 0.899 ± 0.034;

the OD value of the treated short-haired algae polysaccharide of 1mg/ml is 0.912 +/-0.028, the P value is 0.627, and the difference has no statistical significance;

the OD value of the treated sargassum breviatilis polysaccharide is 1.020 +/-0.054, the P value is 0.030 and is less than 0.05, and the difference has statistical significance;

the OD value of the treated 20mg/ml brevifibrum polysaccharide is 1.175 +/-0.047, the P value is 0.001 and less than 0.05, and the difference has statistical significance;

the OD value of 50mg/ml treated short-haired algae polysaccharide is 1.329 +/-0.035, the P value is 0.0001 and is less than 0.05, and the difference has statistical significance;

the OD value after the treatment of the short hair algae polysaccharide of 100mg/ml is 1.390 plus or minus 0.030, the P value is less than 0.0001 and less than 0.05, and the difference has statistical significance;

the combination of the above results shows that when the concentration of the added brevifibrum polysaccharide is more than 10mg/ml, the cell proliferation capacity of the umbilical cord mesenchymal stem cells can be effectively promoted.

Example 3

Detection of regulation and control of brevifibrum polysaccharide on umbilical cord mesenchymal stem cell proliferation by EDU (enhanced data Unit) experiment

(1) Inoculating umbilical cord mesenchymal stem cells of generation P3 into a 96-well plate, wherein each well has 4000/100 ul cells, after the cells are attached to the wall, the control group does not add the short-hair algae polysaccharide, and the experimental group adds the short-hair algae polysaccharide with the concentration of 100 mg/ml;

(2) after 48h of treatment, adding EDU dye A solution, and continuing to incubate for 6 h;

(3) after the incubation is finished, the dye solution is discarded, and 0.3% BSA is added to clean the cells for 2 times, 5min each time;

(4) adding 4% formaldehyde, and fixing at room temperature for 15 min;

(5) after the fixation is finished, 0.3% BSA is added for washing for 2 times, and 0.5% TritonX-100 is added for incubation for 20min at room temperature;

(6) after removal, 0.3% BSA is added for cleaning for 2 times, a proper amount of 1 XCLICICK-iT cocktail reaction solution is added, and a horizontal shaking table is incubated for 30min in a dark place at room temperature;

(7) after removal, 0.3% BSA is added for washing for 2 times, and Hoechst33342 is added to a dark horizontal shaker for incubation for 30 min;

(8) after washing with PBS 2 times, photographs were taken under a fluorescence microscope, and the proportion of positive cells was the number of proliferating cells/the total cell number × 100%.

The difference is statistically significant, and the result further indicates that the short-hair alga polysaccharide prepared by the invention can effectively promote the proliferation of umbilical cord mesenchymal stem cells.

Example 4

Western Blot experiment for detecting regulation and control of short-haired algae polysaccharide on umbilical cord mesenchymal stem cell proliferation related protein

(1) Inoculating the P3 generation umbilical cord mesenchymal stem cells into a 6-hole cell culture plate, after the cells are attached to the wall, adding no short-hair algae polysaccharide in a control group, and adding the short-hair algae polysaccharide with the concentration of 100mg/ml in an experimental group;

(2) placing a 6-hole plate in a cell culture box, culturing for 72h, adding 100ul of protein lysate into each hole to lyse cells, scraping the cells by using a cell scraper, collecting the cells into a 1.5ml EP tube, and placing the EP tube on ice to lyse for 30 min;

(3) after the cracking is finished, placing the EP tube in a centrifuge for centrifugation, taking a supernatant after the centrifugation, detecting the protein concentration by using a BCA method, adding a loading buffer solution, and boiling in boiling water for 5min to fully denature the protein;

(4) preparing electrophoresis gel, adding 10ul of protein sample and protein Marker into each hole, adjusting the voltage to 90V, keeping the voltage constant until the protein Marker is separated, and then adjusting the electrophoresis voltage to 120V until bromophenol blue reaches the bottom of the electrophoresis gel;

(5) taking out the glue, installing an electric rotating clamp according to a classic 'sandwich' model, adjusting the current to 250mA, and electrically rotating for 1.5 h;

(6) after the electrotransformation is finished, taking out the membrane, placing the membrane in a sealing solution, and sealing for 1h at room temperature;

(7) incubating SCF, p21 and GAPDH primary antibody, incubating at 4 ℃ overnight, sucking the primary antibody, washing the membrane for 3 times by using TBST, and incubating corresponding secondary antibodies;

(8) after 1h incubation, the secondary antibody was removed and development exposure was performed.

The obtained experimental results are shown in fig. 3, wherein it can be seen that the protein expression level of the SCF protein in the experimental group is significantly higher than that of the control group, while the expression level of the p21 protein in the experimental group is significantly lower than that of the control group. The results show that the brevifolia polysaccharide can effectively promote the protein expression of the cord mesenchymal stem cell proliferation promoting protein SCF, and can effectively inhibit the protein expression of the cord mesenchymal stem cell proliferation inhibiting protein p 21.