CN111304166A - Method for improving proportion of CD106 positive subset purified from human umbilical cord mesenchymal stem cells - Google Patents
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Abstract
The invention discloses a method for improving the proportion of CD106 positive subgroups purified from human umbilical cord mesenchymal stem cells, which comprises the following steps: step one, carrying out primary separation and amplification on umbilical cord mesenchymal stem cells in a human umbilical cord, and carrying out subculture; step two, obtaining primary shunted CD106 positive mesenchymal stem cells from the P2 generation umbilical cord mesenchymal stem cells by a flow sorting method; step three, carrying out adherent culture on the primary shunted CD106 positive umbilical cord mesenchymal stem cells obtained in the step two to a P4 generation, and obtaining secondary shunted CD106 positive umbilical cord mesenchymal stem cells by using a flow sorting method again; step four, carrying out adherent culture on the secondarily shunted CD106 positive umbilical cord mesenchymal stem cells obtained in the step three until the generation of P6, and obtaining the thirdly shunted CD106 positive umbilical cord mesenchymal stem cells by using a flow sorting method again. The invention can obtain the human umbilical cord mesenchymal stem cells with high expression of CD 106.
Description
Technical Field
The invention belongs to the technical field of biology, relates to a cell purification method, and particularly relates to a method for improving the proportion of CD106 positive subgroups purified from human umbilical cord mesenchymal stem cells.
Background
Mesenchymal Stem Cells (MSCs) are a class of adult Stem Cells derived from mesoderm and have the potential for self-renewal and multidirectional differentiation, and have been isolated and cultured from tissues such as bone marrow, fat, umbilical cord, amniotic membrane and placenta, and many studies have confirmed that Mesenchymal Stem Cells derived from various tissues can be differentiated into Cells of 3 germ layer (endoderm, ectoderm and mesoderm) lineages, such as adipocyte, osteoblast, chondroblast, hepatocyte and neural cell, and the like.
CD106 is also called vascular cell adhesion molecule 1(VCAM-1), its gene expression is mainly regulated by transcription factor NFK B VCAM-1 is one of the main adhesion molecules expressed by bone marrow stromal cells, is expressed by stromal cells in the blood vessel endothelial cells and bone marrow hematopoietic microenvironment, can also be expressed by epithelial cells, macrophages and dendritic cells, can mediate adhesion between cells and extracellular matrix, is involved in hematopoietic cell development, stimulates activation and proliferation of T cells, etc., VCAM-1 may play a role in the development and progression of diseases such as arteriosclerosis and rheumatoid arthritis, in bone marrow α 4 β 1 (surfactant 4)/CD106 mediates the interaction between HSC and mesenchymal stem cells, transmits intercellular information, the interaction between HSC and mesenchymal cells and the adhesion between HSC and stem cell niche plays a crucial role in regulating HSC fate, granulocyte colony stimulating factor mobilizing stem cells is to promote the mobilization of HSC-expressing CD 12 by attenuating human stromal cell adhesion molecule-1 (VCAM-1) and CD 12, and the mobilizing of HSC by reducing the expression of bone marrow stromal cell adhesion molecule-1 (VCAM-1) and CD 12) and the chemotactic factor 106 is reported in patients with the literature that the loss of bone marrow stromal cell expressing the CD106+Cell subsets, and CD106－The subpopulation of MSCs cells did not support hematopoiesis and maintain CD34 well+Cell dryness, further indicating CD106+The subpopulation of MSCs cells is one of the crucial cellular components in the bone marrow stem cell niche.
Compared with mesenchymal stem cells from bone marrow tissues, the human umbilical cord mesenchymal stem cells have the advantages of wide source, convenient collection, non-invasive operation, no ethical dispute and the like, can be industrially amplified and stored in a large quantity to be widely concerned, have stronger proliferation strong performance and lower immunogenicity, and can be used for xenotransplantation. The human umbilical cord mesenchymal stem cells not only express higher vascular endothelial growth factors and genes related to a P13K/NFKB pathway, have stronger capacity of promoting angiogenesis, but also have the functions of inhibiting lymphocyte proliferation, inhibiting proinflammatory T cell subsets, improving immunosuppressive T cell subsets and reducing immunoregulation function of secretion of immune cell inflammatory factors, and provide a basis for the effectiveness of clinically applying the human umbilical cord mesenchymal stem cells to treat the immunoinflammatory diseases.
The expression of CD106 of MSCs from different sources is different, the expression of CD106 in umbilical cord mesenchymal stem cells is low (4.06 Shi 2.19%), and the CD106 is purified by immunoadsorption+Subgroup, efficiency is very low. How to improve the proportion of the CD106 positive subset of the human umbilical cord mesenchymal stem cells and obtain more CD106+ subset cells is of great importance for basic research and clinical transformation of the stem cells. At present, no relevant report of an experimental method for improving the proportion of the positive subgroup of the human umbilical cord mesenchymal stem cells CD106 exists.
Disclosure of Invention
The invention provides a method for improving the proportion of CD106 positive subgroups purified from human umbilical cord mesenchymal stem cells, which overcomes the defects of the prior art.
To achieve the above object, the present invention provides a method for increasing the proportion of CD106 positive subpopulations purified from human umbilical cord mesenchymal stem cells, comprising the steps of: step one, carrying out primary separation and amplification on umbilical cord mesenchymal stem cells in a human umbilical cord, and carrying out subculture; step two, obtaining primary shunted CD106 positive mesenchymal stem cells from the P2 generation umbilical cord mesenchymal stem cells by a flow sorting method; step three, carrying out adherent culture on the primary shunted CD106 positive umbilical cord mesenchymal stem cells obtained in the step two to a P4 generation, and obtaining secondary shunted CD106 positive umbilical cord mesenchymal stem cells by using a flow sorting method again; step four, carrying out adherent culture on the secondarily shunted CD106 positive umbilical cord mesenchymal stem cells obtained in the step three until the generation of P6, and obtaining the thirdly shunted CD106 positive umbilical cord mesenchymal stem cells by using a flow sorting method again.
Further, the present invention provides a method for increasing the proportion of CD106 positive subpopulations purified from human umbilical cord mesenchymal stem cells, which may also have the following characteristics: the specific method of the first step comprises the following steps: collecting human umbilical cord under aseptic condition, cleaning umbilical cord blood stain, removing umbilical cord driven vein blood vessel, cutting, culturing adherent to wall, digesting, centrifuging, resuspending, inoculating, culturing until 80-90% fusion, digesting cells, carrying out passage amplification culture, and using umbilical cord mesenchymal stem cells in logarithmic phase of generation 2 for later use, namely umbilical cord mesenchymal stem cells of generation P2.
Further, the present invention provides a method for increasing the proportion of CD106 positive subpopulations purified from human umbilical cord mesenchymal stem cells, which may also have the following characteristics: the specific method of the second step is as follows: observing the adherent growth of the umbilical cord mesenchymal stem cells of the generation P2 under a microscope until the adherent growth reaches more than 80 percent, sucking the original culture medium in a culture bottle, and cleaning the surface of the cells; then adding enzyme digestive juice for digestion for 2-3min, centrifuging after digestion, and then re-suspending by using a basal medium DMEM; adding phycoerythrin mouse anti-human monoclonal antibody CD106, mixing, and incubating in dark for 30 min; adding a basal medium DMEM, centrifuging, adding a basal medium DMEM containing 1% streptomycin to resuspend cells, loading on a machine, and carrying out flow-type sorting on the CD106 positive subgroup to obtain the primary shunted CD106 positive mesenchymal stem cells.
Further, the present invention provides a method for increasing the proportion of CD106 positive subpopulations purified from human umbilical cord mesenchymal stem cells, which may also have the following characteristics: the specific method of the second step is as follows: observing the adherent growth of the umbilical cord mesenchymal stem cells P2 generation under a microscope until the adherent growth reaches more than 80%, sucking the original culture medium in a culture bottle, adding PBS to clean the cell surface, and washing the cells for 2 times; removing PBS, adding enzyme digestion solution, covering the bottom of a culture flask, digesting for 2-3min at 37 ℃, adding 3ml of basal medium DMEM to terminate digestion, blowing, transferring to a 15ml centrifuge tube, centrifuging at 1200rpm for 5min, removing supernatant, adding 1ml of basal medium DMEM to resuspend, and counting; according to 1X 106cell number, 5ul phycoerythrinThe concentration of the mouse anti-human monoclonal antibody CD106 and the phycoerythrin mouse anti-human monoclonal antibody CD106 is 25ug/ml, the cells are fully and uniformly mixed by a vortex oscillator after the antibody is added, and the cells are incubated for 30min in a dark place; adding 4ml of basal medium DMEM, centrifuging at room temperature of 1200rpm for 5min, adding 500ul of basal medium DMEM containing 1% streptomycin to resuspend cells, loading on a machine, and carrying out flow-type sorting on the CD106 positive sub-population to obtain the primary shunted CD106 positive mesenchymal stem cells.
Further, the present invention provides a method for increasing the proportion of CD106 positive subpopulations purified from human umbilical cord mesenchymal stem cells, which may also have the following characteristics: the third step is specifically as follows: centrifuging the secondarily shunted CD106 positive mesenchymal stem cells obtained in the step two for 5min at 1200 rpm; discarding the supernatant, adding 5ml of complete culture medium for human mesenchymal stem cells for resuspension, counting, inoculating into a culture bottle according to the number of cells for culture, and marking the cells as P3 generations; the cells grow to more than 80 percent, and the cells are subjected to passage expansion; when the CD106 positive mesenchymal stem cells of the P4 generation are fused to 80-90 percent, the mixture is digested and resuspended according to 1 multiplied by 106Adding 5ul phycoerythrin mouse anti-human monoclonal antibody CD106 into the cells, wherein the phycoerythrin mouse anti-human monoclonal antibody CD106 is 25ug/ml, and incubating for 30min in a dark place; centrifuging, removing supernatant, adding 500ul of basal medium DMEM containing 1% streptomycin for resuspending cells, loading on a machine, and carrying out flow-type sorting on the CD106 positive subset to obtain the secondary-shunted CD106 positive umbilical cord mesenchymal stem cells.
Further, the present invention provides a method for increasing the proportion of CD106 positive subpopulations purified from human umbilical cord mesenchymal stem cells, which may also have the following characteristics: wherein, the complete culture medium formula of the human mesenchymal stem cells comprises 10 percent of MSC-grade fetal bovine serum, DMEM low-sugar culture medium and 1 percent of streptomycin.
Further, the present invention provides a method for increasing the proportion of CD106 positive subpopulations purified from human umbilical cord mesenchymal stem cells, which may also have the following characteristics: the specific method of the step four is as follows: centrifuging the primary shunted CD106 positive mesenchymal stem cells obtained in the third step at 1200rpm for 5 min; discarding the supernatant, adding 5ml of human mesenchymal stem cell complete culture medium for resuspension, and countingAccording to the number of the cells, inoculating the cells into a culture bottle for culture, wherein the generation of the cells is marked as P5 generation; the cells grow to more than 80 percent, and the cells are subjected to passage expansion; when the CD106 positive mesenchymal stem cells of the P6 generation are fused to 80-90 percent, the mixture is digested and resuspended according to 1 multiplied by 106Adding 5ul phycoerythrin mouse anti-human monoclonal antibody CD106 into the cells, wherein the phycoerythrin mouse anti-human monoclonal antibody CD106 is 25ug/ml, and incubating for 30min in a dark place; centrifuging, removing supernatant, adding 500ul of basal medium DMEM containing 1% streptomycin to resuspend cells, loading on a machine, and carrying out flow-type sorting on the CD106 positive subset to obtain the triple-shunt CD106 positive umbilical cord mesenchymal stem cells.
The invention has the beneficial effects that: the invention provides a method for improving the proportion of CD106 positive subgroups purified from human umbilical cord mesenchymal stem cells, which improves the proportion of the umbilical cord mesenchymal stem cells CD106 positive subgroups from 1.4% to 62.5% by flow sorting, and overcomes the defect that little CD106 is obtained due to the low expression of the umbilical cord mesenchymal stem cells CD106+The defects of MSCs (mesenchymal stem cells) can be overcome, and the method can obtain the human umbilical cord mesenchymal stem cells with high expression of CD106, thereby providing a new selection way for basic research and clinical transformation of the stem cells.
Drawings
FIG. 1a is a flow chart of the CD106 positive subset in the umbilical cord mesenchymal stem cells generation P2;
figure 1b is a graph of flow sorting of the CD106 positive subpopulation from the P4 generation CD106 positive mesenchymal stem cells;
figure 1c is a graph of flow sorting of the CD106 positive subpopulation from the P6 generation CD106 positive mesenchymal stem cells;
fig. 2 is a graph of post-flow sorting purity analysis of CD106 positive subpopulations in generation P6 CD106 positive mesenchymal stem cells;
fig. 3 is a morphogram of CD106 positive mesenchymal stem cells generation P7;
fig. 4 is a P7 generation CD106 positive mesenchymal stem cell surface marker analysis plot;
fig. 5 is a graph of adipogenic chondrogenic differentiation of CD106 positive mesenchymal stem cells at P7 generation.
Detailed Description
The present invention is further illustrated by the following specific examples.
As shown in fig. 1, the present invention provides a method for increasing the proportion of CD106 positive subpopulations purified from human umbilical cord mesenchymal stem cells, comprising the steps of:
step one, carrying out primary separation and amplification on umbilical cord mesenchymal stem cells in human umbilical cord, and carrying out subculture.
1) Screening the parturient meeting the health requirement of the biological sample donor to collect the sample, ensuring the safety of the biological sample, eliminating the viruses including HIV, HBV, HCV, CMV, HIV-1&2, EBV and other possible detection viruses and avoiding the risk of transmitting infectious diseases.
2) Umbilical cord origin: with informed consent, the experimental protocol was approved by the ethical committee for stem cell research in the drumhead hospital.
The collection requirements of the method comprise that ① healthy parturients or cesarean sections are subjected to Apgar scoring, the total score is 8-10, ② both ends of the umbilical cord near the fetus end, which is 10-20 cm. long, are cut off by 2cm respectively under the aseptic condition, the umbilical cord is soaked in DPBS containing 2% streptomycin and transported by a medical incubator and treated in 4 h.
3) The staff enters the facility and operates in the biological safety cabinet in the whole process.
4) The umbilical cord was cut into small pieces of about 2cm and washed with DPBS containing 2% penicillin streptomycin until no blood was present.
5) Removing 3 blood vessels (two arteries and one vein) from umbilical cord, and cutting umbilical cord into pieces of about 1mm with ophthalmic scissors3The fine tissue mass of (2).
6) Shearing the tissue, directly sticking the sheared tissue to a T75 culture bottle, inverting for 4h, rightly placing the culture bottle, and adding 10ml of human mesenchymal stem cell complete culture medium. The complete culture medium formula of the human mesenchymal stem cells comprises 10 percent of MSC-grade fetal bovine serum, a DMEM low-sugar culture medium and 1 percent of streptomycin.
7) After about 14 days, the cells climb out to form colonies CFU-F, and the culture flask is gently tapped to drop the tissue mass and discarded.
8) And (3) gently washing the cell surface by PBS, replacing 10ml of fresh human mesenchymal stem cell complete culture medium, placing the cell in a culture box for continuous culture, and carrying out passage when the primary cells (P0 generation cells) grow to 40-50% of fusion.
9) The old culture solution was discarded, DPBS left at room temperature was added and washed once, and DPBS was aspirated off. Adding proper amount of Tryple, and incubating and digesting for 3min at 37 ℃.
10) Gently blowing and beating the cells, collecting the cell suspension into a centrifuge tube, centrifuging for 5min at room temperature at 1200r/min, and discarding the supernatant. Adding cell culture solution of complete culture medium of human mesenchymal stem cells, re-suspending the cells, gently blowing and beating uniformly, and inoculating with density of about 1.5 × 104/cm2。
11) Standing at 37 deg.C for 5% CO2And (3) performing static culture in an incubator until 80-90% of cells are fused, marking the generation of cells as P1 generation, and performing a new round of digestion and passage to obtain the umbilical cord mesenchymal stem cells of P2 generation.
And step two, obtaining primary shunted CD106 positive mesenchymal stem cells from the P2 generation umbilical cord mesenchymal stem cells by a flow sorting method.
1) Observing the adherent growth of the umbilical cord mesenchymal stem cells of the generation P2 under a microscope until the adherent growth reaches more than 80%, sucking the original culture medium in a culture bottle, adding PBS (phosphate buffer solution) to gently clean the cell surface, and washing the cells for 2 times.
2) Removing PBS, adding enzyme digestion solution, covering the bottom of the culture flask, digesting for 2-3min at 37 ℃, adding 3ml of basal medium DMEM to terminate digestion, lightly blowing, transferring to a 15ml centrifuge tube, centrifuging at 1200rpm for 5min, removing supernatant, adding 1ml of basal medium DMEM to resuspend, and counting.
3) According to 1X 106Adding 5ul of Phycoerythrin (R-Phytoerythrothrin, R-PE) mouse anti-human monoclonal antibody CD106 into the cells, wherein the concentration of the Phycoerythrin mouse anti-human monoclonal antibody CD106 is 25ug/ml, adding the antibody, mixing the cells with a vortex oscillator, and incubating for 30min in a dark place.
4) Adding 4ml of basal medium DMEM, centrifuging at room temperature of 1200rpm for 5min, adding 500ul of basal medium DMEM containing 1% streptomycin to resuspend cells, loading on a machine, and carrying out flow-type sorting on the CD106 positive sub-population to obtain the CD106 positive mesenchymal stem cells subjected to primary flow splitting, wherein the positive proportion is 1.4%, and the flow-type sorting diagram is shown in figure 1 a.
Step three, carrying out adherent culture on the primary shunted CD106 positive umbilical cord mesenchymal stem cells obtained in the step two to a P4 generation, and obtaining secondary shunted CD106 positive umbilical cord mesenchymal stem cells by using a flow sorting method again.
1) And D, centrifuging the secondarily shunted CD106 positive mesenchymal stem cells obtained in the step two at 1200rpm for 5 min.
2) Discarding the supernatant, adding 5ml of complete culture medium of human mesenchymal stem cells for resuspension, counting, inoculating into a proper culture flask according to the number of cells, and culturing, wherein the number of the cells is marked as P3.
3) And (5) growing the cells to be more than 80%, and carrying out passage expansion on the cells.
4) When the CD106 positive mesenchymal stem cells of the P4 generation are fused to 80-90 percent, the mixture is digested and resuspended according to 1 multiplied by 106And (3) adding 5ul of phycoerythrin mouse anti-human monoclonal antibody CD106 into the cells, wherein the content of the phycoerythrin mouse anti-human monoclonal antibody CD106 is 25ug/ml, and incubating for 30min in a dark place.
5) Centrifuging, discarding supernatant, adding 500ul of basal medium DMEM containing 1% streptomycin to resuspend cells, loading on a machine, and performing flow sorting on the CD106 positive subset to obtain the secondary-shunted CD106 positive umbilical cord mesenchymal stem cells, wherein the positive proportion is 11.5%, and the flow sorting diagram is shown in figure 1 b.
Step four, carrying out adherent culture on the secondarily shunted CD106 positive umbilical cord mesenchymal stem cells obtained in the step three until the generation of P6, and obtaining the thirdly shunted CD106 positive umbilical cord mesenchymal stem cells by using a flow sorting method again.
1) And (4) centrifuging the primary shunted CD106 positive mesenchymal stem cells obtained in the third step at 1200rpm for 5 min.
2) Discarding the supernatant, adding 5ml of complete culture medium of human mesenchymal stem cells for resuspension, counting, inoculating into a proper culture flask according to the number of cells, and culturing, wherein the number of the cells is marked as P5.
3) The cells grow to more than 80 percent, and the cells are subjected to passage expansion;
4) when the CD106 positive mesenchymal stem cells of the P6 generation are fused to 80-90 percent, the mixture is digested and resuspended according to 1 multiplied by 106cell number, 5ul phycoerythrin mouse antibodyHuman monoclonal antibody CD106, phycoerythrin mouse anti-human monoclonal antibody CD106 is 25ug/ml, and incubation is carried out for 30min in the dark;
5) centrifuging, discarding supernatant, adding 500ul of basal medium DMEM containing 1% streptomycin to resuspend cells, loading on a machine, and performing flow sorting on the CD106 positive subset to obtain the CD106 positive umbilical cord mesenchymal stem cells which are shunted for three times, wherein the positive proportion is 62.5%, and the flow sorting diagram is shown in figure 1 c.
The mesenchymal stem cells are derived from human umbilical cord, are separated, cultured and amplified in a GMP-grade clinical stem cell preparation chamber, and are subjected to strict detection of the quality control and safety evaluation system of the mesenchymal stem cells, so that the immunogenicity is low, no ethical dispute exists, and a new support is provided for the clinical transformation application of the stem cells.
Centrifuging the three-time shunted CD106 positive umbilical cord mesenchymal stem cells at 1200rpm for 5 min; discarding the supernatant, adding 5ml of complete culture medium of human mesenchymal stem cells for resuspension, counting, inoculating into a proper culture flask according to the number of cells, and culturing, wherein the number of the cells is marked as P7. Biological property testing was performed for P7 generation:
as shown in fig. 3, the cell morphology results show that: cells grow adherently under an inverted phase contrast microscope and present a fibroblast shape.
As shown in fig. 4, the surface marker detection results show that: CD73, CD90 and CD105 ≧ 95%; CD14, CD19, CD34, CD45 and HLA-DR ≦ 2%.
As shown in fig. 5, adipogenic osteogenic hose differentiation capacity: cultured in vitro in adipogenic, osteogenic and chondrogenic differentiation induction medium for 21 days. After adipogenic differentiation, after staining with oil red, obvious oil drops can be observed in both cells; after osteogenic differentiation, obvious calcium salt deposition can be observed by staining with alizarin red; after chondrogenic differentiation, the staining of alisnew blue was seen as blue chondrocytes.
The results show that the CD106 positive subset cells obtained after the P2 generation umbilical cord mesenchymal stem cells are subjected to three-time flow sorting, namely the three-time split CD106 positive umbilical cord mesenchymal stem cells obtained by the method meet the definition of the international society for stem cell research and the international society for cell therapy on the universal standard of mesenchymal stem cells.
Claims (7)
1. A method for improving the proportion of CD106 positive subgroups purified from human umbilical cord mesenchymal stem cells is characterized in that:
the method comprises the following steps:
step one, carrying out primary separation and amplification on umbilical cord mesenchymal stem cells in a human umbilical cord, and carrying out subculture;
step two, obtaining primary shunted CD106 positive mesenchymal stem cells from the P2 generation umbilical cord mesenchymal stem cells by a flow sorting method;
step three, carrying out adherent culture on the primary shunted CD106 positive umbilical cord mesenchymal stem cells obtained in the step two to a P4 generation, and obtaining secondary shunted CD106 positive umbilical cord mesenchymal stem cells by using a flow sorting method again;
step four, carrying out adherent culture on the secondarily shunted CD106 positive umbilical cord mesenchymal stem cells obtained in the step three until the generation of P6, and obtaining the thirdly shunted CD106 positive umbilical cord mesenchymal stem cells by using a flow sorting method again.
2. The method of increasing the proportion of CD106 positive subpopulations purified from human umbilical cord mesenchymal stem cells according to claim 1, wherein:
the specific method of the first step comprises the following steps: collecting human umbilical cord under aseptic condition, cleaning umbilical cord blood stain, removing umbilical cord driven vein blood vessel, cutting, culturing adherent to wall, digesting, centrifuging, resuspending, inoculating, culturing until 80-90% fusion, digesting cells, carrying out passage amplification culture, and culturing umbilical cord mesenchymal stem cells in logarithmic phase of generation 2 for later use, namely the umbilical cord mesenchymal stem cells of generation P2.
3. The method of increasing the proportion of CD106 positive subpopulations purified from human umbilical cord mesenchymal stem cells according to claim 1, wherein:
the specific method of the second step is as follows:
observing the adherent growth of the P2 umbilical cord mesenchymal stem cells to be more than 80% under a microscope, sucking the original culture medium in a culture bottle, and cleaning the cell surface;
then adding enzyme digestive juice for digestion for 2-3min, centrifuging after digestion, and then re-suspending by using a basal medium DMEM;
adding phycoerythrin mouse anti-human monoclonal antibody CD106, mixing, and incubating in dark for 30 min;
adding a basal medium DMEM, centrifuging, adding a basal medium DMEM containing 1% streptomycin to resuspend cells, loading on a machine, and carrying out flow-type sorting on the CD106 positive subgroup to obtain the primary shunted CD106 positive mesenchymal stem cells.
4. The method of increasing the proportion of CD106 positive subpopulations purified from human umbilical cord mesenchymal stem cells according to claim 1, wherein:
the specific method of the second step is as follows:
observing the adherent growth of the P2 umbilical cord mesenchymal stem cells to be more than 80% under a microscope, sucking the original culture medium in a culture bottle, adding PBS (phosphate buffer solution) to clean the cell surface, and washing the cells for 2 times;
removing PBS, adding enzyme digestion solution, covering the bottom of a culture flask, digesting for 2-3min at 37 ℃, adding 3ml of basal medium DMEM to terminate digestion, blowing, transferring to a 15ml centrifuge tube, centrifuging at 1200rpm for 5min, removing supernatant, adding 1ml of basal medium DMEM to resuspend, and counting;
according to 1X 106Adding 5ul phycoerythrin mouse anti-human monoclonal antibody CD106 into the cells, wherein the concentration of the phycoerythrin mouse anti-human monoclonal antibody CD106 is 25ug/ml, adding the antibody, fully and uniformly mixing the cells by using a vortex oscillator, and incubating for 30min in a dark place;
adding 4ml of basal medium DMEM, centrifuging at room temperature of 1200rpm for 5min, adding 500ul of basal medium DMEM containing 1% streptomycin to resuspend cells, loading on a machine, and carrying out flow-type sorting on the CD106 positive sub-population to obtain the primary shunted CD106 positive mesenchymal stem cells.
5. The method of increasing the proportion of CD106 positive subpopulations purified from human umbilical cord mesenchymal stem cells according to claim 1, wherein:
the third step is specifically as follows:
centrifuging the secondarily shunted CD106 positive mesenchymal stem cells obtained in the second step at 1200rpm for 5 min;
discarding the supernatant, adding 5ml of complete culture medium for human mesenchymal stem cells for resuspension, counting, inoculating into a culture bottle according to the number of cells for culture, and marking the cells as P3 generations;
the cells grow to more than 80 percent, and the cells are subjected to passage expansion;
when the CD106 positive mesenchymal stem cells of the P4 generation are fused to 80-90 percent, the mixture is digested and resuspended according to 1 multiplied by 106Adding 5ul phycoerythrin mouse anti-human monoclonal antibody CD106 into the cells, wherein the phycoerythrin mouse anti-human monoclonal antibody CD106 is 25ug/ml, and incubating for 30min in a dark place;
centrifuging, removing supernatant, adding 500ul of basal medium DMEM containing 1% streptomycin for resuspending cells, loading on a machine, and carrying out flow-type sorting on the CD106 positive subset to obtain the secondarily shunted CD106 positive umbilical cord mesenchymal stem cells.
6. The method of increasing the proportion of CD106 positive subpopulations purified from human umbilical cord mesenchymal stem cells according to claim 5, wherein:
the complete culture medium formula of the human mesenchymal stem cells comprises 10% of MSC-grade fetal bovine serum, a DMEM low-sugar culture medium and 1% of streptomycin.
7. The method of increasing the proportion of CD106 positive subpopulations purified from human umbilical cord mesenchymal stem cells according to claim 1, wherein:
the specific method of the step four is as follows:
centrifuging the primary shunted CD106 positive mesenchymal stem cells obtained in the third step at 1200rpm for 5 min;
discarding the supernatant, adding 5ml of complete culture medium for human mesenchymal stem cells for resuspension, counting, inoculating into a culture bottle according to the number of cells for culture, and marking the cells as P5 generations;
the cells grow to more than 80 percent, and the cells are subjected to passage expansion;
when the CD106 positive mesenchymal stem cells of the P6 generation are fused to 80-90 percent, the mixture is digested and resuspended according to 1 multiplied by 106Adding 5ul phycoerythrin mouse anti-human monoclonal antibody CD106 into the cells, wherein the phycoerythrin mouse anti-human monoclonal antibody CD106 is 25ug/ml, and incubating for 30min in a dark place;
centrifuging, removing supernatant, adding 500ul of basal medium DMEM containing 1% streptomycin for resuspending cells, loading on a machine, and carrying out flow-type sorting on the CD106 positive subset to obtain the triple-shunted CD106 positive umbilical cord mesenchymal stem cells.
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