CN113008891A - Construction method and application for screening high-quality osteogenic differentiation potential mesenchymal stem cell quantification standard - Google Patents
Construction method and application for screening high-quality osteogenic differentiation potential mesenchymal stem cell quantification standard Download PDFInfo
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Abstract
The invention discloses a construction method and application for screening a high-quality osteogenic differentiation potential mesenchymal stem cell quantification standard, wherein the construction method comprises the following steps: step one, carrying out primary separation and amplification on umbilical cord mesenchymal stem cells from a plurality of male human umbilical cords, and carrying out subculture until the generation is P5; step two, respectively adding osteogenic differentiation liquid to induce osteoblasts to differentiate; step three, after induction culture for 21 days, respectively carrying out alizarin red staining and collecting pictures; analyzing the picture obtained in the step three by using image J software to obtain the alizarin red coloring area percentage in the picture, and averaging the alizarin red coloring area percentages of a plurality of umbilical cord mesenchymal stem cells to obtain the mesenchymal stem cell quantification standard for screening the high-quality osteogenic differentiation potential. The invention provides a cell selection standard for basic research and clinical transformation of bone tissue regeneration and repair by stem cell therapy so as to improve the treatment effect.
Description
Technical Field
The invention belongs to the technical field of biology, relates to a quantitative standard for screening stem cells, and particularly relates to a construction method and application of the quantitative standard for screening mesenchymal stem cells with high osteogenic differentiation potential.
Background
Mesenchymal Stem Cells (MSCs) can be isolated and cultured from a range of tissues including, but not limited to, bone marrow, fat, umbilical cord, amniotic membrane, placenta, etc., have self-renewal and multipotentiality, and are multipotential Cells that can differentiate into various Mesenchymal Cells such as adipogenic Cells, osteoblastic Cells, chondrogenic Cells, hepatic Cells, and neural Cells. Therefore, the mesenchymal stem cells become a hotspot for research in the field of tissue and organ injury repair and regeneration in basic medicine and clinics.
A large number of researches prove that the MSCs can induce immunosuppression in vivo and can play an anti-inflammatory regulatory role in immune disease treatment through immune regulation, for example, graft-versus-host and rejection reactions in the hematopoietic stem cell transplantation treatment process can be reduced. The immunosuppressive capability of the MSCs is proved in a large number of animal tests and clinical researches, and the mesenchymal stem cells are prompted to have very wide prospects in treating immune diseases. The Human Umbilical Cord Mesenchymal Stem Cells (HUCMSCs) have the advantages of wide sources, non-invasive operation of material collection, non-ethical dispute of cell use and the like, can be industrially amplified and stored in large quantities, have stronger proliferation capacity and lower immunogenicity, and can be widely concerned for xenotransplantation. However, there are some problems, firstly, due to the difference of cell donor sources and the difference of cell separation and amplification methods, the quality of HUCMSCs also has great difference, so that the difference of the immune regulation effect of MSCs in different laboratories is great. In addition, due to the characteristics of cell therapy, there is uncertainty about the effect of MSCs in treating certain autoimmune diseases, for example, there is a great difference in the therapeutic effect of different donor sources of HUCMSCs transplanted into recipients. At present, no HUCMSCs quantitative standard with good immunoregulatory capacity is reported.
Disclosure of Invention
The invention provides a construction method and application for screening a high-quality osteogenic differentiation potential mesenchymal stem cell quantification standard, aiming at overcoming the defects of the prior art.
In order to achieve the above objects, the present invention provides a method for constructing a quantitative standard for screening mesenchymal stem cells having high osteogenic differentiation potential, which has the following characteristics: the method comprises the following steps:
step one, carrying out primary separation and amplification on umbilical cord mesenchymal stem cells from a plurality of male human umbilical cords, and carrying out subculture until the generation is P5;
step two, respectively adding osteogenic differentiation liquid to induce osteoblasts to differentiate;
step three, after induction culture for 21 days, respectively carrying out alizarin red staining and collecting pictures;
analyzing the picture obtained in the step three by using image J software to obtain the alizarin red coloring area percentage in the picture, and averaging the alizarin red coloring area percentages of a plurality of umbilical cord mesenchymal stem cells to obtain the mesenchymal stem cell quantification standard for screening the high-quality osteogenic differentiation potential.
Further, the invention provides a construction method for screening the quantitative standard of the mesenchymal stem cells with high osteogenic differentiation potential, which can also have the following characteristics:
in the first step, the method for separating and amplifying the human umbilical cord mesenchymal stem cells comprises the following steps: collecting human umbilical cord under aseptic condition, cleaning umbilical cord bloodstain, removing umbilical cord driven vein blood vessel, cutting, culturing adherent to wall, digesting, centrifuging, suspending, inoculating, culturing until 80-90% fusion, digesting cells, carrying out passage amplification culture, and culturing umbilical cord mesenchymal stem cells in logarithmic phase of the 5 th generation for later use.
Further, the invention provides a construction method for screening the quantitative standard of the mesenchymal stem cells with high osteogenic differentiation potential, which can also have the following characteristics: in the second step, a plurality of strains of P5 umbilical cord mesenchymal stem cells are added into a 6-well plate for culture after digestion, centrifugation, heavy suspension and counting, and the quantitative cell of each well is 1 multiplied by 105(ii) a Paving 6 holes in each cell, setting 3 holes as a control group when the cell density reaches 60-70% fusion degree, and always replacing the complete culture medium of the umbilical cord mesenchymal stem cells in the culture process; the complete culture medium is 1L of low-sugar MEM, and the following components are added: 10% fetal calf serum, 100U/mL penicillin, 100g/mL streptomycin; in addition, 3 holes are set as an experimental group, and an osteogenic differentiation induction culture medium is always replaced in the culture process; the osteogenic differentiation induction medium is1L of low-sugar MEM was added: the volume ratio is 10 percent of fetal calf serum, 100U/mL penicillin, 100g/mL streptomycin, 10 mu mol/L ascorbic acid, 1.8mmol/L potassium dihydrogen phosphate and 10nmol/L dexamethasone; the medium was changed every three days for a total of 21 days.
Further, the invention provides a construction method for screening the quantitative standard of the mesenchymal stem cells with high osteogenic differentiation potential, which can also have the following characteristics: in the third step, alizarin red staining is carried out on all cells in the wells, and the specific method comprises the following steps: washing all the waste liquid with PBS for 3 times, fixing with 4% paraformaldehyde for 30min, washing with PBS for 3 times, adding alizarin staining solution 1 mL/hole for staining for 15min, and washing with PBS for 3 times; after the staining is finished, photographs are collected under a microscope, 5 pictures are collected in each hole, and 15 pictures are collected in each cell experimental group and each control group.
Further, the invention provides a construction method for screening the quantitative standard of the mesenchymal stem cells with high osteogenic differentiation potential, which can also have the following characteristics: in the fourth step, analyzing the collected pictures by using image J software to obtain the alizarin red staining area percentage of each picture, taking the average value of all pictures in each hole, and then taking the average value of all holes in each cell line to obtain the alizarin red staining area percentage of each cell line; and averaging the alizarin red coloring area percentages of a plurality of cell strains to obtain the quantitative standard for screening the mesenchymal stem cells with high-quality osteogenic differentiation potential.
Further, the invention provides a construction method for screening the quantitative standard of the mesenchymal stem cells with high osteogenic differentiation potential, which can also have the following characteristics: in the fourth step, the number of umbilical cord mesenchymal stem cells is 6, and the average value of the alizarin red staining area percentage of the 6 male umbilical cord mesenchymal stem cells is 40%.
The invention also provides an application of the mesenchymal stem cell quantitative standard for screening the high-quality osteogenic differentiation potential, which has the following characteristics: inducing the cells to be screened to differentiate into osteoblasts, performing alizarin red staining after induction culture, collecting pictures, and analyzing the pictures by using image J software to obtain the alizarin red staining area percentage in the pictures; and comparing the alizarin red coloring area percentage of the cells to be screened with the quantification standard of the mesenchymal stem cells with high-quality osteogenic differentiation potential, wherein the cells larger than the quantification standard are the mesenchymal stem cells with high-quality osteogenic differentiation potential.
The invention has the beneficial effects that: the invention utilizes a plurality of human umbilical cord mesenchymal stem cells to induce osteogenic differentiation, and statistical analysis finds that the mesenchymal stem cells from different donors have obvious heterogeneity and large difference in osteogenic differentiation capacity, and provides a screening standard of high-quality osteogenic differentiation capacity stem cells on the basis of the differentiation standard, the HUCMSCs quantification standard capable of screening high-quality immunoregulation capacity is provided, a cell selection standard is provided for basic research and clinical transformation of bone tissue regeneration and repair of stem cell treatment, so that the treatment effect is improved, the HUCMSCs can be used for promoting the technical field of mesenchymal stem cell osteogenic differentiation, and the blindness of basic research and clinical transformation cell selection of HUCMSCs for treating bone tissue regeneration and repair is overcome.
Drawings
FIG. 1 is a graph showing the staining of osteogenic differentiated alizarin red of 6 male umbilical cord mesenchymal stem cells of example 1 and 6 female umbilical cord mesenchymal stem cells of a control example;
FIG. 2 is a statistical chart of osteogenic differentiation abilities of 6 male human umbilical cord mesenchymal stem cells of example 1 and 6 female human umbilical cord mesenchymal stem cells of a control example;
FIG. 3 is a statistical chart of the sex difference of osteogenic differentiation capacity of umbilical cord-derived mesenchymal stem cells.
Detailed Description
The present invention is further illustrated by the following specific examples.
Example 1
The embodiment provides a construction method for screening a high-quality osteogenic differentiation potential mesenchymal stem cell quantification standard, which comprises the following steps of:
step one, primarily separating and amplifying umbilical cord mesenchymal stem cells from 6 male human umbilical cords, and subculturing to P5 generations.
The method for separating and amplifying the human umbilical cord mesenchymal stem cells comprises the following steps: collecting human umbilical cord under aseptic condition, cleaning umbilical cord bloodstain, removing umbilical cord driven vein blood vessel, cutting, culturing adherent to wall, digesting, centrifuging, suspending, inoculating, culturing until 80-90% fusion, digesting cells, carrying out passage amplification culture, and culturing umbilical cord mesenchymal stem cells in logarithmic phase of the 5 th generation for later use. The method comprises the following specific steps:
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 viruses to be detected, and avoiding the risk of transmitting infectious diseases.
2) Umbilical cord origin: the experimental scheme is approved by the ethical committee of stem cell research in drumhead hospitals, and the acquisition requirements meet the following two points: apgar scoring is carried out on healthy live infants or cesarean after the live infants or cesarean are born, and the total score is 8-10; and secondly, taking the umbilical cord near the fetus end to be about 10-20 cm long under the aseptic condition. The two ends are cut off by 2cm respectively, 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) 3 blood vessels in the umbilical cord, namely two arteries and one vein, are removed. The umbilical cord after blood vessel removal is cut into pieces of about 1mm by 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, adding 10mL of human mesenchymal stem cell complete culture medium, wherein the formula of the complete culture medium is as follows: 10% MSC-grade fetal bovine serum + DMEM low-sugar medium.
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 mesenchymal stem cell complete culture medium, placing the cell in a culture box for continuous culture, and carrying out passage when the primary 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 blow the cells and collect the fine cellsThe cell suspension is put into a centrifuge tube, centrifuged for 5min at the room temperature of 1200r/min, and the supernatant is discarded. Adding cell culture solution of human mesenchymal stem cell culture medium to resuspend cells, gently blowing and beating uniformly, and inoculating density is 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, and performing a new cycle of digestion and passage to obtain the umbilical cord mesenchymal stem cells.
And step two, respectively adding osteogenic differentiation liquid to induce osteoblast differentiation.
The specific method comprises the following steps: digesting, centrifuging, resuspending and counting 6 strains of P5 umbilical cord mesenchymal stem cells, adding the cells into a 6-well plate for culture, wherein the quantitative cell per well is 1 multiplied by 105(ii) a Paving 6 holes in each cell, setting 3 holes as a control group when the cell density reaches 60-70% fusion degree, and always replacing the complete culture medium of the umbilical cord mesenchymal stem cells in the culture process; the complete culture medium is 1L of low-sugar MEM, and the following components are added: 10% fetal calf serum, 100U/mL penicillin, 100g/mL streptomycin; in addition, 3 holes are set as an experimental group, and an osteogenic differentiation induction culture medium is always replaced in the culture process; the osteogenic differentiation induction medium is 1L of low-sugar MEM added with: the volume ratio is 10 percent of fetal calf serum, 100U/mL penicillin, 100g/mL streptomycin, 10 mu mol/L ascorbic acid, 1.8mmol/L potassium dihydrogen phosphate and 10nmol/L dexamethasone; the medium was changed every three days for a total of 21 days.
And step three, after induction culture for 21 days, respectively carrying out alizarin red staining and collecting pictures.
The specific method comprises the following steps: washing all the waste liquid with PBS for 3 times, fixing with 4% paraformaldehyde for 30min, washing with PBS for 3 times, adding alizarin staining solution 1 mL/hole for staining for 15min, and washing with PBS for 3 times; after the staining is finished, photographs are collected under a microscope, 5 pictures are collected in each hole, and 15 pictures are collected in each cell experimental group and each control group. The pictures of the cells of each strain are shown in FIG. 1.
Step four, analyzing the pictures obtained in the step three by using image J software to obtain the alizarin red staining area percentage of each picture, taking the average value of all the pictures of each hole, and then taking the average value of all the holes of each cell strain to obtain the alizarin red staining area percentage of each cell strain, namely the osteogenic differentiation capacity of each cell strain, as shown in figure 2. And averaging the alizarin red staining area percentages of the 6 umbilical cord mesenchymal stem cells to obtain the mesenchymal stem cell quantitative standard for screening the high-quality osteogenic differentiation potential.
The average value of the alizarin red staining area percentage of 6 male umbilical cord mesenchymal stem cells is 40%, and the average value is the quantitative standard for screening the mesenchymal stem cells with the high-quality osteogenic differentiation potential.
Comparative example: and sequentially and respectively carrying out osteoblast differentiation induction on the 6 lines of female umbilical cord mesenchymal stem cells by adopting the same method, carrying out alizarin red staining after induction culture, collecting pictures (shown in figure 1), analyzing each picture by using image J software to obtain the alizarin red staining area percentage (shown in figure 2) in the pictures, and taking the average value of the alizarin red staining area percentages of the 6 lines of female umbilical cord mesenchymal stem cells as 3%.
Fig. 3 is a gender difference statistical chart of osteogenic differentiation abilities of the human umbilical cord-derived mesenchymal stem cells of example 1 and the comparative example, which shows that there is a difference between the osteogenic differentiation of the male umbilical cord mesenchymal stem cells and the osteogenic differentiation abilities of the female umbilical cord mesenchymal stem cells, and the statistical significance of the osteogenic differentiation abilities of the male umbilical cord mesenchymal stem cells is higher than that of the female stem cells (about 10 times).
Example 2
The embodiment provides an application of screening a high-quality osteoblast differentiation potential mesenchymal stem cell quantification standard, which comprises the following steps:
inducing the cells to be screened to differentiate into osteoblasts, performing alizarin red staining after induction culture, collecting the picture, and analyzing the picture by using image J software to obtain the alizarin red staining area percentage in the picture.
And comparing the alizarin red coloring area percentage of the cells to be screened with the quantification standard of the mesenchymal stem cells with high-quality osteogenic differentiation potential, wherein the cells larger than the quantification standard are the mesenchymal stem cells with high-quality osteogenic differentiation potential.
Specifically, when the alizarin red staining area percentage of the cells to be screened is more than 40%, the cells are high-quality mesenchymal stem cells with osteogenic differentiation potential.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Claims (7)
1. A construction method for screening a high-quality osteogenic differentiation potential mesenchymal stem cell quantification standard is characterized by comprising the following steps:
the method comprises the following steps:
step one, carrying out primary separation and amplification on umbilical cord mesenchymal stem cells from a plurality of male human umbilical cords, and carrying out subculture until the generation is P5;
step two, respectively adding osteogenic differentiation liquid to induce osteoblasts to differentiate;
step three, after induction culture for 21 days, respectively carrying out alizarin red staining and collecting pictures;
analyzing the picture obtained in the step three by using image J software to obtain the alizarin red coloring area percentage in the picture, and averaging the alizarin red coloring area percentages of a plurality of umbilical cord mesenchymal stem cells to obtain the mesenchymal stem cell quantification standard for screening the high-quality osteogenic differentiation potential.
2. The construction method for screening the quantification standard of mesenchymal stem cells with good osteogenic differentiation potential according to claim 1, wherein the construction method comprises the following steps:
in the first step, the method for separating and amplifying the human umbilical cord mesenchymal stem cells comprises the following steps: collecting human umbilical cord under aseptic condition, cleaning umbilical cord bloodstain, removing umbilical cord driven vein blood vessel, cutting, culturing adherent to wall, digesting, centrifuging, suspending, inoculating, culturing until 80-90% fusion, digesting cells, carrying out passage amplification culture, and culturing umbilical cord mesenchymal stem cells in logarithmic phase of the 5 th generation for later use.
3. The construction method for screening the quantification standard of mesenchymal stem cells with good osteogenic differentiation potential according to claim 1, wherein the construction method comprises the following steps:
in the second step, theSeveral strains of P5 umbilical cord mesenchymal stem cells are digested, centrifuged, resuspended, counted and added into a 6-well plate for culture, and the quantitative cell of each well is 1 multiplied by 105；
Paving 6 holes in each cell, setting 3 holes as a control group when the cell density reaches 60-70% fusion degree, and always replacing the complete culture medium of the umbilical cord mesenchymal stem cells in the culture process;
the complete culture medium is 1L of low-sugar MEM, and the following components are added: 10% fetal calf serum, 100U/mL penicillin, 100g/mL streptomycin;
in addition, 3 holes are set as an experimental group, and an osteogenic differentiation induction culture medium is always replaced in the culture process;
the osteogenic differentiation induction medium is 1L of low-sugar MEM added with: the volume ratio is 10 percent of fetal calf serum, 100U/mL penicillin, 100g/mL streptomycin, 10 mu mol/L ascorbic acid, 1.8mmol/L potassium dihydrogen phosphate and 10nmol/L dexamethasone;
the medium was changed every three days for a total of 21 days.
4. The construction method for screening the quantification standard of mesenchymal stem cells with good osteogenic differentiation potential according to claim 3, wherein the construction method comprises the following steps:
in the third step, alizarin red staining is carried out on all cells in the wells, and the specific method comprises the following steps: washing all the waste liquid with PBS for 3 times, fixing with 4% paraformaldehyde for 30min, washing with PBS for 3 times, adding alizarin staining solution 1 mL/hole for staining for 15min, and washing with PBS for 3 times;
after the staining is finished, photographs are collected under a microscope, 5 pictures are collected in each hole, and 15 pictures are collected in each cell experimental group and each control group.
5. The construction method for screening the quantification standard of mesenchymal stem cells with good osteogenic differentiation potential according to claim 4, wherein the construction method comprises the following steps:
in the fourth step, analyzing the collected pictures by using image J software to obtain the alizarin red staining area percentage of each picture, taking the average value of all pictures in each hole, and then taking the average value of all holes in each cell line to obtain the alizarin red staining area percentage of each cell line;
and averaging the alizarin red coloring area percentages of a plurality of cell strains to obtain the quantitative standard for screening the mesenchymal stem cells with high-quality osteogenic differentiation potential.
6. The construction method for screening the quantification standard of mesenchymal stem cells with good osteogenic differentiation potential according to claim 1, wherein the construction method comprises the following steps:
in the fourth step, the number of umbilical cord mesenchymal stem cells is 6, and the average value of the alizarin red staining area percentage of the 6 male umbilical cord mesenchymal stem cells is 40%.
7. The use of the mesenchymal stem cell quantitative standard for screening high-quality osteogenic differentiation potential according to any one of claims 1 to 6, wherein:
inducing the cells to be screened to differentiate into osteoblasts, performing alizarin red staining after induction culture, collecting pictures, and analyzing the pictures by using image J software to obtain the alizarin red staining area percentage in the pictures;
and comparing the alizarin red coloring area percentage of the cells to be screened with the quantification standard of the mesenchymal stem cells with high-quality osteogenic differentiation potential, wherein the cells larger than the quantification standard are the mesenchymal stem cells with high-quality osteogenic differentiation potential.
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