CN111534483A - Application of insulin-like growth factor binding protein 7 activator in chondrogenic differentiation of human umbilical cord mesenchymal stem cells - Google Patents

Application of insulin-like growth factor binding protein 7 activator in chondrogenic differentiation of human umbilical cord mesenchymal stem cells Download PDF

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CN111534483A
CN111534483A CN202010444451.XA CN202010444451A CN111534483A CN 111534483 A CN111534483 A CN 111534483A CN 202010444451 A CN202010444451 A CN 202010444451A CN 111534483 A CN111534483 A CN 111534483A
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stem cells
activator
insulin
mesenchymal stem
umbilical cord
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CN111534483B (en
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朱阳晨
赵玲
杨旭
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Guangdong Yijia Regenerative Medicine Research Institute Co.,Ltd.
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Nanjing Fanyida Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1369—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from blood-borne mesenchymal stem cells, e.g. MSC from umbilical blood

Abstract

The invention discloses an application of an insulin-like growth factor binding protein 7 activator in chondrogenic differentiation of human umbilical cord mesenchymal stem cells. The proteoglycan and the type II collagen are cartilage specificity matrix products, the hUC-MSCs in the control induction group have obvious chondrogenic differentiation under the induction of a basic induction culture medium, and the content of the proteoglycan and the type II collagen in the isoalizarin induction group is higher than that in the control induction group, which shows that the isoalizarin in the isoalizarin induction group can further promote the chondrogenic differentiation of the hUC-MSCs and has a dose effect. Isoalizarin may act by activating IGFBP7, a potent IGFBP7 activator. Therefore, the insulin-like growth factor binding protein 7 activator isoalizarin has the function of promoting the chondrogenic differentiation of umbilical cord mesenchymal stem cells in vitro, and can be used for preparing a culture medium for promoting the chondrogenic differentiation of umbilical cord mesenchymal stem cells.

Description

Application of insulin-like growth factor binding protein 7 activator in chondrogenic differentiation of human umbilical cord mesenchymal stem cells
Technical Field
The invention belongs to the field of stem cells, relates to the induced differentiation of umbilical cord mesenchymal stem cells, and particularly relates to an application of an insulin-like growth factor binding protein 7 activator in chondrogenic differentiation of human umbilical cord mesenchymal stem cells.
Background
Cartilage tissue engineering is an important bioengineering approach for treating cartilage damage diseases. In cartilage tissue engineering, the selection of seed cells mainly considers the following points: the cells are easy to obtain and have sufficient sources, and the damage to the material-drawing object is small; lower immune rejection; has induced differentiation potential and strong proliferation and differentiation capacity, etc.; a large number of passage cells can be obtained by in vitro amplification techniques.
Cartilage repair and regeneration have previously focused on chondrocytes, which, despite their ready availability and low immunotype, have acquired a small number of cells and have the disadvantage of dedifferentiation, which limits the development of cartilage repair and regeneration. In the study of inducing chondrogenic differentiation of cells having differentiation potential, cells of various origins have been reported, in which mesenchymal stem cells having an irreplaceable chondrogenic differentiation-inducing ability have been widely noted. Although human mesenchymal stem cells and the like are major targets of cartilage tissue engineering as a stem cell source, an age-limited bone marrow transplantation process is inevitably accompanied by pain and injury, and the utilization rate of bone marrow stromal cells is low, and the proliferation and differentiation ability decreases with age. Compared with other stem cells, the umbilical cord mesenchymal stem cells have low immune prototypes and cartilage forming potential, and can be used as one of cell sources for cartilage tissue engineering.
Human umbilical cord mesenchymal stem cells (hUC-MSCs) are a class of mesenchymal stem cells with multipotential differentiation capacity and self-renewal capacity. The umbilical cord has the advantages of easily-obtained materials, difficulty in pollution, no relation to moral, ethical and legal problems and the like, and is widely used for extracting the hUC-MSCs. Moreover, the in vitro amplification and multidirectional differentiation capacity of the hUC-MSCs is found to be stronger than that of mesenchymal stem cells from other tissues.
The discovery of the von scholar et al shows that the platelet lysate has the function of directionally inducing differentiation of human umbilical cord mesenchymal stem cells into cartilage cells in vitro (the influence of the platelet lysate on in vitro chondrogenic differentiation of human umbilical cord mesenchymal stem cells, journal of Chinese repair and reconstruction surgery, 2011 10).
Luodimei et al found that reduced glutathione has a promoting effect on chondrogenesis induction of human umbilical cord mesenchymal stem cells (influence of reduced glutathione on chondrogenesis induction of human umbilical cord mesenchymal stem cells, journal of bioengineering, China, 2013, 03 phase)
Insulin-like growth factor binding protein 7(IGFBP7) is a secreted glycoprotein, is widely expressed in the body, can be combined with insulin-like growth factors to positively or negatively regulate IGF signal pathway, can also independently play a role, and is involved in regulating important physiological processes of growth differentiation, proliferation, apoptosis, tissue remodeling and the like of cells. The research shows that IGFBP7 has the function of promoting the cartilage differentiation of human umbilical cord mesenchymal stem cells (IGFBP7 promotes the cartilage differentiation of human umbilical cord mesenchymal stem cells, Nature science edition of Nanjing medical university, vol.39, No. 8 of 2019, 8).
Disclosure of Invention
The invention provides an application of an insulin-like growth factor binding protein 7 activator in chondrogenic differentiation of human umbilical cord mesenchymal stem cells in order to overcome the defects in the prior art.
The technical scheme of the invention is as follows:
an application of an insulin-like growth factor binding protein 7 activator in promoting chondrogenic differentiation of umbilical cord mesenchymal stem cells in vitro, wherein the insulin-like growth factor binding protein 7 activator is isoalizarin.
Use of an insulin-like growth factor binding protein 7 activator in preparation of a culture medium for promoting chondrogenic differentiation of umbilical cord mesenchymal stem cells, wherein the insulin-like growth factor binding protein 7 activator is isoalizarin.
A culture medium for preparing chondrocytes from umbilical cord mesenchymal stem cells contains an insulin-like growth factor binding protein 7 activator, wherein the insulin-like growth factor binding protein 7 activator is isoalizarin.
The beneficial technical effects are as follows:
the invention discovers that the insulin-like growth factor binding protein 7 activator rubiadin has the function of promoting chondrogenic differentiation of umbilical cord mesenchymal stem cells in vitro and can be used for preparing a culture medium for promoting chondrogenic differentiation of umbilical cord mesenchymal stem cells.
Drawings
FIG. 1 is a diagram of phenotypic flow assays for hUC-MSCs;
FIG. 2 is a comparison of the content of each of the histones;
FIG. 3 is a graph showing the comparison of collagen type II content in each group;
FIG. 4 is a Western blot chart of the expression level of insulin-like growth factor-binding protein 7(IGFBP 7).
Detailed Description
The following examples are intended to illustrate the essence of the present invention, but should not be construed as limiting the scope of the present invention.
First, test materials
DMEM/F12 medium and fetal bovine serum were purchased from Gibco.
Glutamine and diabody were purchased from Nanjing Senega Biotech, Inc.
The PBS buffer solution is prepared according to the formula, stored at 4 ℃ and used up within 24 h.
Collagenase type iv and pancreatin were purchased from shanghai diligent kang biotechnology limited, and used as described herein.
The purity of the isoalizarin is not lower than 98%.
Second, test method
1. Extraction culture and identification of hUC-MSCs
The hUC-MSCs used are the same as those in patents 2020104333269 and 2020104333714, and the preparation and identification methods are as follows:
collecting umbilical cord of newborn produced by normal full-term caesarean section at about 12cm (storing umbilical cord in PBS buffer solution containing 1% double antibody at 4 deg.C, extracting stem cells within 6h, culturing), washing with PBS buffer solution containing 1% double antibody to remove umbilical artery and vein and umbilical cord adventitia, and shearing to about 1mm3Placing the tissue blocks in a constant temperature shaking instrument at 37 deg.C, adding collagenase type IV and pancreatin, digesting for 60min and 30min respectively to extract cells, resuspending the cells in DMEM/F12 medium containing 20% FBS, 25mmol/L glutamine and 1% double antibody, and adding 1.0 × 106and/mL, inoculating the cells into a cell culture bottle, changing the culture medium after culturing for 4 days, changing the culture medium for 1 time after 2-3 days, and carrying out passage when 80% of the cells are fused. The 5 th generation cells were used for the experiment.
Taking 5 th generation hUC-MSCs, carrying out pancreatin digestion, fully and uniformly blowing to prepare single cell suspension, and adding CD34-PE, CD45-PE, CD73-PE, CD90-PE and CD 105-FITC. Incubating for 30min at room temperature in a dark place, fixing paraformaldehyde, and detecting by a flow cytometer.
2. Induced differentiation of hUC-MSCs into chondrocytes
2.1 grouping
Low concentration of isoalizarin induction group (group a): inducing culture by using a DMEM/F12 culture medium containing 6.25mg/L insulin, 6.25mg/L transferrin, 10 mu g/L transforming growth factor beta 1, 0.1 mu mol/L dexamethasone, 50mg/L vitamin C, 5 mu M isoalizarin (DMSO solubilization), 5% FBS and 1% double antibody;
high concentration of isoalizarin induction group (group B): inducing culture by using a DMEM/F12 culture medium containing 6.25mg/L insulin, 6.25mg/L transferrin, 10 mu g/L transforming growth factor beta 1, 0.1 mu mol/L dexamethasone, 50mg/L vitamin C, 10 mu M isoalizarin (DMSO solubilization), 5% FBS and 1% double antibody;
control induction group (group C): the culture was induced using DMEM/F12 medium (basal induction medium) containing final concentrations of 6.25mg/L insulin, 6.25mg/L transferrin, 10. mu.g/L transforming growth factor beta 1, 0.1. mu. mol/L dexamethasone, 50mg/L vitamin C, 5% FBS and 1% double antibody, to which DMSO vehicle was added in an equal volume to A, B groups.
2.2 Induction culture
Taking 5 th generation well-grown cells hUC-MSCs, digesting with pancreatin, fully and uniformly blowing, preparing into single cell suspension, 2 × 105And/well inoculating in 6-well plate, and when 85% of cells are fused, replacing culture medium according to the above grouping, inducing and culturing for 18d, and replacing corresponding culture medium every 3 d. Each group is provided with 3 multiple holes.
3. Measurement of proteoglycan content
After 18 days of induction, taking 200 mu L of cell supernatant of each group, adding 40 mu L of papain for digestion for 24 hours; adding 100 mu L of 8mol/L guanidine hydrochloride and 750 mu L of 0.25% alisin blue solution, and reacting for 1h at 4 ℃; centrifuging at 12000r/min for 15min, discarding supernatant, adding 150 μ L isopropanol to dissolve precipitate, and measuring absorbance at 600 nm. And (3) preparing a standard curve by using a series of chondroitin sulfate standard solutions with concentration gradients, and substituting the absorbance value into the standard curve to obtain the proteoglycan content.
4. Determination of type II collagen content
After 18 days of induction, 250 mu L of cell supernatant of each group is taken, 50 mu L of hydroxyproline digestive juice is added, 2mL of hydroxyproline reaction solution is added, water bath at 60 ℃ is carried out for 15min, after cooling, 3500r/min is centrifuged for 10min, 150 mu L of supernatant is taken, and the absorbance value is measured at the wavelength of 560 nm. And (4) preparing a standard curve by using hydroxyproline standard solutions with a series of concentration gradients, and substituting the absorbance value into the standard curve to obtain the type II collagen content.
5. Western blot for measuring expression level of IGFBP7
After 18 days of induction, collecting each group of cells, washing 3 times by PBS to remove the culture medium on the cell surface and the components in the culture medium, cracking the lysate, determining the protein concentration in the lysate by using a BCA kit, performing SDS-PAGE electrophoresis on 50 mu g of protein, transferring the protein onto a PVDF membrane, sealing by 5% skimmed milk powder, adding IGFBP7 and GAPDH (internal reference) for primary incubation at 4 ℃ overnight, washing the membrane by TBS-T (TBS-T) washing liquid on the next day, adding secondary antibody for incubation at room temperature for 1h, performing color development and exposure by an ECL method, photographing by a chemiluminescence imaging system, and analyzing the gray scale by using Image analysis software Image J.
6. Data analysis
In SPSS 17.0, data are presented as mean. + -. SD and tested for t, with P < 0.05 indicating that the difference is statistically significant.
Third, test results
1. Extraction culture and identification results of hUC-MSCs
Phenotypic identification of hUC-MSCs flow results As shown in Table 1 and FIG. 1, CD34-PE and CD45-PE negative expression, CD73-PE, CD90-PE and CD105-FITC positive expression, consistent with the phenotypic characteristics of hUC-MSCs.
TABLE 1 phenotypic identification results of hUC-MSCs
Expression rate
CD34-PE 0.35％
CD45-PE 0.47％
CD73-PE 95.2％
CD90-PE 96.8％
CD105-FITC 98.5％
2. Results of proteoglycan content measurement
As shown in table 2 and fig. 2, the content of proteoglycan in the low-concentration rubiadin-induced group (group a) and the content of proteoglycan in the high-concentration rubiadin-induced group (group B) are significantly increased and have a significant dose dependence compared to the control-induced group (group C), and the group B is higher than the group a.
TABLE 2 content of each of the histones
Group of Content (μ g/mL)
Control Induction group (group C) 20.9±3.1
Low concentration of rubiadin inducer group (group A) 35.2±3.4
High concentration of Rubiadin Induction group (group B) 58.5±3.2
3. Determination of type II collagen content
The collagen type ii content in each group is shown in table 3 and fig. 3, and compared with the control induction group (group C), the collagen type ii content in the low-concentration isoalizarin induction group (group a) and the high-concentration isoalizarin induction group (group B) is significantly increased, and there is significant dose dependence, and the group B is higher than the group a.
TABLE 3 collagen content of each group II
Group of Content (μ g/mL)
Control Induction group (group C) 197.5±18.2
Low concentration of rubiadin inducer group (group A) 320.6±21.3
High concentration of Rubiadin Induction group (group B) 572.8±23.5
4. Measurement of expression level of IGFBP7
As shown in fig. 4, compared with the control induction group (group C), the Western blot results show that the content of IGFBP7 protein in the low-concentration isoalizarin induction group (group a) and the high-concentration isoalizarin induction group (group B) is significantly increased, and there is a significant dose dependence, and the group B is higher than the group a.
As known to those skilled in the art, proteoglycan and type II collagen are cartilage specific matrix products, hUC-MSCs in the control induction group have obvious chondrogenic differentiation under the induction of a basic induction culture medium, and the content of proteoglycan and type II collagen in the isoalizarin induction group is higher than that in the control induction group, which indicates that the isoalizarin in the isoalizarin induction group can further promote chondrogenic differentiation of hUC-MSCs and has a dosage effect. Isoalizarin may act by activating IGFBP7, a potent IGFBP7 activator. Therefore, the insulin-like growth factor binding protein 7 activator isoalizarin has the function of promoting the chondrogenic differentiation of the umbilical cord mesenchymal stem cells in vitro, and can be used for preparing a culture medium for promoting the chondrogenic differentiation of the umbilical cord mesenchymal stem cells.

Claims (3)

1. An application of an insulin-like growth factor binding protein 7 activator in promoting chondrogenic differentiation of umbilical cord mesenchymal stem cells in vitro, wherein the insulin-like growth factor binding protein 7 activator is isoalizarin.
2. Use of an insulin-like growth factor binding protein 7 activator in preparation of a culture medium for promoting chondrogenic differentiation of umbilical cord mesenchymal stem cells, wherein the insulin-like growth factor binding protein 7 activator is isoalizarin.
3. A culture medium for preparing chondrocytes from umbilical cord mesenchymal stem cells, comprising an insulin-like growth factor binding protein 7 activator, wherein the activator comprises: the insulin-like growth factor binding protein 7 activator is isoalizarin.
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CN112391341A (en) * 2020-11-30 2021-02-23 张川 Application of SDF-1 protein activator in promoting human umbilical cord mesenchymal stem cell proliferation and differentiation in vitro
CN113249317A (en) * 2021-06-17 2021-08-13 北京国卫生物科技有限公司 Isolated culture and amplification method and system for human umbilical cord mesenchymal stem cells

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