CN110564679A - Preparation method and application of improved mesenchymal stem cells - Google Patents
Preparation method and application of improved mesenchymal stem cells Download PDFInfo
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- CN110564679A CN110564679A CN201910700905.2A CN201910700905A CN110564679A CN 110564679 A CN110564679 A CN 110564679A CN 201910700905 A CN201910700905 A CN 201910700905A CN 110564679 A CN110564679 A CN 110564679A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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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
- C12N5/0662—Stem cells
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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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
Abstract
The invention discloses a preparation method and application of mesenchymal stem cells. The disclosed method comprises: and (3) culturing the mesenchymal stem cells by using a culture medium added with the FSP-1. The MSCs cell product prepared by the preparation method can be applied to treatment of inflammation and fibrosis of liver and other organs. Meanwhile, the preparation method can promote the directional migration of the MSCs to the injured viscera in vivo, and improve the homing efficiency and the immunoregulation capability of the MSCs.
Description
Technical Field
the invention belongs to the technical field of biological pharmacy, and relates to a preparation method and application of Mesenchymal Stem Cells (MSCs).
Background
MSCs are a type of non-hematopoietic adult stem cells of mesodermal origin, which have a strong self-renewal capacity and are capable of differentiating into mesodermal-derived chondrocytes, osteocytes, adipocytes and various ectodermal and endodermal-derived cells. MSCs are present in almost all tissues, and can be isolated from tissues in organs such as bone marrow, adipose tissue, liver, muscle, and lung, and expanded in vitro. MSCs of different tissue origin have generally similar biological properties: adherent growth, expression of CD73, CD90 and CD105 specific surface markers, general non-expression of hematopoietic related surface markers such as CD14, CD34 and CD45 (except that adipose-derived MSCs express CD34), can be induced into osteoblasts, chondrocytes and adipocytes in vitro.
In nature, MSCs are MHC class I+、MHC II-、CD40-、CD80-、CD86-Cells with low immunogenicity exert immunoregulation effects on various cell types such as T cells, B cells, NK cells, macrophages and the like through paracrine cytokines or intercellular interaction MSCs. Clinical trials of treatment with MSCs began in 2004 at the earliest, and there were over 800 clinical trials of MSCs registered worldwide (May 2019, clinicalrials. gov). With the progress of clinical trials, the effectiveness of MSCs therapy has been demonstrated in a variety of diseases (various tissue injuries to the liver, brain, lung, spinal cord, etc.).
Although it is generally considered that MSCs have a certain therapeutic effect in the treatment of various diseases at present, there are still problems to be solved from clinical popularization and application, such as low migration rate of MSCs to injured tissues. The low mobility and colonization rates of MSCs at the damaged site may have a direct impact on the efficacy of the treatment. Researchers observe the distribution of the MSCs in vivo through an animal living body tracing technology, and the result shows that the MSCs can be dispersed and distributed in a plurality of organs such as liver, lung, spleen and the like in vivo, and only part of cells can directionally migrate to the damaged part. It has been demonstrated in multiple liver injury treatment models that a reduction in the homing rate of MSCs greatly affects their therapeutic efficacy. Therefore, how to improve the directional migration ability of MSCs to the damaged part in the body and reduce the distribution of MSCs to other 'ineffective' organs is an urgent problem to be solved for improving the curative effect of MSCs.
Disclosure of Invention
Follistatin-like protein 1 (FSTL 1) is a TGF-beta 1-induced extracellular secreted glycoprotein; during the progression of fibrosis, FSTL1 may promote downstream Smad signaling activation, promoting fibrotic gene expression, by promoting myofibroblast surface TGF- β ligand-receptor binding. And the FSTL1 recombinant protein added into the culture medium can activate TGF-beta signals and promote the expression of downstream target genes.
The research process of the inventor unexpectedly discovers that the mesenchymal stem cells added with FSTL1 in the culture process can greatly improve the hepatic fibrosis treatment effect of MSCs when being used for treating a CCl 4-induced mouse hepatic fibrosis treatment model.
the inventor further researches and discovers that the mesenchymal stem cells after being improved by the invention have high expression of CXCR4, and the cells have stronger migration capability; the expression of inflammatory factors IL-6, IL-1 beta and TNF-alpha is reduced by more than 30 percent, and the proportion of the inflammatory cells which are cultured together is reduced. Therefore, when the modified MSCs are injected during the progressive stage of the disease, the cells migrate to the damaged organs in a targeted manner, the immunoregulatory ability is enhanced, and the disease treatment effect of the MSCs can be further improved.
Based on the discovery, the invention provides a preparation method of an improved mesenchymal stem cell, which comprises the following steps: and (3) culturing the mesenchymal stem cells by using a culture medium added with the FSP-1.
Optionally, the mesenchymal stem cell is not limited to human primary umbilical cord mesenchymal stem cell, wherein the animal is rat, mouse, guinea pig, and the domesticated animal is rabbit, cat, dog, cow, sheep, pig, horse, primate is monkey and human. Further, the mesenchymal stem cell is not limited to umbilical cord origin, but may be a mesenchymal stem cell derived from bone marrow, adipose tissue, synovial tissue, lung tissue, umbilical cord blood or peripheral blood.
Alternatively, the follistatin-like protein 1 is not limited to human proteins, but may be derived from: mouse, rat, guinea pig, rabbit, cat, dog, cow, sheep, pig, horse, or primate such as monkey.
The MSCs improved by the method are used for optimizing and treating hepatic fibrosis, and cell products can be applied to preparing medicaments for treating inflammatory diseases and fibrotic diseases.
On the basis of not changing cell surface markers related to the identification of the MSCs, the preparation method can promote the directional migration of the MSCs to the damaged viscera in vivo, so that the MSCs have targeting and immunoregulation properties in treatment, and the treatment effect of the MSCs is obviously improved.
Drawings
FIG. 1 shows the detection of the expression of the modified cell surface markers CD105, CD90 and CD34 of MSCs by flow cytometry; MSCs (F) represent MSCs of the invention;
FIG. 2 shows that the effect of the addition of FSTL1 on the expression of MSCs endogenous FSTL1 is detected by real-time quantitative PCR (A) and Western blot (B);
FIG. 3 MSCs added with FSTL1 during culture treatment of CCl4 induced liver fibrosis model in mice; a is pathological staining, B is statistical analysis of fibrosis area, and C is liver function index;
FIG. 4 Effect of MSCs added to FSTL1 during culture on macrophage phenotypic transformation in a liver fibrosis mouse model; panel A shows macrophage M1 (iNOS), and panel B shows macrophage M2 (CD 206);
FIG. 5 distribution of MSCs with FSTL1 added during culture in organs of a CCl 4-induced liver fibrosis mouse model (two hours (2h), day one (D1), day 7 (D7) on day three (D3)).
FIG. 6 the effect of FSTL1 addition during culture on the proliferation of MSCs was examined by CCK-8 assay;
FIG. 7 shows the effect of FSTL1 addition during the culture process on the migration of MSCs by cell scratching, Transwell assay; the A picture is the cell scratch, and the B picture is Transwell;
FIG. 8 shows that the expression level of MSCs CXCR4 added with FSTL1 during the culture process is detected by real-time quantitative PCR (shown in A) and Western blot (shown in B); detecting the level changes of TNF-alpha, IL-6 and IL-1 beta in MSCs added with FSTL1 in the culture process by real-time quantitative PCR (shown in a C picture);
FIG. 9 the effect of MSCs added to FSTL1 on macrophage phenotypic transformation during culture was examined by flow cytometry.
Detailed Description
The culture medium of the invention is a culture medium which can be used for culturing MSCs, and is also called a special culture medium for MSCs. In the invention, the follistatin-like protein 1 is added into a culture medium to culture the mesenchymal stem cells.
For a detailed description of the technical solutions and advantages of the invention, reference is made to the following detailed description of the patent with the accompanying drawings and detailed description.
Example (b):
The sources of materials used in the examples illustrate:
SPF grade C57BL/6 at 6-8 weeks of age was purchased from Nanjing university model animal institute;
Human umbilical cord MSCs were supplied by Tianjin Oncai;
flow-through antibodies were purchased from BioLegend;
Dir lipofectamine was purchased from Thermo Fisher Scientific, Transwell cell from Corning, usa, and human FSTL1 recombinant protein was purchased from R & D;
Media specific for human MSCs was purchased from R & D, and other cell culture reagents were purchased from Life Technology.
Preparation and in vitro characterization of the improved MSC of the invention.
37℃5％CO2in the incubator, human umbilical cord MSCs (containing 1% penicillin and 1% streptomycin) are cultured by using a special culture medium for the human MSCs, and when the confluence reaches 90%, the ratio of 1: 2, passage, and continuously culturing until the confluence reaches 50% -60%; the fresh culture medium was replaced and human FSTL1 recombinant protein 1 (final concentration 100ng/ml) was added, and cells were harvested after 24h of culture.
And detecting the expression of the related markers of CD90, CD105 and CD34 by using a flow cytometer. The results suggest that the type of MSCs major surface markers was not altered after incubation with FSTL1 addition (fig. 1); meanwhile, the change of the endogenous FSTL1 level is detected by using real-time quantitative PCR and Westernblot, and the result shows that: there was no significant change in endogenous FSTL1 levels (fig. 2).
The inventor researches to find that the modified MSCs are used for treating mouse hepatic fibrosis induced by CCl 4.
CCl 4-induced liver fibrosis model establishment for mice
Selecting 8-week male C57BL/6 mice to carry out intraperitoneal injection administration for 12 weeks continuously twice a week; CCl4 was dissolved in olive oil to formulate an inducer at 20% concentration for injection at 7 ml/kg/mouse. Control mice were injected with an equal volume of 7 ml/kg/volume of olive oil. Randomly dividing the molded mice into 5 groups, a control group, a normal MSCs treatment group, an FSTL1 injection group, an MSCs + FSTL1 treatment group and an improved MSCs treatment group; 12 weeks after CCl4 administration, MSCs treated by conventional and modified MSCs treated groups were injected into tail vein of MSCs (1 x 10)6/only), MSCs + FSTL1 treatment groups for tail vein injection of MSCs (1 x 10)6One) and simultaneously injecting FSTL1 recombinant protein (2 ug/one), FSTL1 injection group injecting FSTL1 recombinant protein (2 ug/one), control group injecting PBS with equal volume; the MSCs + FSTL1 treatment group and FSTL1 injection group were then injected every other day with FSTL1 recombinant protein (2 ug/mouse). Blood and liver tissue analysis-related indices were collected at day 14 post-MSCs injection: blood is taken from inner canthus of eyeball for liver function measurement; dislocation of cervical vertebrae, and taking liver tissue for H&E staining, sirius red staining. The results show that: h&E staining shows that inflammatory cell infiltration in the liver sink zone of the treatment group is reduced, the ratio of sirius red staining fibrosis area to liver tissue is reduced, and the results have statistical difference compared with the control group of mice; in particular, the improved MSCs-treated group showed significantly more significant and statistically different inflammatory cell infiltration (fig. 3A) and improved fibrosis compared to the conventional treated group (fig. 3B). Compared with the conventional treatment group of the MSCs, the liver function index of the improved treatment group of the MSCs is more obvious (figure 3C); the inflammatory cell infiltration and fibrosis degree of the FSTL1 injection group are obviously increased compared with the control group, and the MSCs + FSTL1 treatment groupthe improvement is not obvious compared with the conventional treatment group of the MSCs.
2. Effect of altered secretion factors from modified MSCs on polarization of M1/M2-type macrophages
During the development of hepatic fibrosis, inflammatory response and fibrosis progression are closely related. Macrophages of different subpopulations exert key regulatory effects both in the progression and regression phase of fibrosis: m1 type macrophage (expressing iNOS) can secrete various fibrosis promoting factors (TGF-beta, PDGF, CCL-2 and the like) and promote the proliferation, differentiation and extracellular matrix deposition of Hepatic Stellate Cells (HSCs) in the process of fibrosis progression; m2 type macrophages (expressing CD206) have strong phagocytosis ability and can secrete MMPs, and then play an important role in promoting HSCs apoptosis and reversing fibrosis. MSCs exert an anti-fibrotic effect by promoting the polarization of M1 to M2 macrophages. The results show that: the number of macrophages of M1 type was significantly reduced in the modified MSCs treated group compared to the MSCs treated group (FIG. 4A), and the number of macrophages of M2 type was significantly increased (FIG. 4B).
3. organ distribution of CCl 4-induced liver fibrosis mice of improved MSCs
At 12 weeks after CCl4 molding, mice after molding were randomly divided into 2 groups, conventional treatment group of MSCs, modified treatment group of MSCs, marked with Dir for MSCs, at 5 × 106only tail vein injection was performed. And dynamically observing the permanent planting condition of the MSCs in each organ by using a small animal living body imager 2h, 1, 3 and 7 days after the injection. The results show that: the localization of modified MSCs in damaged liver tissue was significantly increased relative to the conventional group of MSCs (figure 5).
based on the above findings, the present invention has been further studied as follows.
1. Effect of modified MSCs on cell proliferation and migration Capacity
the effect of the modification on the proliferative capacity of MSCs was examined by CCK 8. Inoculation of 5X 10 in 96-well plates4Human umbilical cord MSCs, 5% CO at 37 ℃2culturing in an incubator overnight, replacing a fresh culture medium, adding FSTL1 (the final concentration is 100ng/ml), culturing for 24h, and detecting the influence of a new preparation process on the proliferation capacity of the MSCs by using a CCK8 kit; the results show that: after the culture with the addition of FSTL1, the proliferation capacity of the MSCs is not obviousSignificant impact (fig. 6).
The influence of the new preparation method on the proliferation capacity of the MSCs is detected through cell scratching and Transwell experiments. Cell scratching: inoculation of 5 x 10 in six well plates5Scratching the cells until the confluence degree reaches more than 90%, changing to a serum-free culture medium, adding FSTL1 recombinant protein, culturing for 12h, and observing and photographing; transwell: starvation treatment is carried out on the cells, and cell precipitates are collected centrifugally after digestion; resuspending with serum-free medium and adjusting the cell density to 1X 105Per ml; adding 200ul cell suspension into the upper Transwell chamber, and adding culture medium containing 15% fetal calf serum into the lower chamber; culturing for 24 h; taking out the small chamber, slightly wiping off cells on the inner layer of the microporous membrane of the small chamber by using a cotton swab, and fixing for 5min by using absolute ethyl alcohol; adding crystal violet solution for dyeing, and standing overnight; photographing and counting after dyeing is finished; the cells were lysed with methanol and the OD at 570nm was determined. The results show that: the addition of FSTL1 resulted in a significant increase in the migratory capacity of MSCs (fig. 7).
2. The expression levels of CXCR4, TNF-alpha, IL-6 and IL-1 beta in the MSCs prepared by the invention;
Inoculation of 5 x 10 in six well plates5when the confluence degree reaches 50-60%, replacing a fresh culture medium, adding human FSTL1 recombinant protein (the final concentration is 100ng/ml), culturing for 24 hours, collecting cells, and detecting CXCR4 expression; the results show that the expression of the MSCs CXCR4 prepared by the invention is increased; the real-time quantitative PCR was used to detect the transcription level of immune-related factors of the new preparation method, and the results showed that the proinflammatory factors (TNF-alpha, IL-1 beta, IL-6) were significantly reduced (FIG. 8).
3. FSTL1 influences the secretion of MSCs inflammation-related factors to inhibit M1 type macrophage polarization
Human umbilical cord-derived MSCs were cultured in 6-well (transwell) or six-well plates and MSCs were treated with FSTL1 recombinant protein (100ng/ml) for 24 h; (ii) isolating bone marrow-derived macrophages and identifying a F4/80(+) CD11b (+) double positive rate of greater than 95%; separately inducing macrophages of M1 type (LPS + IFN gamma), co-culturing MSCs, MSCs + FSTL1(100ng/ml) and MSCs (MSC (F)) of the invention with induced macrophages of M1 type, and analyzing the positive proportion of CD86(+) in each group by flow cytometry after 24 h. The results show that: the proportion of CD86(+) cells of the MSCs prepared by the invention is obviously reduced, which indicates that the MSCs prepared by the invention can inhibit the polarization of M1 type macrophages (figure 9).
Claims (7)
1. A preparation method of an improved mesenchymal stem cell is characterized by comprising the following steps: and (3) culturing the mesenchymal stem cells by using a culture medium added with the FSP-1.
2. The method for preparing improved mesenchymal stem cells according to claim 1, characterized in that the mesenchymal stem cells are cultured by a culture medium added with FSN 1, and the obtained mesenchymal stem cells highly express CXCR 4.
3. The method of preparing improved mesenchymal stem cells according to claim 1, wherein the mesenchymal stem cells are derived from: rat, mouse, guinea pig, rabbit, cat, dog, cow, sheep, pig, horse, monkey or human.
4. the method for preparing improved mesenchymal stem cells according to claim 3, wherein the mesenchymal stem cells are derived from umbilical cord, bone marrow, adipose tissue, synovial tissue, lung tissue, umbilical cord blood or peripheral blood.
5. The method for preparing improved mesenchymal stem cells according to claim 1, wherein the FSP-like protein 1 is derived from: a mouse, rat, guinea pig, rabbit, cat, dog, cow, sheep, pig, horse, monkey or human.
6. Use of mesenchymal stem cells prepared according to claim 1, 2, 3 or 5 for the preparation of a medicament for the treatment of inflammation.
7. Use of the mesenchymal stem cells prepared according to claim 1, 2, 3 or 5 for the preparation of a medicament for the treatment of liver fibrosis.
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