CN115925925A

CN115925925A - Method for inducing differentiation of umbilical cord mesenchymal stem cells and producing cell factors

Info

Publication number: CN115925925A
Application number: CN202211009917.9A
Authority: CN
Inventors: 张化杰; 王振
Original assignee: Beijing Yiyuan Biotechnology Co ltd
Current assignee: Jiangsu Xinchao Biotechnology Group Co ltd
Priority date: 2022-08-23
Filing date: 2022-08-23
Publication date: 2023-04-07
Anticipated expiration: 2042-08-23
Also published as: CN115925925B

Abstract

The invention relates to a method for inducing differentiation of umbilical cord mesenchymal stem cells and producing cell factors. The monoclonal antibody for specifically inhibiting the activity of Wnt-1 in a Wnt 1/beta-catenin pathway is prepared, the antibody can promote umbilical cord mesenchymal stem cells to be differentiated into liver-like cells, the differentiated liver-like cells can highly express cytokines rich in IL6, and the cytokines have good biological activity, can be used for preparing corresponding medicaments, and have wide application prospects.

Description

Method for inducing differentiation of umbilical cord mesenchymal stem cells and producing cell factors

Technical Field

The application relates to the field of biology, in particular to a method for inducing differentiation of umbilical cord mesenchymal stem cells and producing cytokines.

Background

Cytokines are a large class of proteins that are important for intercellular signaling. They are produced by a variety of cells, including stem cells, immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. The release of cytokines will have an effect on the surrounding cellular activities. Cytokines may include interleukins, interferons, growth factors, tumor necrosis factors, chemokines and colony stimulating factors.

Interleukins (Interleukins), also referred to simply as ILs, are cytokines that play important roles in regulating cellular processes (cell growth, differentiation and movement) and stimulating immune responses. Originally found in leukocytes, they have now been found to be produced by a wide variety of cells, including macrophages, lymphocytes, and the like, which have a solid structure and function. Mainly includes CXCL8, IFNL3, IL10, IL11, IL12, IL12A, IL13, IL15, IL15RA, IL16, IL17A, IL17B, IL17F, IL17RA, IL18, IL19, IL1A, IL1B, IL1RL2, IL1RN, IL2, IL20, IL20RA, IL21, IL22, IL23, IL23R, IL3, IL31, IL33, IL36A, IL36B, IL36G, IL36RN, IL37, IL4, IL4R, IL5, IL6, IL7, IL9, etc. Interferons (interferons), abbreviated IFNs, are known for their ability to protect cells from viral infection by "interfering" with viral replication because host cells produce and release a set of signal proteins against a variety of existing viruses. Interferons belong to a large class of cytokine proteins used in the communication between cells to trigger protective defenses of the immune system to eliminate pathogens. Growth factors also belong to a protein or steroid hormone, are naturally occurring substances that stimulate cell growth, differentiation, survival, inflammation and tissue repair. Growth factors play an important role in regulating diverse cellular processes and may be secreted by neighboring cells, distant tissues and glands, and even by tumor cells themselves. Normal cells maintain proliferation and viability by exhibiting a need for several growth factors. Growth factors may exert their stimulatory effects through endocrine, paracrine, or autocrine mechanisms.

There are many methods for cytokine production. For example, CN106520689A provides a method for preparing mesenchymal stem cell cytokine, which comprises the following steps: extracting mesenchymal stem cells; culturing; continuing culturing under a low-oxygen condition; digesting with 0.25% pancreatin EDTA solution; centrifuging, and washing with 0.9% physiological saline; adjusting the cell concentration by using 0.9% physiological saline, and then adding 1 3mg/mL of EDTA and 5 10mg/mL of Vc; freezing and thawing; centrifuging and filtering. However, the method is not directed to high production of the cytokine IL-6.

CN106367389A discloses a preparation method of human umbilical cord mesenchymal stem cell factor, which comprises two steps of primary separation and culture of human umbilical cord mesenchymal stem cells, preparation of freeze-dried powder and activity detection, wherein the cell factor is prepared by centrifuging, incubating, secondary centrifuging and ultrafiltration concentrating cells in P20 generation in a culture bottle to obtain factor concentrated solution, and IL-6 is not highly expressed in the same obtained cell factor. CN105543313A discloses a method for separating and purifying human mesenchymal stem cell factor, which comprises the steps of obtaining cell culture supernatant: taking human mesenchymal stem cells, placing the human mesenchymal stem cells in a mesenchymal stem cell serum-free culture medium for culture, collecting supernatant when the cells grow to reach 75-85% of confluence degree, filtering and concentrating: filtering the supernatant with 0.22 μm filter membrane; and then passing through a 50KD ultrafiltration membrane to collect dialysate, and then passing the dialysate through a 1KD ultrafiltration membrane to collect retentate to obtain cytokine concentrate. The method also produces less IL-6 during the manufacturing process.

Interleukin 6 (IL-6) is the most typical cytokine associated with inflammation. It plays an important role in host defense by modulating immune and inflammatory responses. IL-6 is a small glycoprotein with a molecular weight of 19-28kDa, which forms four alpha helical structures from 184 amino acids, usually in monomeric form, and has an isoelectric point of 5.0. The gene encoding human IL-6 is located on chromosome 7p15-21 and includes 4 introns and 5 exons. IL-6 has 3 receptor binding sites, including 1 specific receptor IL-6R (IL-6R) binding site, and 2 gp130 (signal-transducing protein) binding sites. IL-6 is also a cytokine with pleiotropic activity, and can promote proliferation and differentiation of various cells, accelerate synthesis of acute phase protein of liver cells, inhibit growth of M1 myeloid leukemia cell line, promote maturation and differentiation thereof, and inhibit growth of melanoma and breast cancer cells. Therefore, the efficient production of IL-6 cytokines is an urgent direction of research.

Disclosure of Invention

The present invention provides a method for efficiently producing IL-6 and other cytokines.

In particular, the invention provides a method for producing a cytokine cocktail comprising IL-6 cytokines.

Wnt-1 protein is a secreted glycoprotein composed of 343 amino acids, and most of them act through cell surface Frizzled family receptors, which are essential in the normal development of embryos. Wnt-1 activation leads to a complex cascade of signaling reactions that ultimately leads to the upregulation of multiple gene expression. Wnt-1 is involved in a variety of cellular processes in the human body and affects the normal function of certain systems. Wnt-1 is the most important regulatory protein in the Wnt 1/beta-catenin pathway. By inhibiting the activity of the Wnt-1, the Wnt 1/beta-catenin pathway can be inhibited on the whole.

In one aspect, the invention provides a monoclonal antibody that inhibits the activity of Wnt-1.

The monoclonal antibody for inhibiting the activity of Wnt-1 is Wnt-1-13F, and a light chain variable region VL amino acid sequence:

QIVLSQSPAILSASPGEKVTMTCNTEYRGARGSWYQQKPGSSPKPWIYPWEHQTHGVPFRFSGSGSGTSYSLTISRVEAEDAATYYCIASYMILFLFGGGTKLEIK

heavy chain variable region VH amino acid sequence:

QVKLQESGGGLVQPKGSLKLSCAASGFSFYFIPYPWVRQAPGKGLEWVAVTTVIMWDGPDEWDENLCRRFTISRDDSQSMLYLQMHNLKTDDTAMYYCVRITESAWAIVEFAFWGAGTTVTVSS

the light chain variable region and the heavy chain variable region of the antibody are respectively as follows:

LCDR1	NTEYRGARGS
		LCDR2	PWEHQTH
LCDR3	IASYMILFL
		HCDR1	FIPYP
HCDR2	VTTVIMWDGPDEWDENLCR
		HCDR3	ITESAWAIVEFAF

further, the invention also provides application of the monoclonal antibody for inhibiting the activity of Wnt-1 in preparing a reagent for promoting the differentiation of umbilical cord mesenchymal stem cells into liver-like cells.

Further, the differentiation reagent comprises a three-stage reagent composition, and the three-stage reagent is used in the conditions of inducing the differentiation of the umbilical cord mesenchymal stem cells, wherein the conditions are as follows: culturing umbilical cord mesenchymal stem cells at the current stage 1, namely culturing DMEM-LG +20 mu g/L epidermal growth factor +10 mu g/L basic fibroblast growth factor for 3d; then stage 2 culture, namely DMEM-LG + 1% fetal calf serum with the volume fraction of 20 mu g/L hepatocyte growth factor +10 mu g/L alkaline fibroblast growth factor +0.61g/L niacinamide +10 mu g/L Wnt-1-13F monoclonal antibody, culturing for 6 days, and changing the liquid for 1 time every 3 days; final stage 3 culture: contains fetal calf serum with volume fraction of 5%, human fibroblast growth factor 4 with volume fraction of 10 mug/L, hepatocyte growth factor with volume fraction of 10 mug/L, recombinant oncostatin M with volume fraction of 10 mug/L, wnt-1-13F monoclonal antibody with volume fraction of 10 mug/L, and 10 Wnt-1-13F monoclonal antibody with volume fraction of 10 mug/L ^-7 And (5) culturing the liver-like cells after differentiation for 10 days by using a HBM culture medium containing mol/L dexamethasone.

Further, the present invention provides a method for producing an IL 6-rich cytokine using the differentiated hepatocyte-like cells.

The production method comprises a cell culture method which is conventional in the field, and the cell factor can be obtained by collecting supernatant.

Further, the production method may be such that the density of the liver-like cells induced to differentiate by the monoclonal antibody is inoculated with an α -MEM medium containing 10% fetal bovine serum, the medium is incubated at 37 ℃ with 5% CO ₂ Culturing in a saturated humidity incubator for 5d, centrifuging at 4 ℃ and 1500rpm for 5min to remove cells, and freeze-drying the supernatant to obtain the cytokine composition, and storing at-20 ℃.

Furthermore, the invention also provides application of the prepared cytokine rich in IL6 in preparing medicaments for promoting fibroblast proliferation and treating related diseases such as cancer.

Furthermore, the cell factor prepared by the invention can be used for preparing cell factor preparations, cosmetics and the like.

The pharmaceutical composition of the invention further comprises a buffer, which may be selected from the group consisting of: potassium phosphate, acetic acid/sodium acetate, citric acid/sodium citrate, succinic acid/sodium succinate, tartaric acid/sodium tartrate, histidine/histidine HCl, glycine, tris, glutamate, acetate and mixtures thereof, and is in particular selected from the group consisting of potassium phosphate, citric acid/sodium citrate, succinic acid, histidine, glutamate, acetate and combinations thereof.

The pharmaceutical compositions may optionally comprise one or more additional excipients, as long as they do not reduce or eliminate their advantageous properties as described herein, in particular their stability.

Excipients can be used in the present invention for a wide variety of purposes, such as to adjust the physical, chemical, or biological properties of the formulation (such as to adjust viscosity) and/or the methods of the present invention to further improve effectiveness and/or to further stabilize the formulation and method against degradation and spoilage due to, for example, stresses generated during manufacture, shipping, storage, preparation prior to use, during application, and thereafter. The term "excipient" generally includes fillers, binders, disintegrants, coatings, adsorbents, anti-adherents, glidants, preservatives, antioxidants, flavoring agents, colorants, sweeteners, solvents, co-solvents, buffers, chelating agents, viscosity imparting agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers, and tonicity adjusting agents.

Acceptable excipients are preferably pharmaceutically acceptable, including but not limited to: amino acids such as glycine, alanine, glutamine, asparagine, threonine, proline, 2-phenylalanine include charged amino acids, preferably lysine, lysine acetate, arginine, glutamate and/or histidine.

Further, the primary vehicle or carrier in the pharmaceutical composition may be aqueous or non-aqueous in nature. Suitable vehicles or carriers may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other substances common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are other exemplary vehicles.

Advantageous effects

The monoclonal antibody for specifically inhibiting the activity of Wnt-1 in a Wnt 1/beta-catenin pathway is prepared, the antibody can promote umbilical cord mesenchymal stem cells to be differentiated into liver-like cells, the differentiated liver-like cells can highly express cytokines rich in IL6, and the cytokines have good biological activity, can be used for preparing corresponding medicaments, and have wide application prospects.

Drawings

FIG. 1 shows the result of phage clone ELISA assay

FIG. 2 is a graph showing the result of identifying the specificity of an antibody

FIG. 3 is a graph showing the effect of cytokines on the proliferation of human skin fibroblasts

Detailed Description

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

EXAMPLE 1 preparation of Wnt-1 monoclonal antibody

Human recombinant Wnt-1 (Biovision, cat # 4754-50) was coated on an ELISA plate with 50mmol/L sodium bicarbonate (pH 10.0), PBS/2% milk was blocked and added to a phage library (synthesized by Wuhan Kingkurui bioengineering, inc., fab phage library), gently shaken at room temperature for 1h, washed 6 times with PBS/0.1% Tween, and the bound phage were eluted with an acidic eluent (0.1 mmol/LHCl-Gly, pH2.2,0.1% BSA), and the eluate was neutralized with 1mol/L Tris-HCl (pH 9.1) to measure titer, and amplified phage for the next round of screening. The screening is carried out for 3 rounds, the subsequent 2 rounds of screening process is the same as the first round, but the coating antigen amount is reduced, and the Tween concentration is gradually increased to 0.3 percent and 0.5 percent respectively. After 3 rounds of screening, the enrichment index of the finally enriched phage is 109, which indicates that the phage with high affinity to Wnt-1 has been successfully enriched.

42 positive clones obtained by 3 rounds of screening were selected, inoculated into 100. Mu.l of 2YT medium containing 100mg/LAmp, cultured at 37 ℃ until OD600= 0.3-0.4, infected with 25. Mu.l of helper phage, then added with 25. Mu.l of 2YT medium containing 300mg/L Kan, and cultured overnight at 37 ℃. And (3) carrying out specificity identification on each enriched phage clone by adopting an ELISA method, coating human recombinant Wnt-1 100ng in each hole, adding 100 mu l of phage, and developing through an HRP-labeled mouse anti-M13 phage antibody. The blank was zeroed and the absorbance at A450nm was measured with a microplate reader (Multiskan Spectrum microplate thermo 1500). The positive standard was a P/N value (positive/negative) >2.0 and the negative standard was a P/N value <0.5. The results are shown in FIG. 1.

As can be seen from the results in FIG. 1, the 2 mAbs with the best positive effects were Wnt-1-15C and Wnt-1-13F, respectively.

For Wnt-1-13F, the primers were used with 5' ends: aagacaggctatcgatt and 3' end primer: GCCCCCTATTAGCGTTT amplifies Fab gene fragments, and PCR conditions are as follows: 5min at 94 ℃; 15s at 94 ℃, 30s at 50 ℃, 2min at 72 ℃ and 30 cycles; 10min at 72 ℃. The product is sent to Shanghai Biotechnology Limited company for antibody variable region gene sequence determination, and the sequencing result is searched and analyzed by DNAMAN and database. The amino acid sequence of the variable region of the light chain of the coding Wnt-1-13F monoclonal antibody and the amino acid sequence of the variable region of the heavy chain of the coding Wnt-1-13F monoclonal antibody are obtained by sequence comparison, wherein the sequence of the variable region of the heavy chain is shown as SEQ ID NO.1, and the sequence of the variable region of the light chain is shown as SEQ ID NO. 2.

Obtaining an expression Fab antibody by soluble separation in HB2151 bacteria, inducing expression overnight by 0.5mol/L IPTG at low temperature, obtaining soluble Wnt-1-13F monoclonal antibody from bacteria periplasm cavity by osmotic shock method, purifying by nickel column to obtain purified monoclonal antibody, and adjusting the concentration of the monoclonal antibody to 1mg/mL for standby.

Example 2 identification of affinity and specificity of Wnt-1-13F monoclonal antibodies

The binding capacity of the Wnt-1-13F antibody to the Wnt-1 protein is analyzed by applying an SPR technology: wnt-1 protein was coupled to a CM5 sensor chip and injected with Wnt-1-13F mab at 6 different concentrations in the range of 1-100 nM, at a flow rate of 45 μ L/min in all experiments. The regeneration conditions of the chip are glycine-HCl pH1.5. The kinetic parameters KD were calculated using binding curves obtained at different antibody concentrations. By calculation, the results of table 1 were obtained.

TABLE 1 affinity of Wnt-1-13F monoclonal antibodies

Name of antibody	KD(M)
		Wnt-1-13F monoclonal antibody	2.19E-09

As can be seen from the results in Table 1, the affinity KD of the Wnt-1-13F antibody prepared by the present invention for antigen was 2.19X 10 ^-9 M, has better affinity.

And (3) specific identification: the purified Wnt-1-13F monoclonal antibody is subjected to specificity analysis by an ELISA method, each hole is coated with 100ng of human recombinant Wnt-1 protein, wnt-2 protein and BSA, the Wnt-1-13F monoclonal antibody which is diluted in a gradient manner is added, and the mouse anti-Flag antibody marked by HRP is developed. The results are shown in FIG. 2.

The ELISA plate is coated with Wnt-1 protein and control protein (Wnt-2 and BSA), and the analysis result shows that the Wnt-1-13F monoclonal antibody can be specifically combined with the Wnt-1 protein and not combined with other unrelated proteins (figure 2), and the better specificity is shown.

Example 3 preparation and differentiation Induction of umbilical cord mesenchymal Stem cells

The fresh umbilical cord is placed in a culture dish, and the amnion and the blood vessel are removed. Shearing the separated umbilical cord to about 1mm by using an ophthalmic scissors ³ The tissue blocks of the size were taken in appropriate amounts and placed in sterile petri dishes, covering the bottom of 75% of the petri dishes. Adding serum-free culture medium of mesenchymal stem cells, shaking for 1min to disperse tissue mass, and adding 5% CO ₂ Culturing in an incubator at 37 ℃. On day 5, the medium was changed by half, and the culture was continued for about 14 days, and when the cell mass was grown to about 50%, the tissue mass was removed, and at the same time, the cells were collected for subculture. Changing the culture solution once every 3 days, observing the growth change of the cells by using an inverted microscope, carrying out digestion passage when the adherent cells grow to 90% of the bottom of the bottle, adding 0.05% of pancreatin for digestion, carrying out passage according to the proportion of 1. Viable cell density of 5X 10 in serum-free DMEM medium up to passage 3 ⁶ Cell suspension per mL for detection.

The UC-MSCs immunophenotyping is to measure the expression of CD90, CD45, CD105, CD34 and HIA-DR on the surface of the UC-MSCs by a flow cytometer. The results are shown in Table 2.

TABLE 2 mesenchymal stem cell immunophenotyping

As can be seen from the results in Table 2, CD90 and CD105 are positive, the positive rate is more than 98%, while CD34, CD45 and HLA-DR are negative, the positive rate is less than 2%, which indicates that the umbilical cord mesenchymal stem cells are obtained by separation and preparation.

The hUC-MSCs with good growth state of the 4 th generation are selected to be 5 multiplied by 10 per hole ⁴ Cells were uniformly seeded in 6-well plates and when cells grew to 80% confluency, they were cultured in the following order. Stage 1, culturing DMEM-LG +20 mug/L epidermal growth factor +10 mug/L basic fibroblast growth factor for 3 days; stage 2 culture with DMEM-LG + volume fraction of 1% fetal bovine serum +20 μ g/L hepatocyte growth factor

+ 10. Mu.g/L of basic fibroblast growth factor +0.61g/L of niacinamide + 10. Mu.g/L of Wnt-1-13F monoclonal antibody, culturing for 6 days, and changing the solution 1 time every 3 days;

stage 3 culture: contains fetal calf serum with volume fraction of 5%, human fibroblast growth factor 4 with volume fraction of 10 μ g/L, hepatocyte growth factor with volume fraction of 10 μ g/L, recombinant tumor suppressor M with volume fraction of 10 μ g/L, wnt-1-13F monoclonal antibody with volume fraction of 10 μ g/L, and recombinant tumor suppressor M with volume fraction of 10 μ g/L ^-7 Culturing the cells for 10 days in a HBM culture medium containing mol/L dexamethasone to obtain differentiated cells; wnt-1-13F monoclonal antibodies with the end point concentration of 10 mug/L are added in the stage 2 and the stage 3 respectively to be used as enhancement induction groups, and the hUC-MSCs with good growth state in the 4 th generation are used as control groups without induction.

The cells after induction were digested with 0.25% trypsin and collected, respectively, total RNA was extracted with the kit, and cDNA was synthesized using the reverse transcription kit. SYBR slide Master Mix was chosen for the fluorescent quantitative PCR detection, 1. Mu.L of cDNA template was added with 0.5. Mu.L of each primer, 10. Mu.L of reaction system was used, GAPDH gene was chosen as internal reference, and the experimental conditions were 95 ℃ 10min,58 ℃ 2 s,72 ℃ 30s, 40 cycles in total. The specific primer is an AFP upstream primer: GCTGGTGGTGGATGAAAACA, downstream primer TCCTCTGTTATTTGTGGCTTTTG; IL-6 upstream primer:

aaattcggttacatcctcgac, downstream primer: CAGGAACTGGATCAGGACTT; GAPDH is used as an internal reference gene, and an upstream primer: AGCCACATCGCTCAGACAC; a downstream primer: GCCCAATACGACCAAAATCC. The results are shown in table 3 below.

TABLE 3 expression of IL-6 and AFP genes in each group

As can be seen from the results in table 3, the mRNA expression levels of AFP and IL-6 were significantly higher in the induced group than in the control group (. P < 0.05), and after induction, the umbilical cord mesenchymal stem cell phenotype differentiated into liver-like cells, and the differentiated cells had the high expression characteristic of hepatocyte marker AFP, indicating that the induction of differentiation was successful. After the monoclonal antibody is added, the differentiation and maturation of the hUC-MSCs to the liver-like cells are further promoted after the Wnt/p-catenin signal channel is competitively inhibited. And, unexpectedly, the differentiated mature liver-like cells have the characteristic of high expression of the cytokine IL-6.

EXAMPLE 4 production of cytokine composition containing human IL6 by differentiated cells

The liver-like cells obtained in example 3 after the induction of differentiation with Wnt-1-13F monoclonal antibody were sampled at 5X 10 ³ /cm ² Inoculating 15mL of an alpha-MEM medium containing 10% fetal bovine serum at 37 ℃ and 5% of CO ₂ Culturing in a saturated humidity incubator for 5 days, measuring cell density, centrifuging at 4 deg.C and 1500rpm for 5min to remove cells, and freeze drying the supernatant to obtain cytokine composition, and storing at-20 deg.C.

Taking the hUC-MSCs with good growth state of the 4 th generation as 5 multiplied by 10 ³ /cm ² The density of (A) was determined by inoculating 15mL of an alpha-MEM medium containing 10% fetal bovine serum, incubating the medium at 37 ℃ and 5% of CO ₂ Culturing in a saturated humidity incubator for 5 days, measuring cell density, centrifuging at 4 deg.C and 1500rpm for 5min to remove cells, and freeze drying the supernatant to obtain cytokine composition, and storing at-20 deg.C.

And (3) measuring the secretion amount of the IL6 under the same cell density condition by using an ELISA detection kit. The results are shown in Table 4.

TABLE 4 secretion of IL6

Each group consisting of	IL6 secretion amount (pg/mL 1X 10) ⁵ Cell)
		Control group	83.67±7.52
Wnt-1-13F monoclonal antibody enhanced induction group	279.18±12.72*

As can be seen from the results in Table 4, the cells obtained after the enhancement of induction with Wnt-1-13F monoclonal antibody were able to secrete IL-6 at high concentration, and the difference was significant (P) compared to the control group<0.01 At 1X 10) of ⁵ IL-6 can be isolated at a concentration of one cell that produces (279.18. + -. 12.72) pg/mL.

Example 5 cytokine Activity assay

Human skin fibroblast HFF-1 cells were cultured in 10% fetal bovine serum in DMEM/F12 medium, cultured routinely in a saturated humidity incubator at 37 ℃ and 5% CO2, and trypsinized and subcultured when the cells were 85% long. Taking HFF-1 cells in logarithmic growth phase, 1 × 10 per well ⁴ The individual cells were seeded in 96-well culture plates. After 12 hours, the cells were replaced with complete medium containing the final concentrations of 0. Mu.g/mL, 10. Mu.g/mL, 50. Mu.g/mL, and 100. Mu.g/mL of stem cell factor (prepared in example 4), and the complete medium containing 100. Mu.g/mL of stem cell factor (prepared in example 4 as a control) was used as an experimental control, and after further culturing for 48 hours, 20. Mu.L of 5mg/mL CCK-8 solution was added to each well, and incubation was continued at 37 ℃ for 4 hours in the absence of light, and then the culture medium was removed from each well, and 150. Mu.L of DMSO was added thereto, and the mixture was shaken and mixed well to dissolve it sufficiently. Detecting the absorbance OD value at the wavelength of 570nm by using a microplate reader, taking 0 mu g/mL as a blank control hole, and calculating according to the following formula: proliferation rate (%) = (OD assay well-OD blank control well)/OD blank control well × 100%. The results are shown in FIG. 3.

As can be seen from the results in fig. 3, the cytokine production induced by the monoclonal antibody of the present application has concentration dependency, and with the increase of the concentration, the proliferation of human skin fibroblasts can be significantly promoted (P <0.05 compared with the control group), and the dose dependency is presented. The cell factor of 100 mu g/mL prepared by using the cells induced by the monoclonal antibody can improve the proliferation activity of the HFF-1 cells to (264.1 +/-12.9)%, and the cell factor prepared by obtaining the cells without using the monoclonal antibody can improve the proliferation activity of the HFF-1 cells to (196.8 +/-10.5)%, which also indicates that the content and the activity of the cell factor can be obviously improved after the monoclonal antibody is used for induction.

It is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of components set forth in the following description and/or illustrated in the drawings. The invention is capable of embodiments in addition to those described and of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.

Claims

1. A monoclonal antibody that inhibits the activity of Wnt-1 is Wnt-1-13F, whose light chain variable region VL amino acid sequence:

heavy chain variable region VH amino acid sequence:

QVKLQESGGGLVQPKGSLKLSCAASGFSFYFIPYPWVRQAPGKGLEWVAVTTVIMWDGPDEWDENLCRRFTISRDDSQSMLYLQMHNLKTDDTAMYYCVRITESAWAIVEFAFWGAGTTVTVSS。

2. the use of the monoclonal antibody Wnt-1-13F of claim 1 in the preparation of a medicament for inhibiting the activity of the Wnt1/β -catenin pathway by inhibiting the activity of the Wnt1/β -catenin pathway.

3. Use of the monoclonal antibody Wnt-1-13F inhibiting the activity of Wnt-1 according to claim 1 in the preparation of an agent for promoting differentiation of umbilical cord mesenchymal stem cells into liver-like cells.

4. A method for producing IL 6-rich cytokines with high yield comprises culturing umbilical cord mesenchymal stem cells in stage 1 with DMEM-LG +20 μ g/L epidermal growth factor +10 μ g/L basic fibroblast growth factor for 3d; then stage 2 culture, DMEM-LG + 1% fetal calf serum +20 mug/L hepatocyte growth factor +10 mug/L alkaline fibroblast growth factor +0.61g/L niacinamide +10 mug/L Wnt-1-13F monoclonal antibody by volume fraction, culture for 6 days, and changing the liquid 1 time every 3 days; final stage 3 culture: contains fetal calf serum with volume fraction of 5%, human fibroblast growth factor 4 with volume fraction of 10 mug/L, hepatocyte growth factor with volume fraction of 10 mug/L, recombinant oncostatin M with volume fraction of 10 mug/L, wnt-1-13F monoclonal antibody with volume fraction of 10 mug/L, and 10 Wnt-1-13F monoclonal antibody with volume fraction of 10 mug/L ^-7 Culturing the HBM culture medium containing mol/L dexamethasone for 10 days to obtain differentiated liver-like cells;

inoculating the hepatocyte-like cells into an alpha-MEM medium containing 10% fetal bovine serum, and incubating at 37 deg.C for 5% CO ₂ Culturing in a saturated humidity incubator for 5d, centrifuging at the low temperature of 4 ℃ and 1500rpm for 5min to remove cells, and freeze-drying supernate to obtain a cytokine composition rich in IL 6; wherein Wnt-1-13F monoclonal antibody is the monoclonal antibody of claim 1.