CN115925925B - Method for inducing umbilical cord mesenchymal stem cells to differentiate and producing cytokines - Google Patents
Method for inducing umbilical cord mesenchymal stem cells to differentiate and producing cytokines Download PDFInfo
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Abstract
The invention relates to a method for inducing umbilical cord mesenchymal stem cells to differentiate and producing cytokines. The invention prepares the monoclonal antibody which specifically inhibits the Wnt-1 activity in the Wnt 1/beta-catenin channel, the antibody can promote the differentiation of umbilical cord mesenchymal stem cells to liver-like cells, and simultaneously the differentiated liver-like cells can highly express cytokines rich in IL6, and the cytokines have better biological activity, can be used for preparing corresponding medicaments, and have wide application prospects.
Description
Technical Field
The present application relates to the field of biology, and more particularly to a method for inducing differentiation of umbilical cord mesenchymal stem cells and production of cytokines.
Background
Cytokines are a large class of proteins 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. Cytokine release will have an effect on surrounding cellular activity. Cytokines may include interleukins, interferons, growth factors, tumor necrosis factors, chemokines, and colony stimulating factors.
Interleukins (ILs), a cytokine, play an important role in regulating cellular processes (cell growth, differentiation and movement) and stimulating immune responses. Initially found in leukocytes, they are now found to be produced by a wide variety of cells, including macrophages, lymphocytes, and the like, which have a solid structure and function. Mainly comprises 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 and the like. Interferon (IFN) is a name given to the production of and release of a set of signal proteins by host cells against a variety of viruses present, as it protects the cells from viral infection and "interferes" with viral replication. Interferons are a large class of cytokine proteins that are used in cell-to-cell communication to trigger protective defenses of the immune system to destroy pathogens. Growth factors, which are also 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 various cellular processes and can be secreted by neighboring cells, distant tissues and glands, and even by tumor cells themselves. Normal cells maintain proliferation and viability by exhibiting the need for several growth factors. Growth factors may exert their stimulatory effects by endocrine, paracrine or autocrine mechanisms.
There are many methods for cytokine production. For example, CN106520689a provides a preparation method of mesenchymal stem cell factor, which comprises the following steps: extracting mesenchymal stem cells; culturing; continuously culturing under the condition of low oxygen; digestion with 0.25% pancreatin EDTA solution; centrifuging, and cleaning with 0.9% physiological saline; adjusting the cell concentration with 0.9% physiological saline, and adding 1.3 mg/mL EDTA and 5.10 mg/mL Vc; freezing and thawing; centrifuging and filtering. However, the yield of the method for cytokine IL-6 is not high. CN106367389A discloses a preparation method of human umbilical cord mesenchymal stem cell factor, which comprises the 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 mainly prepared by centrifuging, incubating, secondary centrifuging and ultrafiltration concentrating cells in the generation P20 in a culture flask to obtain a factor concentrated solution, and IL-6 is not 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 into a serum-free culture medium for culture, collecting supernatant when the cells grow to 75-85% of confluence, filtering and concentrating: filtering the supernatant with 0.22 μm filter membrane; then the dialysate is collected through a 50KD ultrafiltration membrane, and then the dialysate is passed through a 1KD ultrafiltration membrane, and the trapped fluid is collected, so that the cytokine concentrated solution is obtained. The process likewise produces less IL-6 during the preparation.
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 and four alpha helices of 184 amino acids, usually in monomeric form, and an isoelectric point of 5.0. The gene encoding human IL-6 is located on chromosome 7p15-21, comprising 4 introns and 5 exons. IL-6 has 3 receptor binding sites, including 1 specific receptor IL-6R (IL-6 binding receptor protein,IL-6R) binding site, and 2 gp130 (signal-transducing protein) binding sites. IL-6 is also a cytokine with multiple-effect activity, and can promote proliferation and differentiation of various cells, accelerate synthesis of acute phase proteins of liver cells, inhibit growth of M1 myelogenous leukemia cell line, promote maturation and differentiation of the myelogenous leukemia cell line, and inhibit growth of melanoma and breast cancer cells. Therefore, the efficient production of IL-6 cytokines is an urgent direction of current research.
Disclosure of Invention
The invention provides a method for efficiently producing IL-6 and other cytokines.
In particular, the invention provides a method for producing cytokine mixtures containing IL-6 cytokines.
Wnt-1 protein is a secreted glycoprotein composed of 343 amino acids, and most of the protein acts through cell surface Frizzled family receptors, and is essential in the normal development process of embryos. Activation of Wnt-1 causes a complex cascade of signaling reactions that ultimately lead to upregulation of multiple gene expression. Wnt-1 is involved in a variety of cellular processes in humans 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 Wnt-1, the Wnt1/β -catenin pathway can be inhibited as a whole.
In one aspect, the invention provides a monoclonal antibody that inhibits Wnt-1 activity.
The monoclonal antibody inhibiting activity of Wnt-1 is Wnt-1-13F, light chain variable region VL amino acid sequence:
QIVLSQSPAILSASPGEKVTMTCNTEYRGARGSWYQQKPGSSPKPWIYPWEHQTHGVPFRFSGSGSGTSYSLTISRVEAEDAATYYCIASYMILFLFGGGTKLEIK
heavy chain variable region VH amino acid sequence:
QVKLQESGGGLVQPKGSLKLSCAASGFSFYFIPYPWVRQAPGKGLEWVAVTTVIMWDGPDEWDENLCRRFTISRDDSQSMLYLQMHNLKTDDTAMYYCVRITESAWAIVEFAFWGAGTTVTVSS
the light and heavy chain variable regions of the antibody are:
furthermore, the invention also provides the application of the monoclonal antibody for inhibiting the activity of Wnt-1 in preparing an agent for promoting the differentiation of umbilical cord mesenchymal stem cells into liver-like cells.
Further, the differentiation reagent comprises three-stage reagents, and the three-stage reagents are respectively used in the induction of the differentiation of umbilical cord mesenchymal stem cells under the following conditions: culturing umbilical cord mesenchymal stem cells in the current stage 1, namely DMEM-LG+20 mug/L of epidermal growth factor+10 mug/L of basic fibroblast growth factor, and culturing for 3d; culturing in stage 2, namely culturing for 6 days, wherein the volume fraction of DMEM-LG+1% fetal bovine serum+20 mug/L hepatocyte growth factor+10 mug/L basic fibroblast growth factor+0.61 g/L nicotinic acid amine+10 mug/L Wnt-1-13F monoclonal antibody, and changing liquid for 1 time every 3 days; and (3) culturing in a final stage 3: contains 5% of fetal calf serum, 10 mu g/L of human fibroblast growth factor 4, 10 mu g/L of hepatocyte growth factor, 10 mu g/L of recombinant tumor suppressor M, 10 mu g/L of Wnt-1-13F monoclonal antibody and 10 mu g/L of human fibroblast growth factor -7 The differentiated liver-like cells are obtained by culturing 10 d of HBM culture medium of mol/L dexamethasone.
Further, the invention also provides methods of using the differentiated liver-like cells for the production of IL 6-enriched cytokines.
The production method includes a method of culturing cells, which is conventional in the art, and collecting the supernatant to obtain the cytokine.
Further, the production method can be that the density of liver-like cells induced and differentiated by monoclonal antibodies is used for inoculating alpha-MEM culture medium containing 10% of fetal bovine serum, and the culture medium is placed at 37 ℃ and 5% of CO 2 Culturing in saturated humidity incubator for 5d, centrifuging at 4deg.C and 1500rpm for 5min to remove cells, lyophilizing supernatant to obtain cytokine composition, and preserving at-20deg.C.
Furthermore, the invention also provides the application of the prepared IL 6-enriched cytokine in preparing medicaments for promoting fibroblast proliferation and treating related diseases such as cancers and the like.
Furthermore, the cytokine prepared by the invention can be used for preparing cytokine 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 in particular selected from 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, provided that they do not reduce or eliminate their advantageous properties as described herein, in particular their stability.
Excipients may be used in the present invention for a wide variety of purposes, such as to tailor the physical, chemical or biological properties of the formulation (such as to tailor the viscosity) and/or the method of the invention to further improve the effectiveness and/or to further stabilize the formulation and method against degradation and spoilage due to stresses generated, for example, during manufacture, shipping, storage, pre-use preparation, administration and thereafter. The term "excipient" generally includes fillers, binders, disintegrants, coatings, adsorbents, anti-adherent agents, 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, including 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 invention prepares the monoclonal antibody which specifically inhibits the Wnt-1 activity in the Wnt 1/beta-catenin channel, the antibody can promote the differentiation of umbilical cord mesenchymal stem cells to liver-like cells, and simultaneously the differentiated liver-like cells can highly express cytokines rich in IL6, and the cytokines have better biological activity, can be used for preparing corresponding medicaments, and have wide application prospects.
Drawings
FIG. 1 phage clone ELISA assay results
FIG. 2 shows a diagram of the results of specific identification of monoclonal antibodies
FIG. 3 is a graph showing the proliferation effect of cytokines on 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 that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
EXAMPLE 1 preparation of Wnt-1 monoclonal antibodies
Human recombinant Wnt-1 (Biovision, cat# 4754-50) was coated on ELISA plates with 50mmol/L sodium bicarbonate (pH10.0), PBS/2% milk was blocked, phage libraries (synthesized by Wohan Jin Kairui Biotechnology Co., ltd., fab phage libraries) were added, and the plates were incubated at room temperature for 1h, washed 6 times with PBS/0.1% Tween, and the bound phage eluted with acidic eluent (0.1 mmol/LHCl-Gly, pH2.2,0.1% BSA), and the eluate was neutralized with 1mol/L Tris-HCl (pH9.1), and titers were measured, and the amplified phages were used for the next round of screening. A total of 3 rounds of screening were performed, the latter 2 rounds of screening were identical to the first round, but the amount of coated antigen was reduced and the Tween concentration was gradually increased, 0.3% and 0.5%, respectively. After 3 rounds of screening, the enrichment index of the final enriched phage was 109, indicating that phages with high affinity for Wnt-1 have been successfully enriched.
Selecting 42 positive clones obtained by 3 rounds of screening, inoculating into 100 mu L of 2YT culture solution containing 100mg/LAmp, culturing at 37 ℃ until OD600 = 0.3-0.4, adding 25 mu L of auxiliary phage for infection, then adding 25 mu L of 2YT culture solution containing 300mg/L Kan, and culturing at 37 ℃ overnight. The enriched individual phage clones were specifically identified by ELISA, 100ng of human recombinant Wnt-1 was coated per well, 100 μl phage was added, and the HRP-labeled murine anti-M13 phage antibody was developed. A450nm absorbance was measured with a blank zeroing and microplate reader (Multiskan Spectrum microplate thermo 1500). The positive standard is P/N value (positive/negative) >2.0, and the negative standard is P/N value <0.5. The results are shown in FIG. 1.
As can be seen from the results of FIG. 1, the 2 strains of monoclonal antibodies with the best positive effect are Wnt-1-15C and Wnt-1-13F, respectively.
For Wnt-1-13F, 5' terminal primer: AAGACAGCTATCGCGATT and 3' -terminal primers: GCCCCCTTATTAGCGTTT Fab gene fragments were amplified and PCR conditions: 94 ℃ for 5min;94℃15s,50℃30s,72℃2min,30 cycles; and at 72℃for 10min. The product was sent to Shanghai Biotechnology Co., ltd for antibody variable region gene sequencing, and the sequencing result was retrieved and analyzed using DNAMAN and a database. The amino acid sequence of a light chain variable region of the Wnt-1-13F monoclonal antibody and the amino acid sequence of a heavy chain variable region of the Wnt-1-13F monoclonal antibody are obtained through sequence comparison, wherein the heavy chain variable region sequence is shown as SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 1.
And (3) carrying out soluble separation in HB2151 bacteria to obtain an expressed Fab antibody, carrying out low-temperature induction expression on 0.5 mol/L IPTG overnight, obtaining soluble Wnt-1-13F monoclonal antibody from a bacterial periplasm cavity by using an osmotic shock method, purifying by using a nickel column to obtain a purified monoclonal antibody, and regulating the concentration of the monoclonal antibody to 1mg/mL for later use.
Example 2 affinity and specificity identification of Wnt-1-13F monoclonal antibodies
SPR technique was used to analyze the binding capacity of Wnt-1-13F antibodies to Wnt-1 protein: wnt-1 protein was coupled to a CM5 sensor chip and Wnt-1-13F mab was injected at 6 different concentrations in the range of 1-100 nM, at a flow rate of 45. Mu.L/min in all experiments. The regeneration condition of the chip is glycine-HClpH1.5. The kinetic parameters KD were calculated using binding curves obtained at different antibody concentrations. The results of Table 1 were obtained by calculation.
TABLE 1 affinity of Wnt-1-13F mab
As can be seen from the results in Table 1, the affinity of the Wnt-1-13F antibody prepared according to the present invention to the antigen had a KD of 2.19X10 -9 M has better affinity.
Specificity identification: the purified Wnt-1-13F monoclonal antibody is subjected to specificity analysis by ELISA method, each hole is coated with human recombinant Wnt-1 protein, wnt-2 protein and BSA 100ng respectively, gradient diluted Wnt-1-13F monoclonal antibody is added, and HRP marked mouse anti-Flag antibody is developed. The results are shown in FIG. 2.
The Wnt-1 protein and the control protein (Wnt-2 and BSA) coat the ELISA plate, and the analysis result shows that the Wnt-1-13F monoclonal antibody can specifically bind to the Wnt-1 protein and not bind to other non-related proteins (figure 2), and the better specificity is shown.
Example 3 preparation of umbilical cord mesenchymal Stem cells and differentiation Induction
Fresh umbilical cord was placed in a petri dish and amniotic membrane and blood vessels were removed. Cutting the separated umbilical cord into pieces of about 1mm with an ophthalmic scissors 3 The tissue blocks with the size are taken and put into a sterile culture dish, and the tissue blocks are covered on the bottom of the culture dish with the size of 75 percent. Adding serum-free medium of mesenchymal stem cells, shaking for 1min to disperse tissue block uniformly, and adding 5% CO 2 Culturing in an incubator at 37 ℃. Half-quantity medium replacement is carried out on the 5 th day, the culture is continued for about 14 days, when the cell mass grows to about 50%, the tissue mass is poured out, and meanwhile, the cells are collected for subculture. Changing culture solution every 3 days, observing cell growth change by using an inverted microscope, performing digestion and passage when adherent cells grow to 90% of the bottle bottom, adding 0.05% pancreatin for digestion, performing passage according to the ratio of 1:4, and separating and purifying the cells while the passage. Viable cell density 5×10 with serum-free DMEM medium at passage 3 6 individual/mL of cell suspension for detection.
The UC-MSCs immunophenotype detection 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 immunophenotype identification
As can be seen from the results in Table 2, CD90 and CD105 were expressed positively with a positive rate of >98%, while CD34, CD45 and HLA-DR were expressed negatively with a positive rate of <2%, indicating that umbilical cord mesenchymal stem cells were obtained by isolation.
Selecting hUC-MSCs with good growth state of the 4 th generation, wherein each hole is 5 multiplied by 10 4 Cells were inoculated uniformly into 6-well plates and, when the cells were grown to 80% confluence, cultured sequentially according to the following procedure. Culturing in stage 1, namely culturing DMEM-LG+20mug/L epidermal growth factor+10mug/L basic fibroblast growth factor for 3d; phase 2 culture DMEM-LG+ volume fraction1% fetal bovine serum+20 mug/L liver cell growth factor+10 mug/L basic fibroblast growth factor+0.61 g/L niacinamine+10 mug/L Wnt-1-13F monoclonal antibody, culturing for 6d, and changing liquid 1 time every 3d;
stage 3 cultivation: contains 5% of fetal calf serum, 10 mu g/L of human fibroblast growth factor 4, 10 mu g/L of hepatocyte growth factor, 10 mu g/L of recombinant tumor suppressor M, 10 mu g/L of Wnt-1-13F monoclonal antibody and 10 mu g/L of human fibroblast growth factor -7 The differentiated cells are obtained by culturing 10 d of HBM culture medium of mol/L dexamethasone; and adding Wnt-1-13F monoclonal antibodies with the end point concentration of 10 mu g/L in the stage 2 and the stage 3 respectively to obtain an enhanced induction group, wherein hUC-MSCs with good growth state of the 4 th generation are not induced and are used as a control group.
The induced cells were digested with 0.25% trypsin and collected, total RNA was extracted from the kit, and cDNA was synthesized using reverse transcription kit. SYBR Slect Master Mix is selected for fluorescence quantitative PCR detection, 1 mu L cDNA template is taken, 0.5 mu L of each primer is added, a reaction system of 10 mu L is adopted, and GAPDH gene is selected as an internal reference, wherein experimental conditions are as follows, 95 ℃ for 10min, 58 ℃ for 20s, 72 ℃ for 30s, and the total circulation is 40 times. Specific primers are AFP upstream primers: GCTGGTGGTGGATGAAACA, downstream primer TCCTCTGTTATTTGTGGCTTTTG; IL-6 upstream primer: AAATTCGGTACATCCTCGAC, downstream primer: CAGGAACTGGATCAGGACTT; GAPDH is taken as a reference gene, and an upstream primer: AGCCACATCGCTCAGACAC; a downstream primer: GCCCAATACGACCAAATCC. The results are shown in Table 3 below.
TABLE 3 expression of IL-6 AFP Gene groups
As can be seen from the results in table 3, the mRNA expression levels of AFP and IL-6 were significantly higher in the induction group than in the control group (< 0.05) and, after induction, the umbilical cord mesenchymal stem cell phenotype differentiated towards liver-like cells, the differentiated cells having the high expression characteristics of the hepatocyte marker AFP, indicating successful induction differentiation. After the monoclonal antibody is added, the differentiation and maturation of hUC-MSCs to liver-like cells are further promoted after the Wnt/p-catenin signal pathway is competitively inhibited. And, unexpectedly, the differentiated and matured liver-like cells have the characteristic of highly expressing the cytokine IL-6.
Example 4 production of cytokine compositions containing human IL6 by differentiated cells
Example 3 Induction of differentiation with Wnt-1-13F mab was performed on 5X 10 liver-like cells 3 /cm 2 15mL of alpha-MEM medium containing 10% fetal bovine serum was inoculated at 37℃and 5% CO 2 Culturing in saturated humidity incubator for 5d, measuring cell density, centrifuging at 4deg.C and 1500rpm for 5min to remove cells, lyophilizing supernatant to obtain cytokine composition, and preserving at-20deg.C.
Taking hUC-MSCs with good growth state of the 4 th generation at 5 multiplied by 10 3 /cm 2 15mL of alpha-MEM medium containing 10% fetal bovine serum was inoculated at 37℃and 5% CO 2 Culturing in saturated humidity incubator for 5d, measuring cell density, centrifuging at 4deg.C and 1500rpm for 5min to remove cells, lyophilizing supernatant to obtain cytokine composition, and preserving at-20deg.C.
The secretion of IL6 was measured under the same cell density conditions using ELISA assay kit. The results are shown in Table 4.
TABLE 4 secretion of IL6
As can be seen from the results in Table 4, cells obtained after induction enhancement with Wnt-1-13F mab were able to secrete IL-6 at high concentrations, the differences and significance (P<0.01 At 1×10) 5 IL-6 production (279.18.+ -. 12.72) pg/mL can be isolated at individual cell concentrations.
Example 5 cytokine Activity assay
Human skin fibroblast HFF-1 cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum, and subjected to conventional culture in a saturated humidity incubator containing 5% CO2 at 37℃until the cells were 85%, and digested with pancreatin, and subcultured. Taking HFF-1 cells in logarithmic growth phase, 1×10 cells per well 4 Inoculating individual cells to96-well culture plates. After 12 hours, the culture medium was replaced with a complete medium containing the stem cell factor (prepared by the method of example 4) at a final concentration of 0. Mu.g/mL, 10. Mu.g/mL, 50. Mu.g/mL, and 100. Mu.g/mL, and the complete medium was used as an experimental control for further culturing for 48 hours, and after adding 20. Mu.L of CCK-8 solution per well at 37℃for further incubation for 4 hours at a dark place, 150. Mu.L of DMSO was added to the culture medium per well, and shaking and mixing were performed to obtain a complete solution. The enzyme label instrument detects the absorbance OD value at 570nm wavelength, takes 0 mug/mL as a blank control hole, and calculates according to the following formula: proliferation (%) = (OD experimental well-OD blank well)/OD blank well x 100%. The results are shown in FIG. 3.
As can be seen from the results of fig. 3, the cytokines produced by the cells induced by the monoclonal antibodies of the present application have concentration dependence, and as the concentration increases, proliferation of human skin fibroblasts can be significantly promoted (P <0.05 compared with the control group), and dose dependence is exhibited. The proliferation activity of HFF-1 cells can be improved to (264.1+/-12.9)% by using 100 mug/mL of cytokine prepared from the cell induced by the monoclonal antibody, and the proliferation activity of HFF-1 cells can be improved to (196.8 +/-10.5)% by using the cytokine prepared from the cell obtained without the monoclonal antibody, which also shows that the content and the activity of the cytokine can be remarkably improved after the monoclonal antibody is induced.
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 or illustrated in the drawings. The invention is capable of embodiments in addition to those described and of being practiced or of being 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 (3)
1. A monoclonal antibody that inhibits the activity of Wnt-1 is Wnt-1-13F, whose light chain variable region VL amino acid sequence is:
QIVLSQSPAILSASPGEKVTMTCNTEYRGARGSWYQQKPGSSPKPWIYPWEHQTHGVPFRFSGSGSGTSYSLTISRVEAEDAATYYCIASYMILFLFGGGTKLEIK
the heavy chain variable region VH amino acid sequence is:
QVKLQESGGGLVQPKGSLKLSCAASGFSFYFIPYPWVRQAPGKGLEWVAVTTVIMWDGPDEWDENLCRRFTISRDDSQSMLYLQMHNLKTDDTAMYYCVRITESAWAIVEFAFWGAGTTVTVSS。
2. use of the monoclonal antibody Wnt-1-13F that inhibits Wnt-1 activity of claim 1 in the preparation of an agent that promotes differentiation of umbilical cord mesenchymal stem cells into liver-like cells.
3. A method for producing IL 6-enriched cytokines, which comprises culturing umbilical cord mesenchymal stem cells in stage 1, wherein DMEM-LG+20 μg/L epidermal growth factor+10 μg/L basic fibroblast growth factor is cultured for 3d; culturing in stage 2, namely culturing for 6 days, wherein the volume fraction of DMEM-LG+1% fetal bovine serum+20 mug/L hepatocyte growth factor+10 mug/L basic fibroblast growth factor+0.61 g/L nicotinic acid amine+10 mug/L Wnt-1-13F monoclonal antibody, and changing liquid for 1 time every 3 days; and (3) culturing in a final stage 3: contains 5% of fetal calf serum, 10 mu g/L of human fibroblast growth factor 4, 10 mu g/L of hepatocyte growth factor, 10 mu g/L of recombinant tumor suppressor M, 10 mu g/L of Wnt-1-13F monoclonal antibody and 10 mu g/L of human fibroblast growth factor -7 Culturing 10 d of HBM culture medium of mol/L dexamethasone to obtain differentiated liver-like cells;
inoculating the liver-like cells with alpha-MEM medium containing 10% fetal bovine serum, and placing at 37deg.C and 5% CO 2 Culturing in a saturated humidity incubator for 5d, centrifuging at 4 ℃ and 1500rpm for 5min to remove cells, and freeze-drying supernatant to obtain the cytokine composition rich in IL 6; wherein the Wnt-1-13F monoclonal antibody is the monoclonal antibody of claim 1.
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