CN114736302A - Serum-free culture medium for stem cells - Google Patents

Serum-free culture medium for stem cells Download PDF

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CN114736302A
CN114736302A CN202210516474.6A CN202210516474A CN114736302A CN 114736302 A CN114736302 A CN 114736302A CN 202210516474 A CN202210516474 A CN 202210516474A CN 114736302 A CN114736302 A CN 114736302A
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monoclonal antibody
rnd1
amino acid
mug
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CN114736302B (en
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亓爱杰
李少波
陈清轩
李斌斌
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Nuosa Union Beijing Biomedical Technology Co ltd
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Abstract

The invention relates to a serum-free culture medium for stem cells. The invention specifically provides a monoclonal antibody aiming at Rho GTP enzyme, the antibody can specifically inhibit the activity of the Rho GTP enzyme, further regulate and control the expression of downstream ROCK1 protein, the antibody can promote the proliferation of stem cells, and can inhibit the invasion and migration capacity of cancer cells, so that the monoclonal antibody has good application value.

Description

Serum-free culture medium for stem cells
Technical Field
The invention relates to the field of biology, and particularly relates to a serum-free culture medium for stem cells.
Background
Rho GTPase belongs to Ras superfamily, and is involved in activities such as cell migration, phagocytosis, contraction and adhesion. ROCK is also called Rho-associated kinase (Rho-associated kinase), and is the Rho downstream effector molecule with the most detailed function research at present. Rho/ROCK signal channel induces cytoskeleton recombination, cell migration and stress fiber formation, is related to multiple physiological functions such as endothelial permeability, tissue contraction and growth, participates in the occurrence of diseases such as diabetes, eye diseases, tumors, heart diseases, nerve injury diseases, hypertension, radiation injury, leukemia and the like, and is more and more concerned by people as a drug research and development target.
The Rho/ROCK signaling pathway activates downstream ROCK through Rho binding to GTP and further phosphorylates ROCK downstream substrates, remodels cytoskeleton, induces actin filament stabilization and actin-myosin contraction, combines actin network and myosin fibers, regulates microtubule dynamics. Rho and ROCK are widely distributed in vivo, so that the potential success of the medicine taking the pathway as a target is large.
The movement of tumor cells in the stroma consists of 4 cyclic steps, i.e., formation and extension of the cephalic pseudopoda, establishment of new adhesion sites, contraction of the cell body and retraction of the tail, migrating forward through 4 processes that are repeated. The molecular mechanisms that precisely regulate this process are complex and involve multiple intracellular signaling pathways. In a plurality of signal cascade reaction paths, RhoGTP enzyme, particularly RhoA, Rac1 and Cdc42, is a key regulatory factor, and is mainly involved in the regulation of cell morphology change, cell and matrix adhesion and cytoskeleton recombination, and the regulation of the invasion and metastasis process of tumor cells. Cellular morphological changes pseudopodogenesis and cellular morphological changes are the initial steps of invasive metastasis. Rac induces plasma membrane processes to form lamellar pseudopodia like sheet, while Cdc42 induces filopodia like processes to form finger-like processes. In highly aggressive and metastatic tumor cells, an aggressive pseudopodogenesis is also seen, which may be the main pseudopodogenesis due to its close association with extracellular matrix degradation. The layered pseudopoda forms adhesive connection with surrounding matrix to generate an anchoring site for forward movement of cells; the filopodia facilitate adaptation of the cell to the surrounding environment and determine the direction of cell migration. Inhibition of rhogtpase therefore helps to inhibit tumor cells.
Researchers found that Rho gtpase Rnd1 is a potential tumor metastasis suppressor in basal-like and triple negative breast cancers. The Rnd1 protein is knocked out to destroy the adhesion and polarity of epithelial cells, so that epithelial-mesenchymal cell transformation is caused, meanwhile, the intracellular c-Myc expression is disordered, the tumor suppressor factor p53 is inhibited, and the cells are also caused to generate tumor transformation. Studies have shown that Rnd1 inhibits Ras signaling by activating the GAP domain of the PlexinB1 protein, thereby inhibiting the function of Rap 1. Inhibition of Rap1 in mammary epithelial cells, however, led to mice that developed undifferentiated but highly invasive tumors. In contrast, normal expression of Rnd1 could suppress spontaneous lung metastasis of mouse breast cancer. Genomics data indicate that the function of the RND1 gene is suppressed in human breast cancer tissues, the main mechanism being deletion of the gene through epigenetic silencing, or very rare point mutations. These experimental results reveal to us that a previously unimagined mechanism for the development of mammary cancer- -Rnd1 limits the Ras-MAPK signaling pathway. The new member of Rho GTP enzyme family can provide a new drug development target for treating breast cancer. By inhibiting the activity of RND1 in other cancers, studies have also been possible to treat cancer.
The division cycle of eukaryotic cells is a fundamental biological process that requires very precise molecular events to ensure DNA accuracy and maintain cellular homeostasis. It has been shown that the ROCK protein plays a role in regulating cell proliferation, and ROCK is activated during the G1/S phase of cell proliferation as well as during mitosis. Overexpression or activation of ROCK i or ROCK2 can cause cell proliferation. In addition, the ROCK activation can induce the expression increase of catenin and a transcription target c-myc thereof, enhance the proliferation of cell strains and mouse epidermal cells, inhibit the activity of ROCK, delay the cytokinesis and promote the abnormal separation of centromere in G1 phase, so that the premature centrosome migration occurs in the mitosis process, and the ROCK activity plays an important role in the regulation of the cell cycle. Cell cycle stage G1 is a restriction node that assesses DNA integrity and determines whether a cell is in the cell cycle or reverts to the quiescent GO stage. Ectopic ROCK expression in NIH-3T3 fibroblasts stimulates progression of the S-phase of the cells and increases the levels of G1/S phase dependent cyclin (cyclin) D1. In contrast, inhibition of ROCK activity in corneal epithelial cells impairs the G1/S phase transition, and it was also demonstrated in cardiac myocytes that inhibition of ROCK reduces the expression of cyclin d3 and cyclin-dependent kinases (CDKs) 6 and P27. Furthermore, activation of ROCK protein during cytokinesis leads to the formation of an actin-rich cleavage groove accompanied by the breakdown of IF under the cleavage groove. Inhibition of ROCK activity delays the division of progeny cells, suggesting that precise regulation of ROCK activity is important for the completion of cell division.
The existing research shows that on the basis of the existing stem cell amplification, the application of a specific Rho kinase inhibitor can promote the synthesis of DNA in the process of stem cell division, promote the proliferation and division of stem cells, realize the rapid in-vitro amplification of the stem cells to the maximum extent and obtain a large amount of seed cells. However, there are not enough Rho kinase inhibitors, especially antibody inhibitors with better biological activity and safety.
Disclosure of Invention
In one aspect of the invention, a Rho gtpase monoclonal antibody RND1-23 monoclonal antibody was prepared from mice using Rho gtpase 1(RND1) as the immunogen.
The affinity dissociation constant (Kd) of RND1-23 monoclonal antibody was 5.17X 10 by conventional dissociation constant determination methods in the art－10And belongs to high affinity antibodies.
The amino acid sequence of the variable region of the light chain of the monoclonal antibody RND1-23 is shown in SEQ ID NO. 1:
DLVMTQTAPSVPVTPGESVSISCRSTCLGIVIYYDYALYWFLQRPGQSPQLLIYLNSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCEFYYMQDEDFGSGTKLEIK
the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2:
VKPGGSLKLSCAASCGNDLRPIMSWVRQTPDKRLEWVAIGLGMREWHYYPDSVKGRFTISRDQDKQTLYLQMSSLKSEDTAMYYCVDMEHPYHVSKWGQGTTVTVS。
in one embodiment, provided herein is an isolated nucleic acid encoding an antibody heavy and/or light chain variable region of an antibody of any one of the embodiments of the invention. In some aspects, the light and heavy chain sequences of the antibody comprise amino acid sequences at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO 2 and/or 2.
The monoclonal antibody prepared by the invention has good activity of inhibiting Rho GTP enzyme, has good binding specificity, and is not specifically bound to other proteins such as BSA, albumin, globulin, fibrinogen and the like.
In particular, related aspects include variants of monoclonal antibodies or fragments thereof comprising one or more conservative amino acid substitutions, wherein the functional activity associated with binding of the antibody or fragment thereof to a Rho gtpase 1 epitope is retained. Related aspects also include truncated or fusion variants of the monoclonal antibody comprising one or more amino acid insertions or deletions wherein the functional activity associated with binding of the antibody or fragment thereof to a Rho gtpase 1 epitope is retained. Related aspects also include variants comprising one or more combinations of conservative amino acid substitutions, insertions and deletions, particularly wherein the number of residues altered by substitution, insertion or deletion is less than the number of residues in the parent antibody molecule, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, 11-15, 16-20 and 21-25 residues. Related aspects also include molecules with larger insertions or deletions of one or more amino acid residues or polypeptide domains that do not alter the functional binding activity of the antibody to a desired epitope in a target molecule.
The invention provides a serum-free culture medium for promoting proliferation of stem cells, which consists of a basic culture medium and an additive component added in the basic culture medium, wherein the basic culture medium is a DMEM/F12 culture medium, and the additive component is L-glutamine, non-essential amino acid, L-ascorbic acid, sodium selenite, fibronectin, ethanolamine, hydrocortisone, a trypsin inhibitor, human transferrin, human insulin, bFGF, TGF-beta 1, PDGF-BB, coenzyme A, sodium pyruvate, EGF and RND1-23 monoclonal antibody; wherein the concentration of L-glutamine is 1-2mM, the concentration of non-essential amino acid is 1-2mM, the concentration of L-ascorbic acid is 40-60mg/L, the concentration of sodium selenite is 10-18 mug/L, the concentration of fibronectin is 15-30mg/L, the concentration of ethanolamine is 1-4mg/L, the concentration of hydrocortisone is 7-15mg/L, the concentration of trypsin inhibitor is 1-1.5mg/L, the concentration of human transferrin is 7-15mg/L, the concentration of human insulin is 10-15mg/L, the concentration of bFGF is 20-30 mug/L, the concentration of TGF-beta 1 is 3-7 mug/L, and the concentration of PDGF-BB is 7-15 mug/L, the concentration of coenzyme A is 80-120mg/L, the concentration of sodium pyruvate is 1-5mM, the concentration of EGF is 5-20 mu g/L, and the concentration of RND1-23 monoclonal antibody is 10-100 mg/L.
Another aspect relates to compositions comprising any of the above antibodies, including compositions comprising at least one antibody targeting Rho gtpase and one or more pharmaceutical excipients.
Other types of antibodies contemplated in the present disclosure are those primarily intended for in vitro use, wherein the antibody is linked to a secondary binding ligand and/or to an enzyme (an enzyme label) that will produce a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) catalase or glucose oxidase. Preferred secondary binding ligands are biotin and avidin and streptavidin compounds.
It is also contemplated to derivatize immunoglobulins by selectively introducing a sulfhydryl group in the Fc region of an immunoglobulin using reaction conditions that do not alter the binding site of the antibody. Antibody conjugates prepared according to the method are disclosed to exhibit improved longevity, specificity and sensitivity. Site-specific linkage of effectors or reporter molecules, wherein the reporter or effector molecules are conjugated to carbohydrate residues in the Fc region, has also been disclosed in the literature. This approach has been reported to produce diagnostically and therapeutically promising antibodies, which are currently in clinical evaluation.
Another aspect relates to a method of treating cancer in a mammalian subject comprising administering to a subject in need thereof an amount of the above antibody sufficient to treat cancer. Related aspects include methods wherein the mammalian subject is selected from the group consisting of human, non-human primate, canine, feline, bovine, equine, and porcine subjects. A preferred aspect relates to a method, wherein the mammalian subject is a human subject.
Related aspects also include the above method, wherein the cancer is selected from colorectal cancer, liver cancer, hepatocellular cancer, and cholangiocarcinoma, pancreatic cancer, gastric cancer, colon cancer, kidney cancer, non-small cell lung cancer, breast cancer, ovarian cancer, cervical cancer, head and neck cancer secondary to human papillomavirus infection, prostate cancer, brain cancer, various forms of glioblastoma, neuroblastoma, retinoblastoma and medulloblastoma, and osteosarcoma.
Antibodies with direct activity against Rho gtpase should be useful for the discovery and development of therapeutic drug products for the treatment of various cancers. These include colorectal, liver, such as hepatocellular and cholangiocarcinoma, pancreatic, gastric, colon, renal, non-small cell lung, breast, ovarian, cervical, head and neck cancer secondary to human papillomavirus infection, prostate, various types of brain cancer, including various forms of glioblastoma, neuroblastoma, retinoblastoma and medulloblastoma, and osteosarcoma.
Advantageous effects
The invention specifically provides a monoclonal antibody aiming at Rho GTP enzyme, the antibody can specifically inhibit the activity of the Rho GTP enzyme, further regulate and control the expression of downstream ROCK1 protein, the antibody can promote the proliferation of stem cells, and can also inhibit the invasion and migration capacity of cancer cells, so that the monoclonal antibody has a good application value.
Drawings
FIG. 1 is a graph showing the results of the proliferation of adipose derived mesenchymal stem cells promoted by monoclonal antibody
FIG. 2 is a graph showing the effect of monoclonal antibody on reducing the migration ability of LS513 human colorectal cancer cells
FIG. 3 is a graph showing the effect of monoclonal antibody on reducing the invasion ability of LS513 human colorectal cancer cells
FIG. 4 is a graph showing the relative expression level of monoclonal antibody-regulated downstream ROCK1
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 preparation of Rho GTPase monoclonal antibody
Rho GTPase 1(RND1) was used as an immunogen (purchased from Shanghai Seawa Biotech Ltd., 5 months 2020).
Using Rho GTP enzyme 1 as immunogen and 5 times diluted MontanideTMEqual volume mixing of GEL 01ST polymer adjuvant, subcutaneous multi-point injection immunization of BALB/c mice, 3 times of total immunization, 2 weeks of each immunization interval, and 100 μ g/mouse. And 7d after the three-immunization, performing impact immunization by using immunogen without adjuvant, and detecting the antibody level by using an indirect ELISA method after 7d after each immunization. And 4d later, taking the spleen cells of the mice with the highest titer for fusion.
Fusing SP2/0 cell and splenocyte under the action of PEG1500, and then using DMEM medium (DMEM dry powder 13.5g/L, NaHCO)33.7g/L, L-glutamine 0.2g/L) was added to HAT and HT for liquid exchange. After the hybridoma cells had grown to about floor area 1/3, the supernatant was aspirated and the antibody titer was determined by indirect ELISA. And selecting the well with strong positive determination for subcloning and screening to obtain the high-activity hybridoma cell strain 2, RND1-5 and RND1-23 capable of secreting the anti-Rho GTP enzyme antibody.
The culture supernatants of hybridoma cells RND1-5 and RND1-23 were collected, and the subtypes of the monoclonal antibodies were determined using a mouse Ig class/subclass identification kit, and the types of the anti-Rho GTPase monoclonal antibodies secreted by strain 2 were IgG1, and the light chains were kappa chains.
Preparation of 2 monoclonal antibodies: the abdomen of the sensitized mice is injected with 1X 106After each hybridoma cell, the abdomen of the mouse is observed to be obviously raised, the supernatant after ascites centrifugation is collected and subjected to indirect ELISA titer determination, and the titer of 2 monoclonal antibodies is 1:256000 for RND1-5 and 1:512000 for RND1-23 respectively. The ascites monoclonal antibody is further purified by Protein G after being crudely extracted by ammonium sulfate salting-out method, and the concentration of 2 monoclonal antibodies is respectively adjusted to 10mg/mL for standby.
The affinity dissociation constant (Kd) of RND1-23 monoclonal antibody was 5.17X 10 by conventional dissociation constant determination methods in the art－10And belongs to high affinity antibodies.
The light and heavy chain sequences of the RND1-23 monoclonal antibody obtained by amplifying the antibody light and heavy chain sequences by a PCR method and identifying through sequencing are respectively as follows:
the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 1:
DLVMTQTAPSVPVTPGESVSISCRSTCLGIVIYYDYALYWFLQRPGQSPQLLIYLNSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCEFYYMQDEDFGSGTKLEIK
the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2:
VKPGGSLKLSCAASCGNDLRPIMSWVRQTPDKRLEWVAIGLGMREWHYYPDSVKGRFTISRDQDKQTLYLQMSSLKSEDTAMYYCVDMEHPYHVSKWGQGTTVTVS
EXAMPLE 2 preparation of a Medium for Stem cell proliferation-promoting serum-free culture
The serum-free culture medium consists of a basic culture medium and an additive component added in the basic culture medium, wherein the basic culture medium is a DMEM/F12 culture medium, and the additive component is L-glutamine, non-essential amino acid, L-ascorbic acid, sodium selenite, fibronectin, ethanolamine, hydrocortisone, a trypsin inhibitor, human transferrin, human insulin, bFGF, TGF-beta 1, PDGF-BB, coenzyme A, sodium pyruvate, EGF and an RND1-23 monoclonal antibody; wherein the concentration of L-glutamine is 2mM, the concentration of unnecessary amino acids is 2mM, the concentration of L-ascorbic acid is 50mg/L, the concentration of sodium selenite is 10 mug/L, the concentration of fibronectin is 20mg/L, the concentration of ethanolamine is 2mg/L, the concentration of hydrocortisone is 10mg/L, the concentration of trypsin inhibitor is 1mg/L, the concentration of human transferrin is 10mg/L, the concentration of human insulin is 10mg/L, the concentration of bFGF is 20 mug/L, the concentration of TGF-beta 1 is 5 mug/L, the concentration of PDGF-BB is 10 mug/L, the concentration of coenzyme A is 100mg/L, the concentration of sodium pyruvate is 4mM, the concentration of EGF is 10 mug/L, the concentration of RND1-23 monoclonal antibody is 1 mug/L, 10. 100 and 200 mg/L.
Wherein DMEM/F12 medium, non-essential amino acids and sodium pyruvate were purchased from GIBCO under the accession numbers 11330-032/11140-050/11360070, respectively; l-glutamine, L-ascorbic acid, sodium selenite, trypsin inhibitor, hydrocortisone, ethanolamine, human transferrin, coenzyme A were purchased from Sigma under the respective accession numbers G3126, A4403, S5261, T6522, H0888, E0135, T0665, C4282, fibronectin, EGF were purchased from Hzbscience under the respective accession numbers ZY037Bo011 and ZY560Ca011, human insulin was purchased from Beijing Wakay Biotech limited under the respective accession numbers D11080, bFGF was purchased from Wolcvi under the respective accession numbers TL-401, PDGF-BB was purchased from Peprotech under the respective accession numbers 100-14B, TGF-beta 1 was purchased from Biovision under the respective accession numbers K4342-100.
EXAMPLE 3 preparation of control group of Stem cell proliferation-promoting serum-free Medium
The serum-free culture medium consists of a basic culture medium and an additive component added in the basic culture medium, wherein the basic culture medium is a DMEM/F12 culture medium, and the additive component is L-glutamine, non-essential amino acid, L-ascorbic acid, sodium selenite, fibronectin, ethanolamine, hydrocortisone, a trypsin inhibitor, human transferrin, human insulin, bFGF, TGF-beta 1, PDGF-BB, coenzyme A, sodium pyruvate and EGF; wherein the concentration of L-glutamine is 2mM, the concentration of unnecessary amino acids is 2mM, the concentration of L-ascorbic acid is 50mg/L, the concentration of sodium selenite is 10 mug/L, the concentration of fibronectin is 20mg/L, the concentration of ethanolamine is 2mg/L, the concentration of hydrocortisone is 10mg/L, the concentration of trypsin inhibitor is 1mg/L, the concentration of human transferrin is 10mg/L, the concentration of human insulin is 10mg/L, the concentration of bFGF is 20 mug/L, the concentration of TGF-beta 1 is 5 mug/L, the concentration of PDGF-BB is 10 mug/L, the concentration of coenzyme A is 100mg/L, the concentration of sodium pyruvate is 4mM, and the concentration of EGF is 10 mug/L.
Wherein DMEM/F12 medium, non-essential amino acids and sodium pyruvate were purchased from GIBCO under the accession numbers 11330-032/11140-050/11360070, respectively; l-glutamine, L-ascorbic acid, sodium selenite, trypsin inhibitor, hydrocortisone, ethanolamine, human transferrin, coenzyme A were purchased from Sigma under the respective accession numbers G3126, A4403, S5261, T6522, H0888, E0135, T0665, C4282, fibronectin, EGF were purchased from Hzbscience under the respective accession numbers ZY037Bo011 and ZY560Ca011, human insulin was purchased from Beijing Wakay Biotech Ltd under the accession number D11080, bFGF was purchased from Wolcvi under the accession number TL-401, PDGF-BB was purchased from Peprotech under the accession numbers 100-14B, TGF-beta 1 was purchased from Biovision under the accession number K4342-100.
Example 4 Effect of RND1-23 monoclonal antibody serum-free Medium on proliferation of adipose-derived mesenchymal Stem cells
Adipose-derived mesenchymal stem cells were obtained from (Cat: HTX2309, Shenzhen, luxury Tuo Biotech, Inc.).
The mass concentration is 0.2 percentThe gelatin solution of (4) was added to the petri dish to be coated and incubated at 37 ℃ for 1 hour to obtain a petri dish pre-coated with gelatin. The matrix is prevented from drying out in the incubation process, and the matrix is discarded to inoculate cells when the culture medium is used. According to a seed density of 5.0X 103/cm2Viable cells/cm2Adipose-derived mesenchymal stem cells were seeded on a gelatin pre-coated culture dish, serum-free media provided in examples 2 and 3 were added, respectively, and the obtained mixture was placed in a culture chamber (37 ℃, 5% CO)2) Culturing for 96 hours, removing the culture solution, adding 20 mu L of MTT solution into each hole, incubating for 4 hours in a constant-temperature incubator, terminating the culture, removing the MTT solution, adding 150 mu L of DMSO into each hole, shaking for 10min to fully dissolve crystals, and detecting the A value of each hole at 490nm wavelength of an enzyme linked immunosorbent detector, wherein the result is shown in figure 1.
From the results in fig. 1, it can be seen that, compared to the blank control group without the RND1-23 monoclonal antibody, after the RND1-23 monoclonal antibody is added, the proliferation efficiency of adipose-derived mesenchymal stem cells can be significantly improved, the difference is significant (P <0.05), and the concentration and dose are in a positive correlation relationship, and under the condition that the concentration is 200mg/L, that is, 200ug/mL, AOD490 reaches 3.02 ± 0.10.
Example 5 Effect of the RND1-23 monoclonal antibody on colorectal cancer cells and downstream Signal ROCK
LS513 human colorectal cancer cells (Cat. CL-0640, Procell) were inoculated into DMEM complete medium of 10% fetal bovine serum, placed at 37 ℃ and 5% CO2Culturing in a saturated humidity incubator, changing the culture medium every 3 days, and performing amplification culture to obtain the experimental cells.
(1) The Transwell chamber was inverted, the lower surface of the membrane was coated with fibronectin (400. mu.g/mL, 10. mu.L), exposed to 37 ℃ for 2h, washed once with PBS, and placed in a 24-well plate previously supplemented with 600. mu.l of DMEM complete medium containing 10% fetal bovine serum per well. The cell concentration of LS513 cells was adjusted to 1X 10 with serum-free DMEM complete medium after trypsinization6Perml, 100. mu.l of cell suspension was added to each Transwell chamber and divided into control and different experimental groups as follows:
blank control group: no RND1-23 monoclonal antibody was added;
monoclonal antibody group 1: adding RND1-23 monoclonal antibody with the final concentration of 1 mu g/mL;
monoclonal antibody group 2: adding RND1-23 monoclonal antibody with the final concentration of 10 mug/mL;
monoclonal antibody group 3: adding RND1-23 monoclonal antibody with final concentration of 100 mug/mL;
monoclonal antibody group 4: adding RND1-23 monoclonal antibody with the final concentration of 200 mug/mL;
positive control group: adding 100 mu g/mL of Y-27632;
37℃，5％CO2culturing in a saturated humidity incubator for 18h, taking out the Transwell chamber, wiping off cells on the inner chamber surface of the membrane by using a cotton swab, drying the other surface, dyeing with crystal violet for 20min, washing with clear water for 3 times, observing the cells under a microscope, and counting. The results are shown in FIG. 2.
As can be seen from the results in FIG. 2, the average cell number on the lower surface of the chamber in the blank control group was 181. + -. 9.5, and the difference between the monoclonal antibody group and the positive control group was statistically significant (P < 0.05). Meanwhile, the cell number of the monoclonal antibody group 4, namely the RND1-23 monoclonal antibody with the final concentration of 200 mug/mL is only 40 +/-3.0, so that the migration capability of LS513 human colorectal cancer cells can be obviously reduced.
(2) The upper face of the bottom membrane of the Transwell chamber was coated with Matrigel 1:3 diluted, 10. mu.l of 400. mu.g/ml fibronectin was coated on the outside of the membrane, allowed to solidify at 37 ℃ and washed once with serum-free medium. Then regulating concentration of the cell to 1 × 10 with serum-free medium after trypsinization6Perml, 100. mu.l of cell suspension was added to each Transwell chamber and divided into control and different experimental groups as follows:
blank control group: no RND1-23 monoclonal antibody was added;
monoclonal antibody group 1: adding RND1-23 monoclonal antibody with the final concentration of 1 mug/mL;
monoclonal antibody group 2: adding RND1-23 monoclonal antibody with the final concentration of 10 mug/mL;
monoclonal antibody group 3: adding RND1-23 monoclonal antibody with final concentration of 100 mug/mL;
monoclonal antibody group 4: adding RND1-23 monoclonal antibody with the final concentration of 200 mug/mL;
positive control group: adding 100 mu g/mL of Y-27632;
into a 24-well plate to which 600. mu.l of DMEM complete medium containing 10% FBS had been added, at 37 ℃ and 5% CO2After culturing in a saturated humidity incubator for 20h, wiping off Matrigel and cells on the surface of the inner chamber of the membrane by using a cotton swab, drying the other surface of the membrane, dyeing the membrane with crystal violet for 20min, washing the membrane with clear water for 3 times, observing the cells under a microscope, and counting. The results are shown in FIG. 3.
Since Matrigel can form a structure similar to a natural basement membrane on the surface of a polycarbonate filter, the ability of cells to invade through Matrigel reflects the invasion ability of the cells, and the more cells enter the lower surface of the chamber by counting, the greater the invasion ability of the cells. As can be seen from the results in FIG. 3, the higher the concentration of the monoclonal antibody, the better the inhibition of invasion. The number of the membrane-crossing cells is only 36 +/-3.0 when the monoclonal antibody group 4, namely the RND1-23 monoclonal antibody with the final concentration of 200 mu g/mL is used, so that the invasion capacity of LS513 human colorectal cancer cells can be obviously reduced.
(3) And (3) taking the cultured cells of each group in the step (2), extracting RNA in the cells by adopting a Trizol one-step method, taking 2 mu g of total RNA for reverse transcription, and carrying out PCR amplification on ROCK1 and GAPDH. The specific primer sequence is as follows: upstream of ROCK 1: 5'-GAAGAAAGAGAAGCTCGAGAAGAAGG-3', downstream: 5'-ATCTTGTAGCTCCCGCATCTGT-3', respectively; GAPDH upstream: 5'-GAAGGTGAAGGTCGGAGTC-3', downstream: 5'-GAAGATGGTGATGGGATTTC-3', PCR amplification conditions are pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 1min, extension at 72 ℃ for 1min for 30 cycles, and final extension at 72 ℃ for 10 min. The product was subjected to 1.5% agarose gel electrophoresis, imaged by a gel imaging system, and the relative expression level of ROCK1 was calculated based on the expression level of GAPDH, and the result is shown in fig. 4.
As can be seen from the results in FIG. 4, the RND1-23 monoclonal antibody of the present invention was able to control the activity of downstream ROCK1 by inhibiting the activity of Rho GTPase 1(RND1) itself, and ROCK1 at a concentration of 200. mu.g/mL had only 0.19. + -. 0.01, which was significantly more effective than 1.1. + -. 0.09 of the blank control group.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Sequence listing
<110> Nosai Union (Beijing) biomedical technology, Inc
<120> a serum-free culture medium for stem cells
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Glu Ser Val Ser Ile Ser Cys Arg Ser Thr Cys Leu Gly Ile Val Ile
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Tyr Tyr Asp Tyr Ala Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser
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Pro Gln Leu Leu Ile Tyr Leu Asn Ser Asn Leu Ala Ser Gly Val Pro
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Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile
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Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Glu Phe Tyr
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Tyr Met Gln Asp Glu Asp Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
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Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Cys Gly
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Arg Leu Glu Trp Val Ala Ile Gly Leu Gly Met Arg Glu Trp His Tyr
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Ala Met Tyr Tyr Cys Val Asp Met Glu His Pro Tyr His Val Ser Lys
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Claims (4)

1. A monoclonal antibody RND1-23 specific for Rho GTP enzyme, characterized in that the amino acid sequence of the variable region of the light chain of the monoclonal antibody is shown as SEQ ID NO.1, and the amino acid sequence of the variable region of the heavy chain is shown as SEQ ID NO. 2.
2. The serum-free medium for the adipose-derived mesenchymal stem cells is characterized by consisting of a basic medium and an additive component added in the basic medium, wherein the basic medium is a DMEM/F12 medium, and the additive component is L-glutamine, non-essential amino acids, L-ascorbic acid, sodium selenite, fibronectin, ethanolamine, hydrocortisone, a trypsin inhibitor, human transferrin, human insulin, bFGF, TGF-beta 1, PDGF-BB, coenzyme A, sodium pyruvate, EGF and RND1-23 monoclonal antibody; wherein the concentration of L-glutamine is 1-2mM, the concentration of non-essential amino acid is 1-2mM, the concentration of L-ascorbic acid is 40-60mg/L, the concentration of sodium selenite is 10-18 mug/L, the concentration of fibronectin is 15-30mg/L, the concentration of ethanolamine is 1-4mg/L, the concentration of hydrocortisone is 7-15mg/L, the concentration of trypsin inhibitor is 1-1.5mg/L, the concentration of human transferrin is 7-15mg/L, the concentration of human insulin is 10-15mg/L, the concentration of bFGF is 20-30 mug/L, the concentration of TGF-beta 1 is 3-7 mug/L, and the concentration of PDGF-BB is 7-15 mug/L, the concentration of coenzyme A is 80-120mg/L, the concentration of sodium pyruvate is 1-5mM, the concentration of EGF is 5-20 mu g/L, the concentration of RND1-23 monoclonal antibody is 10-100mg/L, the amino acid sequence of the light chain variable region of the RND1-23 monoclonal antibody is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2.
3. The application of a monoclonal antibody RND1-23 specific to Rho GTP enzyme in preparing a pharmaceutical composition for inhibiting colorectal cancer cell migration and invasion, wherein the amino acid sequence of a light chain variable region of the monoclonal antibody is shown as SEQ ID No.1, and the amino acid sequence of a heavy chain variable region is shown as SEQ ID No. 2.
4. The use according to claim 3, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
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CN101563449A (en) * 2006-09-22 2009-10-21 理化学研究所 Stem cell culture medium and method
US20100009442A1 (en) * 2006-09-22 2010-01-14 Yoshiki Sasai Stem Cell Culture Medium and Method
CN102395672A (en) * 2009-04-13 2012-03-28 加利福尼亚大学董事会 Methods and compositions for stem cell cultures
US20180022794A1 (en) * 2015-02-03 2018-01-25 Inserm (Institut National De La Sante Et De La Recherche Medicale) Anti-rho gtpase conformational single domain antibodies and uses thereof

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