JP2007159429A - Cell proliferation agent and cell proliferation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for the efficient proliferation of cells capable of easily controlling the cell proliferation timing, period, degree, etc., while keeping the characteristic properties of the cell and provide a cell proliferation agent for the method. <P>SOLUTION: The cell proliferation agent contains a complex of a cell period-relating protein and a polyamine. The polyamine is preferably polyethyleneimine. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a cell proliferating agent for proliferating cells and a method for proliferating animal cells.

  Technology for efficiently proliferating cells is an important technology in fields such as regenerative medicine and production of physiologically active substances, and various studies have been conducted.

  Cell division / proliferation is strictly controlled according to a certain cell cycle, and usually does not divide unless cells need to be regenerated due to tissue damage.

  The cell cycle consists of four phases repeated in the order of G1, S, G2, and M, and various proteins are involved in the progression of this cell cycle. For example, pRB normally binds to E2F and regulates the expression of cyclin E by E2F. However, when phosphorylated, it is dissociated from E2F, and as a result, the expression of cyclin E is enhanced by E2F. Cyclin E forms a complex with CDK2, and the cell cycle transitions from the G1 phase to the S phase. The function of the cyclin E / CDK2 complex is inhibited by binding of p27. On the other hand, p27 is phosphorylated and degraded by the cyclin E / CDK2 complex. When entering S phase, cyclin E is degraded and CDK2 forms a complex with cyclin A. Thus, various proteins are involved in the cell cycle, and the progression process of the cell cycle is controlled.

  Attempts have been made to promote cell growth by controlling the cell cycle using proteins involved in the cell cycle (cell cycle-related proteins).

Methods for promoting cell growth include:
(1) A method for stimulating cell growth using a growth factor (2) A method for stimulating cell growth by incorporating a gene encoding a cell cycle-related protein and expressing the cell cycle-related protein inside the cell (3) Examples thereof include a method of stimulating cell proliferation by introducing a cell cycle-related protein into cells.

  The method (1) has a problem that the selectivity of the proliferation action is low. In addition, in the method (2), it is difficult to control the amount of cell cycle-related protein produced in the cell, and furthermore, since the gene is integrated, there is a problem that it is inherited semi-permanently by subsequent generations. .

  The method (3) has the advantage that the amount of cell cycle-related protein introduced into the cell can be easily controlled. In recent years, a method for introducing a protein into a cell using a complex in which a cell cycle-related protein is bound to a TAT peptide has been reported (for example, J. Biol. Chem., 276, 23572-23580 (2001)). ), J. Biol. Chem., 276, 22742-22747 (2001), Mol. Cell. Biol., 21, 4773-4784 (2001), Mol. Cancer Ther., 1, 1043-1049 (2002), Int Immunol., 14, 905-916 (2002)).

  However, in the method using the TAT peptide, the efficiency of introduction into the cell is low or the binding between TAT and the cell cycle-related protein is difficult to break, so that the original function of the cell cycle-related protein is not fully exhibited and the growth efficiency There was a problem such as low.

JP 2004-049214 A Futami et al., 27th Annual Meeting of the Molecular Biology Society of Japan (2004), p. 895 J. Biol. Chem., 276, 23572-23580 (2001) J. Biol. Chem., 276, 22742-22747 (2001) Mol. Cell. Biol., 21, 4773-4784 (2001) Mol. Cancer Ther., 1, 1043-1049 (2002) Int. Immunol., 14, 905-916 (2002)

  An object of the present invention is to provide an efficient method for proliferating cells and a cell proliferating agent therefor that can easily control the time, period, and degree of proliferation of cells while maintaining the original properties of the cells. To do.

  As a result of intensive investigations by the present inventors to solve the above problems, when a complex of a cationized polymer and a cell cycle-related protein is used, the cell cycle-related protein is efficiently introduced into the cell. The inventors found that the growth can be controlled very easily, and completed the present invention.

That is, the present invention includes the following inventions.
(1) A cell proliferation agent comprising a complex of a cell cycle-related protein and a polyamine.
(2) The cell proliferating agent according to (1), wherein the polyamine is polyethyleneimine.
(3) The cell proliferating agent according to (1) or (2), wherein the cell cycle-related protein is an SVLT-derived protein.
(4) The cell proliferation agent according to any one of (1) to (3), wherein the cell cycle-related protein is biotinylated via a disulfide bond, and the polyamine is avidinized.
(5) A method for proliferating a cell, comprising culturing the cell proliferating agent according to any one of (1) to (4) in contact with the cell.
(6) The method according to (5) above, wherein the culture is performed in a medium having a serum concentration of 20 vol% or less.
(7) A complex of an SVLT-derived protein biotinylated through a disulfide bond and avidinized polyethyleneimine.

  According to the method of the present invention, cell growth can be controlled by introducing a cell cycle-related protein into a cell while maintaining the cell in a normal state. Furthermore, the method of the present invention can easily control the degree and period of cell proliferation by controlling the amount and period of the protein to be introduced.

  As used herein, “cell cycle-related protein” refers to all proteins that are involved in the cell cycle progression mechanism repeated in the order of G1, S, G2, and M, and that promote cell cycle progression. Examples of such proteins include, but are not limited to, SV 40 large T antigen (SVLT), E2F, cyclin C-CDK3, cyclin D-CDK4 / 6, cyclin E-CDK2, Skp2, Cks1 , KPC1, KPC2, c-Myc, LMP-1, etc. or a part thereof. How these cell cycle-related proteins are involved in the cell cycle is shown below with specific examples.

  Cyclin D induces gene expression by a signal that promotes proliferation in the G1 phase, binds to CDK4 or 6, activates these kinases, and phosphorylates pRb. Further, among CKI (CDK inhibitors), the Cip / Kip protein group is concentrated on the cyclin D-CDK4 / 6 complex, and cyclin E-CDK2 is activated.

  While the amount of p27 mRNA is almost constant throughout the cell cycle, the amount of protein expression varies greatly. This variation is mainly controlled by a cell cycle-dependent degradation mechanism. In p27, T187 near the C-terminus is phosphorylated by cyclin E-CDK2. In the S-G2 phase, Cks1 binds to Skp2, thereby increasing the binding ability between T187 phosphorylated p27 and Skp2. As a result, p27 is ubiquitinated and degraded by the proteasome. On the other hand, in the G0-G1 phase, the KPC1-KPC2 complex is ubiquitinated and decomposed.

  p16 is one of the CDK inhibitors and binds only to the cyclin D kinases CDK4 and CDK6. The p16 gene has been found to be mutated in many primary human tumors such as visceral cancer, esophageal cancer, and glioma, which are easily malignant melanoma, and is known to be inactivated in about 40-50% of human cancers. ing. Ets1 and Ets2, which are transcription factors activated by MAPK, are involved in the induction of p16 gene expression.

  c-Myc is an oncogene product associated with many human cancers, and its mutation and amplification have been reported.

  LMP1 is an EBV (Epstein-Barr virus) cancer protein known to cause malignant lymphoma, nasopharyngeal cancer, gastric cancer, etc., and export of Ets2 to the nucleus depends on CRM1 which is a nuclear export receptor. Occurs and is inactivated, decreasing the expression of the p16 gene.

  The cell cycle-related protein is meant to include a complex protein to which a sugar chain, a lipid, and / or a phosphate group are bound, and the structure of the protein may be a natural state or a denatured state.

  In the present invention, the cell cycle-related protein is used as a complex with polyamine. A polyamine is a compound having a plurality of (two or more) amino groups, and examples thereof include polyalkylene polyamines, alkylene diamines, and alkylene triamines.

  Examples of the polyalkylene polyamine include the following general formula (I):

（式中、R¹、R²、及びR³はアルキレン基を表し、X及びYはそれぞれ0以上の整数であり、X+Y≧1である。）
で表されるポリアルキレンイミンが挙げられ、直鎖状及び枝分かれ状のどちらであってもよい。

(Wherein R ¹ , R ² and R ³ represent an alkylene group, X and Y are each an integer of 0 or more, and X + Y ≧ 1)
The polyalkyleneimine represented by these may be mentioned, and may be either linear or branched.

The polyalkyleneimine is preferably a polyalkyleneimine in which R ¹ , R ² , and R ³ in formula (I) are the same or different from each other and are an alkylene group having 2 to 4 carbon atoms. Polyethyleneimine in which R ¹ , R ² and R ^{3 in} (I) are an ethylene group having 2 carbon atoms is more preferred.

  Polyethyleneimine (hereinafter also referred to as “PEI”) preferably used in the present invention is represented by the following formula.

（式中、Ｘ及びＹはそれぞれ1以上の整数である。）

(In the formula, X and Y are each an integer of 1 or more.)

  PEI is a water-soluble polymer with a large positive charge density. PEI is also used as a food additive such as kamaboko precipitant, and it has been confirmed that it is highly safe for living organisms.

  In the present invention, a linear PEI or a branched PEI having many branched chains can be used.

で例示されるような枝分かれ構造を有するPEIが、より高い正電荷密度を有するので好ましい。また、分子量は細胞導入効率、取扱い性等を考慮すると、数平均分子量が100〜100,000の範囲のPEIが好ましく、100〜10,000のPEIがより好ましく、200〜3,000の低分子量のPEIが特に好ましい。

PEI having a branched structure as exemplified in the above is preferable because it has a higher positive charge density. In view of cell introduction efficiency, handling property, etc., the molecular weight is preferably PEI having a number average molecular weight in the range of 100 to 100,000, more preferably 100 to 10,000, and particularly preferably low molecular weight PEI of 200 to 3,000.

  Examples of the alkylene diamine include methylene diamine, ethylene diamine, propylene diamine, and butylene diamine.

A method for producing the composite of the present invention will be described.
The complex of the present invention is a combination of a cell cycle-related protein and a polyamine. The form of “bond” here is not particularly limited, and may be based on a weak interaction such as an electrostatic interaction or a hydrophobic interaction in addition to a covalent bond or a hydrogen bond. . The cell cycle-related protein and the polyamine may be directly bound without any intervening therebetween, or may be bound via a spacer or the like using a known bivalent cross-linking reagent or the like. It may be.

  Alternatively, the cell cycle-related protein and polyamine may be biotinylated and avidin, respectively, and the cell cycle-related protein and polyamine may be complexed through specific interaction between biotin and avidin. This method is preferred because it is highly versatile and can be applied to any cell cycle-related protein.

  The cell cycle-related protein is preferably designed to be separated from the complex in the cell so that the original function of the cell cycle-related protein can be fully exerted when introduced into the cell. For example, if a cell cycle-related protein is bound to a complex by a disulfide bond, the disulfide bond is cleaved by intracellular reductive conditions to release the cell cycle-related protein, and the cell cycle-related protein has its original function. Can fully demonstrate.

  In order to form a disulfide bond between a cell cycle-related protein and polyamine or biotin, for example, a reagent such as SPDP (N-succinimidyl-3- (2-pyridyldithio) propionate) is used. A disulfide bond may be formed between the thiol group of the cysteine residue and the amino group of polyamine or biotin. An example of using SPDP and PEI is schematically shown below.

  In addition, as a method of binding a cell cycle-related protein and a polyamine by chemical bonding, for example, a method using EDC, 2-iminothiolane or the like, a lysine residue in a protein molecule or an N-terminal amino group and PEI Thioether bond is included between thiol group of cysteine residue and amino group of polyamine in protein molecule using GMBS (N- (4-maleimidobutyryloxy) succinimide) etc. Examples thereof include a method of forming a covalent bond.

  In addition to the bonding methods listed here, there may be mentioned bonds by ether bonds, ester bonds, imide bonds, carbon-carbon bonds, etc., and literature (for example, Tokyo Chemical Co., Ltd. “Protein IV Structural Function Correlation” Japan Biochemicals Corporation. Various binding methods can be adopted by referring to the Society edition, 1st edition published on March 20, 1991).

  Moreover, you may label the complex used by this invention as needed. Although it will not specifically limit if it is a well-known method as a labeling method, It is preferable that they are a fluorescence label | marker, an autoradiography, a high electron density substance, and a dye insolubilizing enzyme. A particularly preferred form is to label the complex with a covalently bonded fluorescently labeled compound. Although it does not specifically limit as a fluorescent substance used for a fluorescent label, For example, the compound which has fluorophores, such as a pyrene, an anthraniloyl group, a dansyl group, a fluorescein, a rhodamine, a nitrobenzoxadiazole group, is mentioned. Compounds having the above fluorophores are known (see, for example, Hisaki Hiratsuka, “Protein Nucleic Acid Enzyme”, Vol. 42, No. 7 (1997), etc.) and can be introduced into protein molecules or peptides by a conventional method. it can.

  Cells that can be propagated in the present invention are not particularly limited, and any plant or animal can be used. It can also be applied to human cells, for example, various organs such as damaged skin and liver, organs, organs by proliferating cells by applying the method of the present invention to cells of tissues, Applications to the field of regenerative medicine such as regenerating organs, tissues, etc. are preferred. Specific examples of cells to which the method of the present invention can be applied include cells such as hepatocytes (hepatocytes), hematopoietic stem cells, skin cells (keratinocytes), neurons, cardiomyocytes, chondrocytes and corneal cells. Can do.

  Next, a method for introducing a cell cycle-related protein into a cell using the complex of the present invention to proliferate the cell will be described, but the method of the present invention is not limited thereto.

  The complex of the present invention is added to a medium containing cells to be introduced with cell cycle-related proteins. For the culture of the method of the present invention, a container or an apparatus usually used for culture is used. For example, a multiwell plate, a culture flask, a spinner flask, a jar fermenter, a fermenter and the like can be used.

  The growth medium is not particularly limited, and a known medium (for example, DMEM, MEM, BME, DME, IMDM, L-15 medium, etc.) can be used. In the growth medium, other conventional medium components may be added and blended. For example, one or more of amino acids, vitamins, serum, trace elements and the like may be appropriately added. The culture is performed in a serum-free medium or a low-serum medium (for example, the upper limit of the serum concentration is 20 vol% or less, preferably 10 vol% or less, more preferably 5 vol% or less, most preferably 1 vol% or less with respect to the total volume of the medium and serum) ) Is preferable. By culturing in this range, the cell proliferation effect can be improved.

  The culture is performed under conditions suitable for the culture of the cells used. In general, the cells are cultured for about several days to three months at a culture temperature of about 37 ° C. and a pH of about 6.5 to 7.5.

  The degree of cell proliferation can be easily controlled by changing the amount, concentration, addition time, etc. of the complex to be added. The complex of the present invention is presumed to be taken into cells by a mechanism resulting from electrostatic interaction between the positive charge of the complex and the negative charge on the cell surface. In the case of incorporating the complex, it is preferable to carry out the reaction under conditions in which an anionic polymer such as heparin or nucleic acid is not present. In addition to cell growth by culture, a solution containing the complex of the present invention is directly inoculated into a living body by local administration such as injection to the affected area or application to the skin, and the complex is directly applied to cells in the living body. It can also be taken in.

  Embodiments of the present invention are specifically described below, but these examples do not limit the scope of the present invention.

Example 1: Cell Proliferation by Complex of SVLT-NLS and PEI In this example, N-terminal region of SVLT biotinylated as a cell cycle-related protein via a disulfide bond (residue 1-132: “SVLT-NLS "). SVLT (SV40 large T antigen) is one of two T antigens synthesized by siman virus 40 (SV40), a group of DNA viruses belonging to the genus Paomapaviridae polyomavirus. Is an oncogenic protein essential for replication. Malignant transformation by SV40 is said to involve interaction with p53 and pRB family (pRB, p107, p130, etc.) which are tumor suppressor proteins by SVLT. In the presence of SVLT, pRB can no longer bind to E2F, which causes the cell cycle to transition to S phase.

  On the other hand, PEI having a molecular weight of 600 (PEI600) was used as a polyamine and avidin was bound thereto.

  Balb / c 3T3 A31H mouse embryo fibroblasts were used as the cells to be propagated. The cells were used in a state of confluence and contact inhibition (the cell cycle was stopped in the G1 phase and the growth was also stopped).

The culture was performed as follows.
Adjust balb / c 3T3 cells (mouse fetal fibroblasts) to a density of 4 × 10 ⁴ cells / 500 μl / well using DMEM medium containing 10% FBS (fetal calf serum) and seed them in a 12-well plate. It was. The cells were confluent at 37 ° C. for 48 hours in the presence of 5% CO ₂ . In this state, the cells stop growing due to contact inhibition. The medium was replaced with 500 μl of fresh 10% FBS-containing DMEM medium, and a separately prepared mixture of PEI-modified avidin (200 nM) and biotinylated SVLT-NLS (100 nM) was added to the culture medium. After culturing at 37 ° C. for 72 hours in the presence of 5% CO ₂ , BrdU was added to the culture solution to a concentration of 10 μM.

  As described above, SVLT-NLS was introduced into the cells and the cells were observed. As a result, degradation of p27 and accumulation of cyclin A were confirmed. Moreover, it was confirmed by the cell proliferation assay by BrdU (bromodeoxyuridine) and DAPI that the cells were greatly proliferated (growth rate 33%) regardless of the confluent state. The proliferation rate (%) = (the number of BrdU uptake cells) / (the number of DAPI-stained nuclei). Compared with the gene introduction method using SVLT-NLS expression plasmid DNA (growth rate 12%), it was confirmed that the cell proliferating agent of the present invention is extremely useful for efficient cell proliferation (FIG. 1).

Example 2: Cell growth by a complex of SVLT-NLS and PEI
Adjust balb / c 3T3 cells (mouse fetal fibroblasts) to a density of 4 × 10 ⁴ cells / 500 μl / well using DMEM medium containing 10% FBS (fetal calf serum) and seed them in a 12-well plate. It was. The cells were confluent at 37 ° C. for 48 hours in the presence of 5% CO ₂ . In this state, the cells stop growing due to contact inhibition. Next, the culture supernatant was replaced with 500 μl of fresh DMEM medium (without FBS), and a separately prepared mixture of PEI avidin (200 nM) and biotinylated SVLT-NLS (100 nM) was added to the culture medium. After culturing at 37 ° C. for 72 hours in the presence of 5% CO ₂ , BrdU was added to the culture solution to a concentration of 10 μM. The cell proliferation assay using BrdU confirmed that the cells were proliferating significantly (growth rate 33%) despite the confluent state.

  The cell proliferation agent and cell proliferation method of the present invention are very useful techniques in the medical field. For example, when human skin or organ cells are used as cells, the cells are proliferated by the method of the present invention to produce artificial skin. It can also be used as an artificial organ, and is expected to be applied in the field of regenerative medicine. In addition, cells that produce useful physiologically active substances can be proliferated by the method of the present invention to efficiently produce the active substance for production, and the method of the present invention can be widely applied to the pharmaceutical and food fields. it can.

It is the figure which observed the grade of the proliferation of a cell using BrdU and DAPI.

Claims (7)

  A cell proliferation agent comprising a complex of a cell cycle-related protein and a polyamine.   The cell proliferating agent according to claim 1, wherein the polyamine is polyethyleneimine.   The cell proliferation agent according to claim 1 or 2, wherein the cell cycle-related protein is an SVLT-derived protein.   The cell growth agent according to any one of claims 1 to 3, wherein the cell cycle-related protein is biotinylated through a disulfide bond, and the polyamine is avidinized.   A method for proliferating a cell, comprising culturing the cell proliferating agent according to any one of claims 1 to 4 in contact with the cell.   The method according to claim 5, wherein the culture is performed in a medium having a serum concentration of 20 vol% or less.   A complex of an SVLT-derived protein biotinylated through a disulfide bond and avidinized polyethyleneimine.

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