JPH05184357A - Cell proliferation promoting agent and promoting method - Google Patents

Abstract

PURPOSE:To provide the subject promoting agent composed of a oxazopyrroloquinoline compound or its ester and effective for promoting the proliferation of animal cell to increase the production efficiency of valuable products originated from the cell. CONSTITUTION:The objective promoting agent is composed of an oxazopyrroloquinoline compound and/or its ester expressed by formula [R is same as the R' of an alpha-amino acid of the formula R'-CH2(NH2)COOH; R<2> to R<3> are H, alkyl, alkenyl or benzyl]. The compound of formula is produced preferably by reacting a PQQ compound with various alpha-amino acids (e.g. glycine and valine) and methylamine, etc., in the presence of oxygen.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an animal cell growth promoter comprising oxazopyrroloquinolines and / or ester thereof and a method for promoting animal cell growth using the same.

[0002]

BACKGROUND OF THE INVENTION Various useful substances have been produced using animal cells.
At that time, shortening the period required for culturing and increasing the production efficiency of the target substance are the most important factors when considering economical efficiency. Traditionally, serum media have been used to promote the growth of animal cells. However, the serum medium is
Since the mixture is a multi-component mixture, the procedure for isolating and purifying the target substance after culturing becomes complicated, and it is difficult to carry out the culturing stably because the active ingredient is not specified.

[0003]

[Means for Solving the Problems] For the above-mentioned reasons, the inventors of the present invention have conducted extensive research into a drug that promotes the growth of animal cells, and found that oxazopyrroloquinolines or their esters are found in animal cells. It has been found that it has the effect of promoting the growth of. That is, the present invention relates to a growth promoter for animal cells, which comprises an oxazopyrroloquinoline or an ester thereof, and, when culturing an animal cell, adding oxazopyrroloquinoline or an ester thereof to a medium to grow the cells. And the production efficiency of useful products derived from the cells is enhanced.

Oxazopyrroloquinolines (hereinafter abbreviated as OPQs) are also known as 5-substituted 2,8,10-tricarboxy-1H-oxazo [5,4-h] -pyrrolo [2,3].
-F] quinoline and its salt, and OPQs and its ester form have the following general formulas (formula 1).

[0005]

[Chemical 3]

R of the oxazopyrroloquinoline used in the present invention is represented by the general formula R'-CH ₂ (NH ₂ ) -C.
It may be the same as the R'of the α-amino acid represented by OOH, but is preferably the same as the R'of the natural α-amino acid, and more preferably the same as the R'of the α-amino acid constituting the natural protein. Is.

The OPQs used in the present invention are P
It can be easily produced by reacting QQs with various α-amino acids, methylamine and the like in the presence of oxygen.

As the OPQs in the present invention, PQQ
And glycine, threonine, tryptophan, prolyl
, Tyrosine, serine and monomethylamine
OPQ (R = H) obtained from 1 or 1 (Japanese Patent Application No. 1-2)
92459), a hydrolyzate obtained from PQQ and serine
Xymethyl OPQ (R = CH₂OH) (Japanese Patent Application 1-25
8791), 1-Meth obtained from PQQs and valine
Luethyl OPQ (R = CH (CH₃)₂) (Patent application 1-
309479), obtained from PQQs and isoleucine
1-methylpropyl OPQ (R = CH (CH₃) CH
₂CH₃) (Japanese Patent Application No. 1-309480), PQQ
2-Methylpropyl OPQ (R = C obtained from leucine
H₂CH (CH₃)₂) (Japanese Patent Application No. 1-309481),
Methyl OPQ (R = C obtained from PQQs and alanine
H₃) (Japanese Patent Application No. 1-327347), PQQ and gluta
2-Carboxyethyl OPQ (R =
CH ₂CH₂COOH) (Japanese Patent Application No. 1-327351)
No.), 2-carbamo obtained from PQQs and glutamine
Ilethyl OPQ (R = CH₂CH₂CONH₂) (Special
(Heihei 327348), from PQQs and methionine
The resulting 2-methylthioethyl OPQ (R = CH₂CH
₂SCH₃) (Japanese Patent Application No. 1-32749), PQQ
Benzyl OPQ obtained from phenylalanine
1-327350), obtained from PQQs and tyrosine
4-hydroxyphenylmethyl OPQ (Japanese Patent Application No. 2-1
07354), obtained from PQQs and aspartic acid
1-carboxymethyl OPQ (R = CH₂COO
H), 1-carbamo obtained from PQQ and asparagine
Ilmethyl OPQ (R = CH₂CONH₂), PQQ
1- (4-Imidaryl) me obtained from and histidine
4-amino obtained from chill OPQ, PQQs and lysine
Butyl OPQ (R = (CH₂)_FourNH₂), With PQQ
3-guanidinopropyl OP obtained from arginine
1-mercapto obtained from Q, PQQs and cysteine
Methyl OPQ (R = CH₂SH) etc. Also,
The salts of OPQs are alkali metal salts and alkalis, respectively.
Earth metal salts, ammonium salts and substituted ammonium salts
The typical examples are sodium salt and potassium.
Umium salt, magnesium salt, calcium salt, ammonium
Salt, trimethylammonium salt, triethylammoniu
Rum salts, triethanol ammonium salts, etc.

Further, the OPQ in the present invention represented by the formula 1
In the ester form of R¹, R ², R³Are the same
May also be different, hydrogen atom, alkyl group, alkenyl
Group or benzyl group, monoester, diester
There are le or triester. Of these OPQs
Steroids usually contain OPQs or their salts and alcohols.
Can be easily produced by reacting according to the method
And examples of alkyl groups include methyl and ethyl groups.
Examples of the alkenyl group include an allyl group. Also, PQQ
Alternatively, the PQQ salt and alcohols are reacted by a conventional method,
After obtaining the ester form of PQQs, various amino acid
By reacting with ruthenium or methylamine.
It is also possible to obtain an ester form of OPQ.

[0010]

[Examples] Examples are shown below showing the effect of OPQs and their esters on the growth of animal cells according to the present invention, but the present invention is not limited to these examples.

Example 1 HFS-1 fibroblasts derived from human subcutaneous tissue were supplemented with 10% fetal bovine serum and 100 units / mL streptomycin to prepare a modified Dulbecco Eagle.
Medium (DMEM medium) CO ₂ incubator (37 ° C., under an atmosphere of 5% CO ₂ -95% air) using cultured in. The cells were washed with Hanks solution, suspended in serum-free DMEM medium at 4 × 10 ⁵ cells / mL, and placed in a flat-bottom 96-well microplate.
Culturing was performed in an atmosphere of 5 ° C, 5% CO ₂ -95% air. Twenty-four hours after the start of culture, OPQ was not added to each culture medium, 0.01, 0.1, 1.0, 10, 100 or 500 μg / mL was added, and the culture was further continued. 48 hours after the start of culture, ³ H-thymidine was added to 1.0 μm.
After culturing was further added after 3 hours so that the ci / mL, after stopping the addition of 50% trichloroacetic acid solution growth, cells were washed and solubilized the cells in 25M NaOH, ³ cell fluid H was measured with a liquid scintillation counter. At the same time, the protein content in the cell fluid was measured.

The results are shown in Table 1. The numerical values in the table are average values of the amount of ³ H incorporated (dpm) per 1 μg protein in the culture under the same six conditions. Also, Hanks
The method for preparing the liquid and DMEM medium is shown below. Hanks solution Hanks solution (Nissui Pharmaceutical) 475 mL 2% lactalbumin 25 mL 7.5% NaHCO ₃ 1.52 mL Each solution is sterilized at 121 ° C. for 15 minutes and mixed aseptically before use. DMEM medium Modified Eagle medium of Dulbecco (manufactured by Nissui Pharmaceutical Co., Ltd.) 261 mL Fetal Bovium Serum 30 mL 5 × 10 ⁻³ M 2-mercaptoethanol solution 3 mL Penicillin (100 unit / mL) Streptomycin (100 unit / mL) mixed solution 3 mL 200 mM L-glutamine Except for 3 mL Fetal Bovium Serum, filter and mix aseptically at the time of use.

[0013]

TABLE 1 Table 1 OPQ amount ³ H counts / [mu] g protein relative activity (μg / mL) (× 10 3, dpm) (%) No addition 7.07 100 0.01 7.48 106 0.1 9 .56 135 1.0 9.10 129 10 9.73 138 100 11.70 165 500 9.57 135

[0014]

INDUSTRIAL APPLICABILITY By using the animal cell growth promoter of the present invention, it is possible to effectively promote cell growth in animal cell culture and increase the production efficiency of useful cells. In particular, since the OPQs of the present invention are stable low molecular weight substances, the isolation and purification operation of the target substance after culturing is easy as compared with conventional natural products and their extracts,
Its practical value is extremely large.

Claims (2)

[Claims] 1. An animal cell growth promoter comprising an oxazopyrroloquinoline represented by the formula 1 and / or an ester thereof. 2. When culturing an animal cell, the oxazopyrroloquinoline of formula 1 and / or its ester form is added to the medium to promote the growth of the cell and produce a useful product derived from the cell. Method to increase efficiency