JPS6045848B2 - Method for producing human growth hormone - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to human growth hormone (human growth hormone).
hormone9humanS0mat()tr0Pi
n).

Chemical synthesis methods, tissue culture methods using isohydro, and microbial culture methods using genetic recombination are known methods for producing human growth hormone, but all methods have low yields and are extremely expensive to produce. expensive.

In order to supply human growth hormone in large quantities at a low cost, the present inventor continued intensive research and unexpectedly found that human-derived cells capable of producing human growth hormone were obtained by tissue culture in isohydro. We have completed the present invention by discovering that cells grown using warm-blooded animals other than humans produce significantly higher levels of human growth hormone, reaching approximately 2 to 5 hormones per cell.

That is, the present invention involves transplanting human-derived cells capable of producing human growth hormone into the body of a warm-blooded animal other than humans, or injecting a growth hormone-inducing agent into the cells that can proliferate while being supplied with the body fluids of the warm-blooded animal. The present invention relates to human growth hormone, which is characterized in that it is produced by acting on human growth hormone.

Unlike the case of culturing in isohydro, the method of the present invention not only induces and produces a large amount of human growth hormone, but also eliminates or significantly saves on a nutrient medium containing expensive serum. Furthermore, maintenance during cell proliferation is extremely easy.

In other words, if human-derived cells capable of producing human growth hormone are transplanted into the body of a warm-blooded animal other than humans, housed in a chamber that can receive the animal's body fluids, and reared normally, The cells can easily proliferate using body fluids containing nutrients supplied from the blood animal body.Furthermore, the proliferation of these cells is more stable than when cultured in isohydro. , is characterized by a high proliferation rate, a large amount of cells obtained, and a large amount of human growth hormone produced per cell.The human-derived cells used in the present invention have the ability to produce human growth hormone. It is sufficient if it has the following properties and can be transplanted into the body of a warm-blooded animal other than humans and easily proliferate.

For example, eosinophilic cells of the human prepituitary gland or E
Ectopic human growth, such as cells that naturally have the ability to produce human growth hormone, such as neoplastic eosinophil cells obtained from patients with BVirus, X-rays, or anterior pituitary eosinophil adenoma, and lung cancer cells. Cells with hormone-producing ability and cells obtained by culturing these cells are suitable. In addition, the gene capable of producing human growth hormone in these cells can be transformed by means of cell fusion using polyethylene glycol, Sendai virus, etc., DNA ligase, restriction enzyme (nuclease), DNA
The use of cells by introducing them into cultured lymphoblastoid cells that can be more easily subcultivated by genetic recombination using enzymes such as polymerases is useful because their proliferation rate is high. This is particularly advantageous because the ability to produce human growth hormone per cell is increased by about 2 to 1 ka or more.

In addition, the use of cultured cell lymphoblastoid cells is
When transplanted into a warm-blooded animal other than humans, it easily forms a soft tumor that is difficult to mix with the host animal's cells, and is also easy to disperse after removal, making it extremely advantageous for collecting living human lymphoblastoid cells.

Human-derived cell lines derived from human leukemia or human malignant lymphoma are suitable for such human lymphoblastoid cells, such as Namalva cells, BALL cells, etc.
-1 cells, NALL-1 cells, TAl. L-1 cells, J
Known human-derived cell lines such as BL cells can be used particularly advantageously.

The warm-blooded animal used in the method for producing human growth hormone of the present invention may be any animal that can proliferate human-derived cells capable of producing human growth hormone, such as birds such as chickens and pigeons, dogs, cats, etc. , monkey, goat, puta, cow,
Mammals such as horses, rabbits, guinea pigs, rats, hamsters, regular mice, and nude mice can be used.

Transplanting human-derived cells into these animals may cause an unfavorable immune reaction, so in order to suppress such reactions as much as possible, the animals used should be kept as young as possible, i.e. eggs, embryos, fetuses, or newborns. Preferably those from childhood. Alternatively, these animals may be subjected to pretreatment such as irradiation with approximately 200 to 600 rem of X-rays or gamma rays, or injection of antiserum or immunosuppressants to weaken the immune response before transplantation. good.

When the animal used is a nude mouse, the immune response is weak even when the animal is grown, so cultured human-derived cells can be transplanted without the need for these pretreatments, and the cells can be rapidly grown. This is particularly advantageous since it can be multiplied. In addition, human-derived cells can be transplanted into warm-blooded animals other than humans, such as by first transplanting human-derived cells into hamsters and growing them, and then transplanting these cells into nude mice. They are free to further stabilize proliferation and even increase the amount of human growth hormone induced therefrom.

In this case, the transplant may be between the same species, the same genus, the same class, or the same phylum. The site within the animal body to which human-derived cells are transplanted may be freely selected as long as the transplanted cells can proliferate, such as the allantoic cavity, vein, abdominal cavity, subcutaneous cavity, etc.

In addition, porous filtration membranes that can block the passage of animal cells without directly transplanting human-derived cells into the animal body, such as membrane filters, ultrafiltration membranes, or Diffusion chambers of various known shapes and sizes equipped with hollow eye bars etc. are placed inside the animal body.
For example, it is implanted in the peritoneal cavity, and any known cultured human-derived cells can be grown within the chamber while being supplied with body fluids containing nutrients from the animal body.

In addition, if necessary, a diffusion chamber that connects and perfuses the body fluid containing nutrients in this diffusion chamber with that in the animal body can be attached to the surface of the animal body, for example, to allow human-derived cells in the diffusion chamber to proliferate. It is also possible to make it possible to see through the state of the animal, and to make it possible to attach and detach only the diffusion chamber part to allow cells to proliferate to the fullest of the animal's lifespan without killing each animal, further increasing the amount of cells produced per individual animal. can.

Methods using these diffusion chambers not only allow for easy collection of only human-derived cells since human-derived cells do not come into direct contact with animal cells, but also reduce the risk of unwanted immune reactions. There is no need for pre-treatment to inhibit the use of this method, and it has the advantage of being able to be used freely in a variety of warm-blooded animals.

The maintenance and management of transplanted animals is convenient because it is sufficient to continue the normal breeding of the animal, and no special handling is required even after transplantation.

The period for growing human-derived cells is usually 1-20
It's a week. When the cultured cells to be transplanted are tumor cells or lymphoblastoid cells, their proliferation rate is particularly high, and the purpose can usually be achieved within a period of 1 to 5 weeks. It has also been found that the number of human-derived cells obtained in this manner reaches approximately 10 7 to 10 12 or more per animal. In other words, the number of human-derived cells proliferated by the method for producing human growth hormone used in the present invention is approximately 1C of the number of cells transplanted per individual animal.
P~107 times, or even more, when grown in nutrient media in vitro, about 101-103
double or even more, which is extremely convenient for the production of human growth hormone.

Any method can be used to produce human growth hormone by causing a human growth hormone inducer to act on the human-derived living cells grown in this way.

For example, human-derived cells grown in suspension in the ascites in the peritoneal cavity are collected, or a tumor that has grown subcutaneously is removed, dispersed, and collected, and the cells are kept in a nutrient medium kept at about 20 to 40°C. Human growth hormone may be induced and produced by suspending the cells at a concentration of approximately 101 to 1 Cf3/ml and allowing a growth hormone inducing agent to act thereon for approximately 1 to 1 hour.

Growth hormone inducers include, for example, lysine, arginine,
Tryptophan, leucine, casamino acids, L-DOPA
Amino acids such as serotonin, amino acid metabolites such as serotonin, peptides, etc. are used as appropriate. The human growth hormone induced and produced in this way is the same as the human growth hormone standard product and can be obtained using known purification and separation methods such as salting out, dialysis, etc.
It can be easily purified and separated and collected by filtration, centrifugation, concentration, freeze-drying, etc. Furthermore,
If a high degree of purification is required, it is sufficient to further combine known methods such as adsorption/desorption to ion exchangers, gel filtration, affinity chromatography, isoelectric focusing, and electrophoresis. , it is also possible to collect the highest purity human growth hormone. The human growth hormone obtained in this way can be used as a single substance or in combination with one or more other substances, such as injections,
It can be advantageously used not only to promote human growth, but also to prevent and treat human diseases as an external medicine, an oral medicine, a diagnostic medicine, etc.

The amount of human growth hormone produced is determined by S. M. Glick,e
tal. , Nature (LOndOn), VOl. l
It was measured according to the radioimmunoassay method described in 99784 (1963) and expressed as the weight of the American NIH standard product. A few examples will be described below. Example 1 Tumor eosinophilic cells obtained from a patient with anterior pituitary eosinophilic cell adenoma by removal, cutting, and dispersion were subcutaneously transplanted into adult nude mice, and then raised in the usual manner for 3 weeks.

Approximately 10 g of the tumor formed under the skin was removed and cut into small pieces, and then suspended in trypsin-containing physiological saline to disperse the cells.

The cells were grown in Earle medium 199 (PH 7.2) supplemented with 10 V/v% beef tallow serum and glucose-free.
After washing, the cells were suspended in the same medium containing 30rT1M of L-arginine as a growth hormone inducer at a concentration of approximately 1 cell/ml and kept at 37°C for 6 hours to induce the production of human growth hormone. .

Thereafter, when the cells were sonicated and the amount of human growth hormone was measured using the resulting supernatant, it was found that the amount produced was only about 100 r1g per ml of suspension. Example 2 Tumor eosinophil cells and lymphoblastoid Namalva cells obtained from a patient with anterior pituitary eosinophil cell adenoma (
Namalvacell) and 140n1MNac1,
54mM KC1, 1mM NaH2P04, 2rT] Mc
approximately 103/ml each in a saline solution containing acl2.
The saline solution containing the Sendai virus, which had been previously inactivated with ultraviolet rays, was mixed under ice cooling, and after about 5 minutes, the mixture was transferred to a 37°C thermostatic water bath and stirred for about 3 minutes. At the same time, cell fusion occurred, and the ability to produce human growth hormone was introduced into lymphoblastoid Namalva cells.

These lymphoblastoid Namalva cells were intraperitoneally transplanted into adult nude mice, and then raised in the usual manner for 5 weeks.

Approximately 15 g of the resulting tumor was excised and treated in the same manner as in Example 1 except that L-DOPA was used as the growth hormone inducer to induce and produce human growth hormone. The production amount per Mt of suspension was approximately 1800 r1g. As a control, lymphoblastoid Namalva cells subjected to cell fusion were cultured in vitro in the same manner as in Example 1, and when human growth hormone was induced to be produced, the amount produced was only about 100 ng per ML of suspension.

Example 3 Newborn hamsters were previously injected with antiserum prepared from rabbits using a known method to weaken the hamster's immune response, and then human growth hormone producing ability was subcutaneously introduced into the hamsters according to the method of Example 2. The lymphoblastoid JBL cells obtained were transplanted and then reared for 3 weeks in the usual manner.

Approximately 10 g of the resulting tumor was removed and treated in the same manner as in Example 1 to induce the production of human growth hormone.

The production amount per ml of suspension was approximately 2000 r1g. As a control, lymphoblastoid BL cells that had undergone cell fusion were cultured in vitro in the same manner as in Example 1, and human growth hormone was induced to be produced.
The production amount was only r1g. Example 4 Lymphoblastoid Namalva cells into which human growth hormone producing ability had been introduced by the method of Example 2 were intravenously transplanted into neonatal rats, and then reared for 4 weeks in the usual manner.

Approximately 40 g of the resulting tumor was removed and treated in the same manner as in Example 1 to induce the production of human growth hormone.

The production amount per ml of suspension was approximately 1500 ng. In contrast, the control in vitro cultured induced product produced only about 100 ng per ml of suspension. Example 5 A grown normal mouse was irradiated with X-rays at a dose of about 400 rem to weaken the mouse's immune response, and then neoplastic eosinophilic cells prepared in the same manner as in Example 1 were subcutaneously transplanted, and then normal mice were Breed for 3 weeks using the following method. Ta.

Approximately 15 g of the tumor formed under the skin was removed and treated in the same manner as in Example 2 to induce the production of human growth hormone. floating liquid m
The production amount per liter was about 600 r1g. In contrast, the control in vitro cultured induced product produced only about 200 ng/ml of suspension.

Example 6 Lymphoblastoid JBL cells into which human growth hormone-producing ability was introduced by the method of Example 3 were placed in a plastic cylindrical diffusion chamber with an internal capacity of approximately 10 m1 equipped with a membrane filter with a pore size of approximately 0.5 microns. It was suspended in physiological saline and implanted into the abdominal cavity of an adult rat.

After the rats were housed in the usual manner for 4 weeks, the diffusion chambers were removed.

The human-derived cell concentration obtained in this way was approximately 5×1C
P/ml, which was found to be approximately 103 times higher than when cultured in a carbon dioxide gas incubator in vitro. The cells were made into a suspension in the same manner as in Example 1, and human growth hormone was induced to be produced. suspension solution ml
The production amount per unit was approximately 2200 r1g. In contrast, the control in vitro cultured induced product produced only about 200 ng/ml of suspension. Example 7 Lymphoblastoid J produced by introducing human growth hormone producing ability into fertilized chicken eggs kept at 37°C for 5 days by the method of Example 3
After transplanting the BL cells, they were kept at 37°C for one week.

After the eggs were broken, proliferating cells were collected and treated in the same manner as in Example 1 to induce the production of human growth hormone.

The production amount per ml of suspension was approximately 1300 ng. In contrast, the control in vitro cultured induced product produced only about 200 ng/ml of suspension.

Claims (1)

[Claims] 1. Transplanting human-derived cells capable of producing human growth hormone into the body of a warm-blooded animal other than humans, or proliferating them while receiving the body fluids of the warm-blooded animal, and acting on the resulting cells with a growth hormone inducer. A method for producing human growth hormone, characterized by producing human growth hormone.