KR860000896B1 - Producing method of human prolactin - Google Patents

Abstract

Prodn. of human prolactin(I) comprises (a) transplanting human cells capable of producing (I) into any warm-blooded animal other than a human, or by propagating the cells in a device, esp., a diffusion chamber which supplies them with nutrient body fluid directly from warm-blooded animals. (b) the grown cells are then treated with inducer. The (I)-inducer includes reserpine, phenothiazine, chlorpromazine, a hormone such as estrogen, amino acids such as Lys, Arg, or Cys, salts such as NaCl, KCl, or MgSO4 and a mixt. of salts. Suitable human cells include anterior hypophysis acidophilic cells, viral transformed or X-irradiated cells, hypophysis acidophilic adenoma cells, pulmonary carcinoma cells, and cell lines established from these cells. (I) stimulates development of the mammary gland, prostate, and seminal vesicles.

Description

Method of manufacturing human prolactin

The present invention relates to a method for preparing human prolactin.

Human prolactin is a peptide hormone having a molecular weight of about 22,000, as reported, for example, in Biochemistry Data Handbook I, pages 1326-1328, published by Tokyo Chemical Industries, Ltd. (1979.).

Human prolactin is a hormone produced by eosinophilic cells of the anterior pituitary gland of human pituitary gland, and has a function of promoting the development of the mammary gland, the frontal gland, and the seminal vesicle. Methods of mass production of human prolactin have not yet been established.

As a result of intensive research to mass-produce human prolactin at a low price, the present inventors surprisingly note that human-derived cells having human prolactin-producing ability are warm-blooded animals other than the human body than cells obtained by tissue methods in vitro. Cell proliferation using the human body prolactin production is much higher, and found to reach about 2-50 times per cell, and completed the present invention.

That is, the present invention transplants human-derived cells having human prolactin production into warm-blooded animals other than the human body, or proliferates these cells using a device in which the nutrient fluid of the warm-blooded animals is supplied to these cells. The present invention relates to a method for producing human prolactin, which is characterized by producing human prolactin by acting a prolactin inducer on the obtained cells.

Unlike the case of tissue culture in vitro, the method of the present invention does not require any cell proliferation nutrient medium containing a large amount of prolactin and expensive serum, etc., or only a very small amount of cells. Maintenance of growth medium is extremely easy.

In particular, the nutrients supplied from warm-blooded animals when transplanted into a warm-blooded animal other than the human body having a prolactin-producing ability in humans or suspended in a diffusion chamber designed to receive nutrient fluids of these animals are carried out in the normal breeding. By using the body fluids, it is possible to easily proliferate human-derived cells with prolactin-producing ability.

In addition, compared with the case of culturing in vitro, the present method is characterized in that the proliferation of these cells is stabilized and the proliferation rate thereof is increased, and the amount of human prolactin produced per cell is increased.

The human-derived cells used in the present invention can be used as long as they have human prolactin-producing ability and can be easily proliferated by transplantation into warm-blooded animals other than the human body. Eosinophilic cells of the anterior pituitary gland of human body, or cells having the original human prolactin production ability, such as cells transformed with EB virus, X-ray, etc. and eosinophilic tumor cells obtained from patients with pituitary anterior eosinophilic cell adenomas. ; Cells having ectopic human prolactin production, such as lung cancer cells, and cells cultured with these cells are most suitable.

In addition, genes having human prolactin production ability by gene replacement method using enzymes such as DNA ligase, nuclease, DNA polymerase, or cell fusion using drugs such as polyethylene glycol or Sendai virus. By using cultured human lymphocytes (lymphoblastoids) that can be subcultured more easily by introducing them, the growth rate can be remarkably increased when these human-derived cells are transplanted into non-human warm-blooded animals. However, it can increase human prolactin production per cell by about 2 to 10 times or more.

Furthermore, when the cultured human lymphocytes described above are transplanted to warm-blooded animals other than the human body, they form huge tumors, and since the tumors are very difficult and easily disintegrate with the cells of the host animal, the proliferating living human lymph The blast cells can be easily collected.

Any animal that can be used in the present invention can be used as long as human-derived cells can proliferate in the body. For example, birds, such as chickens and pigeons: dogs, cats, monkeys, goats, pigs, cows, horses, rabbits, guinea pigs, mice, hamsters, mice or mammals, such as the mouse, it is advantageous in the present invention Do. Since transplantation of human-derived cells into these animals may cause undesirable border reactions, in order to suppress this immune response as much as possible, newborn or infancy animals; Or it is preferable to use as early as possible animals such as eggs, embryos, fetuses and the like. In addition, in order to suppress the immune response, these animals can be irradiated with X-rays or γ-rays of about 200 to 600 ram, or injected with antiserum or immunosuppressive agents prepared by conventional methods before transplanting the cells.

In the case of using the Nude mouse as a warm-blooded animal other than the human body, even if it is grown, the immune response is weak, so it is possible to transplant and rapidly proliferate human-derived cells without the need for pretreatment. It is convenient.

Cell proliferation by repeating transplantation using a combination of warm-blooded animals other than the human body, such as in the case of first transplanting cells derived from a human body into a hamster to proliferate, and then transplanting the cells back to Nude mice to obtain a target product. It can further stabilize and increase the production of human prolactin that is derived from it.

At this time, of course, the same rape and the same alum may be transplanted.

The site in the animal body where the human-derived cells can be transplanted may be any site as long as the transplanted cells can proliferate.

For example, you can freely choose urine fluid, vein, abdominal cavity, subcutaneous.

In addition, it is possible to prevent the incorporation of host-derived cells into the diffusion chamber without directly transplanting human-derived cells into the animal body, and also to provide a porous filtration membrane that can supply warm-blooded animals to human-derived cells cultured with the nutrient solution. Nutrition supplied from an animal body by embedding a diffusion chamber of various shapes and sizes having a porous thin film filter, an ultrafiltration membrane or a hollow fiber having a thickness of about 10 ^-7 to 10 ^-5 m in an animal body, such as an abdominal cavity. Any body-derived cells having human prolactin-producing ability, which have been precultured in the chamber while using body fluids, can proliferate any one.

Also, if necessary, the diffusion chamber is attached to the surface of the host animal body so that the body fluid in the animal can circulate into the chamber, and the proliferation state of the human-derived cells in the diffusion chamber is visualized through the transparent tactile window provided on the chamber wall. It is possible to design the diffusion chamber so that it can be removed and the test chamber can be removed and testified into a new chamber, thereby increasing the cell proliferation during the life of the animal to a higher level without slaughtering the host animal. Can increase cell production per animal.

Moreover, when using such a diffusion chamber, since human-derived cells do not come in direct contact with animal cells, only proliferated human-derived cells can be easily harvested, and undesirable immune reactions do not occur. In the present invention, various warm-blooded animals can be freely used as host animals.

Breeding of host animals transplanted with human-derived cells can easily proceed according to conventional methods even after these cell transplants and does not require any special handling.

Maximal cell proliferation is achieved about 1 to 20 weeks after cell transplantation. When the cells transplanted into an animal body and cultured are human tumor cells or human lymphocytes, their proliferation rate is particularly high, so that maximal cell proliferation is usually achieved within 1 to 5 weeks after cell transplantation.

According to the present invention, the human derived cells obtained per host amount to about 10 ⁷ to 10 ¹² , or more per animal.

In other words, the number of human-derived cells transplanted into an animal body is increased by about 10 ² to 10 ⁷ times or more, or about 10 to 10 ⁶ times or more of the number obtained by in vitro tissue culture using nutrient medium. By doing so, these cells are conveniently used for human prolactin production.

As a method for inducing human prolactin, any method may be used as long as it can produce human prolactin by interacting the human-derived cells obtained by the aforementioned method with a human prolactin inducer.

For example, the concentration of cells in a nutrient medium in which human-derived cells obtained by proliferating in an intraperitoneal ascites and then collecting them from the ascites or extracting huge tumors formed subcutaneously and dissecting the tumors are maintained at about 20 to 40 ° C. Is suspended from about 10 ⁴ to 10 ⁸ / ml and the prolactin inducer is then reacted for about 1 to 50 hours to induce the production of human prolactin.

Examples of preferred prolactin inducers are organic compounds such as reserpin, phenothiazine and chlorobromine, hormones such as thyroid-stimulating hormone-releasing hormone and estrogen, amino acids such as lysine, arginine and cysteine and sodium chloride, calcium chloride and calcium chloride And inorganic salts such as magnesium sulfate.

In this case, the present invention may simultaneously produce other hormones such as human growth hormone (hGH).

Induction-produced human prolactin in this way can be easily purified by separation using a known purification method, such as salting, dialysis, filtration, centrifugation, concentration and lyophilization.

In addition, when a high degree of purification is required, the highest purity human proloem may be combined with known methods such as adsorption by ion exchange, desorption gel filtration and affinity chromatography, coin point fractionation, and electrophoresis. Lactin can be obtained.

The human prolactin thus obtained is immunologically identical to the NIH standard human prolactin preparation, and therefore these preparations can be used as a single substance or by combining one or two or more other agents thereto, for example, injection, external, oral or As a diagnostic administration agent, it can be advantageously used for the prevention and treatment of human diseases such as reduced milk secretion.

Throughout the specification, the amount of prolactin produced in the human body is determined by P, Hwang et al., Proc. Nat. Acad. Sci. USA, Vol. Measurements were made according to the radioimmunoassay assay described on pages 68, 1902-1906 (1971) and expressed as the corresponding weight of the US NIH standard per milliliter of cell suspension. In addition, the production of co-produced human growth hormone was described in S. M. Glick et al. Measurements were made according to the radioimmunoassays described on pages 199, 784 (1963) and expressed in the corresponding weight of the US NIH standard.

Some embodiments of the invention are described below.

Example 1

Human eosinophilic tumor cells obtained by subcutaneous dissection and transplantation after implantation from a patient of anterior pituitary eosinophilic cell tumor in the subcutaneous of the grown Nude mouse were transplanted, and then used for three weeks in a conventional manner.

About 10 g of a massive subcutaneous tumor was removed, cut and digested, and suspended in physiological saline containing trypsin.

After washing cells with Glucose-Free Earle Medium 199 (pH7.2) supplemented with 10v / v% of bovine fetal serum, these cells were resuspended in the same new medium containing 30 mM L-arginine as a prolactin inducer. A cell concentration of about 10 ⁵ cells / ml was obtained and incubated for 6 hours at 37 ° C. to induce human prolactin. The cells were then sonicated and the human prolactin in the supernatant obtained was measured.

Human prolactin production was about 600 ng per ml of cell suspension. Simultaneous production of human growth hormone (hGH) was about 500 ng per ml of cell suspension.

The control cells obtained by propagating human eosinophil tumor cells in in vitro Earle medium 199 (pH 7.2) supplemented with 10v / v% of bovine fetal serum and incubating at 37 ° C. were treated similarly as described above with prolactin inducers. Induction of human prostatic.

Human prolactin production was only about 100ng per milliliter of cell suspension.

Example 2

Human eosinophilic tumor cells and human leukemia lymphocytes (Namalva) cells obtained from patients with pituitary anterior eosinophilic cell gland tumors were obtained from 140 mM NaCl, 1 mM KCl, 1 mM NaH ₂ PO ₄ 2 mM CaCl ₂ It was suspended in a vessel containing a salt solution containing a corresponding cell concentration of about 10 ³ cells / ml.

The ice-cooled cell suspension was mixed with the same salt solution as described above containing "Sendai virus" which was previously irradiated with ultraviolet rays, and after about 5 minutes of mixing, it was transferred to an incubator at 37 ° C for 30 minutes to carry out cell fusion for human body. Human prolactin production of human eosinophilic tumor cells was introduced into lymphocytes of the leukemia lymphocytes. After the hybrid tumor cell line having human prolactin-producing ability was isolated by a conventional method, the hybrid tumor cell line was transplanted intraperitoneally of the grown Nude mouse and reared for 5 weeks by a conventional method.

About 15 g of each of the giant tumors thus obtained was removed and replaced with 30 mM L-arginine with about 10 ng of thyroid stimulating hormone-releasing hormone and the culture was carried out in the same manner as in Example 1 except that the culture was carried out at 35 ° C. Lactin was induced. Human prolactin production was about 3,300 ng per mL of cell suspension.

The control test proceeded similarly as in Example 1 by culturing the cell-fused leukemia lymphocyte namalba hybrid cells in vitro and exposing the proliferative cells to prolactin inducers.

Human prolactin production was only about 200ng per milliliter of cell suspension.

Example 3

In order to suppress the immune response that may be caused by cell transplantation, the neonatal hamster is pre-injected with antiserum prepared according to a known method from rabbits, and then subcutaneously human prolactin of human eosinophilic tumor cells similarly to Example 2 Human leukemia lymphocytes JBL cells into which the production capacity was introduced were transplanted, and further grown for 3 weeks in a conventional manner. In this manner, about 10 g of each obtained subcutaneous tumor formed subcutaneously was extracted and treated similarly to Example 1 to induce human prolactin.

Human prolactin production was about 1,800 ng per milliliter of cell suspension. Simultaneous production of human growth hormone (hGH) was about 2,000 ng per mL of cell suspension.

The control test proceeded similarly to Example 1 by culturing fused human leukemia lymphocytes JBL hybrid cells in vitro and exposing these proliferating cells to prolactin inducers.

Human prolactin production was only about 200ng per milliliter of cell suspension.

Example 4

Similarly to Example 2, human leukemia lymphocytes and lymphocytes into which human prolactin-producing ability of human eosinophilic tumor cells were introduced were transplanted into a vein of newborn rats, and then grown for 4 weeks in a conventional manner. 40 g of each of the giant tumors thus obtained were extracted and treated in the same manner as in Example 2 to induce human prolactin. Human prolactin production was about 2,700 ng per 1 ml of cell suspension.

The control test proceeded similarly to that in Example 1 by culturing human leukemia lymphocytes or namalba hybrid cells in vitro and acting on these proliferation agents to prolactin inducers.

Human prolactin production was only about 100ng per milliliter of cell suspension.

Example 5

After irradiating 400-lem X-rays to the normal mice, the immune response of the mice was weakened. Human eosinophil tumor cells obtained similarly to Example 1 were transplanted subcutaneously, and grown for 3 weeks in a conventional manner. Thus, 15 g of each obtained subcutaneous tumor formed subcutaneously was extracted and treated similarly as in Example 2 to induce the production of human prolactin.

Human prolactin production was about 800ng per milliliter of cell suspension.

The control trial proceeded similarly to Example 1 by culturing human eosinophil tumor cells in vitro and exposing the proliferating cells to prolactin inducers.

Human prolactin production was only about 200ng per milliliter of cell suspension.

Example 6

Human leukemia lymphocytes JBL incorporating human prolactin production capacity of human eosinophilic tumor cells similarly to Example 3 in a plastic cylindrical diffusion chamber of about 10 ml with a pore diameter of about 0.5 μm. The cells were suspended in physiological saline and embedded in the intraperitoneal cavity of the grown mice. This chamber was taken out after the rats were reared for 4 weeks by the usual method. The human mammary cell density in the chamber obtained by the same method as described above was about 5 × 10 ⁹ / ml, which was about 10 ³ times or more than that obtained by in vitro culture using a CO ₂ incubator. The cells thus obtained were treated similarly to Example 1 to induce human prolactin.

Human prolactin production was about 1,900 ng per milliliter of cell suspension. Simultaneous production of human growth hormone (hGH) was about 2,200 ng per ml of cell suspension.

The control test proceeded similarly to Example 1 by culturing fused human leukemia lymphocytes JBL hybrid cells in vitro and exposing the proliferating cells to prolactin inducers.

Human prolactin production was only about 200ng per milliliter of cell suspension.

Example 7

Similarly to Example 3, human leukemia lymphocytes JBL cells into which human prolactin-producing ability of human eosinophil tumor cells were introduced were implanted into the urine fluid cavity of fertilized embryos treated at 37 ° C. for 5 days. These embryos were further cultured for one week at the same temperature, and then proliferated human derived cells were collected. The cells were treated similarly to Example 2 to induce human prolactin.

Human prolactin production was about 1,700 ng per mL of cell suspension.

The control trial proceeded similarly to Example 1 by culturing the fused human leukemia lymphocytes JBL hybrid cells in vitro and exposing the proliferating cells to prolactin inducers.

Human prolactin production was only about 200ng per milliliter of cell suspension.

Claims (1)

Transplanting neoplastic eosinophils, Namalva cells, or J. B. cells from the anterior lobe of the human pituitary gland with human prolactin-producing ability into the body of a nude mouse, hamster, rat, mouse or chicken Or by propagating using a chamber having a volume of about 10 ml with a diaphragm of about 0.5 micron in diameter, which is capable of supplying the fluids of these animals and releasing L-arginine or thyroid stimulating hormone into the proliferating cells. A method for producing human prolactin, characterized in that by producing a human prolactin having a molecular weight of about 22,000 by acting a hormone.