KR870001417B1 - Human growth hormone producing method by using pta-210 cell line - Google Patents

Abstract

Human growth hormone (HGh) is prepd. by subculaturing the pituitary tumor cell, PTA-210 (KFCC 10311) in RPMI-1640 medium. Thus, PTA-210 cell line (KFCC 10311) is provided by subculturing pituitary tumor tissue in a RPMI-1640 medium contg. 15% fetal calf serum in vivo to produce 450 g HGh/105 cell/24h for 6 months.

Description

Production method of human growth hormone using PTA-210 cell line

The present invention relates to a method for producing hormones necessary for human growth using PTA-210 cell line (KFCC-10311). Human growth hormone is an indispensable hormone that governs normal growth of humans in the growth phase. Growth hormone is a protein secreted by the pituitary gland, and its chemical structure is well known.

To date, research reports show that the biochemical properties of human growth hormone differ from other animals (G.H.L: Perspective Biology and Medicine 11, 1968).

In addition to human growth hormones, humans respond only to growth hormones derived from monkeys, but mice also respond well to animal-derived growth hormones of different species.

Since growth is caused by a combination of factors and processes, it is very difficult to clearly define the mechanism of action of human growth hormone.

So far, growth hormone has been shown to stimulate protein synthesis, promote the metabolism of electrolytes, promote the secretion of storage lipids, and increase the carbohydrate metabolism, thus increasing blood glucose levels, which in turn causes insulin secretion.

In vitro supply of growth hormone may include animal growth hormone, but among them, only growth hormone extracted from monkeys, which are apes, has a similar effect to human growth hormone, but its use is various. Immunologically problematic, it is practically impossible to use.

As an in vitro production method of human growth hormone, first, it has been reported that the human pituitary cells are cultured in vitro to fix the growth hormone secretory cells.

In addition, the method of producing human growth hormone by yeast or bacteria by genetic manipulation has been studied in various institutions recently, but it is hard to reach the practical stage of use, and careful investigation is required for human application. Therefore, the only method currently used is to extract the pituitary gland from the carcass to purify growth hormone. This method is very expensive and has a large supply limitation.

Therefore, human growth hormone is rare and expensive, but growth hormone deprived children must be limited in the growth phase, such a phenomenon is worse in Korea, only one or two cases of the growth hormone is currently administered.

The rareness of human growth hormone is limited in its own research on growth, and very few effects have been reported for children other than hormone deficiency. However, a five-year study by Gail Shiner (Research Resources Reporter.Vol. IV, No. 12, 1980) found that 24 out of 24 children who had normal growth hormone deficiency, as well as children with growth hormone deficiency. Ten people responded strongly and reached normal growth, which means that if growth hormone is available, human growth hormone infusion may be applied to all children under normal levels, even if healthy.

This fact further emphasizes the importance of the development of a method for producing human growth hormone, and thus the present inventors have succeeded in establishing the human pituitary cells and succeeding in fixing a cell line producing human growth hormone in one cell line. The present invention enables the in vitro production of human growth hormone used.

In other words, the present invention succeeded in securing 0.7 g of the pituitary tissue of a patient with an excessive secretion of human growth hormone (29.79 μg / ml of blood or more) in vitro to fix the cell line, and then directly culture it to produce human growth hormone in vitro. It is to provide a way to.

The cell line used in the present invention was a mixed population of chromophobe cells and alpha cells, which are indispensable for growth hormone production, and the daily growth hormone production of 10 ⁵ cells was confirmed to be 450 μg or more by radioimmunological quantitative analysis. We named the PTA-210 cell line.

On the other hand, the present inventors have found that by culturing the PTA-210 cell line directly, it is possible to produce human hormones in vitro, as well as extracting the gene from the PTA-210 cell line, thus making it an optimal cell line that can be used for genetic engineering applications. .

In the following examples the invention is described in more detail. The cells used in this example are tumors of the pituitary gland, which is a growth hormone producing organ, and are tissues extracted from brain tumor patients who continuously secrete growth hormone due to abnormal growth and enhanced performance of growth hormone producing cells. Growth hormone levels were 29.79 μg / ml, which was 6 times higher than that of normal subjects. Pathological results of the tissues removed during surgery were identified as pituitary tumors, and some of the tissues (0.7 g) were aseptically transported.

Example 1

In vitro application of cell lines

Tissue (0.7 g) was suspended in 5 ml of RPMI-1640 (Flow.USA) medium without serum in sterilized Petri dishes with a diameter of 100 mm in a clean room, and the suspended tissue was sized using a surgical knife (Noll). A small tissue section of about 1㎣ was made.

The tissue sections were then placed in a 50 ml centrifuge tube at room temperature and re-separated. The supernatants were placed directly in 100 mm Petri dishes and adjusted to 20% calf serum (Flow.U.S.A.) in RPMI-1640 medium.

As antibiotics, penicillin, streptomycin, and antifungal agents were added so that 100 units and 100 mcg / ml were used. The precipitated tissue was treated again with 0.75% trypsin, and then proteolyzed in a trypsin container at room temperature for 30 minutes, redistributed (20 pieces) in a 10 ml centrifuge test tube, washed three times, and placed in RPMI-1640 medium.

At this time, the container was dispensed into a flask flask (70 ml).

The cultured cells were placed in a saturated humidity incubator (the temperature of the incubator was adjusted to 37 ° C and the carbon dioxide gas was adjusted to 5%), and after 24 hours, the settled cells were observed to grow under a special phase contrast microscope. Was replaced with fresh medium.

After 3 days, the cells adapted to in vitro and the cells which cannot be fixed were centrifuged with Ficoll / Hypage and redistributed.

Example 2

Establishment of Cell Line

After the procedure described in Example 1 was continued for 1 month, a sizing experiment was performed using a 24 wells plastic culture vessel to measure the characteristics of living cells and the minimum survival rate.

Because the grown cells were suspended, no trypsin treatment was required for passage or dispensing.

These cells were adjusted to 10 ⁵ ml by the cell staining method (dye exclusion) and dispensed into 10 ⁴ wells and 10 ⁵ wells in a 24 well culture vessel.

As a result, the number of cells to maintain growth was at least 10 ⁴ wells, and even below, the cells did not necrosis but completely lost division capacity.

As described above, the number of cells whose division was lost was adjusted to 10 ⁴ wells by centrifugation, and the division capacity was regenerated when the concentration of calf serum was increased from 15% to 20%.

As a result, 10 ⁴ cells were dispensed into each well, and the growth form and the number increase of each well were continuously confirmed by a phase contrast microscope, and each supernatant medium was collected and cooled to 20 ° C. for the measurement of growth hormone. The refrigerated harvested cultures were used for quantitative experiments of hormones by radioimmunoassay. In particular, the double-binding of antibodies against human growth hormone labeled with radioactive substances confirmed that the PTA-210 cell line was capable of producing human growth hormone, as well as its production of 450 μg / 10 ⁵ / cell / 24 hours. It was found to be persistent (six months).

Example 3

Response of PTA-210 Cell Line to Culture Conditions

1) Effect of culture medium

RPMI-1640: The PTA-210 cell lines in this medium were suspended in culture and all spherical in shape. The growth rate was 24 hours when fresh medium was added.

McCoy's 5A: Cells attached and cultured in suspension and co-cultured cells were also co-existing, and the shapes of attached cells were also varied, confirming the shape of polygons and spheres.

The doubled time was longer than RPMI-1640 and about 30 hours.

2) Effect of Serum Amount and Type in Medium

The optimal concentration of calf serum was 15%.

The effect of human placental serum rather than calf serum was effective in 15%.

3) Experiment on cell density and area of culture vessel

The vessels used in the experiments were all plastic, with only a different surface area and the optimum density was 10 ⁴ -10 ⁵ as described above and the optimum vessel capacity was 35 mm Petri dish. Through the process of the above embodiments, the PTA-210 cell line has been established that has the best growth ability and can continuously produce human growth hormone.

The following is on the characteristics of PTA-210 cell line.

1) Pathological histology of cultured cells

The cultured PTA-210 cell line was fixed in micro covergrass.

When observed by fixed staining as described above, the main species of the cell is histopathologically two cell populations, the characteristics of which are as follows.

① Chromophobe

It accounts for 25-30% of the total cells and all have a cytoplasm that is almost spherical, and its size is about 4-6μ, and the aggregation of chromosomes with basic staining can be observed. These cells were histologically proliferated in the PTA-210 cell line as a frontier of growth hormone-producing acidophil (Alpha) cells, and their abundance was somewhat different depending on the number of mature Alpha cells.

② Acidophil (Alpha Cell)

It is spherical in shape and has an average size of 14-20μ. It is filled with cytoplasmic acid staining particles unique to growth hormone secretion.

The size of each particle (growth hormone particle) is about 350mμ, secreted by the mature Alpha Cell and accumulated in the medium.

2) In vitro Growth Characteristics of PTA-210 Cell Line

① PTA-210 cell line has infinite growth characteristics of cultured fixed cells,

② Alpha Cells (growth hormone producing cells) can identify the particles easily observed in a phase contrast microscope in the cytoplasm, these particles can be easily observed within 24 hours of culture in the state secreted in the medium.

③ The maximum culture minimum density is 10 ⁴ / mℓ,

④ cell division is replaced with fresh medium within 24 hours, the number of cells doubled and can confirm the secretion of hormone particles. If the medium is not exchanged, the growth rate is less than 10%, and the growth rate and the hormone production capacity are inversely related.

As in the case of normal pituitary cells, it was almost impossible to detect cells in the dividing phase, and none of the pituitary cell karyotypes were determined.

⑤ Cells are frozen and stored in nitrogen at minus 190 ° C. The medium composition is 95% of PRMI 1640 and 5% of dimethyl sulfoxide.

⑥ Approximately 70% of survival rates (selective dyeing method)

⑦ Optimal medium: RPMI 1640, Fetal Calf Serum 15%

⑧ Plating Efficiency: 30%

⑨ Shape: Lymphoid

As described above, the present invention enables the in vitro production of human growth hormone using the PTA-210 cell line and is a novel invention not found in the conventional method.

Claims (1)

Humans characterized by accumulating human growth hormone in cells by culturing PTA-210 cell line (KFCC 10311) obtained by passage of pituitary tissues of patients with excessive secretion of human growth hormone (more than 27.79 μg / mℓ of blood) in vitro Method of production of growth hormone.