JP2691557B2 - Human malignant tumor cell growth inhibitor - Google Patents

Description

TECHNICAL FIELD The present invention relates to a human malignant tumor cell growth inhibitor.

2. Description of the Related Art A human malignant tumor cell growth inhibitor comprising human malignant tumor cells cultured at 30 ° C. to 37 ° C. in a medium supplemented with glucose and extracted by removing the malignant tumor cells from the medium is disclosed.
Known as issue number 59-162889.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention An object of the present invention is to provide a human malignant tumor cell growth inhibitor having a growth inhibitory effect on malignant tumor cells far larger than those shown in the above-mentioned prior art. .

Means for Solving the Problems This invention is to grow human malignant tumor cells by culturing them under normal culturing conditions for higher animal cells, wash them once with a serum-free culture medium, remove the serum content, and a serum-free glucose concentration. 3-5g
Malignant tumor cells without changing the culture medium in the atmosphere of 34 ° -36 ° C (preferably 35 ± 1 ° C), relative humidity 100%, 5% carbon dioxide concentration using the culture medium prepared to 1 / l (culture medium) After culturing until it is killed (about 1 week), it is centrifuged at 10,000 RPM for 10 minutes to remove malignant tumor cells to obtain a stock solution of malignant tumor growth inhibitor.

The present invention has discovered that in order to have a large ability to suppress the growth of malignant tumor cells, it is necessary to culture the tumor cells until they die without replacing the medium, and it was discovered that the tumor cells could not be obtained until the cells died. This is used as a malignant tumor cell growth inhibitor.

Even when culturing malignant tumor cells while exchanging the medium, almost nothing that has the ability to suppress the growth of malignant tumor cells is produced, and even if a physiologically active substance can be produced by sensitizing with Sendai virus, etc. It is something that cannot be generated without it. In addition, the undiluted solution of the malignant tumor growth inhibitor according to the present invention contains amino acids, vitamins, which are nutrient sources used during culture,
It contains only glucose, minerals, trace amounts of fetal bovine serum components, and substances released from malignant tumor cells, and does not particularly contain substances that adversely affect normal cells.

The glucose concentration in the culture broth must be at least 2 g / l to obtain a stable maximum antitumor activity, and the remaining glucose concentration in the inhibitor stock solution should be measured in another experiment (Experiment 2). It has been confirmed that glucose consumption is 2.5 g / l, so glucose concentration is 4-5 g / l.
To prepare and use.

Also, regarding the temperature at the time of culturing, culturing at 35 ° C. shows the highest activity.

This malignant tumor cell growth inhibitor has a high molecular weight of 3000, unlike a high molecular weight substance composed of glycoproteins such as cytokines.
The following substances are extracted after malignant tumor cell death.

The human malignant tumor cell growth inhibitor of the present invention kills malignant tumor cells in vitro, but suppresses the growth of normal cells and does not have a lethal effect.Because of its small molecular weight, it acts as an antigen in vivo. It seems that there are almost no side effects like conventional antitumor agents.

The malignant tumor cell used in the present invention is not particularly limited.

Example 1 Commercially available synthetic media, such as Eagle's MEM supplemented with 10% fetal calf serum, for malignant tumor cells such as human Hela C.
After ells (uterine cancer cell line) is cultured until the container becomes saturated, it is washed once with a serum-free medium to remove the serum content.

Next, a serum-free medium containing glucose in a commercially available synthetic medium Eagle's MEM was added to a concentration of 5 g / l (medium), and the medium was not replaced in air at 35 ° C, 100% humidity and 5% CO ₂ concentration. Incubate for about a week until the cells die.

The medium is collected and centrifuged at 10,000 RPM for 10 minutes to separate and remove malignant tumor cells to obtain a stock solution of malignant tumor cell growth inhibitor.

Example 2 Malignant tumor cells were cultured in a synthetic medium such as Eagle's MEM or in a medium supplemented with glucose at a concentration of 5 g / l and supplemented with 10% fetal bovine serum until the container became saturated. After that, wash once with a serum-free medium to remove the serum content.

Next, a serum-free medium prepared by adding glucose to a concentration of 5 g / l (medium) in commercially available synthetic medium Eagle's MEM was added, and the medium was not exchanged in air at 35 ° C., 100% humidity and 5% CO ₂ concentration. Incubate for about a week until the cells die.

The medium is collected and centrifuged at 10,000 RPM for 10 minutes to separate and remove malignant tumor cells to obtain a stock solution of malignant tumor cell growth inhibitor.

Experiment 1 Difference in malignant tumor cell growth inhibitory ability depending on the presence or absence of medium exchange during serum-free medium culture.

A. Comparative inhibitor The inhibitor that was cultured by the method of Examples 1 and 2 without changing the medium until the cells died.

Inhibitors of Examples 1 and 2, which were cultured for 10 days without changing the culture until the cells died and changing the medium every other day.

B. Assay A commercially available synthetic medium containing 10% fetal bovine serum added to a plastic dish with a diameter of 15 mm, such as 1 ml of Eagle's MEM and Hella.
Put 1 x 10 ⁴ cells and incubate at 35 ℃ for 24 hours,
The medium was replaced with a medium containing each inhibitor, and the medium was replaced with the same medium every other day thereafter, and the growth state of cells was measured on the 6th day.
The number of surviving lla cells was counted.

Also, as a control, the same operation was performed using a medium having the same composition containing no inhibitor.

Table 1 shows the results, and the inhibition rate is Indicated by

Experiment 2 Optimal Glucose Concentration during Malignant Tumor Cell Culture A. Method of Inhibitor Production Hella Cells that had been grown in culture were washed once with serum-free medium to remove the serum content, and 2 x 10 was added to a culture bottle with a bottom area of 150 cm ^2.
7 respectively 50 of serum-free medium supplemented with glucose to a concentration of Hella Cells and medium per 1.2 and 5g of more Ke
ml was added, and the cells were cultured at 37 ° C., 100% humidity, 5% CO ₂ air for about 1 week without changing the medium until the cells died.

Each medium was centrifuged at 10,000 RPM for 10 minutes to remove tumor cells, and cultured at glucose concentrations of 1, 2 and 5 g / l to obtain a sample stock solution.

B. Assay method for inhibitors Commercial synthetic medium containing 10% fetal calf serum added to a plastic dish with a diameter of 15 mm, such as 1 ml of Eagle's MEM and Hell
Add 1 x 10 ⁴ a Cells and incubate at 37 ℃ for 24 hours, then replace with the medium containing the inhibitor produced in A, and then replace the medium with the same medium every other day to check the number of viable cells. It was measured on the 6th day.

Further, as a control, the same operation was performed using a medium having the same composition containing no inhibitor.

C. Results Table 2 shows the results. The control shows the inhibition rate when no inhibitor is added. Based on the glucose concentration of a commercially available synthetic medium at 1 g / l, the cell survival number is 1/3 at 2 g / l and 1 / at 5 g / l.
4, The suppression rate is 3 times and 4 times, respectively.

In another experiment, the required glucose amount was confirmed to be 2.5 g / l medium, so 4 g / l is the optimum amount.

Experiment 3 Optimum temperature for culturing malignant tumor cells A. Production of malignant tumor suppressor at 25 ° C, 35 ° C and 42 ° C Hella Cells grown in culture were washed once with serum-free medium to remove serum, and the bottom area of 150 cm ² Hella C with 50 ml of Eagle's MEM as a commercially available serum-free synthetic medium in a culture bottle with
ells 2 × 10 ⁷ or more, 42 ° C, 35 ° C and 25 ° C for the experimental group
3 groups, control is 37 ℃, relative humidity 90-100%, 5% C
The cells were cultured in O ₂ air for about 1 week until the cells died without changing the medium.

The culture medium was collected from each group, centrifuged at 10,000 RPM for 10 minutes to remove tumor cells, and sample stock solutions cultured at 42 ° C, 35 ° C, 25 ° C and 37 ° C were obtained.

B. Inhibitor assay Hella Cells 10 ⁴ cells were placed in a 15 mm diameter plastic petri dish and cultured in MEM medium containing 10% fetal bovine serum for 24 hours. Replace the medium with vitamins, glucose, etc. added (commercially available as powder), change the medium every other day, and culture for 6 days in air at 100% humidity, 37 ° C, 5% CO ₂ , Count the number of viable cells, The growth inhibition rate was calculated by.

C. Results Table 3 shows the growth inhibition rate at each culture temperature for the samples of the three inhibitors, and Table 4 shows the growth inhibition rate at other temperatures relative to the growth inhibition rate at the culture temperature of 37 ° C. It can be seen that the optimum temperature for culturing malignant tumor cells is around 35 ° C.

Experiment 4 Inhibitory Effect of Inhibitors on Various Cells FIGS. 1 to 4 are growth curves showing the daily changes in cell proliferation in the post-culture medium of various cells. In each experiment, the experimental group was cultured with BME, which is a medium of various malignant tumor cells. After the medium was ultrafiltered with Amicon YM5 (MW10 ³ ), the medium supplemented with glucose, amino acids, vitamins and 10% serum as nutrient sources was used. Used, control group is fresh medium
BME to which the same nutrient source as in the experimental group was added was used.
The initial concentration of cells was set to 1 × 10 ⁴ cells. In the experiment shown in FIG. 1, a culture vessel having a diameter of 35 mm was used, and in the experiment shown in FIGS.
A mm incubator was used.

In the experiment shown in FIG. 1, the changes in human renal cell carcinoma-derived established cell line HRC were examined over time. It was clearly shown that the growth was suppressed in the experimental group containing the substance extracted from the medium.

FIG. 2 shows the changes over time in normal human diploid skin fibroblasts NAS63. In the experimental group, a medium extracted from the culture medium of human gastric cancer-derived established cell line MK was used. From these growth curves, the growth inhibition effect is clearly shown in the experimental group of human gastric cancer-derived established cell line MK as compared with normal cells.

Experiment 5 Inhibitory effect of a mixture of inhibitor and fresh medium Figures 3 and 4 show human renal cell carcinoma-derived established cell line HRC.
Fig. 3 shows the concentration-response curves of human renal cell carcinoma-derived established cell line HRC, and Fig. 4 shows the results of normal human 2
Fig. 6 shows the concentration-response curve of polyploid skin fibroblast MAS63. In each case, after the culture, the inhibitor filtered through Amicon YM2, which is a membrane that screens the molecules with a molecular weight of 100, is used, and the percentage is the ratio of the inhibitor medium to the sum of the inhibitor and the fresh medium. Shows. From these figures, the inhibitor shows a growth inhibitory reaction even in normal cells, but the growth inhibitory effect is more strongly observed in the human renal cell carcinoma-derived established cell line HRC, which is an example of malignant tumor.

D. Acute toxicity test The cells used in Figs. 2 and 4, namely "normal human 2
"Ploidy skin fibroblast NAS63" is a normal cell collected from the precordial skin of a 63-year-old normal male, and as shown in Fig. 2, it is suppressed in proliferation to some extent but does not show a lethal effect.

[Brief description of the drawings]

FIG. 1 is a diagram showing the growth of human renal cell carcinoma-derived established cell line HRC between the inhibitor of the present invention and a fresh medium, and FIG. 2 is a normal human diploid of the inhibitor of the present invention and a fresh medium. Skin fibroblasts
Figure showing proliferation of NAS63, Figure 3 shows human renal cell carcinoma-derived established cell line HRC at various mixing ratios of inhibitor and fresh medium.
FIG. 4 is a diagram showing the concentration response of normal human diploid skin fibroblast NAS63 similar to FIG.

Claims (3)

(57) [Claims] 1. A human malignant tumor cell growth inhibitor, which comprises a medium obtained by culturing malignant tumor cells in a serum-free medium until the cells are killed, from which the malignant tumor cells have been removed. 2. The medium has a glucose concentration of 3 in Medium 1.
5. The human malignant tumor cell growth inhibitor according to claim 1, wherein the inhibitor is 5 gh. 3. The human malignant tumor cell growth inhibitor according to claim 1, wherein the culture temperature is 35 ± 1 ° C.