KR100651170B1 - Method for primary pure neuronal culture using methylthioadenosine - Google Patents

Abstract

The present invention relates to a method for selectively culturing only non-dividing cells by using a characteristic of selectively dividing cells among the dividing cells and the non-dividing cells of methylthio adenosine, more specifically, Applied to the central nervous system and various peripheral nervous system neurons, the present invention relates to a method for culturing only pure neurons by killing other cells in addition to the neurons that divide continuously during primary culture.

In addition, the present invention can obtain the effect of cost reduction by using the antibiotic activity of methylcyanoadenosine as well as selective cell culture and cell culture without additional antibiotics in the culture medium.

Description

Method for primary pure neuronal culture using methylthioadenosine

Figure 1 is a photograph of the morphological change analysis through the optical microscope of neurons after treatment with methylcyano adenosine at 1 mM concentration for 48 hours.

The present invention relates to a method for selectively culturing only non-dividing cells by using a characteristic of selectively dividing cells among the dividing cells and the non-dividing cells of methylthio adenosine, more specifically, Applied to the central nervous system and various peripheral nervous system neurons, the present invention relates to a method for culturing only pure neurons by killing other cells in addition to the neurons that continue to divide during primary culture. In general, three methods are commonly used to cultivate pure neurons. The first is a method using a centrifuge, the second is a method of obtaining a desired neuron using a fluorescence activated cell fractionator (FACS) technique, and the third is adding a compound or a substitute of a special medium component to the culture medium. It is a way to remove the remaining cells leaving only the desired neurons. The third of these is the most commonly used method.

Differentiated neurons no longer grow. However, other cells mixed with neurons, such as glial cells, astroglia, Schwann cells, microglia, ventricular cells, oligodendrocytes, fibroblasts, and vascular endothelial cells continue to undergo cell division. Therefore, when antimitotics are added to the cultures, these cells stop growing and eventually die. Representative antimytotics are fluorodeoxyuridine (FUdR) and cytosine arabinofuranoside (Ara-C). These compounds interfere with DNA synthesis, causing fatal damage to cells that are trying to grow. In the case of FUdR, there are disadvantages that can affect neurons by interfering with the synthesis of RNA as well as DNA. To supplement this, FUdR is used in combination with uridine. However, these known antimytotics are known to be harmful in some neurons even at very low concentrations of 10 ^-8 M. Usually this is because these neurons still have the ability to divide. Therefore, in this case, there is a very difficult problem to cultivate neural cells purely.

The inventors of the present invention, while studying the physiological activity of methylcyanoadenosine, know that this substance causes problems in the mitosis process in the cell cycle of dividing cells and causes cell death. The present invention has been completed by finding out that apoptosis occurs in non-neuronal cells that divide at a concentration of 0.5 mM or more, but does not affect neurons at all.

The present invention solves the above problems, and the object of the present invention is to provide a method for purely culturing only nerve cells.

In order to achieve the above object, the present invention provides a method for culturing nerve cells using methylthio adenosine.

The nerve cell of the present invention is preferably a nerve cell derived from the central nervous system or the peripheral nervous system, and more preferably a nerve cell derived from the optic nerve of eye tissue, which is a kind of peripheral nervous system.

Since the concentration of methylcyanoadenosine used in the present invention does not kill the growing cells at 0.1 mM or less, and 10 mM or more is poorly soluble in the medium and aqueous solution, the range of 0.1-10 mM is preferable.

It has been known for a long time that methylthioadenosine (hereinafter referred to as "MTA") having a structure of the following formula (1) is produced in an organism. In 1912, the first MTA crystals were refined by Mandel and Dunham (Mandel, JA and Dunham, EK (1912) J. Biol. Chem. 11 , 85-88). This nucleoside was found in both prokaryotic and eukaryotic organisms and was found to be one of the main products of S-adenosylmethionine (SAM) metabolism. Biosynthesis from Methionine was demonstrated by Schlenk in 1952 (Smith, RL and Schlenk, F. (1952) Arch. Biochem. Biophys. 38 , 159-162), and previously by Cantoni that SAM was a precursor to MTA. Cantoni, GL (1953) J. Biol. Chem. 204 , 403-408. For many years, MTA has been regarded only as a byproduct of the polyamine biosynthesis process. In recent years, biochemists and pharmacologists have demonstrated the role of MTA in purine salvage and thiomethyl group recycling, and experimental results of growth inhibition have been observed. Interest has been increased (Williams-Ashman, HG and Canellakis, ZN (1979) Perspect. Biol. Med. 22 , 421-453; Williams-Ashman, HG, Seidenfeld, J. and Galletti, P. (1982) Biochem. Pharmacol. 31, 277-288) . Moreover, deficiency of MTA phosphorylase was observed in several malignant tumor cells (Kamatani, N. and Carson, DA (1980) Cancer Res. 40 , 4178-4182; Kamatani, N. Nelson-Rees, NA and Carton, DA (1981). Proc. Natl. Acad. Sci. USA 78 , 1219-1223) There is a continuing interest in the possibility of a link between MTA metabolism and malignancy.

(1)

(One)

On the other hand, by interfering with the methylation process of the modification of the protein of methyl thio adenosine, by stopping the mitosis process in the cell cycle process of dividing cells to cause apoptosis to distinguish between cells that divide and do not divide Provide a method. Therefore, the present invention provides a new effect by utilizing the action of a mechanism different from the compound for inhibiting cell division, which is typically used for neuronal cell culture.

The present invention provides a method capable of purely culturing only nerve cells by adding methylcyanoadenosine that kills only dividing cells in the culture medium in the central nervous system primary culture.

In addition, the present invention provides a condition capable of culturing only pure neurons by adding methylcyanoadenosine to the culture medium in primary nervous system as well as in the peripheral nervous system such as optic nerve cells.

The present invention provides the effect of reducing the cost and procedure of adding antibiotics to the culture separately due to the antibiotic activity effect of methylthio adenosine.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The cancer cell line, U937, divides very well and primary cultured neurons do not divide. Treating these two different cells with methylcyanoadenosine shows that this compound is harmful only to dividing cells, and then treated with methylcyanoadenosine by concentration in this cell line to determine the amount of apoptosis that can be killed. Determined using. In addition, it was determined whether methylthio adenosine inhibits the growth of bacteria and the appropriate concentration.

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples.

Example 1

U937 cells (Korea Cell Line Bank KCLB No. 21593), which are human leukemia lymphocytes, were cultured in RPMI1640 medium containing 10% FBS . Cell culture was performed in an incubator containing 5% CO ₂ at 37 ° C. The prepared cells were treated with various concentrations of methylcyanoadenosine at 0.01, 0.1, 0.5, and 1 mM for 4 days, and the cell growth and survival rate were measured using Trypan Blue intracellular uptake rejection. Indicated.

TABLE 1

Effect of Methylcyanoadenosine on Survival of Dividing U937 Cell Lines

Methylthioadenosine added amount (mM) / 4 days No addition 0.01 0.1 0.5 One Cell growth (cell count / ml) 4.9 × 10 ^-5 4.2 × 10 ^-5 1.0 × 10 ^-5 0.5 × 10 ^-5 0.1 × 10 ^-5 Cell survival rate (%) 95 93 80 57 18

Methylcyanoadenosine induced cell proliferation inhibition and cell death. No effect was observed on the proliferation of U937 cells up to 0.01 mM methylcyanoadenosine concentration. However, when the concentration of methylthio adenosine was 0.1 mM, cell growth inhibition of 80% was achieved.

The cell survival rate was not significantly affected by the concentration of methylcyanoadenosine at 0.1 mM, but the survival rate was only 57% after 4 days treatment at 0.5 mM. At 1 mM of methylcyanoadenosine, 82% of the cells died on day 4. Therefore, it was found that methylcyanoadenosine causes death in dividing cells, and the method seems to be dependent on the amount.

Example 2

In Example 1, the treatment of methylcyanoadenosine on rapidly dividing cells showed that induction of apoptosis from the concentration of methylcyanoadenosine at 0.5 mM, so methylthioadenosine actually affected cell survival in non-dividing neurons. Methylcyanoadenosine at 1 mM concentration was treated in primary neural cells to show this absence. Primary neural cell cultures were performed using mouse embryos at 15 days of gestation, and after brain extraction from mouse embryos under a surgical microscope, the brain membranes were carefully removed, the cerebral cortex was dissected and placed in 0.09% trypsin solution, followed by 37 ° C incubator. Incubated for 30 minutes. After preparing 5% FBS at a density of 3.5 cerebral hemispheres in one culture vessel, the cell suspension is prepared in the culture vessel. After 24 hours of cell culture, 60% of the medium is changed to a preconditioned GCM (glial conditioned medium) and 3 uM of Ara-C solution. Ten days after the start of the culture was used for the experiment. Cell viability was observed using lactate dehydrogenase (LDH) activity and morphological analysis. The results are shown in Table 2 below and in FIG. 1.

TABLE 2

Comparison of Apoptosis in Normal Control and Methylcyanoadenosine-treated Neurons by Time

Survival time 8 24 48 Normal control 90 82 72 Methylcyanoadenosine 1 mM treatment group 92 85 77

Experimental results showed that there was no difference in survival rate between the neurons without methylcyanodenosine treatment and those treated with 1 mM concentration compared to the normal control group over time. Therefore, the treatment of methylcyanoadenosine at this concentration is considered to be able to sufficiently select and culture neurons and non-neuronal cells.

In addition, as can be seen in Figure 1, the histological changes through the microscope of the normal control group not treated with methylthio adenosine and neurons treated at 1 mM concentration for 48 hours was hardly observed.

Example 3

Disc diffusion was performed to verify the antibacterial effect of methylthio adenosine.

A Gram-positive strains are S.aureus ATCC 29231, S.pyogens ATCC 8668, gram-negative strains as was used for P.aeruginosa ATCC27853 increase each of the strain of 0.9% saline to the concentration of the turbidity standard solution grown in medium gyunyong Dilute to The sterilized cotton swabs were sufficiently moistened within 15 minutes in the diluted bacterial dilution solution, and then inoculated evenly by rubbing a cotton swab moistened with the bacterial solution on a dry agar plate medium. Close the plate lid and let it stand for 3 to 5 minutes to let the surface absorb moisture. 100 μl of 10 mM methylthio adenosine was slowly dropped on a blank disk, and then dried sufficiently in a clean bench to prepare a methyl cyano adenosine disk. Using a sterilized tweezers, the control antibiotic disc and the prepared methylcyanodenosine disc were placed on the agar plate evenly inoculated with the bacteria, lightly backed off the center of the disc and completely adhered to the agar surface. After 16 to 18 hours of incubation, the diameter of the growth inhibition zone was measured using a ruler or the like. The results are shown in Table 3 below.

TABLE 3

Methylcyanoadenosine antibacterial effect result in gram positive bacteria and gram negative bacteria

S.aureus  ATCC 29231 S.pyogens  ATCC 8668 P.aeruginosa  ATCC27853 Methyl Cioadenosine 300ug S ^a (18 ^c ) S (19) S (18) Negative Control (Saline) R ^b R R Control antibiotics Ofloxacin 5mcg S (21) S (32) S (19) Ceftriaxone 30mcg S (21) S (41) S (19)

In Table 3, a represents a Sensitive, b represents a Resistance, and c represents a growth inhibition zone diameter (mm).

Experimental results showed that methylcyanoadenosine was effective against gram positive and gram negative bacteria.

The method of the present invention is carried out through a different mechanism from the compound used for the selective culture of neurons, and has a low harmful effect on neurons and also includes an antibiotic function that prevents bacterial contamination. There will be savings.

Claims (6)

Pure neuronal cell culture method using methylthio adenosine. The method of claim 1, The nerve cell is a nerve cell culture method, characterized in that the nerve cells derived from the central nervous system. The method of claim 1, The nerve cell is a nerve cell culture method, characterized in that the nerve cells derived from the peripheral nervous system. The method of claim 3, wherein The neuronal cell culture method, characterized in that the nerve cells derived from the optic nerve of the eye tissue. The method according to any one of claims 1 to 4, The concentration of the methyl cyano adenosine is nerve cell culture method, characterized in that 0.1 to 10mM. The method according to any one of claims 1 to 4, Methylcyanoadenosine is a neuronal cell culture method characterized in that it has an antihytostatic activity and antibiotic activity.

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