JP2000287683A - Muscle-derived component and rna having neuron survival activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a novel muscle-derived component which originates from flesh and skeletal muscle, shows physiological activity, e.g. survival activity or the like, on neuron and is used for the treatment, prevention and test for various diseases, particularly amyotrophic lateral sclerosis or the like, capable of illustrating nerve degenerative disease as a typical instance. SOLUTION: This muscle-derived component is a novel component which is contained in the final fraction on the following purification process and shows neuron survival activity. This component is obtained by initially homogenizing skeletal muscle to extract it with aqueous 2 w/v.% NaCl solution, collecting a supernatant after centrifugal separation, adding acetone into the supernatant in order to adjust the final concentration to 30 w/v.%, in addition adding acetone again into the resultant supernatant in order to adjust the final concentration to 50 wt.%, collecting the formed precipitate, dissolving it into 50 w/v.% ammonium sulfate aqueous solution, adding a phenyl- chloroform mixture into the supernatant, passing the separated aqueous layer through a phenyl sepharose CL-4B column, eluting the resultant adsorbent and finally fractionating and purifying the eluate by gradient elution or the like using 0-25 w/v.% CH3OH on Capcell-Pak C18 column.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a component having a neuronal physiological activity such as a neuronal survival activity.

[0002]

2. Description of the Related Art In the process of embryo formation, the number of nerve cells is reduced after excessive neurons are formed in the initial stage. This process is called natural cell death. For example, 4 in the lumbar spinal cord
It has been observed that about 0% of motor neurons die. It has also been observed that this motor neuron death proceeds in time with the formation of neuromuscular synaptic connections. If the skeletal muscle is experimentally removed before the innervation of the target tissue, skeletal muscle, during the process of embryogenesis, the motor neurons die. Then, when the skeletal muscle is experimentally removed in the process of embryo formation and a limb bud is taken out from another embryo and implanted in the vicinity of the skeletal muscle, very few motor neurons die. Thus, motor neurons compete for a limited amount of trophic factors from the target tissue, so that only those that have acquired enough of the factor form neuromuscular junctions, while failing to acquire that factor. It has been hypothesized that motor neurons that die are dead. Regarding the mechanism of spontaneous cell death of motor neurons, it is generally accepted that the survival depends on the target tissue.

[0003] Nerve growth factor (NGF) is the prototype of a trophic factor and has been found to promote the survival of developing sympathetic and sensory nerve cells. For motoneurons, NGF had no effect on spontaneous cell death, but rather increased cell death caused by axotomy in neonates. NGF that appears to be involved in the peripheral and central nervous systems since the 1970's
Studies have been conducted to isolate other factors. Factors isolated so far include brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3),
Leukemia inhibitory factor (LIF), glial cell line-derived neurotrophic factor (GDNF) and the like have been found to block motor neuron death in vivo or in vitro. None of these have been isolated from skeletal muscle, but have been found to be expressed (present) in skeletal muscle. However, NGF, BDNF, NT-
3. Selective disruption of the mouse gene encoding either LIF or GDNF had little effect on motor neuron survival. The effects of multiple gene deletions are not well known, but the results of these gene knockouts suggest that each of these factors is not a true motor neuron survival factor,
It appears that there is still another molecule to identify in skeletal muscle.

[0004] It has also been hypothesized that a group of neurodegenerative diseases is caused by abnormalities of specific neurotrophic factors, and these neurotrophic factors have enormous potential in the treatment of neurodegenerative diseases. However, these factors are attributed to amyotrophic lateral sclerosis (A), one of the motor neuron diseases.
Although the patient was clinically tested on LS) patients, it is currently determined to be ineffective. Therefore, there is a long-awaited expectation that a completely novel factor (or a complex factor group) capable of reducing ALS or other motor neuron diseases will be obtained as a therapeutic agent.

[0005]

DISCLOSURE OF THE INVENTION The present invention has been achieved in view of the above circumstances, and has an object to have a physiological activity such as a survival activity on neurons and to include neurodegenerative diseases. An object of the present invention is to provide a biologically-derived component useful for treatment, prevention, testing, and the like of various diseases.

[0006]

Means for Solving the Problems The inventors of the present invention have made intensive studies to solve the above-mentioned problems, and have found that a component that has a survival effect on motor neurons can be obtained by using a certain series of column chromatography. Was successfully isolated. When we began our research, we expected that the active ingredient would be a protein or polypeptide, similar to the trophic factors known so far, but the newly isolated active ingredient Neither protein nor polypeptide. And the newly isolated active ingredient is probably a single component (eg, RNA derivative, modified,
Complex), a composite component system containing any of the foregoing, or a mixed component system containing any of these. The present invention has been accomplished based on such knowledge.

According to the present invention, the following purification steps were performed.
Neuronal survival sometimes contained in the final fraction
The active ingredient is provided: In step 1, skeletal muscle is
Modified and extracted with 2% (w / v) NaCl aqueous solution
And centrifuged to collect the supernatant;
Acetone is added to the supernatant obtained in Step 1
Was added to a final concentration of 30% (v / v), and centrifuged.
The supernatant is removed by separation and the supernatant is
Acetone is further applied so that the concentration becomes 50% (v / v).
And centrifugation to collect the precipitate;
In this case, the precipitate obtained in Step 2 was reduced to 50% (w /
v) (NH_Four)_TwoSO_FourDissolve in aqueous solution, centrifuge and top
Collecting the supernatant; in step 4, obtained in step 3
The supernatant to an equal volume of phenol: chloroform mixture
(1: 1, v / v), shake at room temperature,
Perform centrifugation and collect the aqueous phase;
The aqueous phase obtained in Step 4 was mixed with Phenyl Sepharose C
Using an L-4B column, 50% (NH_Four)_TwoSO_FourContent 5
After washing with 0 mM HEPES (pH 7.5),
 Elute with HEPES (pH 7.5) and collect effluent
In step 6, the effluent obtained in step 5
To Capcell-PakC₁₈0-25% CH using column_Three
The gradient elution of OH (pH 6.5) was performed and
Fractions are separated at regular intervals and the most active
In step 7, in step 6
The obtained fraction was applied to a DEAE-5PW column.
0-0.5M NaCl (pH 7.5)
Fractionation at predetermined intervals,
Collect the most active fraction; go to step 8
Then, the fraction obtained in step 7 is
l-PakC ₁₈5-19.25% CH using column_ThreeCN
(PH 6.75)
Fractions are separated at intervals, and the most active fraction
Select the final fraction.

The active ingredient for surviving neurons according to the present invention can be obtained from skeletal muscle or other muscle tissue. It is preferable to homogenize the skeletal muscle in step 1 and then degrease using acetone or the like before proceeding to step 2.

[0009] It is speculated that the above-mentioned active component for survival of neurons is probably RNA, but it cannot be determined at this time. However, in any event, at least a portion of the purified neuronal viable active component is RNA or R
It is composed of a part of the molecular structure of NA.

[0010] The surviving active ingredient according to the present invention may exhibit important physiological activity on nerve cells, and may be used for treatment, prevention and diagnosis of diseases in which nerve cells are involved in some form. . In particular, since the survival active ingredient according to the present invention has been confirmed to exhibit survival activity on spinal cord motor neurons, it has been shown that treatment of amyotrophic lateral sclerosis (ALS), a degenerative disease of spinal cord motor neurons, Use for prevention and diagnosis is greatly expected.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION First, using skeletal muscle of chick embryo, we perform a number of purification steps sequentially by trial and error to obtain a bioactive component having a survival activity on spinal cord motor neurons. Obtained. Then, the physicochemical properties as well as the survival activity of this physiologically active component were investigated. In view of the results of such initial experiments, it was speculated that this component would be RNA, an RNA derivative (for example, a modified product, a complex) or a mixture containing any of these, and the purification method performed at the beginning was modified. Then, the same active ingredient was efficiently purified from skeletal muscle of chicken embryo, and the active ingredient obtained by this modified purification method was identified enzymatically.

A. Purification of the active ingredient in the early stage (1) Experimental materials Fertilized eggs of white leghorn were purchased from the general market and incubated at 37 ° C. for 6 to 19 days in a humidified spatula (this was Hamburger Hamilton stage 29).
Or corresponding to the stage 45).

(2) Preparation of Bioassay System A primary culture system of spinal cord neurons is obtained from the above-mentioned 6-day-old embryo of white leghorn fertilized egg, and is used for a biological assay for screening the activity of each fraction obtained in the purification process. did. Six-day-old embryos were used because spontaneous cell death of motor neurons begins to occur from this stage.

Antibiotics (penicillin (10 units / m
1, Meiji), streptomycin (50 μg / ml, Meiji)) and 10% heat-inactivated calf fetal serum (FBS) (Mitsubishi Kasei) added to Dulbecco's modified Eagle's medium (DM).
The spinal cord of a 6-day-old embryo was placed in ice-cooled (EM) (Gibco BRL), and the cells were dissected using scissors under a stereoscopic microscope. It was converted to 0.25% trypsin-containing Ca
37 ° C. in 10 ml of 2 ⁺ -, Mg ²⁺ -free phosphate buffered saline using a stirred water bath (about 70 cycles / min).
For 15 minutes. The reaction of trypsin is
The suspension was stopped by adding 2 ml of heat-inactivated horse serum. After centrifugation at 1,000 rpm for 5 minutes, the supernatant was removed, and 6 ml of DMEM / TIP [ie, transferrin (5 μg / ml, Sigma), insulin (5 μg / ml, Collaborative Res.) Progesterone (0%) was added to the precipitate. .2 μM, Sigma) and DMEM containing the same antibiotics as described above], and the mixture was stirred by repeating aspiration and discharge with a plastic tip for 20 sets to separate cells from each other. The resulting spinal cord neuron suspension is filtered through a nylon mesh (# 150),
For subsequent culture, the cells were diluted with DMEM / TIP medium to 1 × 10 ⁵ cells / ml. Wells (culture dishes) for tissue culture (15 mm diameter, Sumitomo)
Were precoated with polyethyleneimine for at least 4 hours. The precoated plate was rinsed twice with sterile distilled water and DMEM containing antibiotics was added. Isolated spinal cord neurons were seeded at a concentration of 1 ml / well. This concentration corresponded to 200 cells / mm ² .

A test sample was added to the wells on which the cultured cells had been spread as described above, and an assay was performed. In particular,
Transfer three different volumes of muscle extract or fractionated sample to microcentrifuge tubes (Ultrafree C3GV, Millipore
The mixture was sterilized and filtered by using the method described above, and added to three wells for each volume. Then 5% CO ₂ -95
After incubating for 2 days at 37 ° C. in a humidifier in the presence of% air, a predetermined area (895 μm × 1340 μm) was added to each well.
The number of neurons surviving in m) was counted using a phase-contrast microscope (Nikon). One unit of biological activity is
It was defined as the minimum volume of the test sample that survived at least 10% of the initial number of spinal cord neurons.

In order to test the active ingredient finally obtained by a series of purification steps, a cultured cell line containing a large amount of spinal cord motor neurons (motor neuron culture system) was used. This motoneuron culture system was prepared by a slightly modified method described in Arakawa et. Al. Specifically, first, by performing the same operation as the primary culture system for screening, the spinal cord of the 6-day-old embryo was divided, trypsinized, heat-inactivated horse serum was added, and centrifuged. Cell sediment was obtained. The sediment is washed with Ca 2 ⁺ -, M
g ²⁺ -free glucose (4 mg / l) suspended in 4 ml of cold Hank's balanced salt solution (HBSS),
The cells were separated from each other by stirring by repeating 20 sets of suction and discharge with a plastic chip. The obtained spinal cord neuron suspension was applied to a nylon mesh (# 15
6.8% in a glass tube after filtration through 0)
Metrizamide-containing, 25 mM HEPES-added HBSS
(PH 7.5) Laminated on 4 ml, 400 × at 4 ° C.
Centrifuged at g for 15 minutes. The motoneurons contained in the middle layer were diluted with about 5 times the volume of 10% FBS and DMEM with antibiotics (described above) and
Centrifuged at pm for 5 minutes. The resulting cell sediment is D
Diluted with MEM / TIP. According to the procedure for fractionation of metrizamide, large motoneurons can be contained in the cultured cell line at an abundant rate of 83%.

(3) Purification Procedure In order to purify the viable active ingredient, skeletal muscle derived from a 19-day-old chick embryo was used, and each purification step from step 1 to step 9 as shown in Table 1 below was sequentially performed. I went through trial and error.

[0018]

[Table 1]

The 19-day-old embryo was used because a 6-day-old embryo is too small to collect a large amount of skeletal muscle. And, according to our preliminary experiments, among the test muscles from the age of 4 days to 21 days, skeletal muscles derived from 19-day-old embryos and 20-day-old embryos have the strongest biological activity (unit / wet). weigh
t) was shown to have.

First, about 1 kg of skeletal muscle in a total amount was removed from the hind limb of a 19-day-old embryo and immediately frozen in ethanol / dry ice. Thaw skeletal muscle 50g each, 100%
Homogenized in ice-cold acetone to remove lipids. They were centrifuged at 9,000 rpm for 30 minutes at 4 ° C., the precipitate was filtered off and dried (acetone powder).
In step 1, this acetone powder was extracted twice using 2 liters of a 2% (w / v) aqueous NaCl solution. The two extracts were combined.

In step 2, acetone was added to the extract so that the final concentration of the acetone was 30% (v / v), and the supernatant was collected by centrifugation. Acetone was further added to the supernatant so that the final concentration of acetone was 50% (v / v), and the mixture was centrifuged to obtain a precipitate.

In step 3, the precipitate of acetone was dissolved in distilled water. To this solution was added (NH ₄ ) ₂ SO
_{4 The} aqueous solution is adjusted to a final concentration of (NH ₄ ) ₂ SO ₄ of 30%.
(W / v) and the supernatant was collected by centrifugation. The supernatant has a final concentration of (NH ₄ ) ₂ SO ₄ of 5
An aqueous solution of (NH ₄ ) ₂ SO ₄ was further added so that the concentration became 0% (w / v), and the supernatant was collected again by centrifugation. A series of column chromatography from step 4 to step 9 was sequentially performed on the supernatant thus obtained. The columns, equilibration and washing buffers, and elution conditions used are shown below.

Step 4: Phenyl Sepharose C
L-4B (Pharmacia), 50% (w / v) (NH ₄ )
₂ SO ₄ containing washed twice by 50mM HEPES (pH7.5), elution with 50mM HEPES (pH7.5).

Step 5: DEAE-Sepharose C
L-6B (Pharmacia), 50 mM Tris-HC
Equilibration with 1 (pH 7.5), step elution of 0 M, 0.2 M and 0.5 M NaCl.

Step 6: preparative DEAE-5P
W (21.5 mm ID × 150 mm, tow saw),
Equilibration with 50 mM Tris-HCl (pH 7.5), 0-0.5 M gradient elution of NaCl.

Step 7: preparative POROS-5
0PE (10 mm ID × 15 mm, Perseptive
20% containing 50% (w / v) (NH ₄ ) ₂ SO ₄
Equilibration with Tris-HCl (pH 7.5), (N
H ₄₎ 50-0% Gurajuento elution ₂ SO _4.

Step 8: Preparative Capcell-Pak
C ₁₈ (20 mm ID × 250 mm, Shiseido), 10
equilibration with mM CH ₃ COONH ₄ (pH 8.9),
0-20% (v / v) gradient of methanol.

Step 9: Analytical Capcell-Pak
C ₁₈ (4.6 mm ID × 250 mm, Shiseido), 1
0 mM CH ₃ COONH equilibrated by ₄ (pH8.9), 0-20% methanol (v / v) Gurajuento.

Preparative DEAE-5PW, preparat
ive POROS-50PE and preparative Capce
ll-Pak _C18 is a JASCO HPLC system "Gu
lliver "at a flow rate of 5 ml / m
in. Analytical Capcell-Pak C
₁₈ was operated at a flow rate of 1 ml / min by a Hewlett Packard (Series 1100).

Step 4 (Phenyl Sepharose C)
For L-4B), all eluates were used for the next step. In step 5, the flow does not flow at 0.2M,
The spill from was used for the next step. Step 6
From No. to 8, a fraction (fraction fraction) was continuously collected every 5 minutes, and the fraction having the highest activity in the screening assay was used for the next step. For step 9, the fractions were continuously collected every minute and each fraction was screened for activity. 1/1000 capacity (steps 1 to 7) or 1/1000 respectively
100 volumes (steps 8 and 9) of the extracts or fractions were lyophilized, dissolved in 100 μl of DMEM / TIP and subjected to the biological assays described above.

(4) Results The total biological activity of the extract with a 2% aqueous NaCl solution was estimated to be 4,000,000 units (Table 1).
Immediately after the primary cultured spinal cord neurons for screening were seeded on the wells, they were rounded and suspended in the medium. Under these conditions, few neurons survived after two days. However, when the NaCl extract was added to the medium, the neurons continued to survive after 2 days, some of which extended the processes. The NaCl extract also showed survival activity on a cultured cell line containing a large amount of spinal motoneurons (motor neuron culture system). Therefore, we confirmed that components extracted from chicken skeletal muscle using 2% NaCl had a survival effect on motor neurons. However, it was not always possible to use a motoneuron culture system to analyze a large number of fractions fractionated by a series of column chromatography runs in succession. Therefore, we used the primary culture system described above, which is relatively easy to prepare, as an assay system. As shown in Table 1, half of the initial biological activity was found in the supernatant of 50% (NH ₄ ) ₂ SO ₄ . After the supernatant was desalted with phenyl sepharose CL-4B, a two-step elution consisting of 0.2 M and 0.5 M NaCl was performed using DEAE-Sepharose CL-6B. The eluate was prepared by prep using an HPLC system with a linear gradient of 0-0.5 M NaCl.
passed through arative DEAE-5PW. The fractions that retained activity were divided into three groups, designated A, B and C (Table 1). Fraction A is
The elution was performed when the NaCl concentration was 0.25 M or less, the fraction B was obtained when the concentration was about 0.3 M, and the fraction C was obtained when the concentration was 0.35 M or more. Of these, fraction C showed the strongest biological activity and was subjected to the next step. POROS-5
When 0PE column chromatography is performed, the activity becomes 2
There were two fractions C1, C2. Fraction C
Fraction C1, which has 1.4 times higher activity than 2, was applied to a preparative Capcell-Pak _C18 . As a result, only one prominent peak indicating activity was detected. This fraction is used for the final step, analytical Ca.
Run on pcell-Pak _C18 . The elution profile obtained by UV absorption at 254 nm of the eluate obtained in this last step showed a single peak at a retention time of 30 minutes, which contained all the biological activity ( FIG. 1 (a). All the substances eluted between 25 minutes and 35 minutes showed the same UV spectrum as shown in FIG. 2 (b). Its maximum UV absorption was observed at 258 nm. This result indicates that the active ingredient is neither a protein nor a peptide. Even when the muscle extract was purified by the modification method described below, the finally obtained active ingredient was detected by the above-mentioned Capcell-Pak _C18 for analysis only at the same retention time. When this active ingredient was added to a cultured cell line containing a large amount of spinal motor neurons (motor neuron culture system), the motor neurons continued to survive. This result indicates that the purified component exerts a survival activity on spinal motoneurons.

(5) Consideration on Initial Purification Experiment We have succeeded in purifying a component exhibiting a survival activity on spinal motor neurons from chicken skeletal muscle. This component shows a single peak in the column chromatography elution profile (FIG. 2 (a)),
According to the V absorption profile, the components in the fraction were not peptides (FIG. 2 (b)). During the purification process, we tested various columns and solvents for better results. At each step of the purification procedure, we selected the fraction that showed the highest viability at that step and used it for the next step.
In the last step 9 of Table 1, we obtained the fraction containing the active ingredient. The UV absorption has a maximum at 258 nm, a minimum at 230 nm, and a ratio (A ₂₆₀ / A
₂₈₀ ) was 1.92. Therefore, we concluded that it was quite different from peptides. In view of their absorption properties, the active ingredients we have obtained may be nucleic acids or derivatives of nucleic acids.

The purified component had the following characteristics during the purification process. At the (NH ₄ ) ₂ SO ₄ elution step, viable activity was recovered in the supernatant of 50% (NH ₄ ) ₂ SO ₄ . Therefore, it was suggested that the active ingredient contained in the supernatant would have a small molecular size. Using a DEAE-5PW column to elute the fraction with the highest activity (Fraction C) requires a high ionic concentration (> 0.35 M NaCl) in almost neutral (pH 7.5) conditions Was. The concentrations were even higher than required to elute proteins and peptides. In addition, it was observed in additional experiments that components with viable activity were never adsorbed on the cation exchange column. These results suggest that the active fraction contains an acidic component with a strong negative charge. When we started our purification efforts, we believed that the active ingredient could be a peptide or a protein. Therefore, from the above conclusions, we considered that the purified component would be a small size acidic peptide. However, the following results strongly indicated that the active ingredients were not peptides. That is, in this experiment, Capcell
The-PAK C _18, poor hydrophobic than acetonitrile M
Used in combination with MeOH / CH ₃ COONH ₄ system.
In our preliminary experiments performed using the acetonitrile / trifluoroacetic acid system, we found that only weak acetonitrile concentrations (<5%) compared to the solvents used to elute common proteins or peptides. Was recovered. Therefore, it is suggested that the active ingredient has strong hydrophilicity. In addition, we attempted to analyze the amino acid sequence of the purified component by Edoman's digestion method, but did not obtain a signal. Taken together, the purified components are considered to be neither proteins nor peptides, and all of their characteristics are consistent with those of ribonucleic acids.

In the course of the purification, there were some fractions with viable activity. This suggests that skeletal muscle may contain other active molecules other than the components purified in this study. After separation using a preparative DEAE-5PW column, the active ingredient was found in three different fractions (Table 1). Those fractions
The retention time was different not only when performing DEAE-5PW column chromatography, but also when performing POROS-50PE column chromatography. In view of this, it was considered that these three fractions contained completely different active ingredients. Therefore, it is likely that muscle contains not only one kind but also a plurality of components having survival activity. Several proteins, partially purified from chicken or rat skeletal muscle, have been shown to promote motor neuron survival, which differentiate in vitro to induce morphological and acetylcholine (ACh). It is also recognized to cause.

In the course of the purification work, survival activity on spinal cord motor neurons was assayed using a primary culture of spinal cord neurons derived from 6-day-old chick embryos. Since this primary culture system contains not only motor neurons of the spinal cord but also interneurons of the spinal cord in a relatively large amount, the interneurons may have contributed to the activity value. Survival activity was demonstrated in a culture system rich in motor neurons. Therefore, we argue that the components purified in this study should work to survive motoneurons in vitro, and that they may also show survival activity in other neurons, such as interneurons. I concluded that there was.

Hefti et al. Describe neurotrophic factors as "endogenous, which regulates the survival, growth, morphological plasticity of neurons, or the synthesis of proteins to maintain the differentiated functions of neurons." Soluble protein ". Therefore, the components purified by us have non-peptidic characteristics, and thus do not fall under this definition of neurotrophic factor.

B. Purification by Modification Method and Identification of Active Ingredients (1) Experimental Materials As in the earlier experiments described above, fertilized eggs of white leghorn were purchased from the general market and placed in a humidified pan at 37 ° C.
Embryos incubated for 19 days from day 19 were used.

(2) Preparation of Bioassay System The same assay system used in the earlier experiments described above was used. That is, in order to screen the survival activity of each fraction obtained in the purification process, the assay is performed using the same technique using a primary culture system that is relatively easy to prepare but contains a relatively large amount of other neurons such as interneurons. went. The final purified components were assayed by the above-described technique using a cultured cell line containing a large amount of spinal motoneurons (motor neuron culture system). For all samples, 3 concentrations for each of the three concentrations
The test was performed for each well.

(3) ¹ H-NMR spectrum analysis The final active fraction purified by the same method as performed in the initial experiment, ie, λ max by UV was 258 nm
Fraction in which a single peak of
g) was lyophilized and then dissolved in D ₂ O (99.8%, Merck). About this dissolution solution, JNM-α60
0M using a spectrum analyzer (JEOL)
¹ H-NMR measurement was performed at room temperature (27 ° C.) at a Hz.

(4) Modification of Purification Method From the result of ¹ H-NMR spectrum described later, it was predicted that the active ingredient was RNA, its derivative, or a component group containing any of them, so that it was carried out at an early stage. The purification method used was modified as follows.

That is, the skeletal muscle of a 19-day-old chicken embryo was homogenized in acetone, passed through a filter paper, and the acetone powder was recovered. In step 1, this acetone powder was extracted with a 2% (w / v) aqueous NaCl solution and centrifuged at 12,000 × g at 4 ° C. for 30 minutes (the same centrifugation conditions were: Unless otherwise stated, was employed throughout the purification procedure).

In step 2, the obtained supernatant is
Acetone with a final concentration of 30% (v / v) of acetone
And the supernatant was collected by centrifugation. Acetone was further added to the supernatant so that the final concentration of acetone was 50% (v / v), and the mixture was centrifuged to obtain a precipitate.

In step 3, the acetone precipitate was directly dissolved in a 50% (w / v) (NH ₄ ) ₂ SO ₄ aqueous solution, centrifuged, and the supernatant was collected.

In step 4, this (NH ₄ ) ₂ S
The supernatant of the O ₄ aqueous solution was mixed with an equal volume of a phenol: chloroform mixture (1: 1, v / v), shaken at room temperature, centrifuged, and the aqueous layer was removed.

A series of column chromatography from step 5 to step 8 was sequentially performed on the aqueous layer thus obtained. The columns, equilibration and washing buffers, and elution conditions used are shown below.

Step 5: Phenyl Sepharose C
L-4B column (Pharmacia), 50% (NH_Four)_TwoS
O_FourWashing with 50 mM HEPES (pH 7.5)
Twice, dissolution with 50 mM HEPES (pH 7.5)
Out; Step 6: Capcell-PakC₁₈(20 mm ID × 2
50 mm, Shiseido), 10 mM CH_ThreeCOO (NH_Four)
(PH 6.5), 0-25% CH _ThreeOH included
Available 10 mM CH_ThreeCOO (NH_Four) (PH 6.5) lini
To a gradient (8 ml / min., 100 minutes)
Reverse phase HPLC. Partially purified components (PPS)
Obtained by Step 6. See FIG. 4 of the results).

Step 7: DEAE-5PW (21.5
mmI. D. × 150 mm), 50 mM Tris-HCl (p
H7.5), 0-0.5 M NaCl containing 5
Anion exchange HPLC using a linear gradient (5 ml / min., 120 minutes) of 0 mM Tris-HCl (pH 7.5).

Step 8: Capcell-Pak C ₁₈ (20 mm
I. D. × 250 mm), 5% (v / v) 100 mM triethylamine-acetate (pH 6.75) containing CH ₃ CN
(Equilibration buffer A), 5-19.25
Reverse-phase HPL by linear gradient (8 ml / min. / 150 min) of% CH ₃ CN (pH 6.75)
C. This elution was performed from 100% buffer A to 100%
Of buffer B (85 mM triethylamine-acetate (pH 6.75) containing 19.25% (v / v) CH ₃ CN)
This was done by gradually changing the eluate.

Step 5 (Phenyl Sepharose C)
For L-4B), all eluates were used for the next step. For steps 6 to 8, fractions (fraction fractions) were collected continuously every 5 minutes and the fraction with the highest activity in the screening assay was selected. Extracts or fractions from each step were lyophilized, dissolved in DMEM / TIP and subjected to the biological assays described above.

(5) Reactivity of Partially Purified Component (PPS) to RNase A and Proteinase K The active fraction obtained by step 6 (Reverse phase Capcell-Pak C ₁₈ ) has an elution profile (λmax 258n
m) has a biological activity corresponding to the peak of
It contained PS. This PPS fraction was digested with 10 units of RNase A or 4 units of proteinase K as enzyme. One of the aliquots of the PPS fraction (corresponding to 1.66 g of skeletal muscle) received only distilled water and no enzyme (control). Another preparative sample was prepared without any treatment.
C. (Original). Samples and controls treated with RNase A or Proteinase K
Incubated at 37 ° C. for 3 hours. The enzyme-treated sample was incubated with VP-318 (4.6 mm I.D.).
D. × 150mm, Senshpac) and 10m
Equilibrated with M ammonium acetate (pH 6.5) and subjected to reverse phase HPLC (Hewlett Packard series 1100) by gradient elution (1 ml / min.) Of 0-25% (v / v) methanol to remove digestive enzymes. . Then, the survival activity of each sample was assayed using a cultured cell line containing a large amount of spinal motor neurons (motor neuron culture system).

(6) Muscle crude extract from chicken embryo (CM
Preparation of X) Eighteen-day and nineteen-day-old chick embryos were grown at 2% (w / v)
Homogenized in NaCl aqueous solution, 150,000 ×
g, ultracentrifugation at 4 ° C. for 15 minutes to prepare a crude muscle extract (CMX). The supernatant was washed with an equal volume of phenol: chloroform: isoamyl alcohol (25: 2
4: 1, v / v), 19,000 × g, 4 ° C.
For 10 minutes. The upper aqueous layer (corresponding to 0.1 g of skeletal muscle) was collected, and the number of surviving neurons was counted using a cultured cell line containing a large amount of spinal motoneurons (motor neuron culture system), and the survival activity was assayed. did. As described below, the activity of the final fraction of the modified purification
X activity.

(7) Results The ¹ H-NMR spectrum of the component contained in the final fraction purified by the earlier method showed a peak characteristic of the polymer (FIG. 2). Signals resulting from protons of β-D-ribose, purine bases and pyrimidine bases were observed, but 2 ″ -deoxy-β-D-
No signal resulting from ribose 2′H and 2 ″ H was observed (FIG. 2). This result indicated that the purified component was a ribonucleotide (RNA).

Next, the crude muscle extract (CMX) was extracted with phenol / chloroform / isoamyl alcohol and subjected to a biological assay. After extraction of CMX with phenol / chloroform / isoamyl alcohol, the ability to maintain the survival of cultured spinal cord neurons at each test dose was maintained (FIG. 3), indicating that the biologically active component is not a protein. Was done. This fact reinforces that the ¹ H-NMR spectrum results indicated that the purified component was RNA.

Considering that the active ingredient in question is RNA,
An earlier purification procedure was modified to incorporate phenol / chloroform extraction as one of the purification steps.
The fraction finally obtained by the modified purification method has a λmax 258 in the UV elution profile.
nm peak was observed and 38-40% of the initially seeded cells remained alive by screening assay (the same purification was performed independently using skeletal muscle by 2-40%).
Rounds).

The partially purified component (PPS) obtained by this modified purification method was tested for sensitivity to RNase treatment. RNase A treatment completely abolished the biological activity of PPS (FIG. 4). In contrast, treatment with proteinase K had no significant effect on the biological activity of PPS (FIG. 4). These results suggest that the component involved in biological activity in a biological assay using spinal cord neurons is not a protein but an RNA molecule.

Further, the survival activity of the final fraction obtained by the modified purification method and the survival activity of CMX were assayed using a cultured cell line containing a large amount of spinal motoneurons (motor neuron culture system) and compared. . In the above assay system using the motor neuron culture system, the amount of CMX that gives the greatest survival effect corresponds to 10 mg of skeletal muscle, at which amount almost 100% of the neurons survive. On the other hand, when the purified component contained in the fraction obtained by the modified purification method is added to 1 ml of the medium in an amount corresponding to 384 ng of RNA or 240 μg of skeletal muscle, 110 ± 3. 2
% (Mean ± SEM) survival could be maintained.
In addition, 39 ± 4.8% of CMX-treated neurons (mean ±
(SEM) A considerable survival effect was obtained with a quantity of purified components corresponding to 38.4 ng of RNA.

(8) Consideration on Identification of Purified Components Spectral analysis by ¹ H-NMR revealed that a major component of the purified biologically active component was RNA (FIG. 2). Considering the observation that the components we obtained are effectively purified by phenol / chloroform / isoamyl alcohol (FIG. 3),
The initial purification procedure was modified. Enzymatic digestion of the PPS sample obtained by the modified purification method with RNase A and proteinase K confirmed that the biologically active component was RNA (FIG. 4).

The proton NMR spectrum of DNA can be distinguished from the spectrum of RNA based on the proton signal of ribose residues. In DNA, the chemical shift (δ) of the signal resulting from 2′H and 2 ″ H of 2 ″ -deoxy-β-D-ribose is 1.8 to 3.0 p.
pm, but no noticeable signal was detected in this region (FIG. 2). From this result, it was further confirmed that the purified component was RNA and not DNA.

As a result of summarizing all the characteristics of the purified components, we came to the conclusion that it was an RNA strand (polyribonucleotide) and not a monomer of ribonucleotide. For example: 1) Biological activity was lost by RNase A treatment; 2) ¹ H-NMR spectrum showed a broad peak indicating that it was a polymer; 3) This component was insensitive to heat. Stable (96 ° C., 5 minutes), but ribonucleotide monomer is stable to heat treatment; 4) Molecular weight 17,000-47,000
(By gel permeation chromatography).

[0060] The partially purified component (PPS) is
Nuclease (RNase T₁, RNase U _Two, RNase B. cere
us and RNase Phy M)
No sensitivity and relatively large amount to completely inactivate
Of RNase A (10 units, FIG. 4). On the other hand,
When xonuclease digestion is performed, a small amount of enzyme (0.
04 units of nuclease P₁) Effective biological activity
Disappeared. PP for endoribonuclease treatment
The resistance of S is caused by the potential formation inside the RNA molecule.
It may be due to secondary structure. This resistance is
RNase A treatment or a series of applied production processes
To increase the stability of biologically active ingredients against degradation
Would have contributed.

[0061] Initial experiments did not indicate that the biologically active component was RNA. For example, we have observed that the biologically active components contained in CMX significantly reduce activity upon trypsinization. And a similar result is baker's y
est) was also observed when the assay was performed by HPLC elution profile and polyacrylamide gel electrophoresis using the tRNA of (est). However, we believe that this phenomenon
We consider this to be an artifact due to trace amounts of exonuclease present in the commercially available reagent trypsin.

In the modified purification method, the 50% of the biological activity recovered in the immediately preceding step is used in each purification step.
The fraction with more than% remaining was selected and further purified. This indicates that purified RNA accounts for a significant portion of the biologically active components from CMX. Further research may be needed to identify the potential role of this viable active ingredient in ovo during the time of spontaneous cell death of embryonic spinal motoneurons, but small amounts of RNA may be added. The observation that (384 ng RNA / ml) alone can prevent the death of spinal motoneurons will open up new boundaries for RNA as an intercellular signaling molecule.

[0063]

As described above, it is almost certain that the active ingredient according to the present invention contains RNA as a main component, but it cannot be completely determined at present. Whether the nature of the purified component is RNA alone, an RNA derivative, or a complex component system (for example, a complex, a mixture, etc.) containing RNA or a derivative thereof as one of the constituent elements is still unclear. Unknown.

In any case, it was confirmed that the active ingredient according to the present invention showed a survival activity on spinal cord motor neurons in vitro. Therefore, the active ingredient according to the present invention is considered to have a great possibility of having important physiological activity on spinal motoneurons in vivo, and amyotrophic lateral sclerosis, a degenerative disease of spinal motoneurons The use for treatment, prevention, and diagnosis of ALS is greatly expected.

The active ingredient according to the present invention is also important for nerve cells other than spinal cord motor neurons, in particular, for spinal cord neurons other than spinal cord motor neurons (eg, interneurons) and motor neurons other than the spinal cord. And may be used for the treatment, prevention, and diagnosis of diseases other than ALS in which nerve cells are involved in some form.

[Brief description of the drawings]

FIG. 1 shows the UV absorption properties of the final fraction obtained by an initial purification procedure. FIG. 1 (a)
FIG. 7 shows the UV elution profile of Capcell-Pak _C18 analysis and the corresponding viability. FIG. 1 (b)
FIG. 3 is a view showing a UV absorption spectrum of a purified active ingredient.

FIG. 2 shows the final file obtained by an earlier purification procedure.
The active ingredient contained in the fraction
¹H-NMR spectrum (D_TwoO)
You. Each number in the figure is based on the expected chemical shift.
Indicate the determined positions: 1 ', 2', 3 ', 4', 5 ', and
And 5 '' are, respectively, 1′H, 2′H, 3′H of β-D-ribose,
Corresponds to 4′H, 5′H, and 5 ″ H; 5 and 6 are pyrimidines
Correspond to bases 5H and 6H; 2 and 8 correspond to purine bases
Corresponds to 2H and 8H. HOD, Ac, CD _ThreeOD is
H respectively_TwoHOD, ammonium acetate and
Butyl alcohol, these are HPLC elution buffers.
Used as a fa. Chemical shift is parts / mill
Expressed in ion (ppm).

FIG. 3 is a graph showing the recovery of biological activity when extracted from a crude muscle extract (CMX) using a phenol / chloroform / isoamyl alcohol mixture. Within a predetermined rectangular area (895 μm × 1340 μm, 1.2 mm ² )
The number of surviving spinal cord neurons (mean ± SD) was counted. The amount of sample added was represented by an index when the amount corresponding to 10 mg of skeletal muscle was set to 1. The two different assays revealed the following: 1) The biologically active component migrated to the aqueous phase (CMX / PhOh) after extraction with a phenol / chloroform / isoamyl alcohol mixture (P = 0. 25); 2) Biological activity was independent of sample loading (P <0.05).

FIG. 4 is a view showing the result of inactivating a partially purified component (PPS) with an enzyme. PPS is proteinase K
Alternatively, the cells were treated with RNase A, incubated at 37 ° C. for 3 hours, purified by HPLC, and added to cultured spinal cord neuron cells. Number of surviving spinal cord neurons (mean ± SD)
Was counted after 2 days of culture in serum-free medium. The addition amount of the sample was represented by an index when the amount corresponding to 166 mg of skeletal muscle was set to 1. The two different assays revealed the following: 1) there was a significant difference in the effect of the enzyme treatment (P <0.05); 2) significant changes in the results depending on the amount of sample added. (P <0.05). When the amount of sample added was 1/10 equivalent, there was no statistically significant difference between the four samples (P> 0.05), but when the amount of sample added was 1 equivalent, proteinase K and RNase were added. A had a statistically significant difference (P <0.05).

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Yuhei Miyata 5-6-5 Tsukushino, Abiko-shi, Chiba F-term (reference) 4B024 AA01 AA11 BA80 4C087 AA02 AA04 AA05 AA10 BB47 CA20 NA14 ZA01

Claims (4)

[Claims] The final fraction is obtained when the following purification step is performed.
The active ingredient for survival of neurons contained in
In Step 1, skeletal muscle was homogenized and 2% (w
/ V) Extract with aqueous NaCl solution, centrifuge and collect supernatant
Taking; in step 2, the supernatant obtained in step 1
Then, acetone was added to a final concentration of 30% (v /
v) and the supernatant is removed by centrifugation,
In this supernatant, the final concentration of acetone was 50% (v / v).
Add more acetone and centrifuge
Collecting the precipitate; in step 3, obtained in step 2
The precipitated precipitate is washed with 50% (w / v) (NH_Four)_TwoSO_FourWater soluble
Dissolve in solution, centrifuge and collect supernatant; go to step 4
Then, the supernatant obtained in Step 3 is
A mixture of chloroform and chloroform (1: 1, v / v),
Shake at room temperature, centrifuge and collect the aqueous phase;
In step 5, the aqueous phase obtained in step 4 is
50% using Enil Sepharose CL-4B column
(NH_Four)_TwoSO_FourContaining 50 mM HEPES (pH 7.
5) Wash with 50 mM HEPES (pH 7.5)
Collect the effluent by elution;
The effluent obtained in Step 5 is used for Capcell-PakC₁₈Column
0-25% CH using_ThreeGrade of OH (pH 6.5)
Perform fraction elution and separate fractions at predetermined intervals.
And collecting the most active fraction; step
In 7, the fraction obtained in step 6 is
0-0.5M NaCl using EAE-5PW column
(PH 7.5) gradient elution
Fractions are separated by a gap, and the most active
In step 8, obtained in step 7
The collected fraction is transferred to Capcell-PakC ₁₈Using a column
5-19.25% CH_ThreeGrade of CN (pH 6.75)
Perform fraction elution and separate fractions at predetermined intervals.
The most active fraction to the final fraction
Select as 2. The active neuron survival component according to claim 1, wherein at least a part of the active neuron survival component is constituted by RNA or a part of the molecular structure of RNA. 3. An RNA derived from muscle tissue having neuronal survival activity. 4. The R of claim 3, which is derived from skeletal muscle.
NA.