WO2007102685A1

WO2007102685A1 - Noua novel in vivo perfusion method for inducing differential signaling in an animal

Info

Publication number: WO2007102685A1
Application number: PCT/KR2007/001085
Authority: WO
Inventors: Kang-Yeol Choi; Soung-Hoo Jeon
Original assignee: Industry-Academic Cooperation Foundation, Yonsei University
Priority date: 2006-03-06
Filing date: 2007-03-06
Publication date: 2007-09-13
Also published as: KR100797908B1; KR20070091458A

Abstract

The present invention relates to a novel in vivo perfusion method capable of obtaining a large amount of proteins together with the most ideal control protein sample, wherein the proteins changed specifically to cell signaling are modified into the most similar form to be present under in vivo conditions. More particularly, the present invention relates to a method for facilitating researches on diseases caused by abnormal cell signaling, or screenings of various drugs and in vivo ligands in the similar form to that present under in vivo conditions. Also, this is an innovative method capable of solving the problems, for example the small amount of produced sample and the controls in its production, in the studies on the direct changes in tissues or cells by various compounds (for example, cell growth factors or medical supplies) which are administered to elucidate a cell signaling mechanism and a physiological phenomenon.

Description

A NOVEL IN VIVO PERFUSION METHOD FOR INDUCING DIFFERENTIAL

SIGNALING IN AN ANIMAL

TECHNICAL FIELD

The present invention relates to a perfusion method for inducing modifications

of proteins specific to cell signaling, and therefore changes in protein-protein binding in

a similar manner to in vivo conditions when signal transducers or compounds (medical

supplies) such as growth factors were treated for a short period, wherein the perfusion

method may be widely used in the field of various studies or applications. More

particularly, the present invention relates to a method for facilitating various researches

and developments, for example the animal researches on various diseases including

tumors caused by abnormal cell signaling, reactions to various drugs (cell growth factors,

cell inhibitory factors, compounds or the like), etc. The method is easy to study and

develop in the most physiologically similar form by administrating growth factors,

cytokines or various drugs into organs, each of which is present in at least a pair in one

individual, for a short period to induce changes in proteins by administration of cell

stimulating factors or low molecular weigh molecules into the organs, each of which is

present in a pair under the same genetic and physiological environments. In particular,

this method may be used to easily obtain a large amount of protein extracts of

experimental groups and a control group, which are modified to different extents according to the presence of direct signaling. Therefore, the method may be effectively used in the field of a variety of applications, particularly the research and development

ofproteome. BACKGROUND ART

Generally, researches on the cell signaling associated to cancers or other diseases

is to analyze the mechanism of oncogenesis that results from the overexpression or loss

of certain proteins by the genetic modification. And, certain cell lines have been

widely used in the studies or researches on the cell signaling mechanism. These

researches are mainly carried out with the cell lines, and used to develop certain disease

occurrence mechanisms or therapeutic agents. However, the researches on the

mechanism using the cell lines have been carried out under artificially defined cell

environments, but the artificially defined cell environments do not directly stand for in vivo environments.

In recent years, many researchers have made ardent attempts using small animals

(mice or rats) in order to satisfy the in vivo conditions. However, it is difficult to

distinguish differences by actual experiments from changes by various factors such as

genetic difference, environmental and growth conditions, etc. because the animal

researches are carried out by employing different individuals within the same animals as

an experimental group and a control group. That is to say, the parameters resulting

from individual specificity may not be eliminated since the same tissue of one animal

individual may not be divided into a control tissue and an experimental group tissue for

use in the experiment. Various kinds of cell growth factors or inhibitory factors are used to elucidate an in vivo cell signaling mechanism, and it has been known that the treatment of a laboratory animal is usually carried out by intraabdominal, intravascular

or subcutaneous administration (Coligan J E et al., Current protocols in immunology, Unit 1 (1991)). However, when the cell growth factors or inhibitory factors are

intraabdominally, intravascularly or subcutaneously administered into animals, the

administered factors may have affects on a target tissue and other tissues, and they also

be destroyed by the in vivo rejection to the administered compounds themselves.

Accordingly, there is required an experimental method designed to solve the above

problems, and, in particular, it is urgent to obtain exact experimental data about specific

reaction to the administered compounds. Also, it is important to solve the problems

caused by differences in genetic traits or physiological conditions among animal

individuals when organs and tissues are taken from the animal individuals as an

experimental group and a control group.

Also, proteomics technologies such as protein chips, two-dimensional

electrophoresis and LCMS/MS have been widely used to develop medical supplies and

to screen medicinal agents. The technologies are useful to develop medical supplies by

screening direct target proteins of the medical supplies through the change of protein

and change in its binding by treatment of the medical supplies and studying a

mechanism that the target proteins remove or attenuate the cause of diseases which

leads to the changes in biological activity of cells or tissues. However, the most

extreme obstacles to the technologies are the use of the samples whose physiological

environments are different from those of the cells that are representative of the

physiological conditions as described above, and the small amount of desired proteins obtained through their cell culture. For example, there are a large number of media

and time to obtain proteins required for proteomic analysis in a cell line. Also, because

the absence of a large-scale incubator leads to performing several repeated experiments, it is difficult to obtain proteins of the same quality due to the differences in the cell

cultures and the treatment of compounds. Accordingly, the inventive in vivo perfusion

method is an epochal and innovative method capable of solving the above problems

caused due to the difference in the genetic traits or physiological conditions among the

animal individuals and obtaining a protein sample of the same quality, which are

required for the systemic large-scale experiments, because the method solves the problems appearing when medical supplies are intraabdominally, intravascularly or

subcutaneously administered into animals, and also obtain an experimental group and a

positive control group from one individual treated with the medical supplies.

DISCLOSURE OF INVENTION

Accordingly, the present invention is designed to solve the problems of the prior

art, and therefore it is an object of the present invention to provide a tissue protein

sample which is useful to induce the direct change in expression of target proteins in

each organ, particularly a liver by the presence or absence and suppression of signal

transductions (cell growth factor, cell inhibitory factors, compounds or the like) to

screen proteins that binds differentially to certain proteins with the specific proteins by

the signalling.

The in vivo perfusion method makes possible to obtain a control sample having the same genetic traits and conditions and obtain a large amount of a desired protein.

In order to accomplish the above objects, the present invention provides a novel in vivo perfusion method including a) incising an abdominal region of an animal to

dividedly obtain a liver tissue, an artery and a vein, the liver tissue being composed of multiple lobes to be perfused; b) inserting a blood vessel catheter into the vein and

fixing the catheter; c) connecting the catheter with a perfusion tube; d) circulating

through the tube a solution to be used as a control group; e) separating one of the

multiple lobes as a control tissue after the perfusion using the solution is completed; and

f) removing the control tissue, transferring the perfusion tube to a solution containing

experimental-group compounds and performing a perfusion operation.

The suitable organ in the present invention includes, but is not limited to, an

organ selected from the group consisting of liver composed of multiple lobes, kidney

present in a pair, adrenal gland and lungs. Also, the catheter of the present invention is preferably fixed with suture (for

example, silks or polydioxanones), but the present invention is not limited thereto.

Also, the solution used as the control group preferably includes one selected

from the group consisting of, but is not limited to, salines (for example, Ringer's

solution, or phosphate-buffered saline (PBS)). Also, the experimental group compound of the present invention preferably

includes one selected from the group consisting of, but is not limited to, growth factors,

medicinal agents and other test compounds.

The cell growth-associated signaling molecules of the present invention

preferably includes Wntl, Wnt2, Wnt3a, Wnt5a, Wnt5b, Wnt7a, Wntl l, epidermal

growth factor (EGF), hepatocyte growth factor (HGF), platelet-derived growth factor

(PDGF), fibroblast growth factor (FGF), transforming growth factor (TGF) and insulin-like growth factor (IGF), and all drugs are preferred if they are used as chemical compounds. The compound is preferably selected from the group consisting of, but is not

limited to, medicinal agents, toxic substances, peptides, proteins, lipids,

monosaccharides, disaccharides, polysaccharides, DNA, RNA and food additives.

The compound of the present invention is more preferably selected from the

group consisting of growth factors, lithium chloride, calcium chloride, tautomycetin and

valproic acid (VPA).

Hereinafter, the present invention will be described.

For the purpose of researches on tumors and various diseases caused by the

abnormal cell signaling, the present invention provides a method for inducing

differential signaling (cell growth factors, cell inhibitory factors, compounds or the like) in organs, particularly a liver which is present in a pair in one animal. This

experiment is related to a perfusion method, and to the development of an

experimental method capable of solving problems that may be caused by

experimental differences, and also differences in genetic traits and or physiological

conditions among individuals, that is, different individuals, when a control group is

obtained using molecules showing a changed aspect which is the most close to the in vivo conditions, the molecules being changed in a signaling (cell growth factors,

cytokines, compounds or the like)-specific or -dependent manner.

In order to obtain a target tissue, one embodiment of the present invention

provides a method including steps of incising an animal; administering cell growth factors or inhibitory factors into a target tissue of the animal; extracting the target tissue to prepare an extracting solution; performing electrophoresis on the extracting solution;

and confirming an in vivo effect of the cell growth factors or the inhibitory factors by observing a differentially changed aspect between the control group and the

experimental group through activation of the target proteins after the electrophoresis.

In one embodiment of the present invention, the organs that are present in a pair,

for example a liver tissue or a kidney tissue, are suitably used as the target tissue, and

they are preferred to determine a signaling effect of the target tissue when the drugs are

administered for a long or short time.

Also, most of the signaling molecules may be used in the present invention, and

the cell growth-associated signaling molecules are preferably Wnt such as Wntl, Wnt2,

Wnt3a, Wnt5a, Wnt5b, Wnt7a, and Wntl l, epidermal growth factor (EGF), hepatocyte

growth factor (HGF), platelet-derived growth factor (PDGF), fibroblast growth factor

(FGF), transforming growth factor (TGF) and insulin-like growth factor (IGF), and all

of the drugs are preferred if they are used as chemical compounds.

The present invention is characterized in that a cell growth factor or an inhibitory

factors used in the conventional animal experiments is not injected intraabdominally,

intravascularly or subcutaneously, but saline is injected into a vein of a target liver tissue

in advance to extract a non-stimulated tissue and a cell growth factor or an inhibitory

factor is injected to extract a stimulated tissue. In particular, the in vivo perfusion of

the present invention may be useful to observe reaction to a signal stimulus in an in vivo level, the signal stimulus being able to appear for a very short period of 1 to 10 minutes,

and to exclude differential effects by variations in one individual since a control tissue and an experimental group tissue may be extracted from the one individual at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of preferred embodiments of the present invention will be more fully described in the following detailed description,

taken accompanying drawings. In the drawings:

FIG. 1 is a schematic view showing a novel perfusion method including steps of

injecting PBS into a liver of a mouse; closing a certain liver vein with a knot for use as a

control group; administering various ligands or chemical compounds including growth

factors into the other liver lobe of a live animal for a short period of 15 to 30 minutes;

and extracting the liver lobes immediately.

FIG. 2 is a diagram showing experimental tools used in the perfusion method of

the present invention.

FIG. 3 a ((D) is a diagram showing a liver, a hepatic vein and an artery of a

mouse which is anesthetized with ethyl ether and incised at an abdominal region for

their perfusion, which will be performed later, FIG. 3b (©) is a diagram showing that a

suture is installed under a hepatic vein that a blood vessel catheter will be inserted for its

perfusion, FIG. 3c ((3)) is a diagram showing that the blood vessel catheter is inserted

into the hepatic vein and tied up and fixed with the previously installed suture, FIG. 3d

(©) is a diagram showing that the blood vessel catheter inserted into the hepatic vein is

connected with a perfusion tube, blood in the liver is washed by circulating about 50 ml of previously prepared saline for 10 minutes using an air-compressor pump, and one of three liver lobes is selected and a suture is installed in a bottom of the selected liver lobe

and obtained as a control group during its perfusion, FIG. 3e ((B)) is a diagram showing

that the previously installed suture is tied up and a control liver tissue is separated if the liver lobe is completely perfused with the saline, wherein the separated tissue is

immediately deep-frozen with liquid nitrogen, and will be used later in experiments.

FIG. 3f (©) is a diagram showing that, after the separated control liver tissue is taken

off, the perfusion tube is transferred to a vial containing 50 ml of an experimental-group

solution (EGF, 20 ng/ml) and the control liver tissue is stimulated with the solution for

10 minutes, and FIG. 3g ((D) is a diagram showing that, after the experimental-group

solution is completed, a liver tissue is separated and its lobes are taken and frozen in the

same manner as the control group.

FIG. 4 is a diagram showing that the frozen mouse liver tissues of the control

group (treated with PBS) and the experimental group (treated with EGF) are pulverized

to obtain tissue extracts, each of the resultant tissue extracts is centrifuged

(fractionation) to obtain a cytoplasmic protein fraction, and the cytoplasmic protein

fraction is subject to 10% SDS-PAGE to separate a target protein, and phosphorylated

Raf-1, MEK, ERK and α -tubulin, which are all associated with activation of certain

proteins in a Ras-ERK signaling system, are subject to western blotting using specific

antibodies.

FIG. 5 is a diagram showing that the perfused mouse liver tissues of the control

group (treated with PBS) and the experimental group (treated with EGF or IGF) are

pulverized to obtain tissue extracts, the resultant tissue extracts are centrifuged (fractionated) to obtain a cytoplasmic protein fraction, and the cytoplasmic protein fraction is subject to 10% SDS-PAGE to separate a target protein, and phosphorylated

ERK, phosphorylated Akt and GSK3b, and α -tubulin are subject to western blotting

using specific antibodies, the phosphorylated ERK and the phosphorylated Akt being associated with activation of certain proteins in a Ras-ERK signaling system and an Akt

signaling system, respectively.

FIG. 6 is a diagram showing that the perfused mouse liver tissues of the control

group (treated with PBS) and the experimental group (treated with EGF or LiCl) are

pulverized to obtain tissue extracts, each of the resultant tissue extracts are divided into

a crude tissue cell (WL), a membrane protein fraction (PM), a cytoplasm fraction (Cy),

and a nucleoprotein fraction (Nu), and each fraction is western-blotted using antibodies

specific to phosphorylated ERK, MEK, Akt and α -tubulin, and an antibody specific to

Histone 1.

FIG. 7 is a diagram showing that a mouse NIH3T3 fibroblast cell line is treated

with LiCl (an increasing concentration of 0-50 mM) for 30 minutes and centrifuged to

collect a cell pellet, and the resultant crude cell extract is western-blotted using

antibodies specific to phosphorylated ERK, MEK, Raf and α -tubulin, and an antibody

specific to Histone 1.

BEST MODES FOR CARRYING OUT THE INVENTION

Hereinafter, non-limiting preferred embodiments of the present invention will be

described in more detail with reference to the accompanying drawings.

Example 1 1) Preparation

Mice and rats used in this experiment may be variously several weeks old, and

surgical tools and blood perfusion tools were prepared. The mice used in the present invention were male and 12 weeks old from the ICR birth, and 50 ml of epidermal growth factor (20 ng/ml EGF, 100ng/ml IGF-I or 20 niM LiCl) was administered to the

mice. The surgical tools had a suitable size for small mice, and their kinds are shown

in FIG. 2.

2) Anesthesia and Surgery

Anesthesia of a mouse was carried out by dipping a gauze in ethyl ether and

putting the gauze and a mouse into a closed ether jar to force the mouse to sleep.

Generally, anesthesia time of the mouse was 3 to 5 minutes, and its heart beat should be

maintained within a normal range in this case. When the mouse was anesthetized

completely, four legs of the mouse were fixed, and skin of the mouse was incised along

a lower part of the ribs with surgical scissors to expose digestive organs. Then, a

hepatic vein and an artery buried in the digestive organs were exposed using a cotton

bud. (FIG. 3a ((D))

3) Blood Perfusion and Injection of Growth Factor

A surgical suture was installed in a lower part of the exposed hepatic vein using

a forceps to tie the hepatic vein (FIG. 3b/©). Subsequently, a blood vessel catheter

was inserted into the hepatic vein, and the previously installed suture was tied to fix the

blood vessel catheter. A perfusion tube was inserted into the fixed blood vessel

catheter, and 50 ml of saline was injected for 10 minutes using an air-compressor pump.

At this time, the artery was immediately burst out to perfuse the artery naturally. At

this time, blood in the liver was replaced with saline, and therefore the liver tissue was changed from dark brown to light brown, indicating that the liver is normally perfused

(FIG. 3c ((H)) and FIG. 3d (©)). A suture was installed in a bottom of a right lobe of

the liver while the liver was perfused with saline, and then a blood vessel passed through the right liver lobe was tied up and extracted as a control group, which will be

used later (FIG. 3e/©). After the right liver lobe was extracted, a perfusion tube was

transferred to a vessel containing epidermal growth factor, and 50 ml of the growth

factor (EGF, 20ng/ml) was injected into the perfusion tube for 10 minutes (FIG. 3f/©).

At this time, air should not enter the perfusion tube, but if a space is filled with the air,

the perfusion may be failed since a solution may not enter the space. If the injection of

the cell growth factor is completed, the other liver tissues were extracted and

immediately frozen in liquid nitrogen to preserve proteins in the liver tissue (FIG. 3g

⁽©⁾⁾• 4) Centrifugation (Fractionation)

The extract of the liver tissue was centrifuged at 4 ^°C at a rotary speed of 800 x

g for 10 minutes to separate a precipitated pellet from a supernatant. The supernatant

was centrifuged at 4 ^°C at a rotary speed of 10,000 x g for one hour to separate

supernatant as a cytoplasmic fraction, and the resultant precipitated pellet was mixed

with an NP-40 buffer, and then centrifuged at 4 ^°C at a rotary speed of 15,000 rpm for

25 minutes. The separated supernatant was a cytoplasmic fraction. The precipitated

pellet separated after the primary centrifugation of the cell extract was mixed with

sucrose, centrifuged at 4 ^°C at a rotary speed of 45,000 rpm for 45 minutes, and then

the resultant supernatant was discarded. The resultant precipitate was mixed with PBS,

centrifuged at 4 ^°C at a rotary speed of 800 x g for 10 minutes, and then the resultant

supernatant was discarded. This procedure was repeated twice. And, the extracted solution was added thereto and kept on ice for 30 minutes. The solution was centrifuged at 4 ⁰C at a rotary speed of 15,000 rpm for 25 minutes to separate a

supernatant as a nucleus fraction.

5) Preparation and Electrophoresis of Liver Tissue Extract

The liver tissue stored in liquid nitrogen was ground into powder using a pestle

and mortar, and mixed with the extracted solution, and the resultant mixture was then

kept at 4 ^°C for 30 minutes to dissolve proteins in the solution. Subsequently, the

mixture was centrifuged at 4 ^°C at a rotary speed of 12,000 rpm for one hour to obtain

a supernatant, and the resultant supernatant was electrophoresed.

Examples 2 and 3; Use of IGF and LiCl as Administered Compounds

Examples 2 and 3 were repeated in the same manner as in Example 1, except

that IGF (100 ng/ml, Example 2) and 20 mM LiCl (Example 3) were used as the

administered compound, respectively, instead of the EGF (20 ng/ml) used in Example 1.

Example 4; Change of Target Proteins in Liver Tissue of Rat by EGF

Example 4 was repeated in the same manner as in Example 1 to obtain a cell

extract, except that a mouse was used as the experimental animal instead of the rat used

in the Example 1, and IGF (100 ng/ml) was used as the administered compound instead

of the EGF (20 ng/ml) used in the Example 1. Then, the resultant cell extract was electrophoresed, and the electrophoresed gel was transferred into a protein transfer membrane and western-blotted using a specific antibody to the target proteins to observe the increased target proteins. As a result, it was observed that a level of the target proteins (p-Raf-1, p-MEK and p-ERK and) in the liver tissue was very significantly increased by the epidermal growth factor (EGF). The data was shown in FIG. 4.

Example 5: Change of Target Proteins in Liver Tissue of Rat by EGF and

IGF

Example 5 was repeated in the same manner as in Example 1 to obtain a cell

extract, except that the mouse was used as the experimental animal, and EGF (20 ng/ml)

or IGF (100 ng/ml) was used as the administered compound. Then, the resultant cell

extract was electrophoresed and western-blotted in the same manner as in Example 4 to

observe the increased target proteins. As a result, it was observed that levels of the

target proteins (p-Raf-1, p-MEK and p-ERK and) in the liver tissue were increased by

the EGF and IGF. The data was shown in FIG. 5.

Example 6: Change of Target Proteins in Mouse Liver Tissue by EGF and

LiCl and Change of Target Proteins by Position of Cells

Example 6 was repeated in the same manner as in Example 1 to obtain a cell

or LiCl (20 mM) was used as the administered compound. Then, the resultant cell

extract was directly used, or cell membrane, cytosol, nucleus and the like were separated from the cell extract to prepare protein samples (it was confirmed that the protein

samples are sufficiently separated using the cytosol marker protein α -tublin and the

nucleus marker protein Histone 1). The cell extract or the protein samples were

electrophoresed and western-blotted in the same manner as in Example 4 to observe the

increased target proteins. As a result, it was observed that ERK and MEK were commonly activated, respectively, by EFG which is a ligand in a Ras-ERK signaling

system, and a LiCl which is a regulatory compound in a Wnt/ β -catenin signaling

system, and the activated ERK was migrated into cell nucleus at an increased level,

whereas the activated MEK was positioned in the cytoplasm. The data was shown in

FIG. 6.

Example 5: Change of Target Proteins in Mouse Fibroblast Cell Line by

LiCl

Mouse fibroblast NIH3T3 cell was treated with LiCl (0, 5, 20, 50 mM) for 15

minutes, and then collected to prepare a cell extract. The resultant cell extract was

electrophoresed and western-blotted to observe the increased target proteins. As a

result, it was observed that levels of the activated ERK and MEK, and Raf-1 were increased in a concentration-dependent manner. The data was shown in FIG. 7.

From the results (FIG. 6, right part) obtained by perfusing an animal liver with LiCl, it

was shown that the activation of the Raf-1, MEK and ERK was increased to an

appropriate level, as described from the results (FIG. 6, left part) obtained by treating

the animal liver with EGF which is a ligand in the Ras-ERK signaling system.

Therefore, the above results were proven through the experimental result in the mouse

fibroblast. Also, the perfusion method of the present invention was confirmed to be

effective since the results obtained by perfusing an animal liver with the ligand or the compound was substantially identical to those obtained in treating the cell line with the

ligand or the compound. INDUSTRIAL APPLICABILITY

As described above, the present invention may solve the problems caused by

mutations within various individuals including genetic factors by preparing tissues of

the control group and the experimental group in one individual at the same time, the

problems causing controversies over animal experiments, and provide an epochal

turning point on the development of medical supplies through the direct screening of

target proteins to the medical supplies as well as the in vivo research method, by

providing the success in the signaling (cell growth factors, cytokines, compounds or the

like) from the in vivo liver tissue.

Claims

What is claimed is:

1. A novel in vivo perfusion method, comprising:

a) incising an abdominal region of an animal to prepare a liver tissue, an artery

and a vein, the liver tissue being composed of multiple lobes to be perfused;

b) inserting a blood vessel catheter into the vein and fixing the catheter;

c) connecting the catheter with a perfusion tube;

d) circulating through the tube a solution to be used as a control group;

e) separating one of the multiple lobes as a control tissue after the perfusion

using the solution is completed; and

f) removing the control tissue, transferring the perfusion tube to a solution

containing experimental-group compounds and performing a perfusion operation.

2. The novel in vivo perfusion method according to claim 1, wherein the

organ is selected from the group consisting of liver, kidney, adrenal gland and lungs.

3. The novel in vivo perfusion method according to claim 1, wherein the catheter is fixed with suture.

4. The novel in vivo perfusion method according to claim 3, wherein the suture is a silk suture or a polydioxanon suture.

5. The novel in vivo perfusion method according to claim 1, wherein the solution used as a control group is saline.

6. The novel in vivo perfusion method according to claim 5, wherein the

saline is a Ringer's solution or a phosphate-buffered saline (PBS).

7. The novel in vivo perfusion method according to claim 1, wherein the

experimental-group compound is selected from the group consisting of a growth factor

and a compound that is used in a cell line.

8. The novel in vivo perfusion method according to claim 7, wherein the

growth factor is selected from the group consisting of Wntl, Wnt2, Wnt3a, Wnt5a,

Wnt5b, Wnt7a, epidermal growth factor (EGF), hepatocyte growth factor (HGF),

platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming

growth factor (TGF) vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF).

9. The novel in vivo perfusion method according to claim 7, wherein the

compound is selected from the group consisting of medicinal agent, inorganic salts,

toxic substances, peptide, protein, lipid, monosaccharide, disaccharide, polysaccharide,

DNA, RNA and food additives.

10. The novel in vivo perfusion method according to claim 7 or 9, wherein the compound is selected from the group consisting of growth factor, lithium chloride, calcium chloride, tautomycetin and valproic acid (VPA).