JP4791056B2 - Production method of endogenous antibacterial substances - Google Patents

Description

  The present invention relates to a method for producing endogenous antibacterial substances, especially defensins, which are antibacterial peptides, from panate cells collected from small intestinal crypts of mammals, particularly humans.

  The evolution of multicellular organisms is a history of fighting microorganisms, and many organisms have various means to protect themselves from external enemies. The single layer of the epithelium that forms the gastrointestinal mucosa is the largest place for the host to come into contact with the outside world, and it absorbs nutrients necessary for survival, water, electrolytes, etc., while blocking the invasion of pathogens and toxins that are external enemies. Is functioning as Thus, the gastrointestinal epithelial cells function as a strong first-line barrier that separates the gastrointestinal contents from the host circulatory system. The epithelium that composes the crypts and villi of the small intestinal mucosa is columnar cells, enteroendocrine cells, goblet cells, and paneth cells. It consists of four cell groups.

  From the birth of the host to death, these epithelial cell populations are believed to continue to be supplied from stem cells that are presumed to exist near the base of the crypt. Among these, cells of three lineages other than Panate cells move from the crypt to the direction of the villi (lumen side), and are repeatedly dropped and regenerated every 3 to 4 days. On the other hand, it is known that only Panate cells are located in the most basal part of the small intestine crypt and continue to live there for about 20 to 25 days.

  Paneth cells are granule-secreting epithelial cells that Paneth first reported their existence in the 19th century. He found that these cells exist only in the crypt base of the small intestine and that the function It is reported that it seems to be secreted into the vagina in the tubule. Subsequently, the morphology and biochemical characteristics of Paneth cells have been gradually elucidated using microstructural analysis methods such as electron microscope observation and various histochemical techniques.

For example, observation of panate cells before and after bacterial administration showed microscopically the degranulation of panate cells, and further revealed the Ca ²⁺ kinetics in pancreatic cells in the small intestinal crypts caused by cholinergic stimulation. (Non-Patent Document 1). It is known that the granules secreted by Paneth cells contain lysozyme exhibiting antibacterial activity in vivo, secretory phospholipase A ₂ , and defensins, particularly α-defensins (Non-patent Documents). 2; Patent Documents 1 and 2).

  Among them, α-defensin is an endogenous antibacterial peptide that exhibits strong antibacterial activity, and is a major agent responsible for innate immunity as a biological defense mechanism. Unlike existing antibiotics, resistant bacteria appear. It is known that it exhibits a strong antibacterial, antifungal, antiprotozoal and antiviral activity. Therefore, even though such an endogenous antibacterial substance is useful for the prevention and treatment of various diseases because of its powerful antibacterial action, it can be mass-produced using microorganisms such as Escherichia coli due to its antibacterial activity. There have been disadvantages such as being impossible, and until now, no method has been known for producing such endogenous antibacterial substances in large quantities.

  The present inventors have succeeded in secreting α-defensins by stimulating panate cells already isolated from the crypts of the small intestine with various inducers such as cholinergic substances (Non-patent Document 3). ). Therefore, the practical use of endogenous antibacterial peptides produced in human-type small intestine bioreactors is an extremely important medical issue, and when considering bioreactors for obtaining large quantities of these endogenous antibacterial peptides, Cells that secrete these antibacterial substances need to survive for a long time in an artificial environment.

  However, none of the small intestinal epithelial cells including panate cells have been successfully cultured in the primary, and it is extremely difficult to culture intestinal epithelial cells while preserving their functions. For example, the crypts survive only about 4 hours at 37 ° C. in the medium.

Japanese National Patent Publication No. 8-508165 Japanese Patent No. 3085972 Digestion, 34: 115-121, 1986 FASEB J., 10: 1280-1289, 1996 Nature Immunol., Vol.1, No.2, 113-118 (2000)

  Therefore, in view of the present situation, the present invention can cultivate a small intestinal epithelial cell group containing panate cells for a long period of time, and can produce a large amount of a secretion containing an endogenous antibacterial substance such as α-defensin. It is an object of the present invention to provide a method for producing endogenous antibacterial substances, particularly defensins that are antibacterial peptides.

  In order to solve the above-mentioned problems, the present inventors paid attention to the use of a culture scaffold as a material supplementing a biological matrix function in the construction of a cell culture system. That is, it is generally known that the culture scaffold is extremely important in the construction of a cell culture system that maintains the cell function.

  Based on this concept, the present inventors separated the crypts collected from the small intestine of mammals into individual cells, isolated the Panate cells, and then used the isolated Panate cells as a material supplementing the biological matrix function. By culturing using a culture scaffold, Panate cells survived for a long time, and during that time, they were cultured in the presence of an inducer and succeeded in efficiently secreting endogenous antibacterial substances from the Panate cells. It came to complete.

Therefore, the present invention
(1) Panate cells isolated from crypts collected from the small intestine of a mammal are cultured in the presence of an inducer by using a culture scaffold as a material supplementing the biological matrix function to secrete endogenous antibacterial substances. A method for producing an endogenous antibacterial substance characterized by:
(2) The method for producing an endogenous antibacterial substance according to (1) above, wherein the inducer is a cholinergic substance, a bacterium, or a bacterial antigen;
(3) The cholinergic substance is carbamylcholine, the bacterium is Escherichia coli, Salmonella or Staphylococcus aureus, and the bacterial antigen is lipopolysaccharide, lipotycoic acid or muramyl dipeptide as described in (2) above A method for producing an endogenous antibacterial substance of
(4) The method for producing an endogenous antibacterial substance according to the above (1), wherein the culture scaffold as a material supplementing the biological matrix function is a sheet-like material comprising a porous polymer;
(5) The method for producing an endogenous antibacterial substance according to (4) above, wherein the pore diameter of the porous polymer is 2 to 20 μm;
(6) The method for producing an endogenous antibacterial substance according to any one of (1) to (5) above, wherein the mammal is a human;
(7) The method for producing an endogenous antibacterial substance according to any one of (1) to (6) above, wherein the endogenous antibacterial substance is an antibacterial peptide;
(8) The method for producing an endogenous antibacterial substance according to (7) above, wherein the antibacterial peptide is a defensin;
(9) The method for producing an endogenous antibacterial substance according to (7) above, wherein the antibacterial peptide is α-defensin;
It is.

  The present invention is characterized in that it has found a method capable of culturing while preserving the cell function of a small intestinal epithelial cell group including panate cells, which has been considered impossible until now. This therefore enables the development of bioreactors that produce endogenous antimicrobial substances, in particular endogenous antimicrobial peptides including α-defensins.

  In addition, the production method provided by the present invention enables mass production of endogenous antibacterial substances, particularly endogenous antibacterial peptides including α-defensin, and thus the living intestinal tract is born. It is possible to develop a new treatment method for infectious diseases using an endogenous antibacterial substance that is an effector of innate immunity.

  Furthermore, it leads to clinical application to patients with infectious diseases and refractory inflammatory diseases, and this technology has the advantage that it can be applied to regenerative medicine.

  Endogenous antibacterial substances produced by the production method provided by the present invention include α-Defensin (HD-Human Defensin-5), HD6 (Human Defensin-6), lysozyme (Lysozyme), and secretion. Type phospholipase A2 (Secretory Phospholipase A2; sPLA2) and the like. Among them, α-defensin that is an antibacterial peptide can be preferably produced.

  These endogenous antibacterial substances are substances that activate the forefront innate immune system in organisms ranging from insects to mammals in order to protect themselves from microbial attack in tissues that come into contact with the outside world. The main role of the action is played by endogenous antibacterial peptides such as defensin. Among them, defensin is one of the endogenous antibacterial peptides that have been shown to exist from early on, and is present in intracellular granules of phagocytic cells in blood such as neutrophils and macrophages, and has a killing effect on phagocytosed microorganisms. Involved and contributes to innate immunity.

  Mammalian defensins consist of three families (α-, β-, θ-defensins), and α-defensins are specifically expressed in Paneth cells in the mucosal epithelium of mice and humans. This panate cell-specific α-defensin plays a mucosal protective action by innate immunity against bacterial infection of the intestinal tract.

The method for producing an endogenous antibacterial substance provided by the present invention basically uses panate cells isolated from crypts collected from the small intestine of a mammal, using a culture scaffold as a material supplementing a biological matrix function, In the method for producing an endogenous antibacterial substance, the culture is carried out in the presence of the substance to secrete the endogenous antibacterial substance.
Specifically, Panate cells are isolated from crypts collected from the small intestine of mammals, and the isolated Panate cells are cultured using a culture scaffold as a material that supplements biological matrix functions, and inducers in the Panate cells This is carried out by giving a stimulus by the method to secrete the endogenous antibacterial substance and recovering the endogenous antibacterial substance from the culture solution.
Hereinafter, these methods will be described step by step.

Panate cell isolation :
Panate cells are isolated by isolating panate cells from crypts taken from the mammalian small intestinal mucosa. As such a mammal, a human is preferable.
Collection of crypts from the small intestinal mucosa can be performed according to the method proposed by the present inventors (Non-patent Document 3). For example, the crypts can be isolated and collected by applying uniform vibration to the small intestinal mucosa in the presence of 30 mM EDTA or the like.
The obtained crypts can be separated into columnar cells, gastrointestinal endocrine cells, goblet cells, and panate cells constituting the crypts, for example, by treating with collagenase. This separation may be such that Panate cells are obtained to some extent as single cells, and it is not necessary to completely separate each cell. Furthermore, Panate cells may be appropriately isolated from the culture medium containing these cells using a conventional purification method, or may proceed to the next stage in a state where each cell is mixed.
Therefore, in the production method of the present invention, the isolated “Panate cell” is not necessarily limited to one isolated as a single cell, but includes isolated intestinal epithelial cells containing more Panate cells.

  As a particularly preferred embodiment, a large amount of panate cells can be prepared using a cell sorter (Fluorescence Activated Cell Sorter). For example, an isolated cell fraction obtained from a small intestine crypt is subjected to flow cytometry using a region in which Panate cells are specifically distributed as a function of cell size and internal density. This region accounts for approximately 2-5% of all cells analyzed as isolated cells. Thereby, Panate cells can be selectively isolated from the isolated cell group.

Panate cell culture :
The Panate cells isolated above are cultured. What is necessary is just to select the culture solution in this case by a conventional method. However, a medium that can survive for about one week under the conditions of the present invention is preferred. As will be understood from the examples described later, it is not always necessary to proliferate, and it is important that the Panate cell is a culture system that continues to survive while maintaining its cell function.

In general, it is known that the culture scaffold is extremely important in the construction of a cell culture system that maintains the cell function. Therefore, in culturing Panate cells, it is preferable to use, as a culture scaffold, a material that supplements a biological matrix function that would have a function similar to a crypt where Panate cells continue to survive. It has been found that a sheet-like material made of a porous polymer is preferably used as such a culture scaffold.
That is, it is considered that the Panate cells fit into the pores of the porous polymer, and the Panate cells continue to survive under conditions similar to those in the crypt.

Examples of the porous polymer material used for the culture scaffold include polymers such as polycarbonate, polyacrylate, α-hydroxycarboxylic acid, polycaprolactone, polyhydroxybutyrate, and polyanhydride. Can be preferably used.
Examples of such sheets include commercially available polycarbonate sheets having various pore sizes (for example, Whatman's: Cyclopore / Cyclopore (registered trademark) and Nuclepore / Nuclepore (registered trademark)). Can be used.

  The pore diameter of the porous polymer needs to be about 2 μm to 20 μm in order for Panate cells to survive and be cultured. If this pore size is less than 2 μm, even if it is used as a culture scaffold, Panate cells cannot survive and be cultured, and if it exceeds 20 μm, the function as a culture scaffold is reduced.

Endogenous antimicrobial secretion :
In the panate cell culture described above, the panate cells are stimulated with an inducer to secrete endogenous antibacterial substances including α-defensin, which is an antibacterial peptide.
Such inducers include cholinergic substances (eg, carbamylcholine), bacteria (eg, Escherichia coli, Salmonella, Staphylococcus aureus), bacterial antigens (eg, lipopolysaccharide, lipotycoic acid, muramyl dye). Peptide).
In particular, when human panate cells are used, carbamylcholine (carbacol), bacteria (Salmonella, Escherichia coli, Staphylococcus aureus) and bacterial antigens (lipopolysaccharides) that are cholinergic substances are effective.
The amount of these inducers added is about 1 × 10 ³ CFU / Cell for bacteria such as Salmonella, Escherichia coli, and Staphylococcus aureus, and about 100 ng / mL to 100 μg / mL for bacterial antigens, cholinergic substances ( Carbachol) is preferably about 1 μM to 100 μM.

Recovery of endogenous antibacterial substances :
Thus, the endogenous antibacterial substance secreted from the Panate cell is recovered by culturing the Panate cell by stimulating the inducer, and the recovery recovers the endogenous antibacterial substance from the Panate cell culture medium or its supernatant. Is done. The collection method is not particularly limited. For example, after each culture time, a secretion inducer is added to various Panate cells for stimulation, and then the supernatant is collected after a certain period of time, dialyzed with a cut filter, lyophilized, and then put into physiological saline. It can be obtained by melting. The antibacterial substance obtained as the supernatant may be separated and purified using reverse phase high performance chromatography (HPLC) after passing through dialysis and lyophilization processes.

  Thus, the endogenous antimicrobial substance targeted by the present invention is produced. Among these endogenous antibacterial substances, α-defensin, which is an antibacterial peptide, is a major agent responsible for innate immunity as a biological defense mechanism. Unlike existing antibiotics, resistant bacteria are unlikely to appear. It is useful for the prevention and treatment of various diseases due to its strong antibacterial, antifungal, antiprotozoal and antiviral effects

  EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

Example 1: Panate cell isolation Isolation and recovery of crypts Fresh intestinal mucosa was obtained from human intestinal tract by excision or endoscopic method. Collection of isolated crypts from human intestinal mucosa was performed under informed consent. The method is shown below.
In the presence of 30 mM EDTA, the intestinal mucosa was fractionated continuously at regular intervals of 5 to 10 minutes while applying uniform vibration. In the case of the small intestine, intestinal villi were separated and collected in the fraction immediately after the start of separation, and isolated intestinal crypts were observed in the subsequent fraction. When these were observed under a microscope, the isolated small intestine crypts could be easily distinguished from other cells due to the remarkable contrast of the intracellular granules of Panate cells present in the basal part. Only isolated crypts were collected using a micropipette under a phase contrast microscope. The resulting isolated intestinal crypt is shown in FIG.

2. Isolation of Panate Cells from the Intestinal Crypt Fraction containing a large number of isolated intestinal crypts was extracted with 300 units / mL collagenase IA-S (Sigma, No. C-5894; derived from Clostridium histolyticum) for 37 minutes. By treating at 0 ° C., crypt cells were separated to a single cell, and an isolated cell group was obtained.
This cell fraction is obtained by using a large number of crypts and a small number of villi as materials, and isolated cells from them, and is characterized by no contamination of blood-derived cells. In a single experiment, 5 × 10 ⁶ to 2 × 10 ⁷ isolated intestinal epithelial cells were obtained. In this fractionation, Panate cells were easily distinguished from other cell groups under various microscope observations due to the presence of abundant granules with strong specific contrast. These isolated Panate cells were selectively obtained using a micropipette under a microscope on Petri dishes supplemented with 0.5% agar.

Example 2: Examination of culture system Examination of cell culture medium Live isolated crypts were collected from mouse small intestinal mucosa by the separation method using EDTA described in Example 1 above. In order to determine the optimal conditions for small intestinal crypts and their isolated cell culture, 19 cell culture media were used to examine morphological changes and cell survival.
The crypts obtained from the mouse small intestine were divided into 2.5 × 10 ³ pieces, added to various media, cultured in a CO ₂ incubator at 37 ° C., and the state of the crypts was observed with a phase contrast microscope over time. . Furthermore, the viability of each cell was determined using trypan blue solution.
As a result, it was found that there was a difference in the morphology and survival time of mouse small intestinal crypt cells depending on the medium within one day, and DMEM / F-12 BASE MEDIUM (manufactured by Sigma; Product No. FD-9785) was found to be most preferable. did. Therefore, DMEM / F-12 BASE MEDIUM was used as the basal medium for the culture.

  The composition of DMEM / F-12 BASE MEDIUM is 15 mM HEPES added to a normal DMEM medium. L-glutamine, L-leucine, L-lysine, calcium chloride, magnesium chloride, magnesium sulfate, phenol red, carbonic acid Contains no sodium hydride.

2. Examination of the culture scaffold The influence on the culture result by the difference in the pore size of the porous polymer in the culture scaffold as a biological culture material was examined.
Using isolated cells of crypts obtained from mouse small intestinal mucosa, a sheet of porous polymer having various pore sizes is laid on the bottom of a cell culture plate or a petri dish for culture, and the isolated cells are placed on the sheet In such a state, the cells were cultured using various media, and the survival of the cells was examined. Moreover, the detection of the bactericidal activity of the culture supernatant was also examined by the method according to the examples described later.
The results are summarized in Table 1 below.

  As can be seen from the above results, it was found that cells can survive and be cultured under the condition that the pore size of the porous polymer as the culture scaffold is 3 μm to 20 μm using DMEM / F-12 BASE MEDIUM as the medium. .

Example 3 Confirmation of Optimum Conditions for Culture System for Human Panate Cells Based on the study in Example 2 above, the optimal conditions for the culture system were determined using the human isolated intestinal cell fraction obtained in Example 1. Confirmed.
DMEM / F-12 BASE MEDIUM was used as a medium, and cyclopores having a pore size of 8 μm and 12 μm were used as culture scaffolds as biological tissue culture materials.
As a result, in both the cyclopores having a pore size of 8 μm and 12 μm, an image in which the isolated cells fit into the respective pores was observed.
Such a phenomenon of trapping (insertion) of the isolated cells in the pores has already started to be observed 60 minutes after the start of the culture, indicating that the isolated panate cells are alive in the pores after one week. confirmed. FIG. 1 shows the situation in many cases using 12 μm cyclopores.
Moreover, it was confirmed that many living cells existed after 8 days of culture. The state is shown in FIG. In the figure, fluorescence is a living cell.

Example 4: Measurement of bactericidal activity by stimulation with inducer 5 × 10 ⁵ intestinal epithelial cells containing a large number of isolated panate cells were used as a medium, and DMEM / F-12 BASE MEDIUM was used as a biological tissue culture material. As a culture scaffold, culture was performed using a cyclopore having a pore size of 12 μm, and after 1 day and 7 days of culture, 1 × 10 ³ CFU / cell of Salmonella Typhimurium as an inducer, (2) LPS (lipopolysaccharide), a bacterial antigen, is added at 100 ng / mL, 1 μg / mL and 10 μg / mL, and (3) a physiological concentration of 10 μM cholinergic substance (carbacol) is added to the medium for 30 minutes or 1 Supernatants were collected after time and examined for their antibacterial activity.

The bactericidal activity was measured as follows. That is, a supernatant obtained by dialysis and lyophilization of the collected culture supernatant, and a reaction product obtained by culturing Salmonella Typhimurium phoP-1 1 × 10 ³ CFU / cell at 37 ° C. for 1 hour are added to a nutrient medium (Triptic soy agar ), And cultured overnight at 37 ° C. to count the number of viable bacterial colonies. The results were examined by comparison with the number of surviving bacterial colonies of Salmonella Typhimurium PhoP- not exposed to the supernatant.

  As a result, bactericidal activity against Salmonella Typhimurium phoP- was observed in the culture supernatant after 1 day. The resulting bactericidal activity was observed in all of the supernatants of isolated intestinal epithelial cell cultures stimulated with S. Typhimurium, LPS and cholinergic substances. The results are summarized in Table 2 below.

  In addition, an increase in bactericidal activity was observed depending on the concentration by the addition of LPS. This bactericidal activity was not observed in a medium in which isolated intestinal cells were not cultured. A similar bactericidal activity was also observed in the culture supernatant after 7 days due to cell stimulation.

Example 5: Confirmation of endogenous antibacterial secretion from Paneth cells
Secretion of α-defensin (HD-5), an endogenous antibacterial peptide, in the culture supernatant of small intestinal epithelial cells by stimulation with Salmonella typhimurium (1 × 10 ³ CFU / cell) was examined by Western blotting. The results are shown in FIG.
It can be seen that α-defensin (HD-5) is secreted from panate cell granules by bacterial stimulation, bacterial antigen stimulation or cholinergic stimulation. Secretion of α-defensin (HD-5) was not observed in control supernatants that were not exposed to bacteria.

  As described above, when the production method of the present invention is used, a large number of panate cells are prepared from a large number of isolated intestinal cells, and various inducers (bacteria) are cultured under the culture conditions using the porous polymer as a culture scaffold. By using stimulation, bacterial antigen stimulation, and cholinergic substance stimulation, a system (bioreactor) capable of producing a large amount of endogenous antibacterial substances including active α-defensin (HD5) can be established. it can.

It is a microscope picture which shows the Panate cell culture | cultivated for 1 week using a 12 micrometer cyclopore in Example 3. A state in which Panate cells are vertically placed in the pores is observed from above. 4 is a photomicrograph showing the presence of viable cells after 8 days in culture in Example 3. In the figure, fluorescence is a living cell. It is a figure which shows the western blot of (alpha) -defensin (HD5) which the cultured panate cell in Example 5 secreted.

Claims (6)

A Paneth cell isolated from a crypt collected from the small intestine of a mammal is used as a culture scaffold as a material for supplementing a biological matrix function, using a sheet-like material made of a porous polymer having a pore diameter of 2 to 20 μm , and cholinergic A method for producing an endogenous antibacterial substance, comprising culturing in the presence of an inducer which is a substance, a bacterium or a bacterial antigen, and secreting the endogenous antibacterial substance. 2. The endogenous antibacterial according to claim 1 , wherein the cholinergic substance is carbamylcholine, the bacterium is Escherichia coli, Salmonella or Staphylococcus aureus, and the bacterial antigen is lipopolysaccharide, lipotycoic acid or muramyl dipeptide. A method for producing a chemical substance. The method for producing an endogenous antibacterial substance according to claim 1 or 2 , wherein the mammal is a human. The method for producing an endogenous antibacterial substance according to any one of claims 1 to 3 , wherein the endogenous antibacterial substance is an antibacterial peptide. The method for producing an endogenous antibacterial substance according to claim 4 , wherein the antibacterial peptide is a defensin. The method for producing an endogenous antibacterial substance according to claim 4 , wherein the antibacterial peptide is α-defensin.

Images