WO2015133459A1 - Agent for preventing, suppressing or treating corneal disease or corneal damage, cell sheet, cell culture aid, and cell culture method - Google Patents

Abstract

This drug for preventing, suppressing or treating corneal disease or corneal damage is characterized by containing, as an effective ingredient, a polypeptide including a region comprising a polypeptide selected from the group consisting of the following (a)-(c): (a) a polypeptide formed from 70 or more continuous amino acids in the amino acid sequence represented by SEQ ID NO: 4, and including the region 166aa-235aa among SEQ ID NO: 4; (b) a polypeptide formed from 70 or more continuous amino acids in the amino acid sequence represented by SEQ ID NO: 2, and including the region 166aa-235aa in SEQ ID NO: 2; and (c) a polypeptide having 80% or more sequence homology with the polypeptide of (a) or (b).

Description

Preventive, suppressive or therapeutic agent for corneal disease or corneal injury, cell sheet, cell culture aid, and cell culture method

The present invention relates to a preventive, suppressive or therapeutic agent for corneal disease or corneal damage containing semaphorin 3A as an active ingredient. The present invention also relates to a cell sheet cultured in the presence of semaphorin 3A, a cell culture adjuvant containing semaphorin 3A as an active ingredient, and a cell culture method of culturing under semaphorin 3A.
This application claims priority on March 3, 2014 based on Japanese Patent Application No. 2014-041017 filed in Japan, the contents of which are incorporated herein by reference.

The cornea is an avascular transparent tissue located on the outermost surface of the eyeball. In order to obtain a healthy visual function, the cornea must be transparent without scar tissue or blood vessel invasion. Corneal endothelial cells located in the innermost layer of the cornea play an important role in maintaining the transparency of the cornea. Corneal endothelial cells are cells that control the amount of water in the corneal stroma by means of a barrier function and a pump function, and it is thought that humans have little ability to divide in vivo.
It is reported that the density of about 3000 cells per square millimeter at birth usually decreases by 0.5% / year with aging. At this pace, corneal endothelial cells have a life span of 200 years. It will be. However, the rate of corneal endothelium reduction is high because of contact lens wear, various eye surgery, corneal infections, corneal genetic diseases such as Fuchs corneal endothelial degeneration, glaucoma, and uveitis. When about 400 corneal endothelial cells are cut per square millimeter, water control of the corneal stroma cannot be performed by the corneal endothelial cells, so that the entire cornea falls into edema and exhibits frosted glassy turbidity. This condition is called bullous keratopathy. The turbidity of bullous keratopathy is irreversible, and in such a state, the patient presents a very strong visual dysfunction with pain, resulting in social blindness.
Currently, there is no drug treatment targeting the corneal endothelium, and the only method for bullous keratopathy is corneal transplantation such as full-thickness corneal transplantation and corneal endothelial transplantation. Bullous keratopathy is currently the leading cause of corneal transplantation. However, it has been pointed out that full-thickness keratoplasty for bullous keratopathy has a poor prognosis because the graft has a shorter life span than other diseases. In Japan, donor cornea supply is inadequate, and even corneal transplantation, which is the only treatment at the present time, cannot be performed sufficiently, and many patients suffer from impaired visual function due to corneal endothelial dysfunction. I'm in pain. From such a background, it is desired to develop a therapeutic method for the purpose of restoring the function of the corneal endothelium and preventing bullous keratopathy, but there is no current situation.

Human corneal endothelial cells (HCEC) are said not to divide in vivo, and attempts have been made to overcome this.
In recent years, separation of cells having the characteristics of stem cells from mouse corneal stroma has been reported (Non-patent Document 4). The stem cells derived from the neural crest have the ability to differentiate into nerve cells, fat cells and the like by using a differentiation induction medium. It has been reported that tissue stem cells / progenitor cells can be induced to differentiate into corneal endothelial cells by adhesion culture in a medium supplemented with TGFb (Patent Document 4). The presence or absence of a function (for example, a pump function) is not shown.
So far, it has been reported that aggregates (Sphere) obtained by suspension culture of corneal endothelial cells have the properties of corneal endothelial precursor cells (Patent Document 5 and Non-Patent Document 3). However, the aggregate obtained by this method does not express the neural crest stem cell marker p75, which is the origin of the corneal endothelium, and its undifferentiation is unknown.
Corneal endothelial cells are said not to divide in vivo, but can proliferate to some extent in vitro. Attempts have also been made to grow corneal endothelial cells in vitro and use them for treatment (Non-patent Document 1 and Patent Documents 1 to 3). However, in the conventional culture method using serum, when corneal endothelial cells are cultured for a long period of time, the cells change and the corneal endothelial function is lost, and finally the growth is completely stopped. It can only be grown to 7 (Non-Patent Document 2).
In addition to the development of cell infusion treatment using cultured corneal endothelial cells, and technology for proliferating cultured corneal endothelial cells such as gene transfer technology and rho kinase inhibitor instillation treatment, the cell state is poor, only a small amount Due to inability to produce, high technical requirement level, high cost, etc., cultured corneal endothelial cells have not been transplanted and are currently difficult for clinical application.

A typical semaphorin molecule Sema3A whose function in the immune system has been clarified has been energetically analyzed around the world for its function as a guidance factor in the nervous system (Non-patent Document 5).
There have been several reports on the relationship between Sema3A and the cornea in recent years. Non-Patent Document 6 shows that Sema3A is expressed in each of the corneal epithelium, corneal stroma, and corneal endothelium. In addition, it has been reported that expression of Sema3A is enhanced by corneal wound or inflammation (Non-patent Documents 7 and 8).
However, there are no reports to date that have clarified the action and function of Sema3A in the cornea.

International Publication No. 2006/092894 International Publication No. 2010/084970 International Publication No. 2013/012087 International Publication No. 2013/051722 International Publication No. 2011/096593

The present invention has been made in view of the above circumstances, and provides a preventive, suppressive or therapeutic agent having a preventive, suppressive or therapeutic effect on corneal diseases or corneal damage, and promotes good culture of corneal endothelial cells. It is an object of the present invention to provide a cell culture auxiliary agent, a cell sheet containing corneal endothelial cells, and a cell culture method for corneal endothelial cells.

The present inventors have found that semaphorin 3A (Sema3A), which has been conventionally known as a guidance factor in the nervous system, has an excellent proliferation promoting action on corneal endothelial cells, and has completed the present invention. That is, the present invention is as follows.

(1) Prevention or suppression of corneal disease or corneal damage, comprising as an active ingredient a polypeptide comprising a region comprising any polypeptide selected from the group consisting of the following (a) to (c): Or a therapeutic agent.
(a) a polypeptide comprising at least 70 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 4 and comprising a region from 166aa to 235aa in SEQ ID NO: 4,
(b) a polypeptide comprising a region of 166aa to 235aa in SEQ ID NO: 2 consisting of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2,
(c) A polypeptide having a sequence homology of 80% or more with the polypeptide of (a) or (b) (2) The corneal disease is a disorder involving a decrease in the number of corneal endothelial cells or a malfunction (1) ) For preventing, suppressing or treating corneal disease or corneal damage.
(3) It is obtained by culturing corneal endothelial cells in the presence of a polypeptide containing a region consisting of any polypeptide selected from the group consisting of (a) to (c) below, and contains corneal endothelial cells: A cell sheet characterized by.
(a) a polypeptide comprising at least 70 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 4 and comprising a region from 166aa to 235aa in SEQ ID NO: 4,
(b) a polypeptide comprising a region of 166aa to 235aa in SEQ ID NO: 2 consisting of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2,
(c) a polypeptide having 80% or more sequence homology with the polypeptide of (a) or (b) (4) consisting of any one of the following polypeptides selected from the group consisting of (a) to (c) A cell culture aid for corneal endothelial cells, comprising a polypeptide containing a region as an active ingredient.
(a) a polypeptide comprising at least 70 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 4 and comprising a region from 166aa to 235aa in SEQ ID NO: 4,
(b) a polypeptide comprising a region of 166aa to 235aa in SEQ ID NO: 2 consisting of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2,
(c) a polypeptide having a sequence homology of 80% or more with the polypeptide of (a) or (b), (5) any one selected from the group consisting of the following (a) to (c) A cell culture method comprising a step of culturing in the presence of a polypeptide comprising a region comprising a polypeptide.
(a) a polypeptide comprising at least 70 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 4 and comprising a region from 166aa to 235aa in SEQ ID NO: 4,
(b) a polypeptide comprising a region of 166aa to 235aa in SEQ ID NO: 2 consisting of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2,
(c) a polypeptide having 80% or more sequence homology with the polypeptide of (a) or (b)

According to the preventive, suppressive or therapeutic agent for corneal disease or corneal damage of the present invention, it is possible to prevent, suppress or treat corneal disease or corneal damage in vivo which has not been conventionally performed. Moreover, according to the cell culture aid of the present invention, corneal endothelial cells can be cultured well. Furthermore, according to the present invention, a high-quality cell sheet containing corneal endothelial cells and a cell culture method suitable for culturing corneal endothelial cells can be provided.

It is a figure which shows the result of the human corneal endothelial primary cell culture in the culture experiment 1. FIG. It is a figure which shows the result of the human corneal endothelial primary cell culture in the culture experiment 1. FIG. It is a figure which shows the proliferation result of the human corneal endothelial cell in the culture experiment 2-1. It is a figure which shows the proliferation result of the human corneal endothelial cell in the culture experiment 2-2. 6 is a photograph showing the appearance of corneal endothelial cells after 12 hours of culture in cell culture experiment 2-1. 4 is a photograph showing the results of a Wound healing experiment in culture experiment 3. It is a photograph which shows the result of the Wound healing experiment in the culture experiment 4. 5 is a graph showing the speed of wound healing in culture experiment 4. It is a photograph of the anterior eye part of a mouse showing the results of a prevention and / or treatment experiment for corneal oxidative stress. It is the observation photograph of a corneal endothelial cell which shows the result of the prevention and / or treatment experiment with respect to corneal oxidative stress. It is a photograph of the corneal tissue after BrdU immunostaining showing the result of a prevention and / or treatment experiment against corneal oxidative stress.

Hereinafter, preferred examples of the present invention will be described, but the present invention is not limited to these examples. Additions, omissions, substitutions, and other modifications can be made without departing from the spirit of the present invention.
≪Preventive, suppressive or therapeutic agent≫

The preventive, suppressive or therapeutic agent for corneal disease or corneal damage of the present invention contains semaphorin 3A as an active ingredient.

The active ingredient semaphorin 3A may be a natural type or a mutant. Specific examples of natural semaphorin 3A or semaphorin 3A mutant include a region consisting of any of the following polypeptides (a) to (c), and have preventive, suppressive or therapeutic activity for corneal disease or corneal damage: The polypeptide which has can be mentioned.
(a) Consists of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing, and 166aa to 235aa in SEQ ID NO: 4 (meaning "166th amino acid to 235th amino acid") A polypeptide comprising the region of
(b) A polypeptide comprising a region of 166aa to 235aa in SEQ ID NO: 2 consisting of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.
(c) A polypeptide having 80% or more sequence homology with the polypeptide of (a) or (b).

Here, the “prevention, suppression or therapeutic activity of corneal disease or corneal damage” means an activity of improving the symptom of the corneal lesion of corneal disease or corneal damage or preventing exacerbation of the symptom. Whether or not a polypeptide has a preventive, suppressive or therapeutic activity for corneal disease or corneal damage can be examined by in vitro experiments, for example, administration of the polypeptide to corneal endothelial cells and cell proliferation Or it can be examined by promoting migration. The proliferation or migration of corneal endothelial cells can be determined by a Wound healing experiment as shown in <Culture experiment 3> and <Culture experiment 4> in [Example] described later. Alternatively, whether or not the polypeptide has the activity may be examined by an in vivo experiment. For example, the polypeptide is administered to an animal that has developed a corneal disease or corneal damage by instillation, subconjunctival administration, or the like. It can be examined by evaluating whether or not the symptoms of the lesion are improved. As the corneal disease or corneal-damaged animal, for example, a model animal such as a mouse with UV keratitis described in the following examples can be used. This mouse developed ultraviolet keratitis by irradiating the eye with ultraviolet rays. Evaluation of the prevention, suppression, or therapeutic activity of corneal disease or corneal damage using this mouse can be carried out by, for example, applying polymorphism for a certain period of time by instillation and subconjunctival administration to the corneal lesion of the mouse, as specifically described in the following Examples. This can be done by administering the peptide and evaluating the presence or absence of improvement in the symptoms of the lesion. The presence or absence of improvement of symptoms in the conjunctival disease area can be evaluated using, for example, scar, corneal transparency, cell morphology, etc. as an index, as described in the following examples. For example, when the average corneal inflammation score of the keratitis onset group administered with a polypeptide for a certain period is significantly lower than the average corneal inflammation score of the untreated UV keratitis onset group Or, for example, when the average score of the polypeptide administration group is about two-thirds or less, preferably about one-half or less of the average score of the untreated individuals, It can be judged to have preventive, suppressive, or therapeutic activity for disease or corneal damage.

In the present invention, “polypeptide” refers to a molecule formed by peptide bonding of a plurality of (two or more) amino acids, and includes not only high molecular weight molecules having a large number of amino acids but also the number of amino acids. Low molecular weight molecules (oligopeptides) and proteins are also included.

In the present invention, “having an amino acid sequence” means that amino acid residues are arranged in such an order. Thus, for example, “a polypeptide having the amino acid sequence represented by SEQ ID NO: 2” means a Met７７Gly Trp Leu ... (omitted) ... Pro Arg Ser Val amino acid sequence having a size of 771 amino acid residues. Means polypeptide.

The amino acid sequences shown in SEQ ID NOs: 2 and 4 are semaphorin 3A protein sequences (human and chicken, respectively), and are registered in GenBank as accession numbers NM006080 and U02528, respectively. Semaphorins are proteins that have been identified as molecules that guide nerve axon elongation. It is also called collapsin because it was discovered based on the activity of collapsing the growth cone at the tip of the neurite. Semaphorins constitute a secreted or transmembrane protein family, and more than 30 members have been identified to date from invertebrates such as nematodes to vertebrates such as humans. Semaphorin 3A (Sema3A) is a secreted protein member of semaphorin and is a protein molecule known as a repulsive nerve axon guidance molecule. Secreted semaphorins such as Sema3A have three structural domains: a semaphorin (sema) domain of ~ 500aa amino terminus, a C-2 type immunoglobulin (Ig) domain, and a basic terminal domain. It is known that the region necessary for Sema3A to exert physiological activities such as nerve elongation-inhibiting action is a region of 70 amino acids corresponding to the 166th to 235th of the sema domain (Koppel et al., Neuron , Vol. 19, 531-537, 1997). The 70 amino acid region (hereinafter referred to as “70aa region”) is a region of 166aa to 235aa in both SEQ ID NO: 2 and SEQ ID NO: 4. Sema3A has been identified from various animals such as humans, chickens, mice and rats. The homology of these Sema3A is as high as about 85% when compared as a whole protein, and is even higher as about 95% or more when compared only with the above 70 amino acid region. In the chicken Sema3A having the amino acid sequence of SEQ ID NO: 4, the sema domain is 61aa to 567aa, the Ig domain is 592aa to 655aa, and the basic terminus is 726aa to 772aa (Feiner et al., Neuron, Vol.19, 539) -545, 1997). In human Sema3A having the amino acid sequence of SEQ ID NO: 2, the sema domain is 61aa to 567aa, the Ig domain is 591aa to 654aa, and the basic terminal domain is 726aa to 771aa.

The polypeptide (a) is a polypeptide comprising a region containing at least the 70aa region of the chicken Sema3A protein having the amino acid sequence shown in SEQ ID NO: 4. Specifically, from the full length of SEQ ID NO: 4 Or a fragment thereof containing the 70aa region. One example of the polypeptide contained as an active ingredient in the preventive, suppressive or therapeutic agent of the present invention includes a region comprising the polypeptide of (a), and has a preventive, suppressive or therapeutic activity for corneal disease or corneal damage (Hereinafter referred to as “active ingredient polypeptide a” for convenience). The active ingredient polypeptide a may be composed only of the polypeptide of (a) as long as it has a preventive, suppressive or therapeutic activity for corneal disease or corneal damage, and one end of the polypeptide of (a). Alternatively, an amino acid or polypeptide added at both ends may be used. The length of the amino acid sequence constituting the active ingredient polypeptide a is not limited, but is, for example, 70 to 10,000 aa, preferably 70 to 3000 aa, more preferably 70 to 1500 aa.

Specific examples of the active ingredient polypeptide a consisting only of the polypeptide (a) are not particularly limited, and examples thereof include a polypeptide consisting of the entire amino acid sequence shown in SEQ ID NO: 4 (ie, chicken Sema3A protein). . The fact that chicken Sema3A protein has preventive, suppressive or therapeutic activity for corneal disease or corneal damage is as specifically described in the following examples. Further, even if it is a fragment lacking a part of the N-terminal or C-terminal of chicken Sema3A protein, the active ingredient polypeptide a is within the scope of the present invention as long as it has the activity of preventing, suppressing or treating corneal disease or corneal damage. Is included.

In addition, specific examples of the active ingredient polypeptide a in which a polypeptide or the like is added to the polypeptide of (a) are not particularly limited. For example, the 70aa region of SEQ ID NO: 4 corresponds to a secreted semaphorin other than Sema3A. Examples include chimeric proteins constructed by replacing the regions, and polypeptides in which various tags such as His tags, FLAG tags, and myc tags are added to the polypeptide (a). Examples of secretory semaphorins other than Sema3A include, but are not limited to, collapsin 2, collapsin 3, and collapsin 5. The amino acid sequences of collapsin 2, collapsin 3 and collapsin 5 are known as described in Feiner et al., Neuron, vol. 19, 539-545 (1997). As disclosed in the above paper by Koppel et al., The chimeric protein constructed by replacing the region containing at least the 70aa region of Sema3A with the corresponding region of collapsin 2 retains the DRG retraction activity of Sema3A, and is similar to Sema3A. Demonstrate physiological activity. Therefore, such a chimeric protein is also included in the scope of the present invention as an active ingredient polypeptide a because it has a preventive, suppressive or therapeutic activity for corneal diseases or corneal damage. Furthermore, Sema3A lacking the Ig domain also has DRG regression activity (Koppel et al., 1997), but such a polypeptide is also present in the region of Sema3A containing 70aa at the N-terminal side of the Ig domain. A polypeptide to which a basic domain is added is included in the active ingredient polypeptide a and is within the scope of the present invention. The specific examples described above are merely examples, and any amino acid or polypeptide added to the polypeptide of (a) can be used as long as it has preventive, suppressive or therapeutic activity for corneal disease or corneal damage. The active ingredient polypeptide a is included in the scope of the present invention.

The polypeptide of (b) above is a polypeptide comprising a region containing at least the 70aa region of the human Sema3A protein having the amino acid sequence shown in SEQ ID NO: 2, specifically, from the full length of SEQ ID NO: 2. Or a fragment thereof containing the 70aa region. Another example of the polypeptide contained as an active ingredient in the preventive, suppressive or therapeutic agent of the present invention includes a region consisting of the polypeptide (b), and prevents, suppresses or treats corneal disease or corneal damage. A polypeptide having activity (hereinafter referred to as “active ingredient polypeptide b” for convenience). The homology between chicken Sema3A (SEQ ID NO: 4) and human Sema3A (SEQ ID NO: 2) is 86% over the entire protein length, and is even higher at 95% when compared only with the 70aa region important for the physiological activity of Sema3A. As described in the Examples below, since chicken Sema3A can prevent, suppress or treat corneal disease or corneal damage in mice that are not chickens, chicken sema3A can be used to treat corneal diseases other than chickens such as humans and mice. Alternatively, it is possible to prevent, suppress or treat corneal damage. Therefore, the active ingredient polypeptide b such as human Sema3A protein is also the same as the above active ingredient polypeptide a such as chicken Sema3A protein. Prevention, suppression or treatment of corneal diseases such as bullous keratopathy, UV keratitis or corneal damage Useful for the preparation of agents. Similarly to the active ingredient polypeptide a described above, the active ingredient polypeptide b may be composed only of the polypeptide of (b) as long as it has a preventive, suppressive or therapeutic activity for corneal disease or corneal damage, In addition, an amino acid or polypeptide may be added to one or both ends of the polypeptide (b). The length of the amino acid sequence constituting the active ingredient polypeptide b is not limited, but as an example, it is 70 to 10,000 aa, preferably 70 to 3000 aa, more preferably 70 to 1500 aa. Specific examples of such a polypeptide are the same as those of the active ingredient polypeptide a. For example, although not particularly limited, a polypeptide comprising the full length of SEQ ID NO: 2 (ie, human Sema3A protein), corneal disease or corneal damage Examples thereof include fragments thereof having preventive, suppressive or therapeutic activity, chimeric proteins according to the above embodiment with secretory semaphorins other than Sema3A, and polypeptides obtained by adding various tags to the polypeptide (b). These specific examples are also merely illustrative, and any amino acid or polypeptide added to the polypeptide of (b), as long as it has preventive, suppressive or therapeutic activity for corneal disease or corneal damage, The active ingredient polypeptide b is included in the scope of the present invention.

The polypeptide (c) is a polypeptide in which a small number (preferably 1 to several) amino acid residues of the polypeptide (a) or (b) are substituted, deleted and / or inserted. Peptide, which is a polypeptide having homology with the original sequence of 80% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more. In general, a protein may have almost the same function as the original protein even when a small number of amino acid residues in the amino acid sequence of the protein are substituted, deleted, or inserted. Are widely known to those skilled in the art. Therefore, since the region consisting of the polypeptide of (c) can also bring about the physiological activity of the Sema3A protein in the same manner as the 70aa region described above, it includes the region consisting of the polypeptide of (c) above, and the corneal disease or cornea A polypeptide having preventive, suppressive or therapeutic activity for damage (hereinafter referred to as “active ingredient polypeptide c” for convenience) is also prevented, suppressed or treated according to the present invention in the same manner as the active ingredient polypeptides a and b. Useful for the preparation of agents.

Here, the “homology” of the amino acid sequences means that both amino acid sequences are aligned so that the amino acid residues of the two amino acid sequences to be compared match as much as possible, and the number of matched amino acid residues is defined as the total number of amino acid residues. It is the percentage divided by the number. In the above alignment, a gap is appropriately inserted in one or both of the two sequences to be compared as necessary. Such sequence alignment can be performed using a known program such as BLAST, FASTA, CLUSTAL W, and the like. When gaps are inserted, the total number of amino acid residues is the number of residues obtained by counting one gap as one amino acid residue. When the total number of amino acid residues counted in this way is different between the two sequences to be compared, the homology (%) is the total number of amino acid residues in the longer sequence, and the number of amino acid residues matched. Is calculated by dividing. The 20 amino acids constituting the natural protein are neutral amino acids having low side chains (Gly, Ile, Val, Leu, Ala, Met, Pro), and neutral amino acids having hydrophilic side chains (Asn). , Gln, Thr, Ser, Tyr Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), aromatic amino acids (Phe, Tyr, Trp) It is known that the properties of a polypeptide often do not change as long as it can be grouped and the substitution between them. Therefore, when substituting amino acid residues in the polypeptide of (a) or (b) above, by substituting between these groups, corneal disease or cornea in the conjunctiva of the polypeptide used as the active ingredient It is more likely that damage prevention, suppression or therapeutic activity can be maintained.

The polypeptide (c) is specifically a polypeptide having a sequence homology of 80% or more with a polypeptide consisting of the full length of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 (ie, Sema3A protein) Or a polypeptide having a sequence homology of 80% or more with a Sema3A protein fragment containing a 70aa region. Therefore, the active ingredient polypeptide c is a polypeptide consisting only of these polypeptides, or an amino acid or polypeptide added to one or both ends of the polypeptide of (c), and the cornea It is a polypeptide having preventive, suppressive or therapeutic activity for disease or corneal damage. Specific examples thereof are the same as those of the active ingredient polypeptides a and b, and are not particularly limited. For example, the polypeptide having the full length of SEQ ID NO: 2 or 4 (ie, Sema3A protein) has a sequence homology of 80% or more. A polypeptide having a sequence homology of 80% or more with a Sema3A protein fragment having preventive, suppressive or therapeutic activity for corneal disease or corneal damage, having a sequence homology of 80% or more with a region containing 70aa region Examples include chimeric proteins in which a region and other regions of secreted semaphorins other than Sema3A are fused, polypeptides in which various tags are added to the polypeptide of (c), and the like. These specific examples are also merely examples, and any amino acid or polypeptide added to the polypeptide of (c), as long as it has a preventive, suppressive or therapeutic activity for corneal disease or corneal damage, The active ingredient polypeptide c is included in the scope of the present invention.

The active ingredient polypeptide c is particularly preferably a polypeptide whose homology in the 70aa region is higher than the homology of the entire region comprising the polypeptide (c). For example, when the active ingredient polypeptide c is a polypeptide having 80% homology with the polypeptide having the amino acid sequence of SEQ ID NO: 2, those having a homology in the 70aa region of more than 80% are preferable. Polypeptides having homology in the region of 90% or more, more preferably 95% or more, further preferably 98% or more, and more preferably 70aa region are identical. Further, for example, the active ingredient polypeptide c is a chimeric protein in which a region having 80% sequence homology with the sema domain of human Sema3A protein and a region other than the sema domain of secreted semaphorin other than Sema3A are fused. In some cases, the homology in the 70aa region in the sema domain is preferably more than 80%, in particular, the homology in the 70aa region is 90% or more, more preferably 95% or more, more preferably 98% or more, more preferably Chimeric proteins having the same sequence in the 70aa region are preferred.

Among the active ingredient polypeptides a to c described above, in particular, 80% or more, preferably 90% or more, more preferably 95% or more, and further preferably, a polypeptide having the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4. Is preferably a polypeptide having a homology of 98% or more. Among them, as described above, a polypeptide having a homology in the 70aa region higher than the homology of the entire polypeptide is preferable. Most preferably, the polypeptide contained as an active ingredient in the preventive, suppressive or therapeutic agent of the present invention is a polypeptide having the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4.

In general, in a pharmaceutical comprising a polypeptide, a sugar chain or a polyethylene glycol (PEG) chain is added to the polypeptide or at least a part of the amino acids constituting the polypeptide in order to increase the stability of the polypeptide in vivo. Techniques using D-form amino acids or adding Fc domains are widely known and used. By adding sugar chains or PEG chains, or by using D-form amino acids as at least part of the amino acids that make up the polypeptide, it is less susceptible to degradation by peptidases in vivo, reducing the polypeptide in vivo by half. The period becomes longer. The polypeptide used in the present invention may be subjected to these known modifications for in vivo stabilization as long as it has preventive, suppressive or therapeutic activity for corneal disease or corneal damage. The term “polypeptide” in the claims and in the claims is used to include those modified for in vivo stabilization, unless the context clearly indicates otherwise.

Glycosylation to polypeptides is well known, for example, Sato M, Furuike T, Sadamoto R, Fujitani N, Nakahara T, Niikura K, Monde K, Kondo H, Nishimura S., "Glycoinsulins: dendritic sialylolulins displaying a prolonged blood-sugar-lowering activity. ", J Am Chem Soc. 2004 Nov 3; 126 (43): 14013-22, Sato M, Sadamoto R, Niikura K, Monde K, Kondo H, Nishimura S," Site- specific introduction of sialic acid into insulin. ", Angew Chem Int Ed Engl. 2004 Mar 12; 43 (12): 1516-20. The sugar chain can be bound to the N-terminus, C-terminus, or an amino acid therebetween, but is preferably bound to the N-terminus or C-terminus so as not to inhibit the activity of the polypeptide. The number of sugar chains to be added is preferably 1 or 2, and preferably 1. The sugar chain is preferably monosaccharide to tetrasaccharide, more preferably disaccharide or trisaccharide. The sugar chain can be bound to a free amino group or carboxyl group of the polypeptide directly or via a spacer structure such as a methylene chain having about 1 to 10 carbon atoms.

The addition of PEG chains to polypeptides is also well known, e.g. Ullbricht K, Bucha E, Poschel KA, Stein G, Wolf G, Nowak G., "The use of PEG-Hirudin in chronic hemodialysis monitored by the Ecarin Clotting influence on clotting of the extracorporeal system and hemostatic parameters. ", Clin Nephrol. 2006 Mar; 65 (3): 180-90. and Dharap SS, Wang Y, ChandnahandP, Khandare JJ, Qiu B, Gunaseelan S, seeSinko Stein S, Farmanfarmaian A, Minko T., "Tumor-specific targeting of an anticancer drug delivery system by LHRH peptide.", Proc Natl Acad Sci USA. 2005 Sep 6; 102 (36): 12962-7. " The PEG chain can be attached to the N-terminus, C-terminus, or the amino acid between them, and usually one or two PEG chains are attached to a free amino group or carboxyl group on the polypeptide. The molecular weight of the PEG chain is not particularly limited, but is usually 3000 to 7000. Degrees, preferably those of about 5000 is used.

A method in which at least a part of amino acids constituting a polypeptide is D-form is also known, for example, BrennemanneDE, Spong CY, Hauser JM, Abebe D, Pinhasov A, Golian T, Gozes I., "Protective peptides that are orally active and mechanistically nonchiral. ", J Pharmacol Exp Ther. 2004 Jun; 309 (3): 1190-7 and Wilkemeyer MF, Chen SY, Menkari CE, Sulik KK, Charness ME.," Ethanol antagonist peptides: structural specificity without "J. Pharmacol. Exp. Ther. 2004. Jun; 309 (3): 1183-9. A part of the amino acids constituting the polypeptide may be D-form, but since the activity of the polypeptide is not inhibited as much as possible, all of the amino acids constituting the polypeptide are D-form amino acids rather than only a part of the amino acids. It is preferable that

A method for adding an Fc domain to a polypeptide is also well known, and for example, it can be produced using an Fc fusion protein expression vector such as commercially available pFUSEN-Fc (manufactured by InvivoGen).

Semaphorin 3A used as an active ingredient in the present invention can be easily prepared by a conventional method using, for example, a commercially available peptide synthesizer. Moreover, it can prepare easily using a well-known genetic engineering method. For example, a Sema3A gene cDNA is prepared by RT-PCR from RNA extracted from a tissue expressing the Sema3A gene, and the full length or a desired part of the cDNA is incorporated into an expression vector and introduced into a host cell. The desired polypeptide can be obtained. RNA extraction, RT-PCR, cDNA integration into a vector, and introduction of a vector into a host cell can be performed by known methods. Further, vectors and host cells to be used are well known, and various types are commercially available. A method for producing a chimeric protein is also well known in this field. For example, a chimeric protein can be produced by the method described in the above-mentioned paper by Koppel et al. The stabilization modification can also be easily performed by a known method as described in each of the above documents.

Semaphorin 3A, which is an active ingredient in the preventive, suppressive or therapeutic agent of the present invention, is effective in preventing, suppressing or treating corneal diseases or corneal damage.

The cause of the corneal disease is not particularly limited, and may be a corneal disease that occurs locally in the cornea or a corneal disease associated with a disease other than the cornea.
The corneal disease is preferably a corneal endothelial disorder accompanied by a decrease or malfunction of corneal endothelial cells. When a decrease or malfunction of corneal endothelial cells occurs, it becomes impossible to control the water of the corneal stroma by the corneal endothelial cells, which may be an obstacle to not only the corneal endothelium but also the entire cornea. In addition, by enhancing the function of corneal endothelial cells, it can be effective in preventing, suppressing or treating disorders of the entire cornea.
Corneal diseases associated with corneal endothelial cell loss or dysfunction include bullous keratopathy, desquamation syndrome (PE), desquamating keratopathy, pseudosquamous corneal endotheliopathy, corneal herpes congenital hereditary inherited corneal endothelial dystrophy Corneal endothelial degeneration, corneal endothelial degeneration, corneal endothelial dystrophy, corneal endothelial dystrophy, posterior polymorphic corneal dystrophy, iris corneal endothelial dystrophy, corneal endothelial dystrophy Cytomegalovirus corneal endotheliitis, herpes simplex virus corneal endotheliitis), bacterial corneal infection, corneal mycosis, amoeba keratitis, rejection after corneal transplantation, and the like.

External factors that can cause corneal endothelial damage include corneal uveitis, keratitis, and the like. In addition, physical damage that can cause corneal endothelium damage includes trauma, surgical damage, rapid increase in intraocular pressure represented by glaucoma attacks, long-term contact lens wear, degeneration due to aging, oxidative stress due to ultraviolet rays, etc. Etc. Trauma includes birth trauma, traces of infectious diseases, and chemical trauma caused by the intrusion of alkaline or acidic liquids.
Surgery techniques that can cause corneal endothelium damage include, but are not limited to, intraocular surgery, laser therapy, and the like. Specific examples of surgery include corneal transplant surgery, retinal and vitreous surgery, cataract surgery, postoperative treatment for PE patients, glaucoma surgery (filtering surgery, peripheral iridotomy, laser therapy), laser treatment for secondary cataract ( YAG), laser iridotomy (LI), laser anguloplasty (LGP), corneal adhesion dissociation (GSL), laser myopia surgery, excimer laser tissue transpiration, and the like.
Due to corneal endothelial dysfunction caused by the above-mentioned causes, bullous keratopathy is finally caused.

The subject of administration of the preventive, suppressive or therapeutic agent of the present invention is a mammal, and examples thereof include humans, dogs, cats, rabbits and hamsters. In addition, it is considered that the use of semaphorin 3A derived from the same species as the patient to be prevented, suppressed, or treated is considered to have a higher prevention, suppression, or therapeutic effect, and is desirable from the viewpoint of safety in clinical application. . Therefore, for example, when the subject to which the preventive, suppressive or therapeutic agent of the present invention is administered is a human, the prophylactic, suppressive or therapeutic agent containing the above-mentioned active ingredient polypeptide b as an active ingredient is particularly preferable.

The preventive, suppressive or therapeutic agent of the present invention may consist of semaphorin 3A alone, or a pharmaceutically acceptable excipient, stabilizer, preservative, buffer, suitable for each dosage form, Additives such as solubilizers, emulsifiers, diluents, tonicity agents and the like can be mixed as appropriate to prepare a preparation. Formulation methods and usable additives are well known in the field of pharmaceutical preparations, and any method and additive can be used.

The preventive, suppressive or therapeutic agent of the present invention is used by administering to the corneal part to be prevented or the corneal lesion to be treated. Examples of the administration method include local administration (instillation on the cornea, subconjunctival injection, etc.) and the like. Examples of the dosage form include a contact lens type, an eye ointment, an injection, and an eye drop.
In the contact lens type, semaphorin 3A can be contained in a transparent film having a contact lens-shaped spherical surface, and semaphorin 3A released from the film can be administered to the cornea.
The dosage is appropriately selected according to symptoms, age, body weight, administration method and the like, and is not particularly limited. In the case of eye drops, the amount of active ingredient is usually about 6000 to 60000 U per day for the target animal, preferably It is around 4000-40000 U (see International Publication No. 2013/005603).
In the case of the subconjunctival injection amount, the amount of active ingredient is usually about 500 to 5000 U per day, preferably about 500 to 5000 U per day, and is administered once or several times. For example, the amount of subconjunctival injection can be about 0.001 to 10 ng per day, about 0.05 to 5 ng, for example, 2 μl for each eye at a concentration of 10 ng / ml, 100 ng / ml, 300 ng / ml, 1000 ng / ml, preferably Depending on the degree of improvement, it is administered once or several times every day or every other day for several days to several months.
In the case of ophthalmic administration or subconjunctival injection, for example, the daily dosage may be about 1 to 3000 ng, about 20 to 900 ng, about 50 to 900 ng, about 50 to 500 ng, about 80 to 500 ng, for example 10 ng / ml , 100 ng / ml, 300 ng / ml, 1000 ng / ml, 2 μl for each eye, preferably once or every day for several days to several months, or 1 every few days, depending on the degree of symptom improvement It is administered periodically on a daily basis or several times.

The preventive, suppressive or therapeutic agent of the present invention may be used alone or in combination with other preventive, suppressive or therapeutic agents. Other preventive, suppressive or therapeutic agents include antibiotics, anti-inflammatory agents, antiviral agents, cell growth promoters, wound therapeutic agents, extracellular matrix components, vitamins and the like.
Taking the above eye ointment as an example, for example, Sema3A / hyaluronic acid ointment, Sema3A / neomedrol EE ointment, Sema3A / Lindron A ophthalmic ophthalmology, Sema3A / flavitan tongue ointment 0.1% can be exemplified. Sema3A / hyaluronic acid ointment can be used as a preparation for hyaluronic acid ophthalmic surgery. The above-mentioned neomedolol EE ointment is a combination of antibiotics and synthetic corticosteroids and can be expected to have a high anti-inflammatory effect when used in combination with Sema3A. The same applies to the Linderon A ointment. In addition, you may apply by reducing suitably the component of a corticosteroid from the component in neomedolol EE ointment.
The preventive, suppressive or therapeutic agent of the present invention can be used in combination with substances such as ascorbic acid, ascorbic acid derivatives, or salts or hydrates thereof, Rock inhibitors or related substances thereof. As the ascorbic acid derivative, a phosphate ester derivative of ascorbic acid such as ascorbic acid-2-phosphate is preferable.

It is known that the amount of semaphorin 3A in the body is stabilized by a feedback action. Therefore, semaphorin 3A acts gently in vivo. Moreover, since administration of semaphorin 3A can activate mitotic ability without causing a noticeable morphological change of corneal endothelial cells, the preventive, suppressive or therapeutic agent of the present invention has little risk of side effects.

In one embodiment, the present invention provides a method for preventing, suppressing or treating a corneal disease or corneal damage comprising the step of administering semaphorin 3A to a mammal.

In one embodiment, the present invention provides semaphorin 3A for prevention, suppression or treatment of corneal disease or corneal damage.

In one embodiment, the present invention provides the use of semaphorin 3A for producing a preventive, suppressive or therapeutic agent for corneal disease or corneal damage.

≪Cell culture aids≫
The cell culture aid for corneal endothelial cells of the present invention contains semaphorin 3A as an active ingredient. As semaphorin 3A, the same thing as what was shown by above-mentioned << prevention, suppression, or a therapeutic agent >> can be used. As corneal endothelial cells, in addition to corneal endothelial primary cells and corneal endothelial stem cells isolated from the cornea, corneal endothelial cells after subculture of the corneal endothelial cells, corneal endothelial progenitor cells, or any other cell that has been induced to differentiate. It is used to include corneal endothelium-like cells that exhibit the same properties as endothelial cells, corneal endothelial stem cells, corneal endothelial progenitor cells, or corneal endothelial cells. Whether a cell is the corneal endothelial cell can be determined by examining whether the known corneal endothelial cell has at least one of various phenotypic characteristics that are remarkable.
In addition to the semaphorin 3A, the cell culture aid of the present invention is used as a conventionally known cell culture aid such as a nutrient component, growth factor, cell growth factor, differentiation inducing factor, antibacterial agent, antifungal agent and the like. It may further contain components, and may further contain substances such as anti-inflammatory components such as corticosteroid hormones, ascorbic acid, ascorbic acid derivatives or salts or hydrates thereof, Rock inhibitors or related substances thereof. As the ascorbic acid derivative, a phosphate ester derivative of ascorbic acid such as ascorbic acid-2-phosphate is preferable.
The cell culture aid of the present invention may be used by adding an appropriate amount to the culture medium of corneal endothelial cells to be cultured. The cell culture aid contains a medium component and provides the cell culture aid itself as a medium. May be. The concentration of semaphorin 3A contained in the cell culture aid or medium can be appropriately selected according to the type, state, amount, etc. of the target corneal endothelial cells using the adjuvant, but 1 per medium. -3000 ng / mL, preferably 20-900 ng / mL, more preferably 50-900 ng / mL, still more preferably 50-500 ng / mL, and 80-500 ng / mL Particularly preferred is mL.

The medium is not particularly limited as long as it can culture corneal endothelial cells, and can be, for example, a DMEM medium, a BME medium, an IMDM medium, or a mixed medium thereof.
Examples of the above-mentioned medium components include components contained in normal medium for animal cells, nutrients such as glucose, sodium chloride, vitamins and minerals, amino acids, growth factors, cell growth factors, differentiation inducing factors, antibacterial agents, Examples include antifungal agents.
According to the cell culture aid of the present invention, corneal endothelial cells in a state close to the original corneal endothelial cells can be cultured with high productivity.

≪Cell culture method, cell sheet≫
The cell culture method of the present invention includes a step of culturing corneal endothelial cells in the presence of semaphorin 3A. As semaphorin 3A, the same thing as what was shown by above-mentioned << prevention, suppression, or a therapeutic agent >> can be used. As the corneal endothelial cells, the same cells as those shown in the above-mentioned << cell culture adjuvant >> can be used. The presence of semaphorin 3A includes a medium containing semaphorin 3A, and the concentration of semaphorin 3A contained in the medium is appropriately selected according to the type, state, amount, etc. of corneal endothelial cells to be cultured. However, it is preferably 1 to 3000 ng / mL, more preferably 20 to 900 ng / mL, further preferably 50 to 900 ng / mL, more preferably 50 to 500 ng / mL per medium. More preferably, it is particularly preferably 80 to 500 ng / mL.

In the culture, in addition to the semaphorin 3A, it may further contain components used for culturing conventionally known corneal endothelial cells such as nutrient components, growth factors, cell growth factors, differentiation-inducing factors, antibacterial agents and antifungal agents. Good. Further, it can be used in combination with components such as anti-inflammatory components such as corticosteroid hormones, ascorbic acid, ascorbic acid derivatives, salts or hydrates thereof, Rock inhibitors or related substances thereof. As the ascorbic acid derivative, a phosphate ester derivative of ascorbic acid such as ascorbic acid-2-phosphate is preferable.
According to the cell culture method of the present invention, corneal endothelial cells in a state close to the original corneal endothelial cells can be cultured with high productivity.

In one embodiment, the present invention provides corneal endothelial cells obtained by culturing in the presence of semaphorin 3A.

The cell sheet of the present invention is obtained by culturing corneal endothelial cells in the presence of semaphorin 3A, and preferably contains corneal endothelial cells. The cell sheet of the present invention can be obtained by the cell culture method of the present invention. Therefore, in one embodiment, the present invention provides a method for producing a cell sheet, comprising a step of culturing corneal endothelial cells in the presence of semaphorin 3A. The cell sheet of the present invention may be composed only of cells, or may further contain a scaffold such as a collagen gel or a transplant carrier. The phrase “the cell sheet contains corneal endothelial cells” means that the ratio of the corneal endothelial cells in the cells constituting the cell sheet is preferably 10% or more, 30% or more, 50% or more in terms of volume, and 80% or more. Is more preferable, and it is still more preferable that it is 90% or more.
The cell sheet of the present invention is of a high quality that can be suitably used as a corneal tissue for transplantation, and is an extremely useful cell sheet that has never been used.

The culture of corneal endothelial cells contained in the cell sheet can be performed, for example, under conditions of 37 ° C. and 5% CO ₂ . In culturing corneal endothelial cells on a culture substrate, the concentration of corneal endothelial cells in the culture solution can be, for example, in the range of 1 × 10 ³ to 6 × 10 ³ cells / ml. Corneal endothelial cells cultured on the culture substrate adhere to each other to form a cell sheet. The cell sheet can be detached from the culture substrate and used for transplantation. Alternatively, among the culture substrates, a cell culture substrate that can be transplanted in vivo may be used as a transplant carrier, and corneal endothelial cells may be cultured thereon and used for transplantation surgery. The culture period may be appropriately determined depending on the degree of cell proliferation, but may be about 1 to 3 weeks. Transplantation can be performed by the same method as conventional donor-derived corneal transplantation.
The culture substrate is not particularly limited as long as it is for cell culture. For example, polymer materials derived from natural products such as fibronectin, collagen, gelatin, hyaluronic acid, alginic acid, laminin, polystyrene, polyester, polyethylene, polypropylene, etc. Synthetic polymer materials, biodegradable polymer materials such as polylactic acid and polyglycolic acid, inorganic materials such as glass, quartz and hydroxyapatite, and amniotic membrane.
The corneal endothelial cell density in the cell sheet is not particularly limited as long as the cell sheet can be formed, and the corneal endothelial cell density is in the range of 1.0 × 10 ⁴ to 5.0 × 10 ⁶ cells / cm ² . It is preferable to be in the range of 1.0 × 10 ⁵ to 1.0 × 10 ⁶ cells / cm ² . It is preferable that the corneal endothelial cells have a density that allows them to function well in the transplantation eye.
The cell sheet can be suitably used for corneal transplantation. The thickness of the cell sheet is not particularly limited, but is preferably a thickness that can be used as a graft. Therefore, the thickness of the cell sheet is preferably about the same as the thickness of the cornea, and more preferably about the same as the thickness of the corneal endothelium layer. More specifically, the thickness of the cell sheet is preferably in the range of 10 to 200 μm, preferably in the range of 10 to 100 μm, and preferably in the range of 15 to 50 μm.

Next, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples.

≪in vitro≫
Sema3A used in the following in vitro experiments is Recombinant Human Semaphorin 3A / Fc Chimera (R & D Systems).
<Culture experiment 1>
Human corneal endothelial cells were cultured in the presence of Sema3A.
(Sema3A group)
150 (7.5 / cm ² ) corneal endothelial primary cells were seeded on the bottom surface of a well containing DMEM Low Glucose medium containing Sema3A and cultured in a 37 ° C. incubator for 25 days. The medium was made to contain Sema3A at a concentration of 100 ng / mL. Corneal endothelial primary cells were obtained from Sight Life or Rocky Mountain Lions Eye Bank.
(Control group)
Human corneal endothelial cells were cultured for 25 days in the same manner as in the Sema3A group except that a medium containing no Sema3A was used.

After culturing for 25 days, the number of cells was counted using a hemocytometer. The results are shown in FIGS. 1A and 1B. The number of cells after 25 days in the Sema3A group was significantly higher than the number of cells in the control group. This shows that Sema3A has the effect of promoting the proliferation of corneal endothelial cells. Since the cell growth rate is within the range of normal cell growth rate, high safety was shown.

<Culture experiment 2-1>
The effects of corneal endothelial cell culture adjuvant and Sema3A, which are conventionally used, on human corneal endothelial cells were compared.
(Sema3A-10 group)
Corneal endothelial cells after passage 3 to 4 times at a cell density of 4000 / cm ² were seeded on the bottom of a 6-well plate containing 10 ng Sema3A per mL and containing DMEM Low Glucose medium, and cultured in a 37 ° C. incubator.
(Sema3A-100 group)
The culture was performed in the same manner as described above (Sema3A-10 group) except that a medium containing 100 ng of Sema3A per mL was used.
(Sema3A-1000 group)
The culture was performed in the same manner as described above (Sema3A-10 group) except that a medium containing 1000 ng of Sema3A per mL was used.
(ROCK inhibitor group)
In place of Sema3A, culturing was carried out in the same manner as described above (Sema3A-10 group) except that 10 μM ROCK inhibitor (Y-27632) per medium was used as a cell culture auxiliary.
(ASC group)
The culture was performed in the same manner as described above (Sema3A-10 group) except that 0.3 mM ascorbic acid-2-phosphate per medium was used as a cell culture auxiliary in place of Sema3A.
(Control group)
The culture was performed in the same manner as in the above (Sema3A-10 group) except that medium containing no Sema3A and other cell culture aids was used.

The cell growth-promoting effect on human corneal endothelial cells was compared between each culture aid and Sema3A. For the control group, ASC group, Sema3A-10 cage group, Sema3A-100 cage group, Sema3A-1000 cage group, and Rock inhibitor group, the number of cells at 3 days after the start of culture and after 6 days from the start of culture, And measured.

The results are shown in FIG. At any time point 3 days after the start of culture and 6 days after the start of culture, the growth rate of corneal endothelial cells was the same as that of the control group in the Rock inhibitor group and Sema3A-10- group. On the other hand, in the ascorbic acid-2-phosphate group, the Sema3A-100 group, and the Sema3A-1000 group, the proliferation rate of corneal endothelial cells was remarkably improved as compared with the control group.

<Culture experiment 2-2>
The cell growth promoting effect on human corneal endothelial cells was compared between a conventionally used corneal endothelial cell culture adjuvant and Sema3A.
(Sema3A-100 group)
Corneal endothelial cells after passage 3-4 times at a cell density of 4000 / cm ² were seeded on the bottom of a 12-well plate containing DMEM Low Glucose medium containing 100 ng of Sema3A per mL, and cultured in a 37 ° C. incubator.
(Sema3A-300 group)
The culture was performed in the same manner as described above (Sema3A 100 group) except that a medium containing 300 ng of Sema3A per mL was used.
(Sema3A-1000 group)
The culture was performed in the same manner as described above (Sema3A-100 group) except that a medium containing 1000 ng of Sema3A per mL was used.
(ASC group)
Culture was performed in the same manner as described above (Sema3A-100 group) except that 0.3 mM ascorbic acid-2-phosphate per medium was used instead of Sema3A as a cell culture aid.
(Sema3A-100 + ASC group)
The culture was performed in the same manner as described above (Sema3A-100 group), except that medium containing 100 ng Sema3A and 0.3 mM ascorbic acid-2-phosphate per mL was used.
(Sema3A-1000 + ASC group)
The culture was performed in the same manner as described above (Sema3A-100 group) except that a medium containing 1000 ng of Sema3A and 0.3 mM of ascorbic acid-2-phosphate was used.
(Control group)
The culture was performed in the same manner as in the above (Sema3A-100 group) except that Sema3A and other medium not containing cell culture auxiliary were used.

For the Sema3A-100 group, the Sema3A-300 group, the Sema3A-1000 group, the ASC group, the Sema3A-100 + ASC group, the Sema3A-1000 + ASC group, and the control group, the cell count at the time point 3 days after the start of culture (12 well plate) It measured using the board.

The results are shown in FIG. In each case of using Sema3A, ascorbic acid-2-phosphate, Sema3A and ascorbic acid-2-phosphate, the number of cells was remarkably increased as compared with the control group.

<Check cell status>
Next, FIG. 4 shows the state of corneal endothelial cells after 12 hours of culture in the control group, ASC group, Sema3A-100 group, and Rock inhibitor group in <Cell culture experiment 2-1>.
In the Rock inhibitor group and the ASC group, the morphology and size of corneal endothelial cells were irregular, and many cells forming pseudopods were also observed. Moreover, the tight junction formation state and cell adhesion state were somewhat poor.
On the other hand, in the Sema3A-100 group, the morphology and size of the corneal endothelial cells were paving stones and excellent in regularity, and there were few cells forming pseudo feet and the cell morphology was also good. Moreover, the adhesion state between cells was also good.
The corneal endothelial cell morphology of the Sema3A-100 group showed a morphology closest to the original corneal endothelial cell state as compared with the group using the conventionally used corneal endothelial cell culture auxiliary agent examined this time. . In addition, since the tight junction is well formed, it is presumed that the barrier function and the pump function, which are important functions of the corneal endothelial cell, are also excellent.

From the above, it is possible to improve the growth rate of human corneal endothelial cells by administering ascorbic acid-2-phosphate, but the cell state produced thereby is superior when Sema3A is administered. It has been found. By using Sema3A, it is possible to culture human corneal endothelial cells faster in a state close to the original corneal endothelial cells. Moreover, it was shown that a high-quality cell sheet of human corneal endothelial cells can be obtained with high productivity by culturing human corneal endothelial cells in the presence of Sema3A.

<Culture experiment 3>
Wound healing experiment of human corneal endothelial cells in the presence of Sema3A was performed.
(Sema3A group)
Corneal endothelial cells after 3 to 4 passages were seeded on the bottom of a well containing DMEM Low Glucose medium containing Sema3A at a cell density of 4000 / cm ² and cultured in a 37 ° C. incubator for 5 days. The medium was made to contain Sema3A at a concentration of 100 ng / ml per mL. Cells were scraped in a circular shape from the bottom of the well to expose the bottom (FIG. 5 (Wound healing day 0, Ctrl)). Thereafter, the culture was continued, and the state of the cells 3 days after the bottom surface was exposed was observed.
(Control group)
Culturing was performed in the same manner as in the above (Sema3A group) except that medium containing no Sema3A and other cell culture aids was used.

The state of human corneal endothelial cells in the control group is shown in FIG. 5 (Wound healing day 3, Ctrl). As shown in FIG. 5 (Wound healing day 3, Ctrl), cells were scattered sparsely in the region where the bottom surface was exposed in a circular shape, and the bottom surface remained exposed particularly in the central part of the region. .
The state of human corneal endothelial cells of the Sema3A group is shown in FIG. 5 (Wound healing day 3, Sema3A). As shown in FIG. 5 (Wound healing day 3, Sema3A), the entire region with the bottom exposed in a circle was covered with human corneal endothelial cells.
From this, it became clear that proliferation and migration of human corneal endothelial cells were promoted by using Sema3A.

<Culture experiment 4>
Furthermore, a comparative experiment of wound healing of human corneal endothelial cells was performed with corneal endothelial cell culture adjuvant and Sema3A.
(Sema3A group)
Corneal endothelial cells after 3 to 4 passages were seeded on the bottom of wells containing DEMA Low Glucose medium containing Sema3A at a cell density of 4000 / cm ² and cultured in a 37 ° C. incubator for 24 hours. The medium was made to contain Sema3A at a concentration of 100 ng / ml per mL. After culturing, the cells were scraped off from the bottom of the well to expose the bottom (FIG. 6 (Wound healing)). Thereafter, the culture was continued, and the state of the cells 24 hours after the bottom surface was exposed was observed.
(ROCK inhibitor group)
In place of Sema3A, the culture was performed in the same manner as in the above (Sema3A group) except that 10 μM ROCK inhibitor (Y-27632) per medium was used as a cell culture auxiliary.
(ASC group)
The culture was carried out in the same manner as in the above (Sema3A group) except that 0.3 mM ascorbic acid-2-phosphate per medium was used as a cell culture auxiliary in place of Sema3A.
(Sema3A + ASC group)
The culture was performed in the same manner as in the above (Sema3A group), except that a medium containing 100 ng Sema3A and 0.3 mM ascorbic acid-2-phosphate per mL was used. (Control group)
The culture was performed in the same manner as in the above (Sema3A group) except that Sema3A and other mediums containing no cyst culture auxiliary were used.

The state of human corneal endothelial cells in the control group is shown in FIG. 6 (Ctrl). The cell density was low in the region where the bottom surface was exposed in a band shape, and the bottom surface remained exposed particularly in the central portion of the region.
The appearance of human corneal endothelial cells in the ROCK inhibitor group is shown in FIG. 6 (ROCK inhibitor). The cell density was low in the region where the bottom surface was exposed in a band shape, and the bottom surface remained exposed particularly in the central portion of the region.
A state of human corneal endothelial cells of the ASC group is shown in FIG. 6 (ASC). The entire region where the bottom surface was exposed in a band shape was covered with human corneal endothelial cells. However, there were variations in cell morphology and size.
The state of human corneal endothelial cells of the Sema3A group is shown in FIG. 6 (Sema3A). The entire region where the bottom surface was exposed in a band shape was covered with human corneal endothelial cells. Cell morphology and size were uniform.
The state of human corneal endothelial cells of the Sema3A + ASC group is shown in FIG. 6 (Sema3A + ASC). The entire region where the bottom surface was exposed in a band shape was covered with human corneal endothelial cells. Cell morphology and size were uniform.

The speed of wound healing in each group is shown in FIG. The vertical axis of the graph of FIG. 7 is the exposed area of the bottom surface in the region exposed in a strip shape. The initial exposed area is shown as 100%, and the state where the entire region is covered with cells is 0%.
From the graph of FIG. 7, it was revealed that the use of Sema3A, ascorbic acid-2-phosphate, and Sema3A and ascorbic acid-2-phosphate promoted proliferation and migration of human corneal endothelial cells. When the conventional cultured cell adjuvant and Sema3A were compared, the wound healing speed of Sema3A was the most excellent.

≪in vivo≫
Sema3A used in the following in vivo experiments is Recombinant Mouse Semaphorin 3A / Fc Chimera (R & D Systems).
<Experiment for prevention and / or treatment of UV keratitis>
(Sema3A administration group)
Experiments were performed using C57BL6 mice (white, 4-week-old male, each group N = 8) as the target animals. In order to induce UV corneal inflammation, UV-A 3.9 J was applied once a day to the eyes of mice after anesthesia with a UV lamp (5 times, 0, 2, 4, 6 and 8 days in total). Irradiated. After each UV-A irradiation, 2 μl of PBS containing Sema3A (100 ng / ml or 300 ng / ml) was injected subconjunctivally. On the 10th day, anterior eye photography and eyeball extraction were performed.
(Control group)
The experiment was conducted in the same manner as the Sema3A administration group except that PBS without Sema3A was injected subconjunctivally.

<Confirmation of corneal condition>
A photograph of the anterior segment of the mouse 10 days after the start of the experiment is shown in FIG. In the control group, formation of new blood vessels was confirmed, and the scar area was> 100 ng / ml group. On the other hand, in the Sema3A administration group, formation of new blood vessels and scars were not confirmed, and the corneal transparency was also good.

Corneal tissue was stained by the Whole mount method.
First, alizarin red staining was performed to observe corneal endothelial cells. A photograph is shown in FIG. In the control group, formation of new blood vessels and cell loss were confirmed. In the Sema3A administration group, the state of the cell arrangement was good. Little formation of new blood vessels and cell loss were observed.

Next, cell division in the tissue was detected by bromodeoxyuridine (BrdU) immunostaining. The staining results are shown in FIG. When comparing the portion where inflammation was thought to be induced by UV irradiation (the inner portion of the broken line in FIG. 10), a clear fluorescent signal was observed in the corneal tissue of the Sema3A administration group compared to the control group, and cell proliferation was confirmed. .
Similarly, as a result of nuclear staining with DAPI, a clear fluorescent signal was observed in the corneal tissue of the Sema3A administration group as compared with the control group, and the repair of the corneal endothelial tissue was confirmed.

From the above results, it was suggested that Sema3A has a function of promoting cell proliferation in the cornea and repairing damage to the cornea due to ultraviolet rays. It was shown that the prevention and / or treatment of ultraviolet keratitis was performed by Sema3A administration in vivo.

The preventive, suppressive or therapeutic agent, cell sheet, cell culture aid, and cell culture method according to the present invention can be widely applied to the fields of medicine, pharmacy, biology, and biotechnology including regenerative medicine.

<SEQ ID NO: 1>
SEQ ID NO: 1 shows the DNA sequence of the human semaphorin 3A gene.
<SEQ ID NO: 2>
SEQ ID NO: 2 shows the amino acid sequence of human semaphorin 3A.
<SEQ ID NO: 3>
SEQ ID NO: 3 shows the DNA sequence of the chicken semaphorin 3A gene.
<SEQ ID NO: 4>
SEQ ID NO: 4 shows the amino acid sequence of chicken semaphorin 3A.

Claims (5)

1. A preventive, suppressive or therapeutic agent for corneal disease or corneal injury, comprising as an active ingredient a polypeptide comprising a region comprising any polypeptide selected from the group consisting of the following (a) to (c): .
   (a) a polypeptide comprising at least 70 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 4 and comprising a region from 166aa to 235aa in SEQ ID NO: 4,
   (b) a polypeptide comprising a region of 166aa to 235aa in SEQ ID NO: 2 consisting of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2,
   (c) a polypeptide having 80% or more sequence homology with the polypeptide of (a) or (b)
2. The agent for preventing, suppressing or treating corneal disease or corneal damage according to claim 1, wherein the corneal disease is a disorder accompanied by a decrease in the number of corneal endothelial cells or dysfunction.
3. A cell sheet obtained by culturing corneal endothelial cells in the presence of a polypeptide comprising a region comprising any polypeptide selected from the group consisting of the following (a) to (c):
   (a) a polypeptide comprising at least 70 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 4 and comprising a region from 166aa to 235aa in SEQ ID NO: 4,
   (b) a polypeptide comprising a region of 166aa to 235aa in SEQ ID NO: 2 consisting of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2,
   (c) a polypeptide having 80% or more sequence homology with the polypeptide of (a) or (b)
4. A cell culture aid for corneal endothelial cells, comprising as an active ingredient a polypeptide comprising a region consisting of any polypeptide selected from the group consisting of the following (a) to (c):
   (a) a polypeptide comprising at least 70 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 4 and comprising a region from 166aa to 235aa in SEQ ID NO: 4,
   (b) a polypeptide comprising a region of 166aa to 235aa in SEQ ID NO: 2 consisting of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2,
   (c) a polypeptide having 80% or more sequence homology with the polypeptide of (a) or (b)
5. A cell culture method comprising culturing corneal endothelial cells in the presence of a polypeptide comprising a region comprising any polypeptide selected from the group consisting of the following (a) to (c).
   (a) a polypeptide comprising at least 70 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 4 and comprising a region from 166aa to 235aa in SEQ ID NO: 4,
   (b) a polypeptide comprising a region of 166aa to 235aa in SEQ ID NO: 2 consisting of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2,
   (c) a polypeptide having 80% or more sequence homology with the polypeptide of (a) or (b)

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