JP2002350436A - In vitro arterialization measuring method and in vitro arterialization measuring kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new arterialization measuring method capable of suppressing a background low, and having high sensitivity. SOLUTION: In a system for performing coexistent culture of human- originated vascular endothelial cells and human fibroblasts in a culture medium for cell culture, this in vitro arterialization measuring method includes a process for adding material for suppressing specifically the activity of VEGF-A into the system, a process for adding an arterialization factor excepting VEGF-A into the system, a process for culturing the system, and a process for observing lumen formation.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention is preferably used for screening an angiogenesis inhibitor or an angiogenesis promoter.
The present invention relates to a method for measuring in vitro angiogenesis. Furthermore, the present invention
The present invention relates to an in vitro angiogenesis measurement kit used for performing the method.

[0002]

BACKGROUND OF THE INVENTION Vertebrates have a closed vasculature and most tissues of the body rely on the close interaction of parenchymal cells with the vasculature. The construction of such a vasculature is played by pre-embryonic angiogenesis followed by narrow angiogenesis, i.e., the formation of new blood vessel branches from existing blood vessels, and the remodeling of various blood vessels. As one of the important factors that specifically control these factors, in recent years, Vascular Endothelial Growth Factor-
A, VEGF-A).

On the other hand, abnormal angiogenesis and vascular hyperpermeability have been observed in the growth process of solid tumors, diabetic retinopathy, rheumatoid arthritis and many other diseases, and the factors that cause them have been studied for many years. It has been revealed that VEGF-A is involved as a very important protein (Masashi Shibuya, Therapeutics vol. 34 pp35
9-364 (2000)).

[0004] VEGF-A is composed of two tyrosine kinase type receptors, Flt-1 (VEGFR-1) and KDR (V
Although it binds to EGFR-2) with high affinity, it has been found that the kinase activity of Flt-1 is weak and its signaling ability is low, whereas the kinase activity of KDR is about 10 times higher. From these findings, it is considered that the proliferation signal of vascular endothelial cells is mainly transmitted through KDR. Therefore, when considering the development of an angiogenesis inhibitor, the target of the first step is considered to be KDR itself and signal transmission therefrom.

On the other hand, in order to screen for angiogenesis inhibitors and angiogenesis factors, an in vitro culture system closer to the individual level is important. For many years, there has been no such stable in vitro tube formation assay, and the development of such a method has long been desired. Recently, a co-culture system of human umbilical vein endothelial cells (HUVEC) and fibroblasts has been developed (WO 99/17116). This system is an excellent system that, by adding vascular endothelial cell growth factor, induces not only the proliferation of vascular endothelial cells but also the formation of a lumen similar to the construction of the vascular system, and can measure the degree of the formation.

However, there are also problems with this system. The formation of a lumen was observed even without adding a cell growth factor such as VEGF-A or FGF (fibroblast growth factor) to the system, and this became a background value, making determination difficult. That is, the tube formation induced by the addition of VEGF-A is not more than 2% of the background.
Double or less. This background tube formation is thought to be largely due to various growth factors secreted from the cells themselves in culture, but the details have not been clarified. Therefore, WO99
V17 as a cell growth factor in the system of
When screening for an angiogenesis inhibitor using EGF-A, even if the formation of a lumen is partially inhibited by adding a candidate substance for an angiogenesis inhibitor, it can inhibit VEGF-A and its receptor system. It was not possible to sufficiently determine whether the inhibition was due to inhibition or due to suppression of growth factor secretion from each cell.

[0007]

DISCLOSURE OF THE INVENTION The present invention relates to a novel method for measuring angiogenesis, which is an improvement of a conventionally known co-culture system of vascular endothelial cells and fibroblasts. It is an object of the present invention to provide a method for measuring angiogenesis, which enables high-level detection. Another object of the present invention is to provide a method for screening a substance having an angiogenesis inhibitory / promoting action.

Another object of the present invention is to provide a kit for measuring angiogenesis, which is suitably used in the above method.

[0009]

Means for Solving the Problems The present inventors have found that a background spontaneous lumen which is a problem in the prior art method of measuring angiogenesis using a co-culture system of vascular endothelial cells and fibroblasts has been proposed. The inventors found that the formation was due to endogenous VEGF-A produced by each cultured cell, and completed the present application.

[0010] That is, the present invention relates to a system for co-culturing human-derived vascular endothelial cells and human fibroblasts in a cell culture medium, wherein a substance that specifically inhibits the activity of VEGF-A is introduced into the system. Adding angiogenic factors into the system,
Provided is a method for measuring in vitro angiogenesis, which comprises the steps of adding VEGF-A, excluding VEGF-A, culturing the system, and observing tube formation.

According to the present invention, by adding a substance that specifically inhibits the activity of VEGF-A into the system, spontaneous lumen formation that occurs without the addition of an angiogenic factor was successfully suppressed. . On the other hand, even if a substance that specifically inhibits the activity of VEGF-A is added, there is no effect on the KDR receptor itself, and the KDR-dependent tube formation action is maintained. Therefore, angiogenic factors other than VEGF-A,
In particular, when an angiogenic factor acting on the tyrosine kinase receptor KDR is added to the system, tube formation is induced in a form close to in vivo.

The method of measuring angiogenesis in vitro according to the present invention comprises:
It can be used for screening a substance having an angiogenesis inhibitory action. In this embodiment, in the method of measuring angiogenesis of the present invention, a candidate substance of an angiogenesis inhibitor is added simultaneously with or before or after the addition of an angiogenic factor. After culturing the system, the formed lumen may be observed and the degree of the lumen formation may be compared with a positive control to which the substance has not been added. When the angiogenesis measurement method of the present invention is used, screening with high sensitivity can be performed because the background value of tube formation is very low.

[0013] That is, the present invention further relates to a system for co-culturing human-derived vascular endothelial cells and human fibroblasts in a cell culture medium, wherein a substance that specifically inhibits the activity of VEGF-A is introduced into the system. Adding, adding angiogenic factors into the system, except for VEGF-A, adding candidate angiogenesis inhibitors into the system, culturing the system, and observing tube formation A method for screening an in vitro angiogenesis inhibitor is provided.

The method of measuring angiogenesis in vitro according to the present invention comprises:
It can be used for screening for a substance that promotes angiogenesis. In this embodiment, in the method of measuring angiogenesis of the present invention, a candidate substance for promoting angiogenesis is added in place of an angiogenic factor, culture is performed, and tube formation is observed.
In the method of the present invention, background lumen formation is almost suppressed, and screening with high sensitivity is possible.

That is, the present invention further provides a system for co-culturing human-derived vascular endothelial cells and human fibroblasts in a cell culture medium, wherein the substance specifically inhibits the activity of VEGF-A in the system. , A method of adding a candidate substance for promoting angiogenesis into a system, a step of culturing the system, and a step of observing tube formation.

In another aspect of the present invention, there is provided a kit useful for performing the method of the present invention. That is, a cell culture vessel in which human-derived vascular endothelial cells and human fibroblasts are co-cultured in a cell culture medium, and VEGF-
A kit for measuring in vitro angiogenesis, comprising a substance that specifically inhibits the activity of A.

In the present invention, the substance that specifically inhibits the activity of VEGF-A is not particularly limited, and any conventionally known substance or any substance to be found in the future may be used. Specifically, an anti-VGEF-A neutralizing antibody, solubilized Flt-1 (Proc. Natl. Acad. Sci. USA., Vol. 9)
0, 10705-10709, 1993; Jpn. J. Cancer. Res., Vol 88,
867-876, 1997.) and the like.

The angiogenic factor used in the method of the present invention, when added to a co-culture system of human-derived vascular endothelial cells and fibroblasts, induces the proliferation and differentiation of vascular endothelial cells and induces angiogenesis. Refers to a factor. The angiogenesis factor is not particularly limited as long as it is not affected by a substance that specifically inhibits the activity of VEGF-A, other than VEGF-A, but is preferably a tyrosine kinase type of vascular endothelial cells. Those that act through the KDR receptor are used. Although it is not particularly limited as a substance acting through the KDR receptor, VGEF-E (NZ-7) (J.
Biol. Chem., Vol. 273 pp. 31273-31282 (1988)), VG
EF-E (D1701) (EMBO J., Vol. 18, pp. 363-3
74, (1999), VEGF-E (NZ-2) (also known as CRFV2-V
EGF) (Proc. Natl. Acad. Sci. USA., Vol. 96, pp.
3071-3076, 1999).

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION As human vascular endothelial cells used in the present invention, cells obtained from human samples and cultured in an appropriate medium may be used. Umbilical vein vascular endothelial cells are most frequently used and easily available as human-derived vascular endothelial cells, and it is convenient to use them also in the present invention, but cells derived from other blood vessels may be used. Methods for isolating and culturing umbilical vein vascular endothelial cells and other vascular endothelial cells are well known to those skilled in the art, and may be obtained using any method. Cultured cells derived from umbilical vein vascular endothelial cells are commercially available, and it is convenient to use them.

The angiogenesis measurement method of the present invention includes, for example,
% Of umbilical cord-derived vascular endothelial cells subcultured in a commercially available medium containing endothelial cell growth medium supplemented with fetal bovine serum at a concentration of 2% to 6% is particularly preferable. Used.

In the present invention, human fibroblasts derived from human adult skin are preferably used. Methods for isolating and culturing fibroblasts from human adult skin tissue are well known to those skilled in the art, and may be obtained by any method. Similarly to vascular endothelial cells, cultured fibroblasts are commercially available, and it is convenient to use them.

In the method of measuring angiogenesis of the present invention, for example, a culture obtained by subculturing a commercially available Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum is preferably used.
Cultured cells that have been passaged for 6 to 10 passages from the collection are particularly preferably used.

In the method of the present invention, both vascular endothelial cells and fibroblasts are cultured under normal culture conditions, for example, 37
℃, 5% CO ₂ Incubator is collected from each cultivation still, counted the number of cells, respectively, mixed at a predetermined ratio immediately before the test, and inoculated into a cell culture container filled with a suitable cell culture medium do it.

In the method of the present invention, the ratio of the number of human-derived fibroblasts to the number of vascular endothelial cells differs depending on the state of the cells such as the individual from which the vascular endothelial cells were collected and the number of passages. Preferably, it is in the range of 8: 1. The optimum cell number ratio may be determined within this range.

Since the optimal cell density in co-culture varies depending on the type of vascular endothelial cells, the collected individuals, and the like, the optimal cell density may be determined by preliminary culture.
Determination of such optimal cell density is routinely made by those skilled in the art.

In the method of the present invention, the co-culture of vascular endothelial cells and fibroblasts may be performed using, for example, a commercially available medium dedicated to angiogenesis (for example, KZ-1400 manufactured by Kurashiki Boseki Co., Ltd.).

A kit for measuring angiogenesis in which human umbilical vein vascular endothelial cells and adult human fibroblasts are co-cultured at a fixed ratio in a cell culture medium is commercially available (for example, manufactured by Kurashiki Boseki Co., Ltd.). KZ-1000), the method of the present invention may be carried out using such a kit.

In the method of the present invention, a substance that specifically inhibits the activity of VEGF-A is added to the cell culture medium. The substance that specifically inhibits the activity of VEGF-A is not particularly limited, as long as it inhibits the activity of VEGF-A but does not affect the function of the tyrosine kinase-type KDR receptor. Specifically, anti-VEGF-A neutralizing antibody and soluble Flt-1 are exemplified. Anti-VEGF-A
Neutralizing antibodies are commercially available, but may be prepared by a conventional method using known VEGF. Methods for making antibodies are well known to those skilled in the art.

In the present invention, the substance that specifically inhibits the activity of VEGF is added to the cell culture medium in such an amount that the formation of a lumen is substantially not observed when no angiogenic factor is added. Such an amount differs depending on various factors such as the antibody titer, cell vitality, cell density, etc. in the case of an antibody, and thus needs to be determined each time a new test system is started.

In the method of measuring angiogenesis of the present invention,
Angiogenic factors that induce endothelial cell proliferation and differentiation, provided that
Except for VEGF-A. As an angiogenic factor
Interacts with the tyrosine kinase receptor KDR
Working substances are preferably used. As such a substance,
VEGF-E is known. As VEGF-E
Is currently three types, VEGF-E (NZ-7), VEGF
-E (D1701), VEGF-E (NZ-2) (also known as CR
FV2-VEGF) is known. VEGF-E (N
Z-7) was isolated from the NZ-7 strain of the Orf virus.
VEGF family of angiogenic factors
VEGF is known to have a potent effect ₁₆₅Comparable to
Is known to exhibit a strong angiogenic effect (J.
Biol. Chem. Vol. 273 pp. 31273-31282 (1998)). VE
The gene / amino acid sequence of GF-E (NZ-7) was
It is shown in the sequence listing.

In the method of the present invention, the culture conditions are not particularly limited, and normal animal cell culture conditions, for example, 3
7 ℃, 5% CO _2, may be performed in a humidified. The period from the start of the culture to the tube formation varies depending on various conditions. For example, when VEGF-E is used as an angiogenic factor, tube formation is usually observed from about day 7 to day 10, and the culture is 7-1 to -1.
It is used for observation from day 0.

In the method of the present invention, the observation of the formation of a lumen is preferably performed by staining the lumen. Any known method may be used for staining the lumen. For example, WO99 / 17
The enzyme immunostaining method described in No. 116 is suitably used. That is, a culture cultured for a predetermined number of days is divided into a primary antibody targeting a human vascular endothelial cell-specific CD31 factor and von Willebrand factor, and a secondary antibody-enzyme complex targeting the primary antibody. What is necessary is just to visualize using the used enzyme immunostaining method, to observe and evaluate this. The enzyme immunostaining method is well known to those skilled in the art, and an antibody may be appropriately selected according to the type of vascular endothelial cells. Various antibodies used for the enzyme immunoassay are commercially available.

For quantification of lumen formation, for example, the lengths of lumens generated in a certain visual field range are measured and totaled, and the number of lumens having a certain length or more is counted using the total length as an index. ,
Various methods can be adopted, such as using the area where each lumen is projected on the image as an index.

According to the method of measuring angiogenesis of the present invention, angiogenesis can be induced in a manner close to in vivo with high sensitivity.

In one embodiment of the present invention, the method of the present invention is applied to screening for an angiogenesis inhibitor. In the method for screening an angiogenesis inhibitor of the present invention,
A candidate substance is added together with a certain amount of angiogenic factors or before or after the addition of the angiogenic factors, and the cells are cultured as described above. To determine whether a candidate substance has an inhibitory effect on angiogenesis by comparing with the tube formation by a negative control without addition of an angiogenic factor and a positive control with addition of an angiogenic factor such as VEGF-E (NZ-7). Can be.

In particular, if VEGF-E (NZ-7) is employed as an angiogenic factor, it is useful for screening for a substance that inhibits any stage of the signal transduction system for cell proliferation and morphogenesis caused by KDR. is there. As described above, since the signal transmission via KDR is considered to play an important role in the proliferation and lumen formation of vascular endothelial cells, the angiogenesis inhibitor found in the screening system of the present invention is an extremely useful drug. Candidate substances.

In another embodiment of the present invention, the method of the present invention is applied to screening for an angiogenesis promoting substance. In this embodiment, a candidate substance for promoting angiogenesis is added in place of an angiogenic factor, and the cells are cultured as described above. Compared with the tube formation by a negative control without addition of an angiogenic factor and a positive control with VEGF-E (NZ-7) added as an angiogenic factor, the angiogenesis promoting effect of the test substance is determined. Good.

In a kit for measuring in vitro angiogenesis provided in still another embodiment of the present invention, a culture vessel for cell culture is inoculated with human-derived endothelial cells and human-derived fibroblasts and co-cultured. And VEGF
-A which specifically suppresses the activity of A.

The culture vessel for cell culture used in the kit of the present invention is not particularly limited. For example, any resin plate usually used for culturing animal cells is suitably used.

In the kit of the present invention, the substance that specifically inhibits the activity of VEGF-A may be added to the co-cultured medium so as to have the above-mentioned appropriate concentration, or may be added during the test. As shown in FIG. The kit of the present invention may further include a suitable cell culture medium. The substance for specifically suppressing the activity of VEGF-A may be added to the cell culture medium in advance, or may be separately attached.

The kit of the present invention preferably further contains the angiogenic factors described above, except for VEGF-A. The kit of the present invention may further contain appropriate reagents for visualizing tube formation. Examples of the reagent include a primary antibody, a secondary antibody-enzyme complex and a substrate used in the enzyme immunoassay described above.

Hereinafter, the present invention will be described in more detail with reference to examples. Note that the present embodiment is for explanation, and does not limit the present invention. Preparation of VEGF-E (NZ-7) VEGF-E (NZ-7) is described in Shibuya et al., The Journal o.
f Biological Chemistry vol. 273, No. 47 pp 31273-3
Prepared based on the method described in 1282. VEGF-E identified in the Orf virus NZ-7 strain genome (N
Z-7) open reading frame sequence (Lyttle
J. Virol. 68, 84-92 (1994)). To facilitate oligonucleotide synthesis, a point mutation that did not affect the amino acid sequence was introduced and PstI and KpnI restriction enzyme sites were created at codons 65/66 and codon 111. The three parts of the sequence were separately synthesized in a conventional manner and then ligated to obtain the full-length cDNA of the NZ-7-derived protein on the pUC18 vector. Preparation of Sf9 Cells Infected with Baculovirus Vector Incorporating VEGF-E (NZ-7) The VEGF-E (NZ-7) full-length cDNA obtained above was subcloned into the BamHI-EcoRI site of pVL1393, and pVEGF-E (NZ-7) was produced. This plasmid is transformed into the linear baculovirus DNA “B
aculoGold ”(Arlington Heights) and Sf9
Cells (Invitrogen) were transfected. Sf9 cells were infected with the virus vector of the culture supernatant, and this was repeated to obtain a high-concentration virus vector solution. Purification of VEGF-E (NZ-7) Sf9 cells infected with a baculovirus vector incorporating VEGF-E (NZ-7) were subjected to serum-free Ex-Ce
The cells were cultured in 11 400 medium (JRH Biosciences). The culture supernatant is passed through an ultrafiltration membrane (Amicon or Millip).
ore) and concentrated with 10 mM sodium chloride and 2 mM
20 mM phosphate buffer containing 0% glycerol (p
(H6.0) overnight. Use the cation S-
The mixture was adsorbed on a P column (Pharmacia) and eluted with a 20 mM phosphate buffer (pH 6.0) containing 10 mM sodium chloride and 20% glycerol to obtain an eluted fraction. Each fraction was subjected to Western blotting (VEGF-E (NZ-
7) Quantification) and silver staining (total protein quantification)
The fraction of E (NZ-7) was collected. (Protein purity 80%).
VEGF-E (NZ-7) thus obtained was used in the following tests.

As a method for preparing VEGF-E (NZ-7), a cDNA in which a tag such as a His tag is bonded to the carboxyl terminal of VEGF-E (NZ-7) is synthesized, and a baculovirus vector is prepared. After expression
VEGF-E (NZ-7) may be purified using a tag. The above protein purification method is used for all VEGF-
E can be applied.

[0044]

Example 1 An angiogenesis kit (KZ-10 manufactured by Kurashiki Boseki Co., Ltd.) in which human umbilical vein vascular endothelial cells and human adult skin-derived fibroblasts are co-cultured on a 24-well plate
00) was used. Medium for angiogenesis (Kurashiki Boseki K
Z-1400) into VEGF-E (NZ-7) 10 n
g / ml to the medium supplemented with anti-human VEGF-A
Neutralizing antibodies were added at concentrations of 0, 250, 500 and 1000 ng / ml and cultured.

Anti-human VEGF-A neutralizing antibodies include An
ti-VEGF, Human, Mouse-Mono (26503.111) (manufactured by R & D)
Catalog No. MAB293) was used.

The culture was performed at 37 ° C. in a 5% CO ₂ incubator. The medium was replaced on day 4, 7 and 9 of culture with one containing the same additives. Start culture 1
On the first day, the medium was removed, and staining was performed using a lumen staining kit (for CD31 antibody staining: KZ-1225 by Kurashiki Boseki Co., Ltd.) according to the following procedure.

A primary antibody (mouse anti-human CD31 antibody ) stained with CD31 (PECAM-1) was blocked with a blocking solution (Dulbecco's phosphate buffer (PBS containing 1% -BSA)).
BS (-)). 0.5 ml of this primary antibody solution was added to each well and incubated at 37 ° C. for 60 minutes. After the incubation, each well was washed three times with 1 ml of the blocking solution.

Next, a secondary antibody solution (goat anti-mouse IgGAlkP complex) diluted 500-fold with a blocking solution
0.5 ml was added to each well, incubated at 37 ° C. for 60 minutes, and then washed three times with 1 ml of distilled water. Meanwhile, two BCIP / NBT tablets were added to 20 ml of distilled water.
And filtered through a filter having a pore size of 0.2 μm to prepare a substrate solution. 0.5 ml of the prepared BCIP / NBT substrate solution was added to each well and incubated at 37 ° C. until the lumen became deep purple (usually 5 to 10 minutes). After completion of the incubation, each well was washed three times with 1 ml of distilled water, and then the washing solution was removed by suction, and allowed to stand for natural drying. After drying, each well was observed under a microscope.

Each well was observed with a microscope of 40 ×, and a photograph of each well was taken. 1 to 4 show the following 40-times enlarged views.

FIG. 1: Negative control FIG. 2: Negative control + anti-VEGF-A antibody (500
ng / ml) Figure 3: VEGF-E (NZ-7) alone Figure 4: VEGF-E (NZ-7) + anti-VEGF-A antibody (500 ng / ml)

Further, a photograph of a 1 mm scale magnified 40 times was taken, and the length of the lumen formed in each visual field was measured based on this scale. The length of the lumen formed in each visual field was summed, and the value divided by the area of the visual field was used as an index of lumen formation. FIG. 4 shows the results. FIG. 5 is a graph showing the average and standard deviation of the length of the lumen in each of the three visual fields.

From the observation results shown in FIGS. 1 to 4 and the length of the formed lumen shown in FIG. 5, the formation of the lumen by each growth factor and the formation of the negative control were prevented by the addition of the anti-VEGH-A neutralizing antibody. VEGF-E (NZ-
It can be seen that lumen formation according to 7) is not affected.
In addition, tube formation of the negative control to which no growth factor was added was almost suppressed by the addition of 500 ng / ml of the anti-VEGF-A neutralizing antibody.

[0053]

Example 2 The same angiogenesis kit and angiogenesis medium as in Example 1 were used. Anti-VEGF-A neutralizing antibody (500
ng / ml) and 10 ng / ml of VEGF-A or 10 ng / ml of VEGF-E (NZ-7) to the medium to which the neutralizing antibody was not added, respectively, or nothing as a control. Without culturing, the cells were cultured for 11 days in the same manner as in Example 1. The lumen was stained in the same manner as in Example 1, the length of each lumen was measured, the total per visual field was calculated and compared. FIG. 6 shows the results.

From the results shown in FIG. 6, it can be seen that the VEGH-A neutralizing antibody almost completely suppresses the formation of a tube by VEGF-A and the control tube,
It can be seen that -E (NZ-7) has no effect on lumen formation.

Example 3 The same angiogenesis kit and angiogenesis medium as in Example 1 were used. Solubilized Flt-1 (sFlt-
1) (500 ng / ml) (Jpn. J. Cancer Res. 88,867)
-876, September 1997)
VEGF-A 10 ng / ml or VEG was added to each of the medium containing sFlt-1 and the medium containing sFlt-1.
After adding 10 ng / ml of FE (NZ-7), the cells were cultured for 11 days in the same manner as in Example 1. The lumen was stained and visualized in the same manner as in Example 1, and observed under a microscope. Photos magnified 20 times are shown in FIGS. Figure 7: Negative control Figure 8: Negative control + sFlt-1 Figure 9: VEGF-A Figure 10: VEGF-A + sFlt-1 Figure 11: VEGF-E Figure 12: VEGF-E + sFlt-1

From the observation results shown in FIGS. 7 to 12, the tube formation by the negative control and VEGF-A was sFlt
Almost completely prevented by the addition of -1 but VEGF-
It can be seen that lumen formation by E (NZ-7) is not affected.

[Sequence List] <110> Kurashiki Boseki Kabushiki Kaisha <120> In vitro assay of angiogenesis and a kit useful for the assay <130> 175 196 <160> 1 <210> 1 <211> 489 <212> DNA <213> Orf virus NZ-7 strain <400> AAGCTTGGAT CCTTTTAAAA CGTCATCACC AGC ATG AAG TTA ACA GCT ACG TTA 54 Met Lys Leu Thr Ala Thr Leu 15 CAA GTT GTT GTT GCA TTG TTA ATA TGT ATG TAT AAT TTG CCA GAA TGC 102 Gln Val Val Ala Leu Leu Ile Cys Met Tyr Asn Leu Pro Glu Cys 10 15 20 GTG TCT CAG AGT AAT GAT TCA CCT CCT TCA ACC AAT GAC TGG ATG CGT 150 Val Ser Gln Ser Asn Asp Ser Pro Pro Ser Thr Asn Asp Trp Met Arg 25 30 35 ACA CTA GAC AAA AGT GGT TGT AAA CCT AGA GAT ACT GTT GTT TAT TTG 198 Thr Leu Asp Lys Ser Gly Cys Lys Pro Arg Asp Thr Val Val Tyr Leu 40 45 50 55 GGA GAA GAA TAT CCA GAA AGC ACT AAC CTG CAG TAT AAT CCC CGG TGC 246 Gly Glu Glu Tyr Pro Glu Ser Thr Asn Leu Gln Tyr Asn Pro Arg Cys 60 65 70 GTA ACT GTT AAA CGA TGC AGT GGT TGC TGT AAC GGT GAC GGT CAA ATA 294 Val Thr Val Lys Arg Cys Ser Gly Cys Cys Asn Gly Asp Gly Gl n Ile 75 80 85 TGT ACA GCG GTT GAA ACA AGA AAT ACA ACT GTA ACA GTT TCA GTA ACC 342 Cys Thr Ala Val Glu Thr Arg Asn Thr Thr Val Thr Val Ser Val Thr 90 95 100 GGC GTG TCT AGT TCG TCT GGT ACC AAT AGT GGT GTA TCT ACT AAC CTT 390 Gly Val Ser Ser Ser Ser Gly Thr Asn Ser Gly Val Ser Thr Asn Leu 105 110 115 CAA AGA ATA AGT GTT ACA GAA CAC ACA AAG TGC GAT TGT ATT GGT AGA 438 Gln Arg Ile Ser Val Thr Glu His Thr Lys Cys Asp Cys Ile Gly Arg 120 125 130 135 ACA ACG ACA ACA CCT ACG ACC ACT AGG GAA CCT AGA CGA TAATGAGAAT TC 489 Thr Thr Thr Thr Pro Thr Thr Thr Arg Glu Pro Arg Arg 140 145

[Brief description of the drawings]

FIG. 1 is a photomicrograph (× 40) showing the result of Example 1 (negative control).

FIG. 2 shows the results of Example 1 (negative control + anti-VE)
It is a microscope picture which shows GF-A antibody (500 ng / ml) (x40).

FIG. 3 shows the results of Example 1 (VEGF-E (NZ-7)).
(X 40).

FIG. 4 shows the results of Example 1 (VEGF-E (NZ-7)).
+ Anti-VEGF-A antibody (500 ng / ml))
It is a micrograph (x40).

FIG. 5 is a graph showing the results of Example 1.

FIG. 6 is a graph showing the results of Example 2.

FIG. 7 is a micrograph (× 20) showing the results of Example 3 (negative control).

FIG. 8 shows the results of Example 3 (negative control + sFl).
It is a micrograph which shows t-1) (x20).

FIG. 9 is a photomicrograph (× 20) showing the results (VEGF-A) of Example 3.

FIG. 10 shows the results of Example 3 (VEGF-A + sFlt).
It is a micrograph which shows -1) (x20).

FIG. 11 shows the results (VEGF-E) of Example 3.
It is a micrograph (x20).

FIG. 12 shows the results of Example 3 (VEGF-E + sFlt).
It is a micrograph which shows -1) (x20).

FIG. 13 shows the gene / amino acid sequence of VEGF-E (NZ-7).

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) (C12Q 1/02 C12N 15/00 ZNAA C12R 1:91)

Claims (16)

[Claims] 1. A system in which human-derived vascular endothelial cells and human fibroblasts are co-cultured in a cell culture medium, a step of adding a substance that specifically inhibits the activity of VEGF-A to the system. Angiogenic factors into the system, except for VEGF-
A method for measuring angiogenesis in vitro, comprising the steps of adding, excluding A, culturing the system, and observing tube formation. 2. In a system in which human-derived vascular endothelial cells and human fibroblasts are co-cultured in a cell culture medium, a step of adding a substance that specifically inhibits the activity of VEGF-A to the system. Angiogenic factors into the system, except for VEGF-
A method for screening an in vitro angiogenesis inhibitor, comprising the steps of: adding A, excluding A, adding a candidate substance for angiogenesis inhibitor into the system, culturing the system, and observing tube formation. 3. In a system in which human-derived vascular endothelial cells and human fibroblasts are co-cultured in a cell culture medium, a step of adding a substance that specifically inhibits the activity of VEGF-A to the system, A method for screening an in vitro angiogenesis promoter, comprising a step of adding a candidate substance for promoting angiogenesis into a system, a step of culturing the system, and a step of observing tube formation. 4. The substance which specifically inhibits VEGF-A activity is an anti-VEGF-A neutralizing antibody.
The method according to any of the above. 5. The method according to claim 1, wherein the angiogenic factor interacts with a KDR receptor. 6. The method of claim 5, wherein the angiogenic factor is VEGF-E (NZ-
The method according to claim 5, which is 7). 7. The method according to claim 1, wherein the human-derived vascular endothelial cell is a human umbilical cord-derived vascular endothelial cell. 8. The method according to claim 1, wherein the human fibroblast is a human adult skin-derived fibroblast. 9. The cell ratio of human-derived fibroblasts to human-derived vascular endothelial cells is 2: 1 to 8: 1.
The method according to any of the above. 10. A cell culture vessel in which human-derived vascular endothelial cells and human-derived fibroblasts are co-cultured in a liquid cell culture medium, and a substance that specifically inhibits the activity of VEGF-A. , An in vitro angiogenesis measurement kit. 11. The substance which specifically inhibits VEGF-A activity is an anti-VEGF-A neutralizing antibody.
The kit as described. 12. The kit according to claim 10, further comprising an angiogenic factor capable of interacting with KDR. 13. The kit according to claim 12, wherein the angiogenic factor capable of interacting with KDR is VEGF-E. 14. The kit according to claim 10, wherein the human-derived vascular endothelial cells are human umbilical cord-derived vascular endothelial cells. 15. The kit according to claim 10, wherein the human fibroblast is a human adult skin-derived fibroblast. 16. The cell number ratio of human-derived fibroblasts to human-derived vascular endothelial cells is from 2: 1 to 8: 1.
The kit according to any one of claims 15 to 15.