JPH0921806A - Tissue examination method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a tissue examination method by which the tumor condition of a tissue is estimated from the tissue extract liquid. SOLUTION: The existing amount of vascular permeability factor in the extract liquid of a human tissue is measured for estimating the tumor condition of the tissue. By measuring the VPF concentration in the extract liquid of a tumor tissue, the quantitative decision of the development of blood vessels can be easily and properly carried out, and this can be applied to the diagnosis of breast cancer, and to the recuperation diagnosis of breast cancer. The diagnosis of breast cancer utilizing this can be clinically very useful in place of minute blood vessel densimetry that has been hitherto used for the diagnosis of the cancer of breast.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a vascular endothelial growth factor or vascular endothelium factor, which is known as a factor for inducing angiogenesis in a tissue extract.
thelial growthfactor or vascular permeability fact
or, hereinafter, abbreviated as VPF), the present invention relates to a method for predicting the tumor state of a relevant tissue by measuring the abundance of a factor, which is used in the pharmaceutical industry, particularly in the test drug industry and further in the biochemical test industry. It is used.

[0002]

2. Description of the Related Art In recent years, it has been known to use a trace amount of a physiologically active substance present in a living body, that is, a substance exhibiting a pharmacological action in a very small amount, as a diagnostic marker, an indicator for judging a therapeutic effect or monitoring after surgery. Widely used. On the other hand, angiogenesis, the proliferation of capillary endothelial cells,
The phenomena of migration and tissue invasion are known to play important roles in physiological or pathological phenomena such as fetal growth, wound healing, and cancer cell proliferation [(Folkman,
J., Cancer Res. 46: 467 (1986)], basic fibroblast growth factor (bFG) acting directly on vascular endothelial cells as a factor inducing angiogenesis.
F), acidic fibroblast growt
h factor, aFGF), platelet-derived endothelial cell growth factor (platele
t-derived endothelial cell growth factor, PD-ECGF)
But also as a substance that acts indirectly on vascular endothelial cells, transforming growth factor-α (TGF-α), transform
ing growth factor-β (TGF-β), angiogenin, tumor ne
crosis factor-α (TNF-α) has been found [Fol
kman, J. & Shing, Y., J. Biol. Chem., 267: 10931 (1992)].

[0003] Among them, VPF is secreted in normal or tumor cell lines of mouse, rat, guinea pig, bovine and human, and is clearly found to exist in brain, pituitary gland, kidney and ovary by tissue. [(Ferrar
a, N., et.al. Endocrine Reviews 13:18 (1992)], human VPF is angiogenesis and metastasis of breast cancer [Weider, N, et.al. N.En.
gl.J.Med. 324: 1 (1991)] and angiogenesis of renal cell carcinoma [Ayumi of Medicine, 168: 231 (1994)], or angiogenesis in retinal diseases [Adamis, AP et.al., Biochem. Biophys.Res.Com
m., 193: 631 (1993)]. Based on these facts, VPF secreted by tumor cell lines
Measuring the amount is considered to be useful for diagnosing cancer, predicting metastasis, or judging the therapeutic effect, and various studies have been made. Furthermore, microvascular density in tumor tissue has been attracting attention as a prognostic factor as a marker of angiogenesis, and it has been reported that patients with high microvascular density also have poor prognosis in breast cancer. In the case of breast cancer, it has been shown that there is a high correlation between the expression of VPF and the density of microvessels (T
oi et al., Jpn J. Cancer Res, vol. 85, p. 1045-104
9, 1994).

However, VPF expression and microvessel density are measured by immunohistochemical staining using specific antibodies against VPF and factor VIII, respectively.
It is very difficult to measure with good reproducibility because of poor quantitativeness.

[0005]

DISCLOSURE OF THE INVENTION As a method effectively used for the diagnosis of breast cancer, in particular, the diagnosis of prognosis, the present inventors have adopted a simpler method instead of measuring the expression of VPF in tumor tissue and the microvessel density. The study was conducted in order to find a quantitative method.

[0006]

[Means for Solving the Problems] The present inventors can predict the tumor state of a tumor tissue by measuring the VPF concentration in the tumor tissue, and further use it for the diagnosis of breast cancer, especially for the diagnosis of prognosis. The present inventors have completed the present invention by finding that they can be applied. That is, the present invention relates to a tissue inspection method characterized by measuring the amount of VPF (vascular permeability factor) present in a human tissue extract to predict the tumor state of the tissue.

Hereinafter, the present invention will be described in detail. In the present invention, it is preferable to use a labeled immunoassay for the measurement of VPF.
An enzyme immunoassay such as a sandwich method using a monoclonal antibody or a polyclonal antibody against F can be mentioned. Regarding human VPF, cDNA for the gene has already been isolated and its nucleotide sequence has been determined, and its amino acid sequence has also been deduced. From this gene, four kinds of proteins with different numbers of amino acid residues (with 121 amino acid residues
, 165, 189, and 206), and 121 amino acid residues (V
PF121) and 165 amino acid residues (VPF16
5) is said to be a mature protein [(Ferrara, N., et.a
l. Endocrine Reviews 13:18 (1992)], the present invention has already obtained a monoclonal antibody and a polyclonal antibody against human VPF121, and the enzyme immunoassay method using the monoclonal antibody and the polyclonal antibody against human VPF121. It has been clarified that VPF can be measured (patent pending “Peptide and monoclonal antibody” filing date June 10, 1994). Furthermore, in the method for detecting VPF in enzyme immunoassay, several pg
We have clarified a method that can detect VPF / ml VPF (patent pending “Measurement method of vascular permeability factor” filing date 5/1995)
(May 16), those antibodies and assay methods are applicable to the present invention and are preferable methods for the present invention. Labels for measuring VPF by labeling immunoassay include erythrocytes, latex, radioisotopes, enzymes, luminescent substances, fluorescent substances, metal molecules, metal gels, bacteriophages, etc., or use luminescent substances. preferable. That is, preferred methods are a measurement method using chemiluminescence, which measures light generated by using a luminescent substrate as a substrate of the enzyme, and a chemiluminescence detection system enzyme immunoassay proposed by the present inventors.

[0008]

According to the present invention, VPF in human tissue extract is
Can be measured with high sensitivity even with a small amount, and the tumor state of the tissue can be predicted from the measured value, and breast cancer can be diagnosed and the prognosis of breast cancer can be predicted by using the measured value.

[0009]

【Example】

(1) Preparation of anti-VPF polyclonal antibody The isolated human VPF cDNA was produced in Escherichia coli as a fusion protein (GST-VPF) with glutathione S-transferase (GST). VPF polyclonal antibodies were produced. The serum of the rabbit with an increased antibody titer was separated, and an IgG fraction of a rabbit anti-VPF polyclonal antibody was obtained by anion exchange column chromatography.

(2) Enzyme Labeling of Anti-VPF Polyclonal Antibody A part of the IgG fraction is digested with pepsin to prepare F (ab ') 2, which is then ligated with alkaline phosphatase (from bovine small intestine) by the hinge method. A phosphatase-labeled rabbit anti-VPF polyclonal antibody was obtained.

(3) Measurement of VPF concentration in tumor tissue VPF concentration in tumor tissue excised from a breast cancer patient was measured by enzyme immunoassay using chemiluminescence detection method as shown below. Tumor tissue (0.2g) was 0.25% Triton X
A sample was prepared by homogenizing and extracting in 50 mM Tris-Cl, pH 7.4 (2 ml) containing -100. 100 μl / well of anti-VPF polyclonal antibody (5 μg / ml) was spread on a 96-well plate and left overnight at 4 ° C., and then washed 4 times with 0.1% bovine serum albumin (BSA) and PBS. 1% B
After blocking with SA, 0.1M sodium chloride, 0.1% sodium azide, 0.2M sodium carbonate buffer pH 9.5 (1 hour at room temperature), 1% bovine serum albumin, 0.4% gelatin and 1 mM. Magnesium chloride, 20 mM
Tumor tissue extract or the same sample diluent diluted 10-fold with 50 mM sodium phosphate buffer containing 7.0% sodium ethylenediamine tetraacetate, 0.1 M sodium chloride and 0.1% sodium azide, pH 7.0 (sample diluent). The dissolved standard VPF was added and left at room temperature for 1 hour. After washing 4 times with Tris buffer pH 7.4 (TBS-T) containing 0.05% Tween 20, 0.14 M sodium chloride, 5 mM potassium chloride, 100 μl / well of alkaline phosphatase-labeled anti-VPF polyclonal antibody was added at room temperature. The reaction was carried out for 1 hour. Tris buffer containing TBS-T four times and 0.14 M sodium chloride and 5 mM potassium chloride
After washing twice with 7.4, 1 mM magnesium chloride, 0.1.
Lumiphos 53 diluted 5 times with 0.1 M diethanolamine buffer pH 10.0 containing 02% sodium azide.
0 (manufactured by Wako Pure Chemical Industries, Ltd.) was put in 100 μl / well and left at 37 ° C. for 30 minutes, and then the emission intensity was measured.

(4) Measurement of VPF Expression in Tumor Tissue Expression of VPF in tumor tissue was carried out by immunostaining a frozen section of breast cancer tissue with a mouse anti-VPF monoclonal antibody by the indirect immunohistological staining method. VPF expression was indicated by + or-according to the intensity of staining.

(5) Measurement of Microvessel Density in Tumor Tissue The microvessel density in tumor tissue was measured by measuring factor VIII antigen in frozen sections of breast cancer tissue by an indirect immunohistological staining method. That is, a frozen section of breast cancer tissue
Monoclonal antibody against factor VIII antigen (Dako Jap
immunostaining at (an, Tokyo) and under the microscope at 1.
The number of stained cells present within the 0 mm ² area was counted.

(6) Correlation between VPF expression in tumor tissue and microvessel density and VPF concentration in tumor tissue extract Using 130 cases of breast cancer tumor tissue, VPF expression in tumor tissue, microvessel density and VPF in tissue extract As a result of measuring the concentration, the results are shown in Table 1. Microvessel density is 100
It is displayed below the count or above 101 counts. A significant difference was observed in the VPF concentration in the tissue extract between the sample having a microvessel density of 100 counts or less and the sample having a microvessel density of 101 counts or more. Similarly, a significant difference was found in the VPF concentration in the tissue extract between the presence or absence of VPF expression and the VPF concentration in the tissue extract. Therefore, instead of measuring microvessel density or VPF expression, VP in tissue extract
It has been found that a method of measuring F concentration can be used.

[0015]

[Table 1]

[0016]

INDUSTRIAL APPLICABILITY According to the present invention, instead of measuring the expression of VPF in tumor tissue and the microvessel density, the concentration of VPF in the tumor tissue extract is measured, whereby the quantification of blood vessel development is easier than both. And can be applied to the diagnosis of breast cancer and the prognosis of breast cancer, and the diagnosis of breast cancer using the method of the present invention is a clinical method as an alternative to the microvessel density measurement which has been used for the diagnosis of breast cancer. Above is very useful.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Iwao Omori Iku Omori, Tsukuba City, Ibaraki Prefecture 2nd Okubo Synthetic Co., Ltd. Tsukuba Research Institute (72) Inventor Katsuhiko Matsuo 2 Okubo, Tsukuba City Ibaraki Toagosei Co. Ltd. Tsukuba Synthetic Co., Ltd.

Claims (1)

[Claims] 1. A tissue examination method, which comprises predicting a tumor state of a tissue by measuring the amount of a vascular permeability factor present in a human tissue extract.