WO2007013517A1

WO2007013517A1 - Evaluation system for lymphangiogenesis

Info

Publication number: WO2007013517A1
Application number: PCT/JP2006/314782
Authority: WO
Inventors: Keiichi Koizumi; Mitsuhiro Matsuo; Masuo Obinata; Masatsugu Ueda
Original assignee: University Of Toyama
Priority date: 2005-07-26
Filing date: 2006-07-26
Publication date: 2007-02-01
Also published as: JPWO2007013517A1

Abstract

Disclosed is a method for establishment of a cell strain derived from a thoracic duct or a vein passing along with the thoracic duct which can be maintained by passage. The method is characterized by performing a selective cloning by using a rat thoracic duct having a large T antigen gene of SV40 temperature-sensitive mutant stain tsA58 introduced therein and an inferior vena cava passing along with the thoracic duct and using markers specific to an endothelial cell and a lymph vessel cell. The resulting lymphatic endothelial cell or vascular endothelial cell is seeded on a gel, and the observation is made with respect to the formation of lumens. Thus, an evaluation system for lymphangiogenesis using the lumen-formation capability in vitro as a measure can be established.

Description

Specification

Evaluation system for lymphangiogenesis

Technical field

[0001] The present invention relates to a rat thoracic lymphatic endothelial cell into which a large T antigen gene of the SV40 temperature-sensitive mutant tsA58 has been introduced, and a venous endothelial cell-derived strain that is parallel to the rat thoracic duct. The present invention relates to a lymphangiogenesis evaluation system characterized by observing the formation of a lumen in a test tube using a modified cell.

Background art

[0002] The vascular system in a living body can be broadly classified into a vascular system composed of vascular endothelial cell force and a lymph vascular system composed of lymphatic endothelial cells. Regarding the vasculature, it has been clarified so far that the function of blood vessels in each organ is organ-specific at the molecular and gene level. It is also known that angiogenesis occurs during menstruation in females under normal conditions, and angiogenesis occurs during wound healing, when rheumatism worsens, when tumors grow, and when metastases are formed. Furthermore, great interdisciplinary efforts have been expended, and it has been elucidated at the molecular and genetic levels that angiogenesis plays an important role in both physiological and pathological aspects. As a result, strategies to target these pathological new blood vessels including tumor growth and metastasis have been devised, and many angiogenesis inhibitors have already been put to practical use in the treatment of rheumatoid tumors. Yes.

We were able to elucidate the vascular function and angiogenesis mechanism, develop pharmaceuticals, and contribute to the welfare of mankind because the vascular endothelial cells were isolated from various organs and their culturing technology improved dramatically. 'The biggest factor is the ability to experiment on the genetic level. For example, vascular endothelial cells are seeded and cultured on a gel to form a lumen that maintains the vascular system function in vivo, which has already been widely used as a model for in vitro angiogenesis. An angiogenesis inhibitor was developed by screening a small molecule organic compound that was randomly synthesized using an in vitro angiogenesis model using the inhibitory effect of vascular endothelial cell lumen formation as an indicator. It has reached practical use.

[0003] On the other hand, the lymphatic system has so far passively excreted waste products in vivo. It has been thought that it exists only to maintain the homeostasis of the living body. Therefore, it has been largely unforeseen that lymphatic angiogenesis contributes positively and actively in disease states. Interdisciplinary progress has been slow compared to vascular and angiogenesis research. There are many pathologies that involve lymphangiogenesis, but it can be roughly divided into abnormalities in lymphangiogenic function and those caused by injured lymphangiogenic function. There is.

(1) Diseases resulting from abnormal enhancement of lymphangiogenic function

Examples include lymph node metastasis of various cancer cells. Regarding the mechanism of lymph node metastasis

Tumor vascular endothelial growth factor (VEGF-C, VEGF-D) induces tumor lymphangiogenesis, resulting in enhanced lymph node metastasis (Non-patent Document 1). Neoplasia and tumor lymph node metastasis are being elucidated at the molecular level. However, this is a major reason why research on the molecular and genetic level of tumor lymphangiogenesis and clinical application research on drug development, etc., do not progress due to a stable lymphangiogenesis evaluation system.

(2) Diseases with impaired lymphangiogenesis function

In many diseases, secondary lymphedema occurs. Lymphedema may be due to a congenital genetic disease or to acquired lymphatic vessel damage. A typical example is lymphedema of the upper limb or lower limb after cancer surgery, which has a therapeutic effect by inducing lymphangiogenesis by gene therapy with the VEGF-C gene ( Non-patent literature 2).

On the other hand, the study of lymphangiogenesis including lymphovascular function specificity and tumor lymphangiogenesis in each organ is mainly from the viewpoint of morphological 'anatomical macro using electron microscopes. However, the research using lymphatic endothelial cells is currently conducted in the primary culture system because the passage and maintenance of lymphatic endothelial cells is extremely difficult. . Under such circumstances, cell lines of the third to fourth passages have been obtained by culturing rat lymphatic endothelial cells in endothelial growth medium EGM-2 (clontecs USA) in hypoxic air (patent document). 1). In addition, a method of isolating lymphatic endothelial cells from microvascular endothelial cells using a specific antibody that recognizes the extracellular domain of VEGFR-3 is known (Patent Document 2). [0004] In addition, primary cells are transformed by introducing oncogenic genes such as ras and cmyc, adenovirus El A gene, SV40 virus large T antigen gene, human papillomavirus HPV16 gene, etc. into primary cells. Attempts have been made to establish an immortalized cell line that retains the vigorous proliferative ability of the cell and does not lose its cell-specific characteristics even by substituting the force. However, even in such an immortal cell line, depending on the target organ, when the primary cells are prepared and these oncogenes and large T antigen genes are introduced, some functions are already lost. It was difficult to obtain an immortalized cell line in a strict sense that retains its original function. In particular, it was extremely difficult to prepare and establish primary cells when the growth rate was very slow or when they originated from micro-organs.

[0005] In contrast, instead of introducing oncogenes and large T antigen genes into individual cells, using these recently established gene transfer technologies for animal individuals, these genes were stably integrated into the chromosome. Immortalized cells are created by creating transgenic animals and preparing primary cells from the organs of animals that already have oncogenes or large T antigen genes in the cells at the time of the individual's development. Methods for establishing stocks have been reported. In particular, transgenic rats that have introduced the large T antigen gene of the SV40 temperature-sensitive mutant tsA58 can easily obtain immortalized cell lines, and the growth and differentiation characteristics of the resulting cells can be obtained. Expression is very effective because it can be manipulated by changing the temperature (Non-patent Document 3), (Non-patent Document 4). As an established cell line from this rat, for example, blood brain Vascular endothelial cells (Patent Document 3), retinal nerve cells (Patent Document 4), periodontal ligament cells (Patent Document 5) and the like are known.

[0006] Patent Document 1: JP 2004-248577 A

Patent Document 2: Special Table 2005-500045

Patent Document 3: International Publication WOOOZ20599

Patent Document 4: JP 2002-112764

Patent Document 5: JP 2002-262862 A

Non-patent literature l: Nature Medcine 7, 186-191 (2001)

Non-Patent Document 2: "Vessel research is powerful", p. 117, Yodosha, 2004 Non-Patent Document 3: Transgenic Research 4, 215—225 (1995)

Non-Patent Document 4: Genes to Cells 2, 235-244 (1997)

Disclosure of the invention

Problems to be solved by the invention

[0007] To promote the elucidation of detailed mechanisms of lymphangiogenesis at the cellular, molecular, and gene levels and to develop drugs that suppress lymphangiogenesis that are involved in various pathological conditions, An Atsei system that matches the rhythm is required. In addition, since the details of the difference between lymphatic vessels and blood vessels are not clear in the construction of the Atsey system, the establishment of an angiogenesis evaluation system, such as the necessity of vascular endothelial cells as a control group at all times, is required. A different approach is needed.

Therefore, an object of the present invention is to construct an evaluation system that can assess the formation of lymphocytes in a test chamber, to establish a cell line used for it, and to inhibit lymphangiogenesis by using the evaluation system. The aim is to find compounds, genes, proteins, etc. that have a promoting effect, and to develop new therapeutics for diseases involving lymphangiogenesis, such as cancer lymph node metastasis and lymphedema.

Means for solving the problem

[0008] The present inventors systemically express the large T antigen gene of SV40 temperature-sensitive mutant ts A58 effective in establishing an immortalized cell line under the control of an endogenous large T antigen gene promoter A collagenase solution is injected into the thoracic duct of Transgenic rats and the accompanying parallel vein, and the exfoliated lymph vessels and vascular endothelial cells are collected, and markers specific to the endothelial cells and lymph vessels are collected. An immortalized cell line was obtained by a method of selective cloning using. By seeding the obtained immortalized lymphatic endothelial cells and vascular endothelial cells on a gel, an evaluation system for lymphangiogenesis in which tube formation was observed in a test tube was constructed, and the present invention was completed. Hereinafter, the present invention will be described in detail.

[0009] The SV40 temperature-sensitive mutant tsA58 large T antigen gene used in the present invention is a transgenic rat that expresses the tsA58 large T antigen gene in all cells. Are described in JP-A-2000-228930. In addition, the modified fluorescent protein (EGFP) gene may be introduced into this rat! To obtain lymphatic endothelial cells and vascular endothelial cells from this rat, ligate the thoracic duct of the rat and the accompanying vein such as the inferior vena cava, and an enzyme solution such as trypsin or collagenase. After injection, the lymph vessels and intravascular cells that were detached in the enzyme solution were collected on a petri dish, and the collected lymphatic endothelial cells and vascular endothelial cells were subcultured twice, followed by colony formation and proliferation. A relatively fast colony is isolated from surrounding cells using a penicillin cup. Repeat this procedure three times to isolate the cell line. In addition, a known or commercially available medium may be used as appropriate for the culture.

[0010] The cultured cells can be identified as lymphatic endothelial cells or vascular endothelial cells by a specific marker. For example, the cell line can be identified as an endothelial cell by assaying the CD31 antigen, which is a marker for endothelial cells, by the FACS method (fluorescence-activated cell sorter). In addition, Prox-1 and LYVE1, which are markers of lymphatic endothelial cells, were assayed by Western blotting and RT-PCR to confirm that they were cell line endothelial cells obtained from the thoracic duct. Can be identified.

[0011] By evaluating the obtained cells on a gel using the lumen forming ability as an index, it can be confirmed that the original functions of lymphatic vessels and vascular endothelial cells are retained.

In addition, by detecting the expression of large T antigen at 33 ° C and disappearance at 37 ° C by Western plotting, it is possible to confirm that both cell lines are temperature sensitive.

[0012] An angiogenic system that is an in vitro lymphatic vessel and a control group includes, for example, the obtained lymphatic endothelial cells or vascular endothelial cells suspended in a medium, seeded on a gel-coated plate, Can be observed with an inverted microscope or the like. For example, the lumen forming ability is obtained by attaching a camera to an inverted microscope, selecting several fields of view randomly from the photograph taken, and analyzing the length of the lumen formed by lymphatic endothelial cells and vascular endothelial cells by image analysis. The ratio of the lymphatic endothelial cell group to the vascular endothelial cell group may be calculated and used as an index.

[0013] In the lymphangiogenesis system of the present invention, when screening for a substance that inhibits lymphangiogenesis, screening of a substance of the present invention that has previously been enhanced with fibroblast growth factor 2 (FGF2) or the like. AMP endothelial cells can be used.

Brief Description of Drawings

[0014] [Fig.1] Lymphatic endothelial cells and accompanying lymphatic and vascular endothelial cells with parallel venous force (Photo 1)

[Figure 2] Confirmation of large T antigen protein in an established cell line (Photo 2)

[Figure 3] Confirmation of CD31 and Prox-1 expression in an established cell line (Photo 3)

[Fig.4] Construction of lymphatic vessels in vitro using an established cell line and an angiogenesis model as a control (Photo 4)

[Fig. 5] Lymphangiogenesis-specific expression gene searched using the model of the present invention (Photo 5) [Fig. 6] Effect of JNK, MEK, PI3K on the ability of FGF-induced cell lines to form cell lumens And the effects of FGFR-TK inhibitors

BEST MODE FOR CARRYING OUT THE INVENTION

[0015] The passable cell line derived from the thoracic lymphatic endothelial cell of the present invention is a thoracic endothelial cell derived from a transgenic mammal into which the large T antigen gene of SV40 temperature-sensitive mutant tsA58 has been introduced. Obtained by sampling. In addition, the passable cell line derived from the vascular endothelial cell of the present invention is parallel to the transmammalian mammalian thoracic duct into which the large T antigen gene of the SV40 temperature-sensitive mutant tsA58 has been introduced. It is obtained by collecting veins. These endothelial cells can be obtained by treatment with collagenase ZEDTA solution to separate the endothelial cells.

After culturing thoracic duct or venous cells, the presence or absence of marker protein expression can be confirmed to identify the obtained cells as lymphatic endothelial cells or vascular endothelial cells. Specifically, for identification of lymphatic endothelial cells, it is confirmed that an endothelial cell marker is expressed and a lymphatic endothelial cell-specific marker is expressed. To identify vascular endothelial cells, it expresses an endothelial cell marker, but confirms that it expresses a lymphatic endothelial cell-specific marker. As the endothelial cell marker, one or more selected from the group consisting of CD31 antigen, Tie-2, VEGFR-2, VEGFR-1, CD34, Notch, PDGFR, EGFR, and FGFR can be used. For example, one or more selected from the group consisting of Prox-1, LYVE-1, VEGFR-3, Podoplanin, and SLC may be used. it can.

[0016] Before confirming the presence or absence of expression of an endothelial cell marker or a lymphatic endothelial cell-specific marker, it is desirable to confirm that the expression of the large T antigen protein is temperature sensitive. That is, it is desirable to confirm that the large T antigen protein is expressed under the culture condition of 33 ° C, but is not expressed under the culture condition of 37 ° C. Such SV40 temperature-sensitive mutant cell lines can control their growth depending on temperature conditions, i.e., retain permanent growth at 33-37 ° C and stop growing at 39 ° C. It is preferable because expression of cell-specific differentiation traits can be controlled.

The cells thus obtained have the lumen forming ability of lymphatic vessels or vascular cells. The lumen forming ability can be confirmed by observation with a microscope or the like.

[0017] The obtained lymphatic endothelial cell line and vascular endothelial cell line can be used for screening for a tube formation inhibitor or promoter. For example, for screening for a tube formation inhibitor, a test substance is added to a medium containing a cell growth factor such as FGF, and the tube formation of the lymphatic endothelial cell line or vascular endothelial cell line of the present invention is performed. This can be done by measuring the degree. In the case where the lumen formation is lower than the control due to the addition of the test substance, the test substance can be a candidate for the tube formation inhibitor. In addition, screening of the luminal formation promoting agent can be performed by adding a test substance in the medium and measuring the degree of luminal formation of the lymphatic endothelial cell line or vascular endothelial cell line of the present invention. it can. In the case where the formation of the lumen is improved as compared with the control by the addition of the test substance, the test substance can be used as a candidate for the lumen formation promoter.

In addition, using the lymphatic endothelial cell line and vascular endothelial cell line of the present invention, the genes or proteins expressed in lymphatic endothelial cells and vascular endothelial cells are compared. By identifying a gene or protein that is not expressed in cells, a lymphatic-related gene or protein or a gene or protein involved in lymphangiogenesis can be identified.

[0018] The following are examples of embodiments of the present invention.

(D SV40 temperature-sensitive mutant tsA58, a cell collected from the thoracic duct of a rat into which a large T antigen gene was introduced, was used with markers specific to endothelial cells and lymphatic vessels. A lymphangiogenesis evaluation system characterized by using a cell line that can be maintained for passage from a thoracic lymphatic endothelial cell obtained by selective cloning, and observing the formation of the lumen of the cell line in a test tube.

(2) SV40 temperature-sensitive mutant tsA58 large T antigen gene-transduced rat thoracic duct and cells that also collected the inferior vena cava force that runs parallel to the thoracic duct were transferred to endothelial cells and lymphatic vessels. A thoracic duct obtained by selective cloning using a specific marker and a cell line that can be maintained for passage from venous endothelial cells that run parallel to the thoracic duct are used. A lymphangiogenesis evaluation system characterized in that formation is observed and compared in vitro

(3) The above-mentioned (1), wherein CD31 antigen is used as a marker for endothelial cells, Prox-l and Z or LYVE-1 are used as markers for lymphatic vessels, and cells are selected using antibodies against them. And the lymphangiogenesis evaluation system according to (2).

(4) SV40 temperature-sensitive mutant tsA58 large rat antigen-transfected rat thoracic duct and the inferior vena cava that accompanies the thoracic duct are used to identify markers specific to endothelial cells and lymphatic vessels. A method for establishing a cell line that can be maintained for passage from a thoracic duct or a vein that runs parallel to the thoracic duct, characterized by performing selective cloning.

(5) As a marker, CD31 antigen and Prox-1 and Z or LYVE-1 are used, and cells are selected using antibodies against them, and veins that run parallel to the established lymphatic endothelial cell or thoracic duct Endothelial cells are obtained, The method as described in said (4) characterized by the above-mentioned.

(6) A cell line derived from a lymphatic endothelial cell obtained by the method for establishing a cell line described in (4) or (5) above, or a cell line derived from a venous endothelial cell that runs parallel to the thoracic duct .

(7) The gene of a rat thoracic duct-derived cell line using the lymphatic vessel evaluation system of (1) to (3) above, and (ii) the venous cutaneous endothelium that runs parallel to the rat thoracic duct A method for identifying or analyzing a gene or protein of a rat lymphatic-related or lymphangiogenesis, which comprises comparing cellular genes or proteins.

Example

The following examples illustrate the present invention in more detail. These are merely illustrative and the present invention is not limited thereto. The main abbreviations used in the examples below:

EDTA: disodium ethylenediamine tetraacetate

PBS: phosphate buffered saline

EGFP: Enhanced Green Fluorescent Protein

Example 1

Isolation of thoracic force lymphatic endothelial cells and concomitant venous vascular endothelial cells in transgenic rats

Separation of vascular endothelial cells from lymphatic endothelial cells with thoracic duct force and veins parallel to the thoracic duct was performed as follows. EGFP- SV40 temperature-sensitive mutant ts A58 large T-transgenic rat was introduced with anesthesia, and the inferior vena cava and its associated lymphatic vessels were detached from the connective tissue. The vein was dissociated using tweezers with a stereomicroscope. Next, the upper ends of each of the thoracic duct and vein were ligated with sutures, and collagenase ZEDTA solution was injected from the lower end with a tuberculin needle. While the collagenase / EDTA solution was contained in the thoracic duct and vein, the upper end from the entry portion of the injection needle was ligated with a suture, and the thoracic duct and vein containing the collagenase ZEDTA solution were removed. The excised thoracic duct and vein are left in a petri dish and left in a 37 ° C carbon dioxide incubator to detach lymphatic vessels and vascular endothelial cells from the thoracic duct and vein with the collagenase ZEDTA solution included. I let you. Then, the encapsulated collagenase ZEDT A solution in which exfoliated lymphatic vessels and vascular endothelial cells are suspended is dispersed in 7 mL of culture medium (Hu-Media EG2, Kurabo Industries, Inc.). The seeds were seeded on a 100 mm φ culture dish (Betaton's Dickinson). Colonies were formed by culturing (primary culturing) in a 37 ° C carbon dioxide incubator (5% carbon dioxide 95% air, saturated humidity). The medium was changed twice a week, and the relatively fast growing colonies that formed colonies after 7-10 days were isolated using the penicillin cup. The obtained cells were again seeded in a 100 mm φ culture dish and cultured in a carbon dioxide gas incubator at 33 ° C to form colonies. Furthermore, colonies with a relatively fast growth rate were isolated using a penicillin cup, and the surrounding cell force was isolated to obtain a thoracic duct-derived lymphatic cell line (B2 TD) and a vein-derived vascular endothelial cell line (P4IVC). (Figure 1Z Photo 1) [0021] Example 2

Large T antigen protein confirmation

Western Plot (Experimental Medicine Bibliography UP Series “Moleculars”) Expression of large ラー antigen protein in thoracic duct-derived lymphatic cell line (B2TD) and venous-derived vascular endothelial cell line (P4IVC) obtained in Example 1 Cancer research protocol by biological approach ”, pages 108-115, Yodosha, 1995). Both cell lines (passage number: 20) were cultured in a 60 mm φ culture dish until saturation. The collected cells are solubilized with 3% SDS-PBS (pH 7.4), centrifuged (10000 rpm, 10 minutes) to remove insoluble fractions, and then flooded (BIO—RAD Protein Assay Kit). The total protein mass was quantified. 20 g of each protein was separated by SDS polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. After further blocking with 3% skim milk solution, anti-SV40 large T antigen antibody (DP02-C, CALBIOCH EM) is used as the primary antibody on the nitrocellulose membrane, and HRP-labeled anti-mouse IgG antibody (Amersham) is used as the secondary antibody. The reaction specific to the large T antigen protein was detected using an ECL Western blotting detection system (RPN2106M1) manufactured by Amersham. In both cell lines, the expression of large T antigen protein at 33 ° C was confirmed to disappear at 37 ° C or higher, and the expression of large T antigen protein was confirmed to be temperature sensitive. It was done. (Figure 2Z Photo 2)

[0022] Example 3

Identification of lymphatic endothelial cells in thoracic duct-derived cell lines

The obtained thoracic duct-derived cell line is a B2TD camper tube endothelial cell. The expression of the endothelial cell marker CD31 antigen was expressed by the FACS method, and the lymphoendothelial cell-specific marker Prox-1 was Western blotted. It was identified by testing by the Ting method.

First, the expression of CD31 antigen was assayed by the FACS method. A thoracic duct-derived lymphatic cell line (B2TD) was cultured in a 60 mm φ culture dish to 80% saturation. Collected cells 1 X 10 ⁶ pieces of lmL of PBS (-) was suspended in, after centrifugation, the pellet O.lmL of PBS (-) was resuspended in the anti-rat CD31 antibody as a primary antibody (Chemicon Inc.) React with FITC-labeled anti-mouse IgG antibody (DakoCytomation) as a secondary antibody to express CD31 antigen-specific expression Was detected using Betasonic FACS.

Furthermore, Prox-1, a lymphatic endothelial cell-specific marker, was tested by Western plotting. Prepared in the same manner as in Example 2, using a trocellulose membrane, anti-rat PROX-1 antibody (AngioBIo) as the primary antibody, and HRP-labeled anti-rabbit IgG antibody (DakoCytomation) as the secondary antibody Was reacted. Prox-1 protein-specific reaction was detected using Amersham ECL Western Blotting Detection System (RPN2106M1). The CD31 antigen positive and Prox-1 positive were identified, and the cell line B2TD of the obtained thoracic duct-derived cell line was identified as a lymphatic endothelial cell. (Fig. 3Z Photo 3) [0023] Example 4

Identification of vascular endothelial cells in venous cell lines

The obtained blood vessel-derived cell line P4IVC is vascular endothelial cell, and the expression of endothelial cell marker CD31 antigen is assayed by FACS method and lymphatic endothelial cell specific marker Prox 1 is assayed by Western blotting method. Was identified.

First, the expression of CD31 antigen was assayed by the FACS method. A blood vessel-derived lymphatic cell line (P41V C) was cultured in a 60 mm φ culture dish to 80% saturation. Collected cells 1 X 10 ⁶ pieces of lmL of PBS (-) was suspended in, after centrifugation, the pellet O.LmL PBS (-) was resuspended, with the primary antibody anti-rat CD31 antibody (Chemicon, Inc. ) Was reacted, and FITC-labeled anti-mouse IgG antibody (DakoCytomation) was reacted as a secondary antibody, and CD31 protein-specific expression was detected with Betaton FACS.

Furthermore, Prox-1, a lymphatic endothelial cell-specific marker, was tested by Western plotting. Using the trocellulose membrane prepared in the same manner as in Example 2, anti-rat Prox-1 antibody (AngioBIo) was used as the primary antibody, and HRP-labeled anti-rabbit Ig G antibody (DakoCytomation) was used as the secondary antibody. ) Was reacted. Prox-1 protein-specific reaction was detected using an ECL Western blotting detection system (RPN2106M1) manufactured by Amersham. Since CD31 antigen was positive and Prox-1 negative, it was identified that the obtained cell line P4IVC, which is a venous cell line, was a vascular endothelial cell. (Fig. 3Z Photo 3) [0024] Example 5

Construction of lymphangiogenesis evaluation system Whether or not both of the obtained cell lines possess lymphatic and vascular functions was tested using the ability to form a lumen on a gel as an index. That is, both cell lines were suspended in DMEM ZF-12 medium containing 0.2% FBS (6 × 10 ⁴ ZmL) and seeded in a 48-well plate (No. 3548, manufactured by Costar) coated with gel (lmgZwell). Every 1 hour, the tube formation ability of lymphatic endothelial cells and vascular endothelial cells on the gel was observed with an inverted microscope and photographed (Fig. 4Z Photo 4). As a result, both cells formed a good lumen. As for the ability to form a lumen, five random fields were selected from the images taken over time after cell seeding, and the length of the lumen formed by lymphatic endothelial cells and vascular endothelial cells as a control group was measured by image analysis. Evaluation was made by calculating the ratio to the control group.

[0025] Example 6

Screening for lymphatic tube formation inhibitors

Serum-starved B2TD cells (1 X 10 ⁴ ) are suspended in FGF2-containing DMEM medium, SP600 125 (JNK inhibitor), U0126 (MEK inhibitor), LY294002 (PIK inhibitor) or SU49 84 (FGFR—TK inhibition) Agent) was pretreated with 20 mM each for 5 minutes and then seeded in a 96-well plate coated with Matrigel. After culturing at 37 ° C for 3 hours, the length of the formed lumen was measured using a map meter. Data are shown as mean (4 holes) standard deviation. * p 0.05 indicates p value compared with FGF2 treatment group. (Figure 6)

Therefore, it was shown that the thoracic duct-derived lymphatic cell line obtained in Example 1 was subject to inhibition of lumen formation by an existing inhibitor, and the thoracic duct-derived lymphatic cell line inhibited lymphatic lumen formation. It proved to be effective for screening substances.

[0026] Search for genes specifically expressed in lymphangiogenesis

The in vitro lymphangiogenesis evaluation system constructed in Example 5 was used to search for lymphangiogenesis-specific expressed genes. Immediately after seeding both cell lines on the gel and at the time of tube formation, both cell lines were collected and subjected to a comprehensive expression analysis using cDNA microarray (Agilent). As a result, about 400 lymphatic vessels and lymphangiogenesis-specific genes were identified. (Figure 5Z Photo 5)

Industrial applicability

[0027] Lymphatic endothelial cells having luminal formation ability and venous blood obtained by the method of the present invention An immortalized cell line of vascular endothelial cells is used to provide a lymphangiogenesis evaluation system characterized by observing the formation of a lumen in a test tube.

The lymphangiogenesis evaluation system provided by the present invention includes drug permeation research in lymphatic vessels, supply of various factors to lymphatic vessels, research on metabolites, and transport mechanism study of selective substance permeation existing in lymphatic endothelial cell membrane. It can be used to study the toxicity of drugs to lymphatic endothelial cells. Furthermore, the evaluation system is an evaluation system that conducts screening on the safety and efficacy of pharmaceuticals, maintenance of in vivo homeostasis, diagnosis of diseases related to dysfunction of lymphoid tissues, development of treatment methods, etc. at the cellular level. Available as

Claims

The scope of the claims

[I] Thoracic force of a transgenic mammal introduced with a large T antigen gene of SV40 temperature-sensitive mutant tsA58 From the inferior vena cava that runs parallel to the collected thoracic lymphatic endothelial cells or thoracic duct A passable cell line derived from collected vascular endothelial cells.

[2] The passable cell line according to claim 1, wherein the cell line is derived from thoracic lymphatic endothelial cells.

[3] The cell line according to claim 1, wherein the mammal is a rat.

[4] The cell line according to claim 1, obtained by screening according to the presence or absence of a marker specific to endothelial cells and lymphatic vessels.

[5] The cell line according to claim 4, wherein the marker force is a CD31 antigen specific to endothelial cells, and the specific marker specific to lymphatic vessels is Prox-1 and Z or LYVE-1.

6. The cell line according to claim 5, wherein the presence or absence of the marker is confirmed by an antibody.

[7] A kit for evaluating lymphangiogenesis by observing tube formation in a test tube, comprising the lymphatic cell according to claim 1 and a cell line derived from Z or vascular endothelial cell.

[8] A kit for evaluating lymphangiogenesis by observing the formation of a lumen comprising the cell line derived from the lymphatic cell according to claim 2 in a test tube.

[9] preparing a transgenic mammal into which the large T antigen gene of SV40 temperature sensitive mutant tsA58 has been introduced;

Obtaining cells comprising endothelial cells from the mammalian thoracic duct;

Screening cells containing endothelial cells based on the presence or absence of markers specific to endothelial cells and lymphatic vessels, and identifying cells expressing markers specific to endothelial cells and lymphatic vessels as lymphatic endothelial cells;

A method for establishing a lymphatic endothelial cell line, comprising:

10. The method for establishing a lymphatic endothelial cell line according to claim 9, wherein the mammal is a rat.

[II] Endothelial cell specific marker force CD31 antigen, wherein the lymphatic vessel specific marker is Prox-1 and Z or LYVE-1 How to establish a cell line.

12. The method for establishing a lymphatic endothelial cell line according to claim 9, wherein the presence or absence of the marker is confirmed with an antibody.

[13] A cell line derived from lymphatic endothelial cells obtained by the method according to claim 9.

[14] preparing a transgenic mammal into which the large T antigen gene of SV40 temperature sensitive mutant tsA58 has been introduced;

Obtaining cells including endothelial cells from the inferior vena cava that runs parallel to the mammalian thoracic duct;

Screening cells containing endothelial cells based on the presence or absence of markers specific to endothelial cells and lymphatic vessels, and the ability to express markers specific to endothelial cells Cells not expressing markers specific to lymphatic vessels Identifying a vascular endothelial cell line, and a method for establishing a vascular endothelial cell line.

15. The method for establishing a vascular endothelial cell line according to claim 14, wherein the mammal is a rat.

[16] The endothelial cell specific to endothelial cell according to claim 14, wherein the marker force is a CD31 antigen specific to endothelial cells, and the marker specific to lymphatic vessels is Prox-1 and Z or LYVE-1 How to establish a stock.

17. The method for establishing a vascular endothelial cell line according to claim 16, wherein the presence or absence of the marker is confirmed with an antibody.

[18] A cell line derived from a vascular endothelial cell obtained by the method according to claim 14.

[19] A screening method using the lymphangiogenesis evaluation kit according to claim 7, wherein the test substance is brought into contact with the cell line and the degree of lumen formation is measured. A screening method for substances or accelerators.

[20] A screening method using the lymphangiogenesis evaluation kit according to claim 8, wherein the test substance is brought into contact with the cell line and the degree of lumen formation is measured. A screening method for substances or accelerators.

[21] A lymphangiogenesis inhibitor obtained by the method for screening a lymphangiogenesis inhibitor or promoter according to [19].

[22] A lymphangiogenesis inhibitor obtained by the method for screening a lymphangiogenesis inhibitor or promoter according to [20]. Using the lymphatic vessel evaluation kit according to claim 7, a comparison is made between (i) a gene or protein expressed in a thoracic duct-derived cell line and (ii) a gene or protein expressed in a vascular endothelial cell-derived cell line. A method of identifying a gene or protein involved in lymphangiogenesis or lymphangiogenesis.