CN102233134A - Slit-Robo-mediated lymph vessel formation and application - Google Patents

Abstract

The invention relates to a method for preventing or treating Slit protein-mediated lymph vessel formation related diseases. The method comprises a step of supplying an effective dose of certain medicament for regulating or inhibiting the combination of a Slit protein and a Robo protein to a certain main body. A synthetic medicament for preventing or treating Slit protein-mediated diseases contains a certain medicament for regulating or disturbing the combination of the Slit protein and the Robo protein. A method for preventing or diagnosing the Slit protein-mediated diseases comprises the following steps of: (1) acquiring a detection sample of a main body to be detected, and detecting the expression level of certain Slit2 and (or) Robo protein in the sample; (2) acquiring a contrast sample of a contrast main body free from suffering from diseases, and detecting the expression level of certain Slit2 and (or) Robo protein in the sample; and (3) comparing the expression level of the Slit2 and (or) Robo in the two samples of (1) and (2) to acquire related results of related Slit protein induced diseases.

Description

The lymphatic vessel of SLIT-ROBO mediation forms and uses

Technical field

The present invention relates to prevention, diagnosis and the treatment of field for Slit-Robo signal conduction relevant disease; The cancer Clinics and Practices that takes place and shift particularly.

Background technology

Cancer transfer late is the main cause of cancer patient death.Existing extensive work devotes research cancer and treatment thereof, especially the growth and the Transfer Mechanism of research regulation and control cancer.But the molecular mechanism of cancer metastasis is still unclear.General cancerometastasis process is such: cancerous cell is impregnated into blood circulation or lymph circulation system from primary tumor, arrives by blood circulation then and enters new tissue, grows into new focus.The high rate of transform of the lymph node of cancerous cell shows that lymphsystem bringing into play important effect in cancer metastasis.

Lymphsystem all has important function to the absorption of the equilibrated maintenance of body fluid in the body, immunoreation and fatty acid.Except these normal physiological actions, lymphsystem also plays an important role to lymphadenectasis and inflammatory reaction.Find that under study for action the patient that cancer metastasis much takes place follows vasculolymphatic increasing, this shows that lymphsystem has important facilitation in the process of cancer metastasis., lymphsystem in cancer metastasis, study carefully how to play a role also do not study clear.

Because lymphsystem is played an important role, tumor lymphatic metastasis is just become a kind of effective means for the treatment of cancer as the treatment target spot in cancer metastasis.

Summary of the invention

One aspect of the present invention is the relevant method that causes prevention and treatment of diseases at Slit albumen.One of them concrete grammar is to utilize a kind of material that can regulate or suppress Slit and Robo protein-interacting to regulate and control vasculolymphatic formation, thereby reaches the purpose of prevention or treatment disease.This material can be at Slit antibody, at the albumen of the antibody of Robo, one section Robo extracellular region or the fusion rotein of one section Robo extracellular region.Slit albumen may be Slit2, and Robo albumen may be Robo1 or Robo4.

Another aspect of the present invention is about at the medicine that can prevent and treat the disease that Slit albumen causes.A kind ofly can regulate or suppress the Slit-Robo signal interaction and regulate and control the material and the drug excipient of vasculolymphatic formation, carry agent and lytic agent thereby wherein a kind of synthetic drug comprises.This material can be at Slit antibody, at the albumen of the antibody of Robo, one section Robo extracellular region or the fusion rotein of one section Robo extracellular region.Slit albumen may be Slit2, and Robo albumen may be Robo1 or Robo4.

One aspect of the present invention is the method about the diagnosis of the disease that causes at Slit albumen.Wherein a kind of method of diagnosis of the disease that causes at Slit albumen is as follows: (a) at first obtain detecting sample from detecting target, detect the expression of independent Slit albumen or the proteic expression of Robo or Slit albumen and Robo albumen; (b) secondly obtain sample in contrast, detect the independent Slit albumen of this sample or the expression of the proteic expression of Robo or Slit albumen and Robo albumen from the normal person masses; (c) compare (a) and (b) detected expression of results then, determine the disease whether this detection target has Slit albumen to cause.Slit albumen may be Slit2, and Robo albumen may be Robo1 or Robo4.The detection of being done can be dna level, rna level and (or) detection of protein level.

The rights and interests of other aspects of the present invention will be illustrated in subsequently description and separate statement.

Description of drawings

Fig. 1 has showed the expression of Slit2 albumen in cancer.(a) detect the Slit2 protein expression situation that many strains derive from the cell strain lysate of various cancers with Slit2 antibody by immunoblot experiment.The Slit2/293 cell strain is as the positive control that detects, and the V/293 cell strain is as the recessiveness contrast that detects.(b) with the Slit2 protein expression situation of Slit2 antibody by immunohistochemistry detection human cancer sample.Arrow indication Slit2 albumen is expressed in tumor cell.The scale of last figure is represented 100 μ m, and the scale of figure below is represented 10 μ m.(c) detect the Slit2 distribution gradient that the human breast carcinoma soaked sample is showed with Slit2 antibody by immunohistochemistry.The trickle tubular structure of arrow indication inside tumor.The scale of last figure is represented 100 μ m, and the scale of figure below is represented 40 μ m.

Fig. 2 shows the structure of Slit2 transgenic mice.(a) be used to do the sketch map of carrier Slit2 (hSlit2) expression carrier of transgenic mice.(b) detected by dot blot hybridization 9# mice is a hSlit2 transgenic positive mice.(c) southern ink dot method detection 9# mice is a hSlit2 transgenic positive mice.(d) with the hSlit2 expression of hSlit2 monoclonal antibody 5A5 by immunoblotting detection hSlit2 transgenic mice pancreas position.The homotypic immunity Lysozyme is as negative control.The HSlit2/293 cell pyrolysis liquid is as positive control.(e) with the hSlit2 expression of FLAG monoclonal antibody M2 by immunoblotting detection Flag-hSlit2 transgenic mice pancreas position.(f) expression of FLAG in the usefulness RT-PCR methods analyst hSlit2 transgenic mice.The C57 mice is as negative control.(g) detect the expression (the picture left above) that the normal mouse pancreatic tissue does not have hSlit2 with immunohistochemical method, remaining figure carries out SABC with serial section, the expression of the insulin of the pancreatic tissue of displaying Rip-Tag2 transgenic mice, the expression of hSlit2, the expression of Robo1 is carried, and the expression of vWF.(h) Slit2 of immunohistochemical analysis hSlit2/Rip1-Tag2 transgenic mice pancreatic tissue expresses the expression that is significantly higher than the Rip1-Tag2 transgenic mice. ^*P＜0.05; Scale: 10 μ m.

Fig. 3 shows that Slit2 promotes the growth of pancreas tumor.(a) the courageous and upright islets of langerhans number that compares Rip1-Tag2 and Slit2/Rip1-Tag2 transgenic mice, (b) gross tumor volume, (c) tumor number, (d) the leukocyte number of pancreas and blood.(e) fluorescence staining of transgenic mice Vipoma.(f) hematoxylin eosin stain of transgenic mice Vipoma.Fluorescence staining and hematoxylin eosin stain have showed that all the Slit2/Rip1-Tag2 transgenic mice has bigger tumor than Rip1-Tag2 transgenic mice, more blood vessel.Immunohistochemical method compares (g) vessel density of Rip1-Tag2 and Slit2/Rip1-Tag2 transgenic mice, (h) proliferation index, (i) apoptotic index, show the Slit2/Rip1-Tag2 transgenic mice and have more new vessels, higher propagation and apoptosis still less than Rip1-Tag2 transgenic mice.Scale: 50 μ m; ^*: p＜0.05; ^*: p＜0.01.

Fig. 4 shows histology's evaluation and the rate of transform analysis that 14 ages in week, Slit2/Rip1-Tag2 transgenic mice tumor lympha shifted.The Slit2/Rip1-Tag2 transgenic mice is showed (a) original position insulinoma, and mesenteric lymph node metastasis and intestinal wall metastasis are dissected sketch map (shown in the arrow), (b-c) hematoxylin eosin stain of metastasis (arrow is depicted as the tumor cell of transfer).(d-h) metastasis of carrying out SABC conclusive evidence mesenteric lymph node and intestinal wall with T-antigen antibody is all formed by islets of langerhans β tumor cell.(i) statistics of Rip1-Tag2 and two kinds of transgenic mice rates of transform of Slit2/Rip1-Tag2, statistical result showed Slit2 crosses and expresses the mesenteric lymph node rate of transform that significantly promotes the pancreatic islet tumor cell.(j) figure shows that the volume of the plain tumor of 14 two kinds of mouse islets in ages in week does not have notable difference, and the rising that the rate of transform be described is not because tumor growth accelerates to cause indirectly that but expression directly causes owing to Slit2 crosses.(k) figure shows that the survival rate of Slit2/Rip1-Tag2 mice obviously descends.

Fig. 5 shows that Slit2 crosses lymphatic vessel new life and Slit2 recombiant protein inductor stranger's lymph endothelial cell migration of expressing insulinoma in the promotion body and forms tubular structure.SABC detects the lymphatic vessel labelled protein, and the result shows that late period Slit2/Rip1-Tag2 mouse tumor lymphatic vessel new life is apparently higher than Rip1-Tag2 mice (a-c).Detect Robo1 with the method for immunofluorescence and on the pancreas lymphatic vessel, express (d).Simultaneously Robo1 is expressed on former generation lymph endothelial cell membrane (e-f).The Slit2.1 albumen of reorganization promotes the tube chamber of lymph endotheliocyte on Matrigel to form (g-h) by acting on its receptor Robo1, and chemotactic lymph endothelial cell migration (i-j); The formation of migration and tubular structure can both be suppressed (g-j) by the R5 specificity."-" is negative control.VEGF-C is a positive control.. ^*：p＜0.05； ^**：p＜0.01。

Fig. 6 shows that (A) hRobo-Fc and hSlit2 recombiant protein interact; (B) R5 (at the monoclonal antibody of first Immunoglobulin-like section of Robo1 albumen) suppresses the interaction of hRobo-Fc and hSlit2 recombiant protein.

Fig. 7 has showed that with labelling the anti-human normal immunoglobulin's detection molecules of the catalatic rabbit of Radix Cochleariae officinalis amount is about 85 daltonian reorganization hRobo-Fc albumen.

Fig. 8 shows that the specificity analyses of Slit2 monoclonal antibody reaches the detection at the various human tumor tissues.Immunoblot experiment is used to detect antibody S1 and S3 to Slit2 albumen total length or segmental identification.(a) S1 is by combining identification total length Slit2 with Slit2 nitrogen end.9E10 and S3 are by combining identification total length Slit2 with the Slit2 carbon teminal.Antibody S1 and S3 can both specific recognition Sli2, and S1 also can discern Slit1 and Slit3 albumen simultaneously.9E10 is as positive control.The S1 monoclonal antibody is used to detect colon cancer and breast carcinoma (b), and original position pulmonary carcinoma is with transfer pulmonary carcinoma (c) and lymph metastasis is arranged and the expression of the middle Slit2 of the colon cancer (d) of no lymph metastasis.MIgG is as negative control (immunohistochemical analysis).Scale: 10 μ m.

The specific embodiment

The present invention is primarily aimed at diagnosis, prevention and the Therapeutic Method of the disease that Slit albumen causes, especially forms relevant disease with lymphatic vessel.The disease that these Slit albumen cause and Slit albumen and its receptor Robo be proteic to be combined relevantly, and these diseases comprise growth of tumor and transfer, such as tumor lymphatic metastasis.

Particularly, first aspect present invention provides a kind of prevention or has treated the method for the disease that is caused by Slit albumen, comprise: grant the scalable of effective in cure dosage or the medicine of blocking-up Slit albumen and a kind of Robo protein-interacting to individuality, it is characterized in that described disease forms relevant with lymphatic vessel.

In a preferred embodiment, described Slit albumen is Slit2.

In another preferred embodiment, described Robo albumen is Robo1 or Robo4.

In another preferred embodiment, described medicine is aimed at the proteic a kind of antibody of Slit.

In another preferred embodiment, described medicine is at the proteic a kind of antibody of Robo.

In another preferred embodiment, described antibody is a kind of monoclonal antibody, preferably humanized antibody.

In another preferred embodiment, described medicine is a certain fragment of Robo protein extracellular.Described fragment preferably derives from proteic first immunoglobulin-like zone of Robo.

In another preferred embodiment, described medicine is a kind of Robo fusion rotein, preferably Robo-Fc fusion rotein.

Second aspect present invention provides a kind of medicine of disease of prophylactic treatment Slit mediation.This medicine comprises medicine and a kind of drug excipient, carrier or the solvent that can regulate or disturb Slit albumen and Robo protein-interacting, and wherein the disease of Slit mediation is to form relevant with lymphatic vessel.

In a preferred embodiment, described Slit albumen is Slit2.

In another preferred embodiment, described energy is regulated or is disturbed the medicine of Slit albumen and Robo protein-interacting to be aimed at Slit albumen or the proteic a kind of antibody of Robo.

In another preferred embodiment, described antibody is a kind of monoclonal antibody, preferred humanized antibody.

In another preferred embodiment, the medicine of described energy adjusting or interference Slit albumen and Robo protein-interacting is a certain fragment of Robo protein extracellular.Described fragment preferably derives from proteic first immunoglobulin-like zone of Robo.

In another preferred embodiment, described energy is regulated or disturbed the medicine of Slit albumen and Robo protein-interacting is a kind of Robo fusion rotein, preferred Robo-Fc fusion rotein.

Third aspect present invention provides a kind of method of diagnosis of the disease that causes at Slit albumen as follows: (a) at first obtain detecting sample from detecting target, detect the level of independent Slit or Robo or the level of Slit and Robo; (b) secondly obtain sample in contrast, detect independent Slit or the level of Robo or the level of Slit and Robo of this sample from the normal person; (c) compare (a) and (b) detected expression of results then, determine the disease whether this detection target has Slit albumen to cause.

In a preferred embodiment, described Slit albumen is Slit2.

In another preferred embodiment, described Robo albumen is Robo1 or Robo4.

In another preferred embodiment, described level is a dna level, rna level, protein level, or comprehensive above-listed 2 or 3 level.

In another preferred embodiment scheme, described disease is the disease relevant with the Slit-Robo signal transduction pathway.

In another preferred embodiment, described disease is cancer or forms relevant disease with lymphatic vessel.

In another preferred embodiment, described disease is a cancer, preferred metastatic cancer.In another preferred embodiment, described cancer is one of following cancer: colon cancer, gastric cancer, esophageal carcinoma, pulmonary carcinoma, hepatocarcinoma, breast carcinoma, and bladder cancer.

Fourth aspect present invention provides the material that can detect Slit albumen or Robo albumen or their pairing mRNA or their encoding gene to be used for detecting the application of the diagnostic reagent of the protein mediated disease of Slit in preparation, and described detection may further comprise the steps:

(a) obtain detecting sample from detecting target, the material that utilization can detect Slit albumen or Robo albumen or their pairing mRNA or their encoding gene detects independent Slit or the level of Robo or the level of Slit and Robo;

(b) obtain sample in contrast from the normal person, the material that utilization can detect Slit albumen or Robo albumen or their pairing mRNA or their encoding gene detects independent Slit or the level of Robo or the level of Slit and Robo of this sample;

(c) relatively (a) and (b) detected expression of results determine the disease whether this detection target has Slit albumen to cause, if the Slit or the Robo level of detection sample are higher than contrast, show that then this detection target suffers from described disease.

In a preferred embodiment, the described material that can detect Slit albumen or Robo albumen or their pairing mRNA or their encoding gene is selected from: with the bonded material of Slit protein-specific, preferably with the bonded antibody of Slit protein-specific, more preferably with the bonded monoclonal antibody of Slit protein-specific; With the bonded material of Robo protein-specific, preferably with the bonded antibody of Robo protein-specific, more preferably with the bonded monoclonal antibody of Robo protein-specific; MRNA or the bonded material of encoding gene (as primer) with Slit or Robo.

Inventor of the present invention is surprised to find that the signal conducting energy of the interaction (effect of mutually combining) between Slit albumen and the Robo albumen (the proteic receptor of Slit) promotes lymphatic vessel to form.Can regulate and control or influence the interactional reagent of Slit albumen and its receptor (Robo albumen) so just might be applied to vasculolymphatic formation or form diagnosis, the treatment of relevant disease, for example lymph metastasis of cancer with lymphatic vessel.

Concrete part more of the present invention is regulation and control or influences Slit albumen and the more interactional reagent of its receptor (Robo albumen).These reagent can be Slit or proteic protein fragments of Robo or homologue (comprising fusion rotein or protein fragments), the perhaps proteic antibody of Slit or Robo (polyclonal antibody, monoclonal antibody or humanized antibody).Other concrete parts of the present invention are method, especially cancer process and cancer metastasiss that interaction causes diagnosis, prevention or the treatment of disease at Slit-Robo.

Use Slit2 as the proteic representative of Slit in the of the present invention concrete example subsequently, use Robo1 and Robo4 as the proteic representative of Robo.Certainly, the present invention also comprises other slit albumen and the Robo albumen that lymphatic vessel forms that influences.

Slit gene code one class very important secretory protein in neurodevelopment.These proteic functions mainly are to induce neural migration.In mammal, known have three Slit gene: Slit1, a Slit2, and Slit3.These genes also have expression (Holmes, G.P.et al.Mech.Dev.79:57-72 (1998)) in non-nervous system.For example, the mRNA of Slit2 and Slit3 also is found (not finding Slit1) (Wu, J.Y.et al.Nature 410:948-952 (2001)) at the rat endotheliocyte.

On the structure, Slit has 4 and is rich in leucic repetitive sequence, 9 epidermal growth factors (EGF) sample functional domain, 1 and is positioned at the 6th and the 7th the ALPS fragment between the EGF functional domain, and 1 cystine knot that is positioned at c-terminus.Though it is Slit is a kind of soluble protein, most of attached to cell surface.

Slit albumen and their receptor Roundabout (Robo) protein binding.Slit and the combined effect of Robo albumen play repulsive interaction (Kidd, T.et al.Cell 92:201-215 (1998) in neural axon guiding and bifurcated and neuronal migration; Wang, K.-H.et al.Cell 96:771-784 (1999); Wu, W.et al.Nature 400:331-336 (1999)).Slit-Robo interacts and can also suppress endogenous leukocyte chemotaxis (Wu, J.Y.et al.Nature 410:948-952 (2001)) in addition.

Because can suppress endogenous leukocyte chemotaxis, Slit albumen (for example Slit2 albumen) is relevant with inflammatory response in the body.(Wu，J.Y.et?al.Nature?410：948-952(2001))。In the inflammation process, leukocyte adhesion passes through capillary wall in vascular endothelial cell, enters tissue or organ that disease is decreased or infected, is the important step in inflammation generation and the evolution, also is a biological phenomena of having studied hundreds of years.Along with biological study method and development of technology, the molecular mechanism to this phenomenon over past ten years has had more clearly understanding.Generally believe that at present leucocyte adherence is by multiple molecule with wearing the wall motion, as chemotactic factor, cell adhesion molecule, cytoskeletal protein and metalloenzyme molecule etc., the coordinative role on time and space is finished.Bibliographical information is arranged, the leukocytic chemotactic motion that Slit2 causes at external energy chemokine inhibiting, and this effect can be blocked by the outer soluble fragments (RoboN) of Robo born of the same parents, proof Slit also is based on the signal path (Wu et al., 2001Nature 410:948) that its receptor Robo is mediated to leukocytic this effect.

The proteic receptor Robo of Slit is transmembrane protein of a class, has found four Robo gene: Robo1, Robo2, Robo3 and Robo4 at present in mammal.Robo albumen also has expression (Piper, M.et al.Mech.Dev.94:213-217 (2000)) outside nervous system.For example, murine interleukin is found Robo1 mRNA (Wu, J.Y.et al.Nature 410:948-952 (2001)).Human endothelial cell is expressed Robo4 (Huminiecki, L.et al.Genomics.79:547-552 (2002)).

As part (for example Slit albumen) combination, the Robo receptor conducts to the generation that finally causes various repellency bioprocesss in the cell with signal, comprises neural axon guiding and bifurcated, neuronal cell migration and the motion of leukocyte chemotaxis.

As above-mentioned, receptor Robo is transmembrane protein of a class.On the structure, Robo1,2 and 3 extracellular fragment have 5 immunoglobulins (Ig) spline structure territory and 3 III Collagen Type VI repetitive sequences, and it is necessary that wherein to be Robo be rich in the leucine repetitive sequence in conjunction with total length Slit and Slit in Ig spline structure territory; Born of the same parents' inner segment contains a plurality of domains, and comprising CC0, CC1, CC2 and CC3 sequence, verified these structures are transmitted relevant with the signal of cell interior.And the extracellular fragment of Robo4 only contains 2 Ig spline structure territories, and its born of the same parents' inner segment only contains CC0 and CC2 sequence.

Slit2 albumen combines (Chen et al., J Neurosci.200121:1548-1556) with first Ig domain of Robo1.The monoclonal antibody R5 of first Ig domain of specific recognition Robo1 can in and the inductive angiogenesis of Slit2 (Wang et al., Cancer Cell.20034:19-29), prove that it is vital that Slit2 combines the inductive angiogenesis of Slit2 with the specificity of Robo1.

Slit and Robo albumen play a role in comprising the multiple biological activity of blood vessel in occurring in.Inventor of the present invention finds Slit-Robo also play a significant role (for example lymphatic vessel generation) that interact in lymph generates under complete objectively condition.The more important thing is and the inventor also find to interact Slit-Robo to generate cancer metastasis closely related for the lymphatic vessel cause.Can destroy prevention and treatment that the interactional material of Slit-Robo just can be used to the prevention and the treatment, particularly cancer metastasis of cancer like this.

Slit2 has high level expression (Fig. 1) in multiple cancerous tissue, then express very low in normal structure or atypical hyperplasia tissue or do not express.Slit2 may play an important role in cancer process and transfer process at the true prompting of the high expressed Slit2 of cancerous tissue.Hereinafter will describe Slit2 plays a significant role in the process of cancer and transfer process really.The possible mechanism that effect is relevant therewith is exactly that Slit2 can induce lymphatic vessel to generate.

The important function of Slit2 in tumor progression and transfer process is detected in some animal models.For example, as shown in Figure 2, the transgenic mice of high expressed Slit2 is fabricated, and Slit2 transgenic mice and Rip1-Tag2 transgenic mice hybridization that can spontaneous Vipoma are obtained Slit2/Rip1-Tag2 double transgenic mice.Relevant Slit2/Rip1-Tag2 double transgenic mice has a detailed description in the example part.These Slit2/Rip1-Tag2 double transgenic mices detect by the immunohistochemical assay method can see that high-caliber Slit2 expresses (Fig. 2 h).Simultaneously, these mouse have shown tangible lymphatic vessel and have generated increase, the increase of tumor size and tumor to mesenteric lymph node transfer phenomena (Fig. 3).These results show that the increase of the increase of Slit2 protein expression level and tumor growth and transfer is closely related.

Slit2 promotes the growth and the transfer of cancer of pancreas.Relatively Rip1-Tag2 and Slit2/Rip1-Tag2 transgenic mice are found, Slit2 can increase number (Fig. 3 a), gross tumor volume (Fig. 3 b), the tumor number (Fig. 3 c), but do not influence leukocyte number (Fig. 3 d) in pancreas and the blood of courageous and upright islets of langerhans.Fig. 3 e has showed the blood vessel fluorescent labeling of transgenic mice insulinoma, Fig. 3 f has showed the hematoxylin eosin stain of transgenic mice insulinoma, can see that the Slit2/Rip1-Tag2 transgenic mice has tumor and the more blood vessel bigger than Rip1-Tag2 transgenic mice.

Can find that by immunohistochemical method analysis comparison Slit2/Rip1-Tag2 transgenic mice and Rip1-Tag2 transgenic mice vessel density (Fig. 3 g) and proliferation index (Fig. 3 h) all increase greatly, apoptotic index then reduces (Fig. 3 i).These results show that the Slit2/Rip1-Tag2 transgenic mice has more new vessels than Rip1-Tag2 transgenic mice, the apoptosis potential of higher multiplication capacity and minimizing.

The expression excessively of Slit2 can increase this phenomenon prompting of tumor number Slit2 may promote neoplasm metastasis.This probability and Slit2 can influence lymphatic vessel to generate this function relevant, because most of neoplasm metastasis is all relevant with lymphsystem.

Fig. 4 has showed that in fact proteic the expression excessively of Slit2 caused vasculolymphatic hypertrophy and tumor lympha to shift in the Slit2/Rip1-Tag2 transgenic mice.The Slit2/Rip1-Tag2 transgenic mice shows the tumor mesenteric lymph node that increases and intestinal wall shifts (Fig. 4 a), H﹠amp; The E method dyes to metastasis, finds that the β cell transfer is between duodenal epithelium layer and the flesh layer; And mesenteric lymph node inside (Fig. 4 b-c).Antibody with T-antigen carries out immunohistochemical staining to the metastasis tissue slice, finds it is the plastidogenetic metastatic tumor of β (Fig. 4 d-h) really.

Rip1-Tag2 and Slit2/Rip1-Tag2 mice to a large amount of 14 ages in week have carried out anatomic observation, find in Rip1-Tag2 mice (32), have only 2 to have only mesenteric lymph node to shift, and 1 intestinal wall shifts; In Slit2/Rip1-Tag2 mice (50), there are 17 mesenteric lymph nodes to shift, 8 intestinal wall shift (Fig. 4 i).There were significant differences for the Chi-square check mesenteric lymph node rate of transform.Antibody with LYVE-1 carries out immunohistochemical experiment and statistical analysis, and the result shows, compares with the Rip1-Tag2 transgenic mice, and the relevant lymphatic vessel of Slit2/Rip1-Tag2 transgenic mice Vipoma forms obvious rising (Fig. 5 a-c).These results show that Slit2 crosses expression and can induce lymphatic vessel to form and the tumor lympha transfer.

The monoclonal antibody R5 of Robo1 (Fig. 5 c-e) or can with the bonded Robo1 extracellular region of Slit2 fragment RoboN (data do not have show) can the inductive lymph endotheliocyte of specific inhibition Slit2 migration and lymphatic vessel form, further confirmed the important function of Slit2 in the tumor lympha pipe generates.Fig. 6 showed recombiant protein (hRobo-Fc) that R5 can specificity destroys Robo1 (Fig. 7) and the combination between the recombiant protein of Slit2 (Fig. 6 A, 6B).

Above-mentioned data clearly demonstrate the important function of Slit-Robo interaction in generation of tumor lympha pipe and tumor lympha transfer process.Therefore, the material that can influence or regulate and control Slit albumen and Robo protein-interacting just can be used as prevention or the treatment that therapeutic agent is applied to certain part cancer.According to research prompting of the present invention, these reagent can comprise the antibody of Slit, the proteic antibody of Robo, and the proteic extracellular region fragment of Robo, or the like.Can be applied to antibody of the present invention can be monoclonal antibody or polyclonal antibody.And this antibody can also be modified to lower immunologic rejection or complication through humanization.

According to of the present invention, the specific antibody of Robo or extracellular region active fragment can be used for the interaction between the competitive inhibition Slit-Robo, thereby suppress the biological function that the Slit-Robo signal transduction is regulated and control.Equally, the proteic specific antibody of Slit also can be used for diagnosis, prevention and the treatment of the disease that the Slit-Robo signal causes.

According to of the present invention, be used for the antibody that Slit albumen causes treatment of diseases and can be made into humanized antibody, at utmost to lower immunological rejection clinically.It is ripe technically to make humanized antibody, and part work of the present invention relates to the method for the material that this class of a large amount of preparations is used for the treatment of.These methods and reagent will be showed in description subsequently.

Except the disease of preventing or treatment Slit albumen causes, the invention still further relates to the diagnosis of this type of disease.As indicated above, the proteic phenomenon that up-regulated is all arranged in the multiple cancer that is expressed in of Slit.Whether so, detect the proteic expression of Slit increases just to may be used to and indicates whether the disease that exists Slit albumen to cause.

Fig. 8 has showed the specificity analyses of Slit2 protein monoclonal antibody and has been applied to the detection of people's tumor sample.This experimental applications three antibody: S1, S3 and 9E10.We use western blotting method to detect antibody S1 and S3 to Slit2 total length or segmental identification situation.Shown in Fig. 8 a, antibody S1 is by identification proteic nitrogen end of Slit2 and Slit2 protein binding, and antibody S3 is by identification proteic carbon teminal of Slit2 and Slit2 protein binding.Antibody S1 and S3 be energy specific recognition Slit2 albumen all, and antibody S1 can also discern people's Slit1 albumen and Slit3 albumen simultaneously.9E10 antibody (purchasing the company in ATCC) is as positive control.

As shown in Figure 8, antibody S1 can be used in and detects human colon carcinoma and breast carcinoma (Fig. 8 b), and original position pulmonary carcinoma and shift pulmonary carcinoma (Fig. 8 c) is followed lymph metastasis or do not had the Slit2 in the colon cancer (Fig. 8 d) of lymph metastasis to express.The proteic specific antibody of the clear demonstration of above-mentioned experiment Slit can be used as a kind of means and comes various cancers to carry out diagnosis forecast.

Below the particular content that present invention will be further described.

Prevention or treatment and Slit2 protein regulation lymphatic vessel form the method for relevant disease

As mentioned above, one aspect of the present invention relates to the method that forms relevant disease at a certain principal prevention or treatment and Slit2 protein regulation lymphatic vessel.Can find method according to the present invention, for example, the regulation and control lymphatic vessel generates caused disease because Slit-Robo interacts to treat this main body can to reach suitable level by the interaction between minimizing or the inhibition Slit-Robo in a certain main body.These diseases may be relevant with transfer with the process of cancer.

Slit2 albumen can be suppressed by a lot of suitable means with the proteic interaction of Robo (for example Slit2 combines with Robo1 or Slit2 combines with Robo4).For example, can reduce duplicating of Slit2 gene by some active substance, reduce duplicating of Slit2 acceptor gene, reduce the Slit2 gene transcription, reduce transcribing of Slit2 acceptor gene, reduce shearing or the translation of Slit2mRNA, reduce the shearing or the translation of Slit2 receptor mrna, reduce the maturation or the intracellular transport of Slit2 precursor, reduce the maturation or the intracellular transport of Slit2 acceptor precursor, or the like.

Described according to another aspect of the present invention, can reduce by the material that applies effective dose to object or suppress that Slit2 albumen and Robo are proteic combines the interaction that reduces Slit2-Robo.Any suitable material can be used for reducing or block the combination between the Slit2-Robo albumen.For instance, these materials can comprise the antibody at Slit2, the antibody of Robo, perhaps can with the extracellular region protein fragment of the bonded Robo of Slit2.Anyly suitable all can reduce or suppress the protein binding of Slit2-Robo at the antibody of Slit2 or Robo, these antibody can comprise polyclonal antibody, monoclonal antibody, Fab or F (ab ') ₂Antibody fragment and their humanized antibody.In addition, can be also included within the direct or indirect bonded receptor antagonist of Robo receptor in.The antagonist of receptor can comprise can be with Robo bonded and do not influence the micromolecule of normal signal transduction or the analog of Slit2.

Method therefor of the present invention can be used for preventing or treat disease that the receptor-mediated lymphatic vessel of Slit2 forms or unusual.The lymphatic vessel that for example can be used for preventing or treat Slit2-Robo1 or Slit2-Robo4 mediation forms relevant disease or unusual.The interaction of Slit2 and Robo1 or Robo4 can be suppressed by any suitable method.Method is to reduce or suppress the interaction that Slit2-Robo or Slit2-Robo4 protein binding reduce Slit2-Robo1 or Slit2-Robo4 by some material that gives effective dose preferably.These typical materials comprise antibody at Slit2, at the antibody of Robo1, at the antibody of Robo4 and can with the extracellular region section albumen of bonded Robo1 of Slit2 or Robo4.

Any antibody at Slit2 is applicable to the present invention, comprises the antibody at Slit2 described in the article of Hu in 1999 on Neuron.Equally, any suitable antibody at Robo1 also can be used, for example the described antibody at Robo1 of the article of Hivert in 2002 on Mol Cell Neurosci.The antibody of Robo1 is preferably the antibody at first Ig district of Robo1 among the present invention, and more preferential is R5 antibody at first Ig district of Robo1.The extracellular segment of any suitable R obo1 also can be used, such as RoboN albumen.

Used material can be individually dosed among the present invention, and method preferably is with pharmaceutically acceptable carrier or excipient give together.。

The present invention is applicable to that the lymphatic vessel of prevention or treatment Slit2 mediation forms relevant disease or unusual, cancer for example, especially metastatic cancer.For instance, cancer can be malignant melanoma, bladder squamous cell carcinoma, neuroblastoma, small cell lung cancer, colon cancer, transitional cell carcinoma of bladder, breast carcinoma, adenoid cystocarcinoma of salivary gland, hepatoma or rhabdomyosarcoma.

The present invention is applicable to that the lymphatic vessel of the Slit2 mediation of any main body forms the prevention or the treatment of relevant disease.Preferential main body is a mammal, and is particularly human.

The main body of some concrete parts is human among the present invention, and respective substance is humanized monoclonal antibody.

Be used to prevent or treat the drug component and the drug regimen of the lymphatic vessel formation relevant disease of Slit2 mediation

On the other hand, concrete part of the present invention is about preventing or treat the drug component of the Slit2 mediation lymphatic vessel formation relevant disease of a certain main body.These drug components comprise the bonded material of inhibition Slit2-Robo of effective dose, may also contain pharmaceutically acceptable carrier or excipient.

The prescription of medicine, dosage and route of administration can be with reference to existing technical scheme (as Remington:The Scienceand Practice of Pharmacy, Alfonso R.Gennaro (Editor) Mack Publishing Company, April 1997; Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Banga, 1999; And Pharmaceutical Formulation Development of Peptides and Proteins, Hovgaard and Frkjr (Ed.), Taylor ﹠amp; Francis, Inc., 2000; Biopharmaceutical Drug Design and Development, Wu-Pong and Rojanasakul (Ed.), Humana Press, 1999).

Lymphatic vessel for the Slit2 mediation forms relevant disease or unusual prevention or treatment, and the proper dosage level is every kilogram of about 0.01～500mg of weight administration every day.Dosage level preferably is 0.1～250mg/kg every day.In some part, dosage level can be optimized at 0.1～20mg/kg every day.Can carry out administration with single or multiple suitable dose.The dosage level of special body and the frequency of administration are variable, depend on multiple factor, for example treatment that standing of the activity of compound used therefor, metabolic stability, compound effects time, main body age, body weight, health level, sex, diet, state and administration time, drainage rate, medication combined, specific environment and patient.

Drug component of the present invention can carry out administration by any suitable forms such as oral, parenteral, suction, topical, rectum, nasal cavity, oral cavity, vagina, heeling-ins.

On the other hand, specific embodiments of the present invention relates to the drug regimen of the lymphatic vessel formation relevant disease of prevention or treatment Slit2 mediation.This drug regimen can comprise 1) reduce or the bonded effective dose of medicine thing of blocking-up Slit2-Robo 2 among the present invention) suppress or the other drug of the effective dose that the blocking-up lymphatic vessel forms.

For the treatment cancer, any cancer therapy drug can with the present invention's use in conjunction together.The example that can be used for the anticarcinogen among the present invention is referring to U.S.patent application Ser.No.2002/044,919.A kind of antitumor and anticancer agent that wherein is used is an angiogenesis inhibitor reagent.Angiogenesis inhibitor reagent can be that matrix membrane degradation inhibitor, cell migration inhibitor, endotheli ocytosis inhibitor and three-dimensional tissue constitute inhibitor.The instantiation of these angiogenesis inhibitor reagent is referring to Auerbach andAuerbach, Pharmacol.Ther., 63:265-311 (1994); O ' Reilly, Investigational New Drugs, 15:5-13 (1997); J.Nat ' l Cancer Instit., 88:786-788 (1996); U.S.Pat.Nos.5,593,990; 5,629,327 and 5,712,291.These available antitumor and anticancer agents are presented as alkylating agent, antimetabolite, natural product, platinum class coordination compound, amerantrone, replacement urea, methyl hydrazine derivant, adrenal cortex inhibitor, hormone and antagonist on the other hand.

The lymphatic vessel of prevention or diagnosis Slit2 mediation forms the method and the test kit of relevant disease

Embodiments more of the present invention relate to the method that is used to prevent or diagnose the lymphatic vessel formation relevant disease of Slit2 mediation, for example, a kind of method in this embodiment may comprise 1) obtain the detection sample of main body to be measured and detect Slit2 in the above-mentioned sample and (or) level of Slit2 receptor (Robo); 2) lymphatic vessel that obtains not take place the Slit2 mediation form relevant disease the contrast main body check sample and detect Slit2 in the above-mentioned sample and (or) level of Slit2 receptor (Robo); 3) relatively 1) and 2) in two kinds of samples Slit2 with (or) level of Slit2 receptor (Robo), compare with contrast main body sample, the Slit2 of main body sample to be measured and (or) rising of its acceptor levels shows that then above-mentioned detection main body the lymphatic vessel that Slit2 mediates has taken place formed relevant disease.The receptor of the Slit2 that detects in the method can be Robo1 or Robo4.Slit2 and (or) its receptor is in the detection of suitable level, as can be at nucleic acid level or protein level.

Embodiment of the present invention relate to the test kit that is used to prevent or diagnose the lymphatic vessel formation relevant disease of Slit2 mediation, and this test kit comprises: the method that 1) obtains main body to be measured or contrast subject detection sample; 2) method of above-mentioned to be measured or check sample Slit2 and/or Slit2 expression of receptor level of assessment; 3) method of Slit2 and/or Slit2 expression of receptor level in more above-mentioned sample to be tested and the check sample.According to concrete part of the present invention, the receptor of the Slit2 that is surveyed can be Robo1 or Robo4.

Embodiment

1) Slit2 expresses on the various human quasi-cancer cell

In order to seek using value, we have detected the human cancer cell strain of different tissue sources and whether have expressed Slit2.Fig. 1 a shows that Slit2 has expression on malignant melanoma A375 cell strain, bladder squamous cell carcinoma SCaBER cell strain, neuroblastoma SK-N-SH cell strain, small cell lung cancer NCI-H446 cell strain, colon cancer LoVo cell strain, transitional cell carcinoma of bladder T24 cell strain, breast carcinoma ZR-75-30 cell strain, adenoid cystocarcinoma of salivary gland Acc-2 and Acc-M cell strain, hepatocarcinoma SMMC-7721 cell strain and rhabdomyosarcoma A673 cell strain.But Slit2 does not express on pulmonary carcinoma A549, epithelium of cervix uteri adenocarcinoma HeLa, breast carcinoma MCF-7 and original renal cell carcinoma 786-O cell strain.

Slit2 is consistent with the result that the detected Slit2 of RT-PCR method expresses on carcinoma of prostate in expression on the various tumor cell strains.In addition, Fig. 1 b shows that Slit2 has expression in people's malignant melanoma, knot rectal mucus adenocarcinoma, IBC and hepatocarcinoma.It should be noted that in the section of breast carcinoma the dyeing of the local Slit2 that tumor cell and blood vessel are intensive is than local strong (Fig. 1 c) of tumor cell and blood vessel rareness.Similar expression pattern is also arranged in the human hepatocellular carcinoma, but in the knot rectal adenocarcinoma, do not have (result does not show).

2) structure of Slit2/Rip1-Tag2 double transgenic Mus

Fig. 2 a is depicted as and has merged the segmental people Slit2 of Flag expression of gene plasmid construction figure under the CMV promoters driven.Set up the pure and mild mice of 3 strain Slit2 transgenic with this expression vector.Confirmed people Slit2 expression of gene in the transgenic mice by dot blot (Fig. 2 b) and the Southern marking (Fig. 2 c) method, western blotting method has been proved conclusively the expression (Fig. 2 d) of people Slit2 protein level.The monoclonal antibody (M2) that utilizes anti-Flag has detected the expression (Fig. 2 e-f) of pancreatic tissue lysate Flag labelled molecule respectively at protein level and dna level by Western immunoblotting and PCR method.These results have proved conclusively the successful foundation of expressing the transgenic mice of Slit2.

The normal C57 mouse islets of Fig. 2 g (up left side) demonstration is not expressed Slit2, and expresses Slit2 (Fig. 2 g, up right side) in the Rip1-Tag2 transgenic mice islets of langerhans in 12 ages in week.Carry out the expressive site (Fig. 2 g, up right side) of Showed by immune group result Slit2 and the expressive site overlapping (Fig. 2 g, last interline) of insulin with serial section.This result shows that Slit2 becomes in the tumor process by up-regulated expression at pancreatic beta cell.In addition, the Showed by immune group result that serial section is carried out, in the Rip1-Tag2 transgenic mice, Robo1 and vWF are not expressed in (Fig. 2 g, descending) on the pancreatic beta cell expressing on the vascular endothelial cell.

In order further to study the function of Slit2 on tumor growth, we produce Slit2/Rip1-Tag2 double transgenic Mus with Slit2 transgenic mice and the hybridization of Rip1-Tag2 transgenic mice.Compare with 10 weeks of the same age, 13 all Rip1-Tag2 mices, the Slit2 protein level of Slit2/Rip1-Tag2 mice obviously increases (Fig. 2 h), shows that the one-tenth merits and demerits of Slit2 among the hybridize mice Slit2/Rip1-Tag2 are expressed.These results suggest pancreatic beta cells are acted on the Robo1 receptor of stably express on the pancreatic blood endothelial cell by secretion inducing Slit2 albumen by the paracrine mode, thereby activate the signal path of insulinoma growth.

3) Slit2 promotes the Vipoma growth

The number that Fig. 3 a shows courageous and upright islets of langerhans in the pancreas in 9 age in week Slit2/Rip1-Tag2 mices apparently higher than the Rip1-Tag2 mice.Fig. 3 b and Fig. 3 c show that the number of 10 ages in week, the plain tumor of 12 Slit2/Rip1-Tag2 mouse islets in age in week and volume are all apparently higher than the Rip1-Tag2 mice.Yet two kinds of mice peripheral blood leucocyte and tumor-infiltrated leukocyte number do not have notable difference (Fig. 3 d).Fig. 3 e shows with the courageous and upright islets of langerhans of Slit2/Rip1-Tag2 mice inside behind the Lectin protein labeling blood vessel that has FITC the more new vessels of horn of plenty densification.Fig. 3 f shows that the Slit2/Rip1-Tag2 mice has more obvious blood vessel to expand and little bleeding.By SABC statistics vessel density, the result shows that the vessel density of the plain tumor of Slit2/Rip1-Tag2 mouse islets inside is apparently higher than Rip1-Tag2 mice (Fig. 3 g).In addition, the hypertrophy index of the plain tumor of Slit2/Rip1-Tag2 mouse islets is apparently higher than Rip1-Tag2 mice (Fig. 3 h), and apoptotic index is starkly lower than Rip1-Tag2 mice (Fig. 3 i).These results show that Slit2 crosses expression and can promote the new vessels of the plain tumor of Rip1-Tag2 mouse islets to form and tumor growth.

Slit2 crosses and expresses the lymphatic metastasis that promotes Vipoma

Rip1-Tag2 is a kind of transgenic mice of spontaneous generation Vipoma, and its tumor is grown tangible stage: normal islets of langerhans stage, courageous and upright islets of langerhans stage, tumor stage and wellability tumor stage; Most Rip1-Tag2 mices die from the pancreas hyperglycemia at 14-15 about age in week.Rip1-Tag2 and Slit2/Rip1-Tag2 mice to a large amount of 14 ages in week have carried out anatomic observation, find in Rip1-Tag2 mice (32), have only 2 to have only mesenteric lymph node to shift, and 1 intestinal wall shifts; In Slit2/Rip1-Tag2 mice (50), there are 17 mesenteric lymph nodes to shift, 8 intestinal wall shift (Fig. 4 i).There were significant differences for the Chi-square check mesenteric lymph node rate of transform.The heart, liver, spleen, lung, kidney etc. are organized and are not all had transfer in addition.And in this stage, the volume of the Vipoma of Rip1-Tag2 and two kinds of mices of Slit2/Rip1-Tag2 and no significant difference (Fig. 4 j).

Antibody labeling lymphatic endothelial cells with LYVE-1, the lymphatic vessel of immunohistochemical analysis Vipoma in late period generates situation, the result shows that with respect to the Rip1-Tag2 mice same period Slit2/Rip1-Tag2 mice has the relevant lymphatic vessel of more tumor to generate (Fig. 5 a-c).Immunofluorescence is label L YVE-1 and Robo1 altogether, finds to have on the lymphatic endothelial cells Robo1 to express (Fig. 5 d).Above result shows that Slit2 crosses expression and can promote lymphatic vessel to generate and lymphatic metastasis.

Recombinant expressed Slit2 albumen can promote external migration of lymph endotheliocyte and tube chamber to form

Method with immunofluorescence detects the marker protein of people's lymph endotheliocyte of former generation and the expression and localization of Robo1.Can see that two marker protein Lyve-1 and Prox-1 are positive, Lyve-1 mainly is expressed on the film, and Prox-1 is positioned in the nuclear as transcription factor.The antibody of used Robo1 is specific recognition Robo1 and the monoclonal antibody R4 of nonrecognition Robo2-4, and the result shows that Robo1 mainly is positioned on the cell membrane, and a little expression is also arranged in the endochylema.This result shows that this lymph endotheliocyte can express the receptor Robo1 of Slit2 in former generation.

Using Boyden Chamber method detects recombinant expressed Slit2.1 albumen the migration of lymph endotheliocyte is had or not function.With VEGF-C albumen as positive control.Fig. 5 i-j shows that Slit2.1 albumen can promote the migration of lymph endotheliocyte in the above concentration of 200pM; The bonded anti-Robo1 monoclonal antibody R5 of excessive blocking-up Slit2 and Robo1 can suppress the migration of the inductive lymph endotheliocyte of Slit2.

Application Matrigel method detection Slit2.1 albumen has or not function to the external tube chamber formation ability of lymph endotheliocyte, equally also uses VEGF-C as positive control.Fig. 5 g-h shows that Slit2.1 albumen can promote the lymph tube chamber to form in the above concentration of 10nM; The lymph endothelium tube chamber that excessive monoclonal antibody R5 can suppress the Slit2 mediation forms.Thereby these results show Slit2 and form by combine the migration and the tube chamber that have promoted the lymph endotheliocyte with its receptor Robo.

Slit2 is the diagnostic substances of cancer patient lymphatic metastasis

The above results prompting Slit2 may become the marker protein of prediction cancer lymphatic metastasis.The result that the Slit2 SABC that application patient tumor sample carries out detects has supported this viewpoint.Fig. 8 a has shown the specificity of the Recognition Protein of used various antibody in the immunohistochemical experiment.Monoclonal antibody S1 at Slit2 can discern proteic total length of Slit2 and N end.9E10 identification is blended in the proteic c-myc fragment of Slit2.Another monoclonal antibody S3 at Slit2 can discern proteic total length of Slit2 and C end fragment.S1 also discerns people's Slit1 and Slit3 albumen, and (Fig. 8 a).Therefore, S1 can be used to detect various Slit albumen.We carry out SABC with S1 to 955 routine patient's tumor samples, and the result has 742 routine Slit to express be positive (table 1, Fig. 8 b-d).What is more important, in colon cancer, gastric cancer and the pulmonary carcinoma of lymphatic metastasis, the expression of Slit is significantly higher than the tumor of the same race (table 2, Fig. 8 c-d) of non-metastatic.

Table 1: carry out SABC with the S1 monoclonal antibody and detect the proteic expression of patient tumor sample Slit

The positive routine number %Slit positive rate of cancer types sample example number Slit

Colorectal cancer 314 256 81.53

Gastric cancer 326 237 72.7

The esophageal carcinoma 89 86 96.63

Squamous cell lung carcinoma 43 38 88.37

Adenocarcinoma of lung 99 100

Hepatocarcinoma 53 45 84.91

Infiltration ductal carcinomas of breast 95 55 57.89

Bladder cancer 26 16 61.54

Table 2: carry out SABC with the S1 monoclonal antibody and detect the proteic expression of Slit in lymphatic metastasis and the non-transfer patient tumor sample

The %Slit The positive expression rate (is drenched

The %Slit The positive expression rate is (non-

Ba Dao shifts Slit in the sample

Shift the positive sample of Slit in the sample

The total routine number positive sample number/lymphatic channel of cancer types sample

Given figure/non-transfer sample number)

Shift sample number)

Colorectal cancer 155 56.06 (37/66) 25.84 (23/89)

Gastric cancer 69 44.64 (25/56) 23.08 (3/13)

Pulmonary carcinoma 49 45.45 (10/22) 29.63 (8/27)

The clear Slit-Robo signal path that shown of above-mentioned example is being regulated the critical function that lymphatic vessel forms and the tumor lympha road shifts.In view of the above, can regulate or disturb the interactional reagent of Slit-Robo, technology and method can be used for treating the lymphatic vessel formation and/or the relevant disease of lymphatic metastasis of the mediation of Slit-Robo signal.

For instance, Robo protein fragments (as Robo extracellular region or its fusion rotein hRobo-Fc etc.) or all can be used to regulate the protein binding of disturbing Slit-Robo at the antibody of Slit, Robo, thus the method at the treatment relevant disease of Slit-Robo signal is provided.In view of the above, proteic fragment of Robo such as extracellular segment can be used as bait protein and combine with the Slit part, thereby stop or minimizing Slit albumen and Robo receptors bind.Except the application of treatment aspect, the protein fragments of Slit and Robo, specific antibody can also serve as a mark, and molecule is used for diagnosing or the prediction disease.For example, the antibody that can use at Slit or Robo detects tumour patient Slit and/or the proteic expression of Robo, thereby whether assessment has lymphatic metastasis.

Concrete part more of the present invention is relevant based on Slit and/or the proteic reagent of Robo and in the method for treatment, diagnosis, prediction Slit-Robo signal path relevant disease.The embodiment of these concrete parts is described below.

(7) structure of the fusion rotein (hRobo-Fc) of outer IgG sample section of people Robo1 born of the same parents and human normal immunoglobulin Fc section

(7a) clone of human normal immunoglobulin Fc section

With the pAc-k-CH3 plasmid available from carrier's immunoglobulin gene constant region of US Biological company is template, select the part of last 687 bases of its heavy chain C-terminal, as primer, prepare human normal immunoglobulin Fc section with hFc forward and hFc reverse by PCR.The sequence of primer hFc forward and hFc reverse is as follows:

hFc?forward：5’-AACCGTGCGGCCGCTGTTGTGACAAAACTCACAC-3’；[SEQ?IDNO：1]

hFc?reverse：5’-CGCGGAGATCTTCATTTACCCGGAGACAGGGAGAGGC-3’。[SEQ?IDNO：2]

With the PCR product that obtains with NotI and BglII double digestion, be connected to same pVL1393 carrier (BD Pharmingen company) then, make up the baculovirus expression plasmid pVL1393-hFc that obtains carrier's immunoglobulin fc region section through NotI and BglII double digestion.The Fc section DNA sequence of confirming this plasmid through order-checking meets design, not sudden change.

(7b) clone of Robo1 constant gene segment C:

With people Robo1 cDNA (nucleotide sequence NM_002941.2 available from Origene company, the article No. SC109739 of Origene company) be template, select its extracellular to comprise the section in five Ig spline structure territories, from start codon ATG up to the 1621st bit base, as primer, prepare Robo1 constant gene segment C cDNA sequence with Robo1 forward and Robo1 reverse by PCR.The sequence of primer Robo1 forward and Robo1 reverse is as follows:

Robo1?forward：5’-GGCGGCCTCTAGAATGAAATGGAAACATGTTCC-3’；[SEQ?IDNO：3]

Robo1?reverse：5’-CTATAAGCGGCCGCCAATGTAAGCACTCCATGTT-3’；[SEQ?IDNO：4]

With the PCR product that obtains with XbaI and NotI double digestion, be connected to same pVL1393-hFc plasmid then, make up the fusion protein expression plasmid pVL1393-hRobo-Fc that obtains outer five Ig spline structure territories of carrier Robo1 gene born of the same parents and human normal immunoglobulin Fc section through XbaI and the preparation of NotI double digestion.The Robo1 genetic fragment DNA sequence of confirming this plasmid through order-checking meets design, not sudden change, and the Robo1 genetic fragment is consistent with the translation reading frame of hFc section, start codon and termination codon correct position.

(7c) hRobo-Fc Expression of Fusion Protein and purification:

Example I: utilize plasmid to take out test kit (Qiagen company) greatly pVL1393-hRobo-Fc is extracted in a large number, the QIAGEN Plasmid Midi Kit service manual that purification process provides according to Qiagen company.The pVL1393-hRobo-Fc plasmid purification well after, according to the baculovirus expression system service manual of BD Pharmingen company, the recombinant baculovirus of hRobo-Fc gene order is carried in preparation.

Carry out plaque titration and recombinant virus amplification, infect Sf9 insect cell (using the Invitrogen IPL-41 of company culture medium culturing) and carry out the hRobo-Fc Expression of Fusion Protein in 27 ℃ Celsius to screening.Utilize horseradish peroxidase-labeled rabbit anti-human igg (Santa Cruz Biotech) that the hRobo-Fc Expression of Fusion Protein is detected, confirm its successful expression (Fig. 7), the hRobo-Fc fusion rotein is a monomer under the reducing condition, and molecular weight is about 85kd.According to the baculovirus expression system service manual of BDPharmingen company, utilize and carry the recombinate shape virus infection Sf9 insect cell of hRobo-Fc gene order, carry out the great expression of hRobo-Fc fusion rotein.The collecting cell culture fluid utilizes ProteinA affinity chromatography medium (GE Healthcare company) purification hRobo-Fc fusion rotein, the nProtein A Sepharose that purification process provides according to GE Healthcare company ^TM4Fast Flow service manual.Carry the Sf9 cell culture fluid of recombinant baculovirus of hRobo-Fc gene order from 1 liter of infection, can purification obtain the hRobo-Fc fusion rotein of several milligrams of levels.Resulting hRobo-Fc is carried out Function detection to be found, hRobo-Fc can combine (Fig. 6 A) with the hSlit2 protein-specific, and at the monoclonal antibody R5 in first Ig spline structure territory in the Robo1 structure, can be special combine (Fig. 6 B) of inhibition hRobo-Fc and hSlit2, proved absolutely by the resulting Robo genetic fragment of the present invention fusion rotein to have good biological activity and specificity.

With above-mentioned specific embodiment is example, has proved absolutely the feasibility and the effectiveness of specific embodiments of the present invention and effect.We based on this invention prepared multiple relate to system not of the same race (as mice Robo gene), different section the people Robo extracellular region section of 1 Ig spline structure territory (as comprise), different fusion rotein (as GST) and different protein expression systems (as CHO-dhfr: Robo genetic fragment fusion rotein dihydrofolate reductase deficiency Chinese hamster ovary cell expression system), for the drug development that carries out disease associations such as tumor that the Slit-Robo signal path participates in and inflammation has been established solid foundation.Wherein part embodiment is described below.

Example II: utilize baculovirus expression system, comprise the great expression of the fusion rotein (mRobo-Fc) of mice Robo1 gene extracellular region and mouse immuning ball protein Fc section.

1. the choosing and cloning of mouse immuning ball protein Fc section:

With mice IgG _2bThe monoclonal antibody gene is a template, selects the part of last 720 bases of its heavy chain C-terminal, uses mFc forward and mFc reverse as primer, prepares the immunoglobulin fc region section by PCR.The sequence of primer mFc forward and mFc reverse is as follows:

mFc?forward：5’-GCACTCTAGACTTGAGCCCAGCGGGCCCAT-3’；[SEQ?ID?NO：5]

mFc?reverse：5’-CTGAGGATCCTCATTTACCCGGAGACCGGG-3’；[SEQ?ID?NO：6]

With the PCR product that obtains with XbaI and BamHI double digestion, be connected to same pVL1393 carrier (BD Pharmingen company) then, make up the baculovirus expression plasmid pVL1393-mFc that obtains carrying mouse immuning ball protein Fc section through XbaI and BamHI double digestion.The Fc section DNA sequence of confirming this plasmid through order-checking meets design, not sudden change.

2. mice Robo1 constant gene segment C choosing and cloning

With mice Robo1 cDNA (nucleotide sequence NM_019413) is template, selection comprises the section of its five Ig spline structure territories and 3 Fibronectin III domains, from start codon ATG up to the 2445th bit base, as primer, prepare mice Robo1 constant gene segment C cDNA sequence with mRobo1forward and mRobo1 reverse by PCR.The sequence of primer mRobo1 forward and mRobo1 reverse is as follows:

mRobo1?forward：5’-ATCGAGATCTATGATCGCGGAGCCTGCTCACT-3’[SEQ?IDNO：7]

mRobo1?reverse：5’-GCTCTCTAGACGCTGCCACCTCCACACTGTA-3’[SEQ?ID?NO：8]

The PCR product that obtains with BglII and XbaI double digestion, is connected to equally then through the pVL1393-mFc plasmid of BglII and the preparation of XbaI double digestion, makes up and obtain fusion protein expression plasmid pVL1393-mRobo-Fc.The mice Robo1 genetic fragment DNA sequence of confirming this plasmid through order-checking meets design, not sudden change, and mice Robo1 genetic fragment is consistent with the translation reading frame of mFc section, start codon and termination codon correct position.

Utilize plasmid to take out test kit (Qiagen company) greatly pVL1393-mRobo-Fc is extracted in a large number, the QIAGEN Plasmid Midi Kit service manual that purification process provides according to Qiagen company.The pVL1392-mRobo-Fc plasmid purification well after, baculovirus expression system service manual according to BD Pharmingen company, the recombinate shape virus infection Sf9 insect cell of mRobo-Fc gene order is carried in utilization, carries out the great expression of mRobo-Fc fusion rotein.The collecting cell culture fluid utilizes Protein A affinity chromatography medium (GE Healthcare company) purification mRobo-Fc fusion rotein, the nProtein A Sepharose that purification process provides according to GE Healthcare company ^TM4Fast Flow service manual.

EXAMPLE III: utilize baculovirus expression system, comprise the great expression of the fusion rotein of people Robo1 gene extracellular region (different length section) and human normal immunoglobulin Fc section.

1. the choosing and cloning of human normal immunoglobulin Fc section:

With human immunoglobulin gene pAc-k-CH3 (US Biological company) constant region is template, selects the part of last 687 bases of its heavy chain C-terminal, prepares the immunoglobulin fc region section by PCR.Primer sequence is as follows:

hFc?forward：5’-AACCGTGCGGCCGCTGTTGTGACAAAACTCACAC-3’[SEQ?IDNO：9]

hFc?reverse：5’-CGCGGAGATCTTCATTTACCCGGAGACAGGGAGAGGC-3’[SEQ?ID?NO：10]

The PCR product that obtains with NotI and BagII double digestion, is connected to equally then through the pVL1393 carrier (BD Pharmingen company) of NotI and BagII double digestion, makes up and obtain baculovirus expression plasmid pVL1393-hFc.The Fc section DNA sequence of confirming this plasmid through order-checking meets design, not sudden change.

2. the choosing and cloning of people Robo1 constant gene segment C:

With people Robo1 cDNA (nucleotide sequence NM_002941.2, the article No. SC109739 of Origene company) be template, selection comprises the section in its four Ig spline structure territories, up to the 1342nd bit base, prepares constant gene segment C cDNA sequence by PCR from start codon ATG.Primer sequence is as follows:

Robo1?forward：5’-GGCGGCCTCTAGAATGAAATGGAAACATGTTCC-3’[SEQ?IDNO：11]

Robo?reverse2：5’-CATTATGCGGCCGCCATCTGTAACTTCCAAATAT-3’[SEQ?IDNO：12]

With people Robo1 cDNA (nucleotide sequence NM_002941.2, the article No. SC109739 of Origene company) be template, selection comprises the section in its three Ig spline structure territories, up to the 1051st bit base, prepares constant gene segment C cDNA sequence by PCR from start codon ATG.Primer sequence is as follows:

Robo1?forward：5’-GGCGGCCTCTAGAATGAAATGGAAACATGTTCC-3’[SEQ?IDNO：13]

Robo1?reverse3：5’-ATAATAGCGGCCGCGAGGTTCTTGAACAGTCAGAGTA-3’[SEQ?ID?NO：14]

With people Robo1 cDNA (nucleotide sequence NM_002941.2, the article No. SC109739 of Origene company) be template, selection comprises the section in two Ig spline structure territory, up to the 829th bit base, prepares constant gene segment C cDNA sequence by PCR from start codon ATG.Primer sequence is as follows:

Robo1?forward：5’-GGCGGCCTCTAGAATGAAATGGAAACATGTTCC-3’[SEQ?IDNO：15]

Robo1?reverse4：5’-ATAATTGCGGCCGCCATCCACAGTTACTGCCAAGTTACT-3’[SEQ?ID?NO：16]

With people Robo1 cDNA (nucleotide sequence NM_002941.2, the article No. SC109739 of Origene company) be template, selection comprises the section in an one Ig spline structure territory, up to the 529th bit base, prepares constant gene segment C cDNA sequence by PCR from start codon ATG.Primer sequence is as follows:

Robo1?forward：5’-GGCGGCCTCTAGAATGAAATGGAAACATGTTCC-3’[SEQ?IDNO：17]

Robo1?reverse5：5’-TTCTATGCGGCCGCAAGGGTTTTGTCTGAAGTCAT-3’[SEQID?NO：18]

The PCR product that above steps is obtained is with XbaI and NotI double digestion, be connected to same pVL1393-hFc plasmid then, make up the fusion protein expression plasmid that obtains outer different sections of carrier Robo1 gene born of the same parents and human normal immunoglobulin Fc section through XbaI and the preparation of NotI double digestion.The Robo1 genetic fragment DNA sequence of confirming this plasmid through order-checking meets design, not sudden change.Different section Robo1 genetic fragments are consistent with the translation reading frame of hFc section, start codon and termination codon correct position.

Utilize plasmid to take out test kit (Qiagen company) greatly pVL1393-hRobo-Fc is extracted in a large number, the QIAGEN P1asmid Midi Kit service manual that purification process provides according to Qiagen company.The pVL1392-hRobo-Fc plasmid purification well after, baculovirus expression system service manual according to BD Pharmingen company, the recombinate shape virus infection Sf9 insect cell of hRobo-Fc gene order is carried in utilization, carries out the great expression of hRobo-Fc fusion rotein.The collecting cell culture fluid utilizes Protein A affinity chromatography medium (GE Healthcare company) purification hRobo-Fc fusion rotein, the nProtein A Sepharose that purification process provides according to GE Healthcare company ^TM4Fast F1ow service manual.

EXAMPLE IV: utilize dihydrofolate reductase deficiency Chinese hamster ovary cell (CHO-dhfr) expression system, comprise the great expression of the fusion rotein of people Robo1 gene extracellular region (different length section) and human normal immunoglobulin Fc section.

1. the choosing and cloning of people Robo1 constant gene segment C:

With people Robo1 cDNA (nucleotide sequence NM_002941.2, the article No. SC109739 of Origene company) be template, selection comprises the section in its five Ig spline structure territories, up to the 1760th bit base, prepares constant gene segment C cDNA sequence by PCR from start codon ATG.Primer sequence is as follows:

Robo1?f1760：5’-GGCCAAGCTTATGAAATGGAAACATGTTCC-3’；[SEQ?ID?NO：19]

Robo1?r1760：5’-TCCACGGAATTCAAATTTGGTTGCC-3’[SEQ?ID?NO：20]

The PCR product that above steps is obtained is with HindIII and EcoRI double digestion, be connected to same pEGFP-N1 carrier (BD Pharmingen) then, make up the plasmid pEGFP-N1-hRobo that obtains outer five the Ig spline structure territories of carrier Robo1 gene born of the same parents through HindIII and the preparation of EcoRI double digestion.The Robo1 genetic fragment DNA sequence of confirming this plasmid through order-checking meets design, not sudden change.

2. the choosing and cloning of human normal immunoglobulin Fc section:

With human immunoglobulin gene pAc-k-CH3 (US Biological company) constant region is template, selects the part of last 714 bases of its heavy chain C-terminal, prepares the immunoglobulin fc region section by PCR.Primer sequence is as follows:

hFc?forward2：5’-AACCGTGAATTCCGTGGACAAGAGAGTTGAGCC-3’；[SEQ?IDNO：21]

hFc?reverse2：5’-TACGGGTCGACTCATTTACCCGGAGACAGGG-3’[SEQ?ID?NO：22]

With the PCR product that obtains with EcoRI and SalI double digestion, be connected to same pEGFP-N1-hRobo plasmid then, make up the plasmid pEGFP-N1-hRobo-Fc that obtains outer five Ig spline structure territories of carrier Robo1 gene born of the same parents and human normal immunoglobulin Fc section through EcoRI and SalI double digestion.The Fc section DNA sequence of confirming this plasmid through order-checking meets design, not sudden change.Different section Robo1 genetic fragments are consistent with the translation reading frame of hFc section, start codon and termination codon correct position.

With the pEGFP-N1-hRobo-Fc plasmid that obtains with HindIII and SalI double digestion, be connected to same p3CI-dhfr carrier then, make up the plasmid p3CI-dhfr-hRobo-Fc that obtains outer five Ig spline structure territories of carrier Robo1 gene born of the same parents and human normal immunoglobulin Fc section through HindIII and SalI double digestion.It is consistent to confirm that through order-checking frame is read in the different section Robo1 of this plasmid genetic fragment and the translation of hFc section, start codon and termination codon correct position.

Utilize plasmid to take out test kit (Qiagen company) greatly the p3CI-dhfr-hRobo-Fc plasmid is extracted in a large number, the QIAGEN Plasmid Midi Kit service manual that purification process provides according to Qiagen company.The p3CI-dhfr-hRobo-Fc plasmid purification well after, according to the Lipofectin service manual of Invitrogen company, infect the CHO-dhfr cell, the screening high-expression clone.The collecting cell culture fluid utilizes Protein A affinity chromatography medium (GE Healthcare company) purification hRobo-Fc fusion rotein, the nProtein A Sepharose that purification process provides according to GE Healthcare company ^TM4 FastFlow service manuals.

EXAMPLE V: utilize baculovirus expression system, comprise the great expression of the fusion rotein of people Robo1 gene extracellular region and human normal immunoglobulin Fc section.

1.pVL1393-hRobo1-Fc structure:

With the p3CI-dhfr-hRobo-Fc plasmid that obtains in the EXAMPLE IV is template, prepares human normal immunoglobulin Fc section by PCR.Primer sequence is as follows:

Robo1?forward’：5’-AGGCGGCCTCTAGAATGAAATGGAAACATGTTCC-3’；[SEQ?IDNO：23]

hFc?reverse3：5’-TACGGGCGGCCGCTCATTTACCCGGAGACAG-3’[SEQ?ID?NO：24]

With the PCR product that obtains with XbaI and NotI double digestion, be connected to same pVL1393 carrier (BD Pharmaingen) then, make up the plasmid pVL1393-hRobo-Fc that obtains outer five Ig spline structure territories of carrier Robo1 gene born of the same parents and human normal immunoglobulin Fc section through XbaI and NotI double digestion.It is consistent to confirm that through order-checking frame is read in the different section Robo1 of this plasmid genetic fragment and the translation of hFc section, start codon and termination codon correct position.

2.hRobo-Fc Expression of Fusion Protein and purification:

Utilize plasmid to take out test kit (Qiagen company) greatly the pVL1393-hRobo-Fc plasmid is extracted in a large number, the QIAGEN Plasmid Midi Kit service manual that purification process provides according to Qiagen company.The pVL1393-hRobo-Fc plasmid purification well after, baculovirus expression system service manual according to BD Pharmingen company, the recombinate shape virus infection Sf9 insect cell of hRobo-Fc gene order is carried in utilization, carries out the great expression of hRobo-Fc fusion rotein.The collecting cell culture fluid utilizes Protein A affinity chromatography medium (GE Healthcare company) purification mRobo-Fc fusion rotein, the nProtein A Sepharose that purification process provides according to GE Healthcare company ^TM4Fast Flow service manual.

Experimental technique

RT-PCR and Northern hybridization

(Geng, J.-G.et al.Nature 343:757-760 (1990)) is described for former generation HUVECs cell culture such as document.(Ma, Y.-Q.and Geng, J.-G.J.Immunol.165:558-565 (2000)) is described for sxemiquantitative RT-PCR experimental technique such as document.The primer is as follows in this research: human Robo1 sense (+4440) 5 '-CCT ACA CAG ATG ATCTTC C-3 ' (SEQ ID NO:25), antisense (4956) 5 '-CAG AGG AGC CTG CAG CTC AGC TTTCAG TTT CCT C-3 ' (SEQ ID NO:26); Human Slit2 sense (+3611) 5 '-GGT GAC GGA TCCCAT ATC GCG GTA GAA CTC-3 ' (SEQ ID NO:27), antisense (4574) 5 '-GGA CAC CTCGAG CGT ACA GCC GCA CTT CAC-3 ' (SEQ ID NO:28); Human β-actin sense (+1) 5 '-ATGGAT GAT GAT ATC GCC GC-3 ' (SEQ ID NO:29), antisense (1127) 5 '-CTA GAA GCA TTTGCG GTG G-3 ' (SEQ ID NO:30).Use ³²The Slit2 of P labelling or Robo1 cDNA fragment detect A375 cell and former generation HUVECs cell total rna.

The preparation of antibody, immunoblotting and immunostaining

Slit2-GST (comprise people Slit2 the 57th to 207 bit bases) and Robo1-GST fusion rotein (comprise the 1st of rat Robo1 to 168 bit bases or the 961st to 1217 bit bases) are built into pGEX-4T-1 carrier (available from Amersham Pharmacia Biotech company).With this fusion rotein is that antigen immune rabbit or mice are in order to preparation anti-Slit2 or anti-Robo1 polyclonal antibody and monoclonal antibody.(Liu, L.-P.et al.Biochem.Biophys.Res.Commun.286:281-291 (2001)) is described for immunohistochemical assay method such as document.

The proteic separation and purification of Slit2 and RoboN

The human embryo kidney (HEK) 293 cell line Slit2/293 of stably express total length people Slit2 albumen (c-terminus merges the myc label) build system, method such as document (Li, H.S.et al.Cell 96:807-818 (1999); Wang, K.-H.et al.Cell 96:771-784 (1999)) described.The collecting cell culture fluid utilizes monoclonal antibody the 9E10 (～1mg/ml of Affi-Gel 10 of anti-myc label; Bio-Rad), use affinity chromatography purification Slit2 albumen.The proteic silver of Slit2 dyes with western blotting method such as document (Ma, L.et al.J.Biol.Chem.269:27739-27746 (1994)) described.

The human embryo kidney (HEK) 293 cell line RoboN/293 of stably express Robo1 extracellular region protein (c-terminus merges hemoagglutinin hemoagglutinin label) build system, method such as document (Li, H.S.et al.Cell 96:807-818 (1999); Wang, K.-H.et al.Cell 96:771-784 (1999)) described.The collecting cell culture fluid, the affinity column purification RoboN albumen of use coupling anti-HA monoclonal antibody HA11 (available from BAbCO company).

Boyden Chamber experiment

Use is carried out cell migration experiment (Terranova, V.P.et al.J.Cell.Biol.101:2330-2334 (1985)) available from 48 hole Boyden Chamber of Neuro Probe company.Before the experiment, cell to be measured is at hungry 4-8 hour (the deciding according to cell state) of EBM/2%FBS.During experiment, the polycarbonate membrane in 8um aperture soaked 45-60 minute in 1% gelatin.In 1% gelatin, soaked 45-60 minute.Add Slit2 or bFGF (available from Sigma company) in the Chamber lower floor; The Chamber upper strata adds the cell suspension (HUVECs, rRobo1/293 and V/293 cell are in the 1%FCS/M199 culture fluid, and density is 500000cell/ml) of certain density.The environment of cell migration is 37 ℃, and incubation time is 4 hours.End to be migrated, carefully take off film from Chamber, and remove the cell that does not move on the film upper strata, the upper strata of film is immersed in 4% paraformaldehyde 4 ℃ downwards and fixedly spends the night, PBS cleans film once and dyeing (room temperature 4-6 hour) in 0.5% crystal violet/PBS then, the cell number of migration is examined under a microscope and added up to PBS flush away surface loose colour.

Directional Migration experiment

The albumen gradient is used document (Hopker, V.H.et al.Nature 401:69-73 (1999)) to mention experimental technique and is produced.Tank of compressed air provides air pressure for pressure injector, and stimulator sends the pulse signal of certain frequency, the controlled pressure drug administration by injection.Use tip diameter to be about 1 micron capillary glass tube administration, spray into 0.15 μ M/picolitre Slit2 or 1 μ M/picolitre bFGF at every turn.Add the Matrigel (available from Becton Dickinson Labware) of 200ul in 24 orifice plates, cell to be measured is joined in the hole that is covered with Matrigel, at 37 ℃ of 5%CO ₂Incubation in the incubator.Capillary glass tube is adjusted to 100 microns of itself and cell distances, the CCD photographing unit (JVC) that use is connected on the Olympus IX70 microscope is noted down image and be kept in the computer, the cell migration situation of using NIH Image or other appropriate software analyses to put at any time.

Tube?Formation?Assay

96 orifice plates and Matrigel are in advance in pre-cooling on ice.Every hole adds the Matrigel (sterile working on ice) of 100ul, place 30 minutes (Malinda for 37 ℃, K.M.et al., Identification of laminin α 1 and β 1 chain peptidesactive for endothelial cell adhesion, tube formation and aortic sprouting, FASEB is (1999) J.13:53-62).After treating that it is frozen into glue, with the cell concentration of 130000Cell/ml with the HUVEC cell dilution in M199/2%FCS solution, the cell suspension that adds 100ul is in 96 orifice plates that are covered with Matrigel.Cell is observed the also length of statistics network spline structure of taking pictures in microscopically after 37 ℃/5%CO2 cultivates 12-18 hour.

The experiment of Xenograft tumor growth

The Lipofectin of A375 cell available from Gibco company ^TMTransfection is also with 400 μ g ml ^-1Hygromycin B (available from Sigma company) screening.Use the antibody of Robo1, Slit2 or tubulin (as last sample contrast), with method validation RoboN/A375_C1, C2, C3 and the V/A375 cell of immunoblotting.Cell suspension is in DME training liquid, at nude mice oxter subcutaneous injection 0.2ml cell suspension (O ' Reilly, M.S.et al., Endostatin:An endogenous inhibitor ofangiogenesis and tumor growth, Cell 88:277-285 (1997)).Suppress experiment for antibody, the mice of injection tumor cell is taked the mode of lumbar injection, the R5 of each every nude mice injection 1mg or contrast IgG _2bAntibody is injected weekly twice (Muller, A.et al.Nature 410:50-56 (2001)).After about 30 to 35 days, nude mice is got the number that the blood lineage counts blood middle leukocytes, puts to death nude mice then.Myeloperoxidase (MPO) MPO method such as the document (Wang, J.-G.et al.Inflamm.Res.51:435-43 (2002)) of measuring the neutrophil cell number in the tumor mass are described.

Cell?Proliferation?Assay

The cell trypsinization of exponential phase, and be suspended in the RPMI-1640 with 5000 cells/ml.The cell suspension of 100 microlitres is joined a hole in 96 orifice plates, be placed on 37 ℃, 5%CO ₂Cultivate in the incubator.At different time points, add the tetrazolium salt of 20 microlitres in every hole, placed 4 hours for 37 ℃, add the 10%SDS/5%isobutanol/ hole cessation reaction of 100 microlitres again.On microplate reader, measure the OD570/630 value.

Other experiments are as immunoprecipitation experiment, and lectin pours into experiment, and HE dyeing and immunofluorescence dyeing are generally acknowledged experimental technique with each laboratory.

The present invention will provide the concrete technical scheme for preparing Robo genetic fragment fusion rotein how effectively in a large number, comprise the selection of Robo genetic fragment, fusion rotein and expression system etc.The present invention can also be by the signal path research that the formation of mediation new vessels, lymphatic vessel formation and tumor lympha are shifted, and exploitation is to treatment of diseases and diagnostic methods such as tumor or inflammation.As suppressing the Slit-Robo signal path by specificity, destroy Slit albumen and the proteic combination of its receptor Robo, and then suppress that new vessels forms, lymphatic vessel forms and tumor lympha shifts, for the drug development that carries out disease associations such as tumor that the Slit-Robo signal path participates in and inflammation has been established solid foundation.

Though the minority embodiment has been set forth in this experiment, this area professional knows that this invention design comprises other embodiments under the condition that does not deviate from the scope of the invention.So the scope of the invention is defined by the additional requirement protection.

Sequence table

＜110〉Shanghai Inst. of Life Science, CAS

 

＜120〉lymphatic vessel of SLIT-ROBO mediation forms and uses

 

<130>102208

 

<160>30

 

<170>PatentIn?version?3.3

 

<210>1

<211>34

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>1

aaccgtgcgg?ccgctgttgt?gacaaaactc?acac 34

 

<210>2

<211>37

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>2

cgcggagatc?ttcatttacc?cggagacagg?gagaggc 37

 

<210>3

<211>33

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>3

ggcggcctct?agaatgaaat?ggaaacatgt?tcc 33

 

<210>4

<211>34

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>4

ctataagcgg?ccgccaatgt?aagcactcca?tgtt 34

 

<210>5

<211>30

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>5

gcactctaga?cttgagccca?gcgggcccat 30

 

<210>6

<211>30

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

<400>6

ctgaggatcc?tcatttaccc?ggagaccggg 30

 

<210>7

<211>32

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>7

atcgagatct?atgatcgcgg?agcctgctca?ct 32

 

<210>8

<211>31

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>8

gctctctaga?cgctgccacc?tccacactgt?a 31

 

<210>9

<211>34

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>9

aaccgtgcgg?ccgctgttgt?gacaaaactc?acac 34

 

<210>10

<211>37

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>10

cgcggagatc?ttcatttacc?cggagacagg?gagaggc 37

 

<210>11

<211>33

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>11

ggcggcctct?agaatgaaat?ggaaacatgt?tcc 33

 

<210>12

<211>34

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>12

cattatgcgg?ccgccatctg?taacttccaa?atat 34

 

<210>13

<211>33

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>13

ggcggcctct?agaatgaaat?ggaaacatgt?tcc 33

 

<210>14

<211>37

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>14

ataatagcgg?ccgcgaggtt?cttgaacagt?cagagta 37

 

<210>15

<211>33

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>15

ggcggcctct?agaatgaaat?ggaaacatgt?tcc 33

 

<210>16

<211>39

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>16

ataattgcgg?ccgccatcca?cagttactgc?caagttact 39

 

<210>17

<211>33

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>17

ggcggcctct?agaatgaaat?ggaaacatgt?tcc 33

 

<210>18

<211>35

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>18

ttctatgcgg?ccgcaagggt?tttgtctgaa?gtcat 35

 

<210>19

<211>30

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>19

ggccaagctt?atgaaatgga?aacatgttcc 30

 

<210>20

<211>25

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>20

tccacggaat?tcaaatttgg?ttgcc 25

 

<210>21

<211>33

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>21

aaccgtgaat?tccgtggaca?agagagttga?gcc 33

 

<210>22

<211>31

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>22

tacgggtcga?ctcatttacc?cggagacagg?g 31

 

<210>23

<211>34

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>23

aggcggcctc?tagaatgaaa?tggaaacatg?ttcc 34

 

<210>24

<211>31

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>24

tacgggcggc?cgctcattta?cccggagaca?g 31

 

<210>25

<211>19

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>25

cctacacaga?tgatcttcc 19

 

<210>26

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

 

<400>26

cagaggagcc?tgcagctcag?ctttcagttt?cctc 34

 

<210>27

<211>30

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>27

ggtgacggat?cccatatcgc?ggtagaactc 30

 

<210>28

<211>30

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>28

ggacacctcg?agcgtacagc?cgcacttcac 30

 

<210>29

<211>20

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>29

atggatgatg?atatcgccgc 20

 

<210>30

<211>19

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉primer

 

<400>30

ctagaagcat?ttgcggtgg 19

Claims (23)

1. material is used for preventing or treats application by the medicine of Slit transporter mediated diseases in preparation, and described material is the medicine that can regulate or block Slit albumen and Robo protein-interacting, and described disease forms relevant with lymphatic vessel.

2. application as claimed in claim 1 is characterized in that, described Slit albumen is Slit2.

3. application as claimed in claim 1 is characterized in that, described Robo albumen is Robo1 or Robo4.

4. as each described application of claim 1-3, it is characterized in that described medicine is selected from: at the proteic antibody of Slit, at the proteic antibody of Robo, can be in conjunction with Slit proteic Robo protein extracellular fragment or Robo fusion rotein.

5. application as claimed in claim 4 is characterized in that described antibody is monoclonal antibody.

6. application as claimed in claim 5 is characterized in that described antibody is humanized antibody.

7. application as claimed in claim 4 is characterized in that, describedly can derive from proteic first immunoglobulin-like zone of Robo in conjunction with the proteic Robo protein extracellular of Slit fragment.

8. application as claimed in claim 4 is characterized in that, described Robo fusion rotein is the Robo-Fc fusion rotein.

9. one kind is prevented or treats the pharmaceutical composition by the Slit transporter mediated diseases, it is characterized in that, described pharmaceutical composition comprises the medicine that can regulate or block Slit albumen and Robo protein-interacting, with pharmaceutically acceptable drug excipient, carrier or solvent, wherein said disease forms relevant with lymphatic vessel.

10. pharmaceutical composition as claimed in claim 9 is characterized in that, described Slit albumen is Slit2.

11. as claim 9 or 10 described pharmaceutical compositions, it is characterized in that described can regulate or blocking-up Slit albumen is selected from the medicine of Robo protein-interacting: at the proteic antibody of Slit, at the proteic antibody of Robo, can be in conjunction with Slit proteic Robo protein extracellular fragment or Robo fusion rotein.

12. pharmaceutical composition as claimed in claim 11 is characterized in that, described antibody is monoclonal antibody.

13. pharmaceutical composition as claimed in claim 12 is characterized in that, described antibody is humanized antibody.

14. pharmaceutical composition as claimed in claim 11 is characterized in that, describedly can derive from proteic first immunoglobulin-like zone of Robo in conjunction with the proteic Robo protein extracellular of Slit fragment.

15. pharmaceutical composition as claimed in claim 11 is characterized in that, described Robo fusion rotein is the Robo-Fc fusion rotein.

16. the material that can detect Slit albumen or Robo albumen or their pairing mRNA or their encoding gene is used for detecting the application of the diagnostic reagent of the protein mediated disease of Slit in preparation, described detection may further comprise the steps:

(a) obtain detecting sample from detecting target, the material that utilization can detect Slit albumen or Robo albumen or their pairing mRNA or their encoding gene detects independent Slit or the level of Robo or the level of Slit and Robo;

(b) obtain sample in contrast from the normal person, the material that utilization can detect Slit albumen or Robo albumen or their pairing mRNA or their encoding gene detects independent Slit or the level of Robo or the level of Slit and Robo of this sample;

(c) relatively (a) and (b) detected expression of results determine the disease whether this detection target has Slit albumen to cause, if the Slit or the Robo level of detection sample are higher than contrast, show that then this detection target suffers from described disease.

17. application as claimed in claim 16 is characterized in that, described Slit albumen is Slit2.

18. application as claimed in claim 16 is characterized in that, described Robo albumen is Robo1 or Robo4.

19. application as claimed in claim 16 is characterized in that, described level is a dna level, rna level, protein level, or comprehensive above-mentioned 2 or 3 level.

20., it is characterized in that described disease is the disease relevant with the Slit-Robo signal transduction pathway as claim 1 or 16 described application or the described pharmaceutical composition of claim 9.

21., it is characterized in that described disease is cancer or forms relevant disease with lymphatic vessel as claim 1 or 16 described application or the described pharmaceutical composition of claim 9.

22. application as claimed in claim 21 or pharmaceutical composition is characterized in that, described cancer is a metastatic cancer.

23. application as claimed in claim 21 or pharmaceutical composition is characterized in that, described cancer is one of following cancer: colon cancer, gastric cancer, esophageal carcinoma, pulmonary carcinoma, hepatocarcinoma, breast carcinoma, and bladder cancer.

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