WO2008011799A1 - NEW USE OF NON-RECEPTOR TYROSINE KINASE c-Ab1 SPECIFIC INHIBITORS - Google Patents

NEW USE OF NON-RECEPTOR TYROSINE KINASE c-Ab1 SPECIFIC INHIBITORS Download PDF

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WO2008011799A1
WO2008011799A1 PCT/CN2007/002113 CN2007002113W WO2008011799A1 WO 2008011799 A1 WO2008011799 A1 WO 2008011799A1 CN 2007002113 W CN2007002113 W CN 2007002113W WO 2008011799 A1 WO2008011799 A1 WO 2008011799A1
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cancer
abl
tumor
cells
gal3
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PCT/CN2007/002113
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French (fr)
Chinese (zh)
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Xiaoming Li
Cheng Cao
Qingjun Ma
Xuan Liu
Yanwen Jin
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Institute Of Bioengineering, Academy Of Military Medical Sciences
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4412—Non condensed pyridines; Hydrogenated derivatives thereof having oxo groups directly attached to the heterocyclic ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/541—Non-condensed thiazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to novel uses of non-receptor tyrosine kinase c-Abl specific inhibitors, particularly to the use of non-receptor tyrosine kinase c-Abl specific inhibitors in the preparation of antitumor drugs.
  • Galectin-3 is a galactosidase-binding protein that has a variety of biological functions and is involved in tumor cell adhesion, proliferation, differentiation, angiogenesis, deterioration and metastasis as a cancer-associated protein.
  • Clinical studies have shown that in many types of tissue cells, Gal3 expression is up-regulated when cells undergo malignant transformation (eg cancer in the colon, thyroid, central nervous system, pancreas, bladder, kidney, stomach, etc.), and Up-regulation of Gal3 expression levels in tumor cells increases the malignancy and metastatic invasiveness of tumors.
  • Gal3 is considered to be a cancer-associated protein and plays an important role in carcinogenesis and tumor progression.
  • the anti-apoptotic activity of Gal3 is manifested by the ability to inhibit drug-induced apoptosis and anchorage-dependent apoptosis to allow cell survival.
  • Gal3 as an indicator of clinical detection of immune cytochemistry, has certain value for the diagnosis of benign and malignant tumor cells and the diagnosis of cancer prognosis. Therefore, Gal3 has a very good application prospect as a new target in the diagnosis and treatment of various types of cancer.
  • Protein tyrosine kinases are a class of proteins with tyrosine kinase activity and can be divided into receptor and non-receptor types, which catalyze the transfer of phosphates on ATP to tyrosine residues of many important proteins. On, make it phosphorylated. Protein tyrosine kinases play an important role in the signal transduction pathways in cells, regulating a series of physiological processes such as cell growth, differentiation and apoptosis.
  • the ABL family of non-receptor protein tyrosine kinases includes two members: Abl and Arg.
  • the Abl gene is located on the long arm of chromosome 9 (9q34.1) and encodes the 145kD Abl protein.
  • c-Abl protein Has tyrosine kinase activity, including SH3, SH2, PTK, DNA binding domain, actin binding domain, etc. (Blume-Jensen P, Hunter T. Oncogenic kinase signalling. Nature, 2001, 411 (6835): 355-65 ).
  • the non-receptor tyrosine kinase c-Abl specific inhibitor can inactivate the ubiquitous Abelson tyrosine kinase (Abl).
  • the application of the present invention is to prepare an antitumor drug or a tumor therapeutic auxiliary drug by using a non-receptor tyrosine kinase c-Abl specific inhibitor as an active ingredient.
  • the non-receptor tyrosine kinase c-Abl specific inhibitors are selected in a variety of ways, specifically STI571, Sunitinib, PKC412, vandetanib Sorafenib, erlotinib, gefitinib, dasatinib and the like.
  • chemotherapeutic drugs such as cisplatin and CTX may be added to the anti-tumor drugs or tumor therapeutic auxiliary drugs prepared by using the non-receptor tyrosine kinase c-Abl specific inhibitor.
  • non-receptor tyrosine kinase c-Abl-specific inhibitors can cause Gal3 to undergo degeneration and aggregation and lysosome degradation by inhibiting the kinase activity of C-Abl, resulting in a significant decrease in Gal3 protein levels in tumor cells, resulting in tumors.
  • the cells die of autophagic cell apoptosis.
  • non-receptor tyrosine kinase c-Abl specific inhibitors have a broad spectrum of anti-tumor effects, including breast cancer, prostate cancer, cervical cancer, lung adenocarcinoma, liver cancer, Colon cancer, thyroid cancer, pancreatic cancer, neuroendocrine tumor, bladder cancer, kidney cancer, stomach cancer, and the like.
  • one or more pharmaceutically acceptable excipients may be added, and the excipients include conventional diluents, excipients, and the like in the pharmaceutical field.
  • a filler, a binder, a wetting agent, an absorption enhancer, a surfactant, a lubricant, a stabilizer, etc., and if necessary, a flavoring agent, a sweetener, a coloring matter, and the like may be added.
  • the antitumor drug or the tumor therapeutic auxiliary drug prepared by the invention can be prepared into a plurality of drug forms such as capsules, tablets, powders, granules, and injections, in addition to the oral solution.
  • the above various dosage forms of the drug can be prepared according to a conventional method in the pharmaceutical field.
  • the oral dose of the drug is generally 400rag/day or 5.55rag/kg. body weight/day, which can be used once or in multiple times for 20 days to 30 days.
  • DRAWINGS Figure 1 shows the results of immunoprecipitation and immunoblotting of Gal3-specific phosphorylation by non-receptor tyrosine kinase c-Abl.
  • Figure 2 shows the results of immunoprecipitation and immunoblotting of C -Abl-specific phosphorylation by non-receptor tyrosine kinase c-Abl specific inhibitor STI571.
  • Figure 3 shows the results of immunostaining for the distribution of Gal3 protein in MCF-7 cells treated with lOuM STI571.
  • Figure 4 shows the results of immunostaining assay for changes in Gal3 protein content in MCF 7 cells treated with 5 uM STI571, 0.5 uM apoptosis-inducing agent STS alone and co-treated.
  • Figure 5 shows the results of immunoblotting for the change of Gal3 protein in MCF-7 cells treated with 5uM STI571, 0.5 uM apoptosis inducer STS alone and co-treated.
  • Figure 6 shows the results of TUNEL assay for apoptosis of MCF-7 cells treated with lOuM STI57K 0. 5uM STS alone and in combination.
  • Figure 7 shows the results of Annexin-V-FLU0S assay for apoptosis of MCF-7 cells treated with lOuM STI571, 0.5 uM STS alone and both.
  • Figure 8 shows the Sub G1 assay for apoptosis of MCF-7 cells treated with lOuM STI571, 4 uM CDDP alone and co-treated.
  • Figure 9 shows the treatment of tumors in nude mice. '
  • Figure 10 shows the body weight statistics of nude mice.
  • Figure 11 shows the results of HE staining of tumor sections.
  • the flag tag will be encoded (a peptide consisting of the following 8 amino acids:
  • the cDNA obtained by reverse transcription of total RNA extracted from normal human breast tissue cells carrying the Gal3 gene was used as a template in primer P1 (upstream primer): 5 '-CGGATCCATGGCAGACAATTTTTCGCTCCA-3 ' (underlined Bases are restriction endonucleases ⁇ 3 ⁇ 4/ ⁇ 1 recognition site) and P2 (downstream primers):
  • the 5'-CGGAATTCTTATATCATGGTATATGAAGCA-3 (underlined base is a restriction endonuclease recognition site), the Gal3 gene was amplified by PCR and the restriction enzymes BainH I and EcoR I were added to both ends of the gene fragment. After the reaction, the PCR amplification product was detected by 1% agarose gel electrophoresis, and a 753 bp DNA fragment was obtained, which was consistent with the expected result. The target fragment was recovered and purified, sequenced, and the sequencing result was obtained.
  • a Gal3 gene fragment (CDS sequence of the Gal3 gene) having a 25'-777 deoxyribonucleotide at the 5' end of GenBank Accession Number: BC001120.
  • the Gal3 gene fragment cloned in step 2 was digested with restriction endonucleases BamH I and EcoR I, and the restriction fragment was ligated with the vector pcDNA 3-flag constructed in step 1 which was digested with the same enzyme, and ligated.
  • the product was transformed into E. coli DH5 ci competent cells by calcium chloride method, positive transformants were screened, plasmids were extracted, and restriction endonucleases/ and I were used for restriction enzyme digestion, and 5400 bp and 753 bp fragments were positively digested.
  • the positive clones were further identified by PCR.
  • the primers were P1 and P2.
  • the PCR products were sequenced.
  • the Gal3 gene can express a fusion protein with a Flag tag under the action of a CMV promoter.
  • a human fetal liver cDNA library (clontech product) carrying the wild type c-Abl gene (GenBank Accession Number: ⁇ -007313) was used as a template in the primer Myc-Abl-1 (upstream primer): 5 '-CGAATTCGGATGG6GCAGCAGCCTGGAA-3 ' (underlined bases are restriction endonuclease I recognition sites) and Myc- (downstream primers):
  • the c-Abl gene was amplified by PCR under the guidance of the ⁇ recognition site, and the restriction endonucleases EcoR I and Bgl II were added to the ends of the gene fragment. After the reaction, the PCR amplification products were subjected to the PCR amplification products. After 1% agarose gel electrophoresis, a 3450 bp DNA fragment was obtained, which was consistent with the expected results. The target fragment was recovered and purified, and sequenced. The sequencing result showed that the 5' end was obtained from GenBank Accession Number: ⁇ -007313.
  • the c-Abl gene fragment of the 440th to 3889 deoxyribonucleotides (the CDS sequence of the c-Abl gene).
  • the c-Abl gene fragment cloned in step 1) was digested with restriction endonucleases EcoR I and ⁇ 3 ⁇ 4 ⁇ _ ⁇ , and the restriction fragment was digested with the vector pCMV-Myc (Clontech) which was digested with the same enzyme. Ligation, the ligation product was transformed into E. coli DH5 C1 competent cells by calcium chloride method, positive transformants were screened, plasmids were extracted, and restriction endonucleases were used to identify the digestive enzymes ⁇ 1 and / / II, and obtained by enzyme digestion. 3800 bp (vector fragment) and 3450 bp fragment were positive clones, and the positive clone plasmid was further identified by PCR.
  • the primers used were Myc-Abl-1 and Myc-Abl-2, and the PCR products were sequenced.
  • the c-Abl gene fragment was inserted between the EcoR I and Bgl II restriction sites of pCMV-Myc, and the sequence was correct.
  • the recombinant expression vector was named pCMV_Myc-c-Abl.
  • the Abl gene can express a fusion protein with a Myc tag under the action of the CMV promoter.
  • the 896 bp Ml fragment and the 2609 bp M2 fragment were obtained, which were consistent with the expected results, recovered and purified.
  • Two target fragments then, 1 ⁇ each of M1 and M2, and mixed, using this as a template, PCR-amplified the full sequence of the c-Abl mutant gene under the guidance of primers Myc-Abl-1 and Myc-Abl-2, After the reaction, the PCR amplification product was subjected to 1% agarose gel electrophoresis, and a DNA fragment of (3450 + 17) bp was obtained, which was consistent with the expected result, and the target fragment was recovered and purified.
  • the c_Abl point mutation gene fragment cloned in step 1) was digested with restriction endonucleases EcoR I and Bgl II, and the restriction fragment was ligated with the vector pCMV-Myc digested with the same enzyme, and the ligation product was used.
  • the calcium chloride method was used to transform E. coli DH5 a competent cells, positive transformants were screened, plasmids were extracted, and restriction endonucleases ⁇ ⁇ and ll were used for restriction enzyme digestion, and 3800 bp and 3450 bp fragments were positively cloned by restriction enzyme digestion.
  • the positive clone plasmid was further identified by PCR.
  • the primers used were Myc-Abl-1 and Myc-Abl-2, and the PCR product was sequenced. The result showed that the c-Abl point mutation gene fragment was inserted into pCMV-Myc. Between the EcoRl and ⁇ /11 cleavage sites, and the sequence is correct, the recombinant expression vector was named pCMV-Myc-c-Abl (K290R).
  • the c-Abl point mutation gene fragment was changed from AAG to AGG from the 5' end of the GenBank Accession Number: NM_007313, and the other was from the 5' end of GenBank Accession Number: ⁇ 007313
  • the base sequence of the deoxyribonucleotide at position 440 to position 3889 is the same, and the Abl point mutation gene can express the fusion protein carrying the Myc tag under the action of the CMV promoter.
  • Abl, Arg double knockout mouse fibroblast MEF (Abl/Arg double knockout mouse embryo) was co-transfected with pcDNA 3-Flag-Gal3 and any of the following three expression vectors, respectively.
  • the transfected cells were lysed and immunoprecipitated with anti-Flag antibody (M5, Sigma). All the procedures should be carried out strictly in ice bath or low temperature conditions. The specific method is: Collect cells, 1000g Centrifuge for 3 minutes and discard the medium. The cells were washed with 5 mL of ice-cold lxPBS, centrifuged at 100 g for 2 minutes, and the supernatant was discarded and washed three times in the same manner.
  • ⁇ 100mm per cell requires 600 ⁇ l of single detergent lysate (50 awake ol/L Tris ⁇ CI, 150 mmol/L NaCl, 0.02% sodium azide, 1% NP-40 and protease inhibitors) (Roche, Inc) 1 tablet / 25 mL), 200 ⁇ l of single detergent lysate was added to each cell of 60 mra. Mix the cells with the lysate, ice bath for 5-10 minutes, and centrifuge at 16000 g for 10 minutes. The cell lysis supernatant was carefully aspirated, and 10-15 ⁇ M anti-Fat antibody cross-linked agarose beads (Sigma) were added and incubated at 4 ° C for 2 hours to perform immunoprecipitation.
  • single detergent lysate 50 awake ol/L Tris ⁇ CI, 150 mmol/L NaCl, 0.02% sodium azide, 1% NP-40 and protease inhibitors
  • the protein was transferred to a nitrocellulose membrane (NC membrane) by: cutting two 2. 5 mm thick filter paper (Bio-Rad, Inc.) and a nitrocellulose membrane into gel size. Soak for 30 minutes in lx transfer buffer (39 mmol/L glycine, 8mraol/L Tris, 0.0037% SDS and 20% methanol). Subsequently, it was placed on the semi-dry transfer apparatus (Bio-Rad, Inc.) from top to bottom in the order of filter paper-gel-nitrocellulose membrane-filter paper, and the air bubbles in the interlayer were exhausted as much as possible. Transfer film voltage 15V, 2- 2.
  • HRP-mouse Ig G horseradish peroxidase
  • step 3 After the NC membrane blocked in step 3 was incubated with primary antibody for 1 hour at room temperature, the membrane was washed three times with lxPBST for 5-10 minutes each time; then the enzyme-labeled secondary antibody was added, and after incubation for 1 hour at room temperature, the membrane was washed with lxPBST. Three times, 5-10 minutes each time. The above incubations were all carried out on a horizontal shaker.
  • the chemiluminescence reaction is carried out by: uniformly dropping the sensitizing liquid WESTERN LIGHTNING (PerkinElmer Life Sciences, Inc.) containing the horseradish peroxidase substrate onto the NC film, and then reacting the coloring solution for 1 minute. Dry, exposed with X-ray film (X-ray film and developer, fixing solution were purchased from Kodak Company).
  • the expression product of pCMV-Myc-c-Abl (K290R) is phosphorylated by Myc_c-Abl (K290R).
  • K290R Myc_c-Abl
  • the reason is that the K290 site of Abl is a key amino acid of the ATP-binding site in the C-Abl kinase domain.
  • c-Abl loses tyrosine kinase activity.
  • Gal3 can specifically Phosphorylated by c-Abl.
  • STI571 Inhibition of c-Abl phosphorylation by non-receptor tyrosine kinase C -Abl specific inhibitor STI571 was detected by transfecting Flag-Gal3 expression plasmid pcDNA 3_Flag-Gal3 into 293 cells, 24 hours later. 10 mol/L STI571 (product of Novartis, trade name: Gleevec, generic name: imatinib mesylate) was transfected with transfected cells for 12 hours to be transfected with an equivalent amount of DMS0 (solvent of STI571) The cells were used as controls, and the cells were lysed, and immunoprecipitation and immunoblotting were carried out in the same manner as in the third step.
  • the Gal3 protein stained secondary antibody was FITC-rabbit (Beijing Zhongshan Jinqiao Co., Ltd.), diluted at a ratio of 1:200 during use, incubated for 1 h at room temperature, and washed three times with PBS for 5 min each time.
  • the lysosomal staining secondary antibody was TRITC-mouse and was stained with Gal3 using the method. Finally, nuclear staining was performed.
  • the nuclear dye Hochest33342 (purchased from Dingguo Biotechnology Co., Ltd.) was used at a concentration of lu g /mL, incubated at room temperature for 30 min, and washed three times with PBS for 5 min each time. Observe under a fluorescence microscope.
  • Fig. 3 from left to right in Fig. A and Fig. B, green fluorescently labeled Gal3 protein, red Mito-Tracker labeled mitochondria, nuclear dye Hochest33342 labeled nuclei, combined images.
  • Figure C from left to right are the green fluorescently labeled Gal3 protein, the red fluorescently labeled lysosome, the nuclear dye Hochest33342 labeled nuclei, and the combined images.
  • strurosporine (sigma, S4400) was treated for 150 min, and the fourth group was treated with 5 uM STI571 for 18 h and then added with 0.5 uu STS for 150 min.
  • the immunostaining method is the same as step one.
  • the MCF-7 cells were inoculated into four 100-well cell culture dishes. The same treatment as the above immunostaining experiments was divided into four groups. The change of Gal3 protein in the cells was further detected by Western blotting. Gal3 detection Resistance to galectin-3 (B-2) (santa cruz).
  • the primary antibody is ⁇ -actin monoclonal antibody ( santa cruz biotechnology, inc)
  • the secondary antibody is HRP -mouse Ig G (santa cruz biotechnology, inc)
  • the above antibodies are purchased from Friendship Zhonglian Biotechnology Co., Ltd.
  • Example 3 Detection of apoptosis of MCF-7 cells treated with ⁇ STI571, 0.5 ⁇ STS alone and both
  • the experiment was divided into four groups: the first group of MCF-7 cells were not treated; the second group of MCF-7 cells were treated with ⁇ STI571 for 18 h; the third group of MCF-7 cells were treated with 0.5 ⁇ of apoptosis inducer STS for 4-5 h, The fourth group of MCF-7 cells were treated with ⁇ STI571 for 18 h and then added with 0.5 ⁇ M STS for 4-5 h.
  • TUNEL method was used to detect the apoptosis of MCF-7 cells treated with ⁇ STI571, 0.5 ⁇ STS.
  • the apoptosis of the four groups of MCF-7 cells treated with different treatments was detected by TUNEL-FLU0S kit (purchased from Roche) and with reference to the kit instructions.
  • the specific method was as follows: using freshly prepared 4% paraformaldehyde at room temperature. The cells were fixed for 30 min, washed with PBS and incubated with blocking agent (3 ⁇ 40 2 methanol solution) for 30 min at room temperature, washed with PBS, and 0.1% Triton X-100 was added and incubated for 2 min in an ice bath.
  • the cells also have apoptosis after being treated with 0.5 uM STS for 4-5 hours, and green fluorescence can be seen in the nucleus.
  • the red Gal3 protein is still distributed in the whole cytoplasm; as shown in Figure 4 in Figure 6, the cells are treated with 10uM STI571 for 18h and then added to 0. 5uM STS for another 4-5h, a strong green fluorescence can be seen. It is expressed in the nucleus, and the nucleus becomes small and shrunken. At this time, the red Gal3 protein is extremely weakly expressed and the apoptosis is remarkable.
  • the apoptosis of the four groups of MCF-7 cells treated with different treatments was detected by using the Annexin-V-FLUOS kit (purchased from Roche) and the kit was as follows: The cells were collected by centrifugation at 500 g for 5 min, and the cells were harvested with PBS. Suspend the cells, wash once, resuspend the cells in a 200 ⁇ l binding buffer (provided with the kit), add ⁇ Annexin-V-FITC and 5 ⁇ 1 ⁇ , mix gently, incubate for 15 min at room temperature, and use flow cytometry. Instrument detection.
  • FIG. 7 top left panel: normal MCF-7 cells, upper right panel: MCF-7 cells treated with 0.5 ⁇ apoptosis inducer STS for 4-5 h, lower left panel: MCF treated with 10 ⁇ STI571 for 18 h- 7 cells, right lower panel: after treatment with 10
  • Apoptosis when treated with STI571 and STS, showed significant apoptosis, further demonstrating that non-receptor tyrosine kinase c-Abl specific inhibitor STI571 can induce tumor cell apoptosis.
  • MCF-7 cells were divided into STI571 (10 ⁇ ) treatment and no treatment. After 18 hours, the two groups of cells were subdivided into two groups treated with the chemotherapy drug cisplatin CDDP (4MM). After 24 hours, the flow was used. Apoptosis analysis was performed using a cytometer.
  • the cells were trypsinized, and the cells were collected with 10-12 mL of PBS into a 15 ml tube, 500 g, and the cells were pelleted by centrifugation for 3 min, the cells were washed with 5 mL of PBS, and the cells were again pelleted by centrifugation. 2 mL of 70% ethanol (70% ethanol in PBS) was added dropwise while shaking. - Incubate overnight at 20 °C. After repeated washing with PBS twice, the pellet was centrifuged, and 2 mL of PC Buffer was added and incubated for 30 min [0. 2 M NaH 2 P0 4 192 mL; 0.2 M Sodium Citrate sodium citrate 8 mL].
  • mice Male, 18-20 g, purchased from the Experimental Animal Center of the Academy of Military Medical Sciences
  • MCF-7 cells in logarithmic growth phase were inoculated into nude mice by pancreatic enzyme digestion at 5 ⁇ 10 6 cells/subcutaneously. After inoculation, nude mice were randomly divided into 5 groups and randomized into groups. 7 only. The first group was the saline group (negative control group), and the nude mice were given saline by intragastric administration twice a day, ⁇ /g body weight twice a day; the second group was STI571 oral administration group.
  • the nude mice were administered by intragastric administration the next day after inoculation of cells, twice a day, 40 mg/kg each time, the solvent was normal saline, and the volume per administration was ⁇ /g body weight; the third group was 1/4 Conventional dose of CTX (cyclophosphamide (product of Jiangsu Hengrui Pharmaceutical Co., Ltd.), intraperitoneal injection group, nude mice were intraperitoneally administered on the 7th day after inoculation of cells, once every two weeks, 25 mg/kg each time, the solvent was Normal saline, each injection volume was ⁇ /g body weight; the fourth group was the combined administration group, and STI571 and CTX were administered in combination according to the second and third groups.
  • CTX cyclophosphamide (product of Jiangsu Hengrui Pharmaceutical Co., Ltd.)
  • CTX full-dose intraperitoneal injection group (Positive control group) nude mice were intraperitoneally administered on the 7th day after inoculation of cells, once every two weeks, 100 mg/kg each time, the solvent was physiological saline, and the volume of each injection was ⁇ /g body weight.
  • the tumor growth inhibition rate was calculated according to the following formula:
  • IR (1 - tumor weight of the treatment group / tumor weight of the negative control group) X 100%.
  • STI571 has a certain anti-tumor effect, and it can play a synergistic anti-tumor effect when combined with the tumor chemotherapy drug CTX.
  • the weight results of the five groups of nude mice are shown in Figure 10.
  • the fourth group of STI571 and 1/4 conventional dose CTX combination group (indicated by "STI571 + CTX (25mg/kg)")
  • the fifth group of CTX full-dose administration group (indicated by "CTX (100 mg/kg)”) showed significant differences in the average body weight of nude mice, but the tumor treatment effect did not exist. Significant differences indicate that STI571, as a combination of tumor chemotherapy drugs, can significantly reduce the side effects of cancer chemotherapy drugs.
  • Control is a negative control group.
  • the present invention provides a novel use of a non-receptor tyrosine kinase c-Abl specific inhibitor, i.e., in the preparation of an antitumor drug or a tumor therapeutic adjuvant.
  • non-receptor tyrosine kinase c-Abl specific inhibitors can cause Gal3 to undergo degeneration and aggregation and lysosome degradation by inhibiting the kinase activity of c-Abl, resulting in a significant decrease in Gal3 protein levels in tumor cells, thereby allowing tumors to The cells die of autophagic cell apoptosis.
  • the non-receptor tyrosine kinase c-Abl specific inhibitor can also make tumor cells extremely sensitive to a variety of tumor therapeutic drugs, and a low concentration of apoptosis-inducing agents can cause all cells to die in a short period of time, thus
  • the specific inhibitor of c-Abl, a tyrosine kinase enhances the therapeutic effect of existing cytotoxic chemotherapeutic drugs, and can greatly reduce the side effects by reducing the dose of chemotherapeutic drugs.
  • Preparation of anti-tumor drugs with non-receptor tyrosine kinase c-Abl specific inhibitor as active ingredient has the following advantages: 1) remarkable curative effect, tumor inhibition rate up to 81.4%; 2) high safety; 3) main The route of administration is oral, and the administration is convenient; 4) the inhibitor is inexpensive, and thus the cost of preparing the antitumor drug is low, thereby reducing the economic burden on the patient.
  • the invention provides a new way for the prevention and treatment of tumors, and has broad application prospects in the field of medicine.

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Abstract

The new use of non-receptor tyrosine kinase c-Ab1 specific inhibitors alone or in combination with cisplatin or cyclophosphamide in preparing antitumor medicaments and / or auxiliary antitumor medicaments. Experiments prove that non-receptor tyrosine kinase c-Ab1 specific inhibitors can inhibit the activity of c-Ab1 kinase to lead the denaturation aggregation and lysosome degradation of Gal3 and to lower the Gal3 protein level in the tumor cells obviously, so as to kill tumor cells in autophagic apoptosis.

Description

非受体酪氨酸激酶 c- Abl特异性抑制剂的新用途 技术领域  New use of non-receptor tyrosine kinase c-Abl specific inhibitors
本发明涉及非受体酪氨酸激酶 c-Abl特异性抑制剂的新用途， 特别是涉 及非受体酪氨酸激酶 c- Abl特异性抑制剂在制备抗肿瘤药物中的应用。  The present invention relates to novel uses of non-receptor tyrosine kinase c-Abl specific inhibitors, particularly to the use of non-receptor tyrosine kinase c-Abl specific inhibitors in the preparation of antitumor drugs.
背景技术 Background technique
根据病人的遗传背景和发病的分子机理进行肿瘤的个体化治疗是肿瘤治 疗的发展方向。 Galectin- 3 (Gal3) 是 —半乳糖苷结合蛋白， 具有多种生 物功能， 作为一种癌症相关蛋白参与肿瘤细胞的黏附、 增殖、 分化、 血管生 成、 恶化及转移。 临床研究表明， 在许多类型的组织细胞中， 当细胞发生恶 性转化时， Gal3的表达量上调（例如在结肠、 甲状腺、 中枢神经***、 胰腺、 膀胱、 肾脏、 胃等器官的癌症） ， 而且， 肿瘤细胞中 Gal3表达水平上调会使 肿瘤的恶性程度和转移侵润能力也随之增强。 此外， 对癌症患者的血清和正 常人血清进行检测发现， GaleCtin3的血清浓度在乳腺癌、 胃肠癌、肺癌、 卵 巢癌、黑素瘤、淋巴瘤中显著增高，而且，伴有癌症转移灶的患者的 GaleCtin3 血清浓度高于原发癌患者， 说明血液中 Galectin3含量与肿瘤转移相关。 因 此认为 Gal3是一种癌症相关蛋白， 在致癌作用和肿瘤进程中起到重要作用。 Gal3的抗凋亡活性表现在能够抑制药物诱导的细胞凋亡和錨着依赖性细胞凋 亡从而使细胞存活。 研究结果显示， 肿瘤细胞能够分泌 Gal3， 分泌的 Gal3 通过与 T细胞表面受体结合可诱导 T细胞的凋亡，从而起到免疫逃避的作用， 这样使得肿瘤细胞在发展进程中能够逃避 T细胞的捕杀。 目前， Gal3作为免 疫细胞化学临床检测的一项指标， 对肿瘤细胞的良恶性诊断和癌症预后诊断 都具有一定价值。 因此， 在多种类型的癌症诊断和治疗方面， Gal3作为一个 新的靶标具有非常好的应用前景。 The individualized treatment of tumor according to the genetic background of the patient and the molecular mechanism of the disease is the development direction of tumor treatment. Galectin-3 (Gal3) is a galactosidase-binding protein that has a variety of biological functions and is involved in tumor cell adhesion, proliferation, differentiation, angiogenesis, deterioration and metastasis as a cancer-associated protein. Clinical studies have shown that in many types of tissue cells, Gal3 expression is up-regulated when cells undergo malignant transformation (eg cancer in the colon, thyroid, central nervous system, pancreas, bladder, kidney, stomach, etc.), and Up-regulation of Gal3 expression levels in tumor cells increases the malignancy and metastatic invasiveness of tumors. Further, normal human sera and serum of cancer patients were found to detect, Gale C tin3 serum concentrations were significantly higher in breast cancer, gastrointestinal cancer, lung cancer, ovarian cancer, melanoma, lymphoma, and, associated with cancer metastasis Gal eC serum concentrations in patients with primary lesions of cancer patients than tin3 described Galectin3 blood content associated with a tumor metastasis. Therefore, Gal3 is considered to be a cancer-associated protein and plays an important role in carcinogenesis and tumor progression. The anti-apoptotic activity of Gal3 is manifested by the ability to inhibit drug-induced apoptosis and anchorage-dependent apoptosis to allow cell survival. The results show that the tumor cells can secrete Gal3, and the secreted Gal3 can induce T cell apoptosis by binding to T cell surface receptors, thus playing a role in immune evasion, thus enabling tumor cells to escape T cells during development. Killing. At present, Gal3, as an indicator of clinical detection of immune cytochemistry, has certain value for the diagnosis of benign and malignant tumor cells and the diagnosis of cancer prognosis. Therefore, Gal3 has a very good application prospect as a new target in the diagnosis and treatment of various types of cancer.
蛋白酪氨酸激酶是一类具有酪氨酸激酶活性的蛋白质， 可分为受体型和 非受体型两种， 它们能催化 ATP上的磷酸基转移到许多重要蛋白质的酪氨酸 残基上， 使其发生磷酸化。 蛋白酪氨酸激酶在细胞内的信号转导通路中占据 了十分重要的地位， 调节着细胞的生长、 分化、 凋亡等一系列生理过程。  Protein tyrosine kinases are a class of proteins with tyrosine kinase activity and can be divided into receptor and non-receptor types, which catalyze the transfer of phosphates on ATP to tyrosine residues of many important proteins. On, make it phosphorylated. Protein tyrosine kinases play an important role in the signal transduction pathways in cells, regulating a series of physiological processes such as cell growth, differentiation and apoptosis.
非受体蛋白酪氨酸激酶中的 ABL家族包括两个成员： Abl和 Arg。 Abl基 因位于 9号染色体长臂 （9q34. 1 ) ， 编码 145kD 的 Abl蛋白。 c-Abl蛋白具 有酪氨酸激酶活性， 含有 SH3、 SH2、 PTK、 DNA结合域、 肌动蛋白结合结构域 等 (Blume- Jensen P， Hunter T. Oncogenic kinase signalling. Nature, 2001， 411 (6835)： 355-65 ) 。 The ABL family of non-receptor protein tyrosine kinases includes two members: Abl and Arg. The Abl gene is located on the long arm of chromosome 9 (9q34.1) and encodes the 145kD Abl protein. c-Abl protein Has tyrosine kinase activity, including SH3, SH2, PTK, DNA binding domain, actin binding domain, etc. (Blume-Jensen P, Hunter T. Oncogenic kinase signalling. Nature, 2001, 411 (6835): 355-65 ).
非受体酪氨酸激酶 c-Abl特异性抑制剂可使普遍存在的 Abelson酪氨酸 激酶 （Abl ) 失活。  The non-receptor tyrosine kinase c-Abl specific inhibitor can inactivate the ubiquitous Abelson tyrosine kinase (Abl).
发明公开 Invention disclosure
本发明的目的是提供非受体酪氨酸激酶 c- Abl特异性抑制剂的新用途， 即 在制备抗肿瘤药物或肿瘤治疗辅助药物中的应用。  It is an object of the present invention to provide a novel use of a non-receptor tyrosine kinase c-Abl specific inhibitor, i.e., in the preparation of an antitumor drug or a tumor therapeutic adjuvant.
本发明所述的应用是以非受体酪氨酸激酶 c-Abl特异性抑制剂为活性成 分， 制备抗肿瘤药物或肿瘤治疗辅助药物。 所述非受体酪氨酸激酶 c- Abl特 异性抑制剂的选择是多种多样的， 具体可为 STI571、 Sunitinib、 PKC412, vandetanib Sorafenib、 erlotinib、 gefitinib、 dasatinib等。  The application of the present invention is to prepare an antitumor drug or a tumor therapeutic auxiliary drug by using a non-receptor tyrosine kinase c-Abl specific inhibitor as an active ingredient. The non-receptor tyrosine kinase c-Abl specific inhibitors are selected in a variety of ways, specifically STI571, Sunitinib, PKC412, vandetanib Sorafenib, erlotinib, gefitinib, dasatinib and the like.
为提高抑瘤效果， 在用非受体酪氨酸激酶 c- Abl特异性抑制剂制备的抗 肿瘤药物或肿瘤治疗辅助药物中， 还可添加化疗药物， 如顺铂、 CTX等。  In order to improve the anti-tumor effect, chemotherapeutic drugs such as cisplatin and CTX may be added to the anti-tumor drugs or tumor therapeutic auxiliary drugs prepared by using the non-receptor tyrosine kinase c-Abl specific inhibitor.
实验证明非受体酪氨酸激酶 c-Abl特异性抑制剂通过抑制 C- Abl的激酶 活性可导致 Gal3发生变性聚集并通过溶酶体降解， 致使肿瘤细胞中 Gal3蛋 白水平显著降低， 从而使肿瘤细胞发生自噬性细胞凋亡而死亡。 研究表明， Gal3在肿瘤细胞中广泛表达， 因而非受体酪氨酸激酶 c- Abl特异性抑制剂具 有广谱的抑肿瘤作用， 包括乳腺癌、 ***癌， ***， 肺腺癌， 肝癌， 结 肠癌、 甲状腺癌、 胰腺癌、 神经内分泌肿瘤、 膀胱癌、 肾癌、 胃癌等。  Experiments have shown that non-receptor tyrosine kinase c-Abl-specific inhibitors can cause Gal3 to undergo degeneration and aggregation and lysosome degradation by inhibiting the kinase activity of C-Abl, resulting in a significant decrease in Gal3 protein levels in tumor cells, resulting in tumors. The cells die of autophagic cell apoptosis. Studies have shown that Gal3 is widely expressed in tumor cells, so non-receptor tyrosine kinase c-Abl specific inhibitors have a broad spectrum of anti-tumor effects, including breast cancer, prostate cancer, cervical cancer, lung adenocarcinoma, liver cancer, Colon cancer, thyroid cancer, pancreatic cancer, neuroendocrine tumor, bladder cancer, kidney cancer, stomach cancer, and the like.
需要的时候， 在本发明所制备的抗肿瘤药物或肿瘤治疗辅助药物中， 还 可以加入一种或多种药学上可接受的辅料， 所述辅料包括药学领域常规的稀 释剂、 赋形剂、 填充剂、 粘合剂、 湿润剂、 吸收促进剂、 表面活性剂、 润滑 剂、 稳定剂等， 必要时还可加入香味剂、 甜味剂及色素等。  When necessary, in the antitumor drug or tumor therapeutic auxiliary drug prepared by the present invention, one or more pharmaceutically acceptable excipients may be added, and the excipients include conventional diluents, excipients, and the like in the pharmaceutical field. A filler, a binder, a wetting agent, an absorption enhancer, a surfactant, a lubricant, a stabilizer, etc., and if necessary, a flavoring agent, a sweetener, a coloring matter, and the like may be added.
本发明所制备的抗肿瘤药物或肿瘤治疗辅助药物除制成口服液外， 还可 以制成胶囊剂、 片剂、 粉剂、 粒剂、 注射液等多种药物形式。 上述各种剂型 的药物均可以按照药学领域的常规方法制备。  The antitumor drug or the tumor therapeutic auxiliary drug prepared by the invention can be prepared into a plurality of drug forms such as capsules, tablets, powders, granules, and injections, in addition to the oral solution. The above various dosage forms of the drug can be prepared according to a conventional method in the pharmaceutical field.
该药物的成人口服用量一般为 400rag/天或 5. 55rag/kg.体重 /天,可以一 次或多次使用， 疗程为 20天一 30天。  The oral dose of the drug is generally 400rag/day or 5.55rag/kg. body weight/day, which can be used once or in multiple times for 20 days to 30 days.
下面结合具体实施例对本发明作进一步详细说明。  The present invention will be further described in detail below in conjunction with specific embodiments.
附图说明 图 1为非受体酪氨酸激酶 c- Abl对 Gal3特异性磷酸化作用的免疫沉淀及 免疫印迹检测结果。 DRAWINGS Figure 1 shows the results of immunoprecipitation and immunoblotting of Gal3-specific phosphorylation by non-receptor tyrosine kinase c-Abl.
图 2为非受体酪氨酸激酶 c-Abl特异性抑制剂 STI571对 C-Abl特异磷酸 化抑制作用的免疫沉淀及免疫印迹检测结果。 Figure 2 shows the results of immunoprecipitation and immunoblotting of C -Abl-specific phosphorylation by non-receptor tyrosine kinase c-Abl specific inhibitor STI571.
图 3为免疫染色法检测经 lOuM STI571处理的 MCF-7细胞中 Gal3蛋白分布 情况的结果。  Figure 3 shows the results of immunostaining for the distribution of Gal3 protein in MCF-7 cells treated with lOuM STI571.
图 4为免疫染色法检测经 5uM STI571、 0. 5uM凋亡诱导剂 STS单独处理及两 者共同处理的 MCF 7细胞中 Gal3蛋白量变化的结果。  Figure 4 shows the results of immunostaining assay for changes in Gal3 protein content in MCF 7 cells treated with 5 uM STI571, 0.5 uM apoptosis-inducing agent STS alone and co-treated.
图 5为免疫印迹法检测经 5uM STI571、 0. 5uM凋亡诱导剂 STS单独处理及两 者共同处理的 MCF-7细胞中 Gal3蛋白量变化的结果。  Figure 5 shows the results of immunoblotting for the change of Gal3 protein in MCF-7 cells treated with 5uM STI571, 0.5 uM apoptosis inducer STS alone and co-treated.
图 6为经 lOuM STI57K 0. 5uM STS单独处理及两者共同处理的 MCF-7细 胞凋亡情况的 TUNEL法检测结果。  Figure 6 shows the results of TUNEL assay for apoptosis of MCF-7 cells treated with lOuM STI57K 0. 5uM STS alone and in combination.
图 7为经 lOuM STI571 , 0. 5uM STS单独处理及两者共同处理的 MCF- 7细 胞凋亡情况的 Annexin- V- FLU0S法检测结果。  Figure 7 shows the results of Annexin-V-FLU0S assay for apoptosis of MCF-7 cells treated with lOuM STI571, 0.5 uM STS alone and both.
图 8为经 lOuM STI571、 4 uM CDDP单独处理及两者共同处理的 MCF- 7细 胞凋亡情况的 Sub G1 检测方法。  Figure 8 shows the Sub G1 assay for apoptosis of MCF-7 cells treated with lOuM STI571, 4 uM CDDP alone and co-treated.
图 9 为裸鼠肿瘤治疗情况。 '  Figure 9 shows the treatment of tumors in nude mice. '
图 10 为裸鼠体重统计结果。  Figure 10 shows the body weight statistics of nude mice.
图 11 为肿瘤切片 HE染色结果。  Figure 11 shows the results of HE staining of tumor sections.
实施发明的最佳方式 The best way to implement the invention
下述实施例中所用方法， 如无特别说明均为常规方法。  The methods used in the following examples are conventional methods unless otherwise specified.
实施例 1、 检测非受体酪氨酸激酶 c-Abl对 Gal3特异性磷酸化作用及非 受体酪氨酸激酶 c- Abl特异性抑制剂 STI571对 c- Abl磷酸化作用的抑制作用 一、 Flag- Gal3表达载体的构建  Example 1. Detection of Gal3-specific phosphorylation by non-receptor tyrosine kinase c-Abl and inhibition of c-Abl phosphorylation by non-receptor tyrosine kinase c-Abl specific inhibitor STI571 Construction of Flag-Gal3 Expression Vector
1、 重组载体 pcDNA 3- Flag的构建  1. Construction of recombinant vector pcDNA 3- Flag
将编码 Flag标签 （由如下 8个氨基酸组成的肽段：  The flag tag will be encoded (a peptide consisting of the following 8 amino acids:
Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys ) 的 DNA序歹 !j DNA sequence of Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys )
( gactacaaggacgacgacgacaag)克隆入载体 pcDNA3 ( Invitrogen公司)多克 隆位点的限制性内切酶 Kpn I和 BamH I酶切位点之间， 得到含有 Flag标签 编码序列的重组载体， 命名为 pcDNA 3- Flag。 2、 Gal3基因的 PCR扩增 (gactacaaggacgacgacgacacaag) was cloned between the restriction endonuclease Kpn I and BamH I restriction sites of the multiple cloning site of vector pcDNA3 (Invitrogen) to obtain a recombinant vector containing the Flag tag coding sequence, which was named pcDNA 3-Fat. 2. PCR amplification of Gal3 gene
以从携带有 Gal3基因 (GenBank Accession Number: BC001120 ) 的正 常人乳腺组织细胞中提取总 RNA反转录得到的 cDNA为模板，在引物 P1 (上游 引物） ： 5 ' -CGGATCCATGGCAGACAATTTTTCGCTCCA-3 ' (带下划线碱基为限制 性内切酶^¾/^ 1识别位点） 和 P2 (下游引物） ：  The cDNA obtained by reverse transcription of total RNA extracted from normal human breast tissue cells carrying the Gal3 gene (GenBank Accession Number: BC001120) was used as a template in primer P1 (upstream primer): 5 '-CGGATCCATGGCAGACAATTTTTCGCTCCA-3 ' (underlined Bases are restriction endonucleases ^3⁄4/^ 1 recognition site) and P2 (downstream primers):
5 ' -CGGAATTCTTATATCATGGTATATGAAGCA-3， (带下划线碱基为限制性内切酶 识别位点）的引导下 PCR扩增 Gal3基因， 并在基因片段的两端分别添 加限制性内切酶 BainH I和 EcoR I识别位点， 反应结束后， 对 PCR扩增产物 进行 1 %琼脂糖凝胶电泳检测，结果获得了 753bp的 DNA片段，与预期结果相 符， 回收并纯化该目的片段， 测序， 测序结果表明获得了具有自 GenBank Accession Number: BC001120的 5 ' 末端第 25位 -777位脱氧核糖核苷酸的 Gal3基因片段 （Gal3基因的 CDS序列） 。  The 5'-CGGAATTCTTATATCATGGTATATGAAGCA-3, (underlined base is a restriction endonuclease recognition site), the Gal3 gene was amplified by PCR and the restriction enzymes BainH I and EcoR I were added to both ends of the gene fragment. After the reaction, the PCR amplification product was detected by 1% agarose gel electrophoresis, and a 753 bp DNA fragment was obtained, which was consistent with the expected result. The target fragment was recovered and purified, sequenced, and the sequencing result was obtained. A Gal3 gene fragment (CDS sequence of the Gal3 gene) having a 25'-777 deoxyribonucleotide at the 5' end of GenBank Accession Number: BC001120.
3、 Flag- Gal3表达载体 pcDNA 3- Flag- Gal3的获得  3. Flag-Gal3 expression vector pcDNA 3- Flag- Gal3
将步骤 2克隆的 Gal3基因片段用限制性内切酶 BamH I和 EcoR I进行双 酶切， 将酶切片段与经相同酶双酶切的步骤 1构建的载体 pcDNA 3- Flag进行 连接， 将连接产物用氯化钙法转化大肠杆菌 DH5 ci 感受态细胞， 筛选阳性转 化子， 提质粒， 用限制性内切酶 / 和 I进行酶切鉴定， 经酶切获 得 5400bp和 753bp片段的为阳性克隆，再对阳性克隆质粒用 PCR的方法做进 一步鉴定， 所用引物为 P1和 P2， 对 PCR产物进行测序， 检测结果表明 Gal3 基因片段已*** pcDNA 3- Flag的 BamH i EcoR I酶切位点之间， 且序列正 确， 将此重组表达载体命名为 pcDNA 3-Flag-Gal3。 Gal3基因可以在 CMV启 动子的作用下表达带有 Flag标签的融合蛋白。  The Gal3 gene fragment cloned in step 2 was digested with restriction endonucleases BamH I and EcoR I, and the restriction fragment was ligated with the vector pcDNA 3-flag constructed in step 1 which was digested with the same enzyme, and ligated. The product was transformed into E. coli DH5 ci competent cells by calcium chloride method, positive transformants were screened, plasmids were extracted, and restriction endonucleases/ and I were used for restriction enzyme digestion, and 5400 bp and 753 bp fragments were positively digested. The positive clones were further identified by PCR. The primers were P1 and P2. The PCR products were sequenced. The results showed that the Gal3 gene fragment was inserted between the BamH i EcoR I restriction sites of pcDNA 3-Fat. And the sequence was correct, and the recombinant expression vector was named pcDNA 3-Flag-Gal3. The Gal3 gene can express a fusion protein with a Flag tag under the action of a CMV promoter.
二、 Myc_c_Abl、 Myc- c- Abl (K290R)表达载体的构建  Construction of Myc_c_Abl, Myc- c- Abl (K290R) expression vector
1、 Myc-c- Abl表达载体 pCMV- Myc-c-Abl的构建  1. Construction of Myc-c-Abl expression vector pCMV- Myc-c-Abl
1 ) c_Abl基因的 PCR扩增  1) PCR amplification of c_Abl gene
以携带有野生型 c- Abl基因 （GenBank Accession Number: 匪— 007313 ) 的人胎肝 cDNA文库 （clontech公司产品）为模板， 在引物 Myc-Abl- 1 (上游 引物） ： 5 ' -CGAATTCGGATGG6GCAGCAGCCTGGAA-3 ' (带下划线碱基为限制性 内切酶 I识别位点） 和 Myc- (下游引物） ：  A human fetal liver cDNA library (clontech product) carrying the wild type c-Abl gene (GenBank Accession Number: 匪-007313) was used as a template in the primer Myc-Abl-1 (upstream primer): 5 '-CGAATTCGGATGG6GCAGCAGCCTGGAA-3 ' (underlined bases are restriction endonuclease I recognition sites) and Myc- (downstream primers):
5， -CGAGATCTCTACCTCTGCACTATGTCACT-3 ' (带下划线碱基为限制性内切酶 ^ΠΙ识别位点）的引导下 PCR扩增 c- Abl基因， 并在基因片段的两端分别添 加限制性内切酶 EcoR I和 Bgl II识别位点， 反应结束后， 对 PCR扩增产物进 行 1 %琼脂糖凝胶电泳检测， 结果获得了 3450 bp的 DNA片段， 与预期结果相 符， 回收并纯化该目的片段， 测序， 测序结果表明获得了具有自 GenBank Accession Number: 匪― 007313的 5 ' 末端第 440位- 3889位脱氧核糖核苷酸 的 c- Abl基因片段（c-Abl基因的 CDS序列） 。 5, -CGAGATCTCTACCTCTGCACTATGTCACT-3 ' (underlined bases are restriction enzymes) The c-Abl gene was amplified by PCR under the guidance of the ΠΙ recognition site, and the restriction endonucleases EcoR I and Bgl II were added to the ends of the gene fragment. After the reaction, the PCR amplification products were subjected to the PCR amplification products. After 1% agarose gel electrophoresis, a 3450 bp DNA fragment was obtained, which was consistent with the expected results. The target fragment was recovered and purified, and sequenced. The sequencing result showed that the 5' end was obtained from GenBank Accession Number: 匪-007313. The c-Abl gene fragment of the 440th to 3889 deoxyribonucleotides (the CDS sequence of the c-Abl gene).
2) Myc-c-Abl表达载体的获得  2) Acquisition of Myc-c-Abl expression vector
将步骤 1 ) 克隆的 c- Abl基因片段用限制性内切酶 EcoR I和 ^¾·_ΖΠ进行 双酶切， 将酶切片段与经相同酶双酶切的载体 pCMV- Myc (Clontech公司）进 行连接， 将连接产物用氯化钙法转化大肠杆菌 DH5 C1感受态细胞， 筛选阳性 转化子， 提质粒， 用限制性内切酶^^ ? 1和^ /II进行酶切鉴定， 经酶切获 得 3800bp (载体片段） 和 3450bp片段的为阳性克隆， 再对阳性克隆质粒用 PCR的方法做进一步鉴定，所用引物为 Myc-Abl- 1和 Myc- Abl- 2， 对 PCR产物 进行测序， 检测结果表明 c- Abl基因片段已*** pCMV-Myc的 EcoR I和 Bgl II酶切位点之间， 且序列正确， 将此重组表达载体命名为 pCMV_Myc- c- Abl。 Abl基因可以在 CMV启动子的作用下表达带有 Myc标签的融合蛋白。  The c-Abl gene fragment cloned in step 1) was digested with restriction endonucleases EcoR I and ^3⁄4·_ΖΠ, and the restriction fragment was digested with the vector pCMV-Myc (Clontech) which was digested with the same enzyme. Ligation, the ligation product was transformed into E. coli DH5 C1 competent cells by calcium chloride method, positive transformants were screened, plasmids were extracted, and restriction endonucleases were used to identify the digestive enzymes ^^ 1 and / / II, and obtained by enzyme digestion. 3800 bp (vector fragment) and 3450 bp fragment were positive clones, and the positive clone plasmid was further identified by PCR. The primers used were Myc-Abl-1 and Myc-Abl-2, and the PCR products were sequenced. The c-Abl gene fragment was inserted between the EcoR I and Bgl II restriction sites of pCMV-Myc, and the sequence was correct. The recombinant expression vector was named pCMV_Myc-c-Abl. The Abl gene can express a fusion protein with a Myc tag under the action of the CMV promoter.
2、 Myc-c-Abl (K290R)表达载体 pCMV- Myc- c- Abl (K290R)的构建  2. Construction of Myc-c-Abl (K290R) expression vector pCMV- Myc- c- Abl (K290R)
1 ) Myc-c-Abl (K290R)点突变基因的 PCR扩增  1) PCR amplification of Myc-c-Abl (K290R) point mutation gene
以上述 pCMV- Myc- c- Abl质粒为模板， 在引物 Myc- Abl - 1 :  Using the above pCMV-Myc-c-Abl plasmid as a template, in the primer Myc-Abl-1:
5， -CGAATTCGGATGGGGCAGCAGCCTGGAA-3， (带下划线碱基为限制性内切酶 EcoR I识别位点）和 K290R- II： 5, -CGAATTCGGATGGGGCAGCAGCCTGGAA-3, (underlined bases are restriction endonuclease EcoR I recognition sites) and K290R-II:
5 ' -GGTGTCCTCCTTCAAGGTCCTCACGGCCACCGTCAGGC-3' (带下划线碱基为突变 位点） 的引导下 PCR扩增 c- Abl的 5' 端序列 （命名为 Ml， 其核苷酸序列除 了自 GenBank Accession Number: 讓― 007313的 5 ' 末端第 1307— 1309位碱 基由 MG改为 AGG夕卜， 其它均与自 GenBank Accession Number为匪— 007313 的 c- Abl基因 5 '末端第 440—第 1327位核苷酸相同），再在引物 Myc- AW - 2: 5， -CGAGATCTCTACCTCTGCACTATGTCACT-3 ' (带下划线碱基为限制性内切酶 Bgl II识别位点）和 K290R-I：  5'-GGTGTCCTCCTTCAAGGTCCTCACGGCCACCGTCAGGC-3' (underlined base is a mutation site) under the guidance of PCR amplification of the 5' end of c-Abl (designated Ml, its nucleotide sequence except from GenBank Accession Number: Jean-007313 The 1307–1309 bases at the 5′ end were changed from MG to AGG, and the others were identical to the nucleotides 440 to 1327 of the 5′ end of the c-Abl gene from GenBank Accession Number 匪-007313). Further in the primer Myc-AW-2: 5, -CGAGATCTCTACCTCTGCACTATGTCACT-3 ' (underlined base is the restriction endonuclease Bgl II recognition site) and K290R-I:
5， -GCCGTACGGTGGCCGTGAGGACCTTGAAGGAGGACACC-3 ' (带下划线碱基为突变 位点） 的引导下 PCR扩增 c-Abl的 3' 端序列 （命名为 M2， 其核苷酸序列除 了自 GenBank Accession Number: 匪― 007313的 5 ' 末端第 1307—1309位碱 基由 AAG改为 AGG外，其它均与自 GenBank Accession Number :醒_007313c- Abl 基因的 5 ' 末端第 1290位 -3889位脱氧核糖核苷酸相同）， 反应结束后， 对上 述 PCR扩增产物进行 1 %琼脂糖凝胶电泳检测， 结果获得了 896bp的 Ml片段 和 2609bp的 M2片段， 与预期结果相符， 回收并纯化两个目的片段， 然后， 取 Ml和 M2各 1 μΐ , 混合， 以此为模板， 在引物 Myc- Abl- 1和 Myc- Abl- 2的 引导下 PCR扩增 c-Abl突变基因的全序列， 反应结束后， 对 PCR扩增产物进 行 1 %琼脂糖凝胶电泳检测， 结果获得了 （3450+ 17) bp的 DNA片段， 与预 期结果相符， 回收并纯化该目的片段。 5, -GCCGTACGGTGGCCGTGAGGACCTTGAAGGAGGACACC-3 ' (underlined base is a mutation site) under the guidance of PCR amplification of the 3' end of c-Abl (named M2, its nucleotide sequence From the GenBank Accession Number: 匪-007313, the 5' end of the 1307-1309 base was changed from AAG to AGG, and the others were from GenBank Accession Number: awake _007313c- Abl gene 5' end 1290-3889 The deoxyribonucleotide was the same. After the reaction, the PCR amplification product was detected by 1% agarose gel electrophoresis. The 896 bp Ml fragment and the 2609 bp M2 fragment were obtained, which were consistent with the expected results, recovered and purified. Two target fragments, then, 1 μΐ each of M1 and M2, and mixed, using this as a template, PCR-amplified the full sequence of the c-Abl mutant gene under the guidance of primers Myc-Abl-1 and Myc-Abl-2, After the reaction, the PCR amplification product was subjected to 1% agarose gel electrophoresis, and a DNA fragment of (3450 + 17) bp was obtained, which was consistent with the expected result, and the target fragment was recovered and purified.
2 ) Myc- c- Abl (K290R)表达载体 pCMV- Myc_c- Abl (K290R)的获得  2) Acquisition of Myc-c-Abl (K290R) expression vector pCMV- Myc_c- Abl (K290R)
将步骤 1 ) 克隆的 c_Abl点突变基因片段用限制性内切酶 EcoR I和 Bgl II进行双酶切， 将酶切片段与经相同酶双酶切的载体 pCMV- Myc进行连接， 将 连接产物用氯化钙法转化大肠杆菌 DH5 a 感受态细胞， 筛选阳性转化子， 提 质粒， 用限制性内切酶 σ ? Ι和 ^ ll进行酶切鉴定， 经酶切获得 3800bp和 3450bp片段的为阳性克隆， 再对阳性克隆质粒用 PCR的方法做进一步鉴定， 所用引物为 Myc-Abl-1和 Myc-Abl-2， 对 PCR产物进行测序， 检测结果表明 c-Abl点突变基因片段已*** pCMV- Myc的 EcoRl和 ^/11酶切位点之间， 且 序列正确，将此重组表达载体命名为 pCMV-Myc-c-Abl (K290R)。该 c- Abl点突 变基因片段除了自 GenBank Accession Number: NM_007313的 5 ' 末端第 1307 —1309位碱基由 AAG改为 AGG夕卜， 其它均与自 GenBank Accession Number: 匪一 007313的 5 ' 末端第 440位- 3889位位脱氧核糖核苷酸的碱基序列相同， Abl点突变基因可以在 CMV启动子的作用下表达带有 Myc标签的融合蛋白。 The c_Abl point mutation gene fragment cloned in step 1) was digested with restriction endonucleases EcoR I and Bgl II, and the restriction fragment was ligated with the vector pCMV-Myc digested with the same enzyme, and the ligation product was used. The calcium chloride method was used to transform E. coli DH5 a competent cells, positive transformants were screened, plasmids were extracted, and restriction endonucleases σ Ι and ll were used for restriction enzyme digestion, and 3800 bp and 3450 bp fragments were positively cloned by restriction enzyme digestion. The positive clone plasmid was further identified by PCR. The primers used were Myc-Abl-1 and Myc-Abl-2, and the PCR product was sequenced. The result showed that the c-Abl point mutation gene fragment was inserted into pCMV-Myc. Between the EcoRl and ^/11 cleavage sites, and the sequence is correct, the recombinant expression vector was named pCMV-Myc-c-Abl (K290R). The c-Abl point mutation gene fragment was changed from AAG to AGG from the 5' end of the GenBank Accession Number: NM_007313, and the other was from the 5' end of GenBank Accession Number: 匪 007313 The base sequence of the deoxyribonucleotide at position 440 to position 3889 is the same, and the Abl point mutation gene can express the fusion protein carrying the Myc tag under the action of the CMV promoter.
三、 检测非受体酪氨酸激酶 c- Abl对 Gal3的特异性磷酸化作用 c-Abl作为非受体酪氨酸激酶， 主要通过使蛋白质酪氨酸磷酸化调节细 胞的生命过程。 现用免疫沉淀和免疫印迹 （Western blot ) 的方法验证 Gal 3 可被 c- Abl特异磷酸化， 并避免另一种细胞内源性非受体酪氨酸激酶 Arg的 磷酸化干扰， 具体方法包括以下步骤：  3. Detection of non-receptor tyrosine kinase c-Abl-specific phosphorylation of Gal3 c-Abl acts as a non-receptor tyrosine kinase that regulates the lifespan of cells primarily by phosphorylating protein tyrosine. Immunoprecipitation and Western blotting have been used to verify that Gal 3 can be specifically phosphorylated by c-Abl and avoid phosphorylation of endogenous non-receptor tyrosine kinase Arg in another cell. The following steps:
1、 共转染  1, co-transfection
将 pcDNA 3- Flag- Gal3分别与下述三种表达载体中的任一种共转染 Abl、 Arg双敲除的小鼠成纤维细胞 MEF (Abl/Arg double knockout mouse embryo fibroblast cells, DKO细胞， 表型为 Abl- /- Arg- /- ) ( Koleske Abl, Arg double knockout mouse fibroblast MEF (Abl/Arg double knockout mouse embryo) was co-transfected with pcDNA 3-Flag-Gal3 and any of the following three expression vectors, respectively. Fibroblast cells, DKO cells, phenotype Abl- /- Arg- /- ) ( Koleske
AJ, Essential roles for the Abl and Arg tyrosine Kinases in neurulation, Neuron 1998 Dec ; 21 (6) ： 1259-72) ： pCMV- Myc-c- Abl、 pCMV-Myc 和 c- Abl负显性突变体的表达载体 pCMV- Myc- c-Abl (K290R)。 AJ, Essential roles for the Abl and Arg tyrosine Kinases in neurulation, Neuron 1998 Dec ; 21 (6) : 1259-72) : Expression of pCMV- Myc-c-Abl, pCMV-Myc and c-Abl negative dominant mutants Vector pCMV- Myc- c-Abl (K290R).
2、 用抗- Flag抗体进行免疫沉淀 （Immunoprecipitation, IP)  2. Immunoprecipitation with anti-flag antibody (Immunoprecipitation, IP)
转染 36-48小时后裂解转染细胞， 用抗 -Flag抗体（M5， Sigma公司）进 行免疫沉淀， 该过程的所有操作应严格在冰浴或低温条件进行， 具体方法为： 收集细胞， 1000g离心 3分钟， 弃尽培养基。 用 5mL冰预冷的 lxPBS洗涤细 胞， lOOOg离心 2分钟， 弃上清， 相同方法洗三次。 Φ 100mm的每皿细胞需加 入 600 μ 1单去污剂裂解液 （50醒 ol/L Tris · CI, 150 mmol/L NaCl, 0. 02% 叠氮钠， 1 %NP- 40以及蛋白酶抑制剂 (Roche, Inc) 1片 /25mL) ， 60mra 的每皿细胞加入 200 μ 1单去污剂裂解液。 将细胞与裂解液混匀， 冰浴 5-10 分钟， 16000g离心 10分钟。小心吸取细胞裂解上清， 加入 10- 15 μ ΐ抗- Flag 抗体交联的琼脂糖珠 （Sigma) , 在 4°C旋转孵育 2小时， 进行免疫沉淀反应。 2小时后， 1000g离心 30秒， 小心弃去上清， 将 IP产物用 250 μ ΐ裂解液洗 涤三次， 尽量将洗液吸净。 向 IP产物中加入 40 μ ΐ lxSDS凝胶上样缓冲液， 100°C水浴 5分钟， 10000g离心 2分钟。取离心上清及 5 μ 1蛋白质预染 Marker ( Invitrogen, Inc) 进行 SDS- PAGE凝胶电泳， 电压设定为 80- 100伏。  After transfection for 36-48 hours, the transfected cells were lysed and immunoprecipitated with anti-Flag antibody (M5, Sigma). All the procedures should be carried out strictly in ice bath or low temperature conditions. The specific method is: Collect cells, 1000g Centrifuge for 3 minutes and discard the medium. The cells were washed with 5 mL of ice-cold lxPBS, centrifuged at 100 g for 2 minutes, and the supernatant was discarded and washed three times in the same manner. Φ 100mm per cell requires 600 μl of single detergent lysate (50 awake ol/L Tris · CI, 150 mmol/L NaCl, 0.02% sodium azide, 1% NP-40 and protease inhibitors) (Roche, Inc) 1 tablet / 25 mL), 200 μl of single detergent lysate was added to each cell of 60 mra. Mix the cells with the lysate, ice bath for 5-10 minutes, and centrifuge at 16000 g for 10 minutes. The cell lysis supernatant was carefully aspirated, and 10-15 μM anti-Fat antibody cross-linked agarose beads (Sigma) were added and incubated at 4 ° C for 2 hours to perform immunoprecipitation. After 2 hours, centrifuge at 1000 g for 30 seconds, carefully discard the supernatant, wash the IP product three times with 250 μM lysate, and drain the wash as much as possible. 40 μL of lxSDS gel loading buffer was added to the IP product, and the mixture was centrifuged at 100 ° C for 5 minutes and centrifuged at 10,000 g for 2 minutes. Centrifugal supernatant and 5 μl protein pre-stained Marker (Invitrogen, Inc) were subjected to SDS-PAGE gel electrophoresis with a voltage of 80-100 volts.
3、 转膜  3, transfer film
电泳结束后， 将蛋白转移至硝酸纤维素膜 （NC膜） ， 具体方法为： 将两 张 2. 5mm厚的滤纸 （Bio-Rad， Inc. ) 及一张硝酸纤维素膜裁剪成凝胶大小， 在 lx转移缓冲液 (39mmol/L甘氨酸， 8mraol/L Tris, 0. 037% SDS以及 20 %甲醇） 中浸泡 30分钟。 随后， 自上而下按照滤纸 -凝胶 -硝酸纤维素膜-滤 纸的顺序在半干转移仪（Bio- Rad， Inc. )上放置好， 尽量赶尽夹层中的气泡。 转膜电压 15V， 2- 2. 5小时后将 NC膜取出， 加入 5mL封闭液（含有 5%脱脂奶 粉的 IxPBST)，放在平缓摇动的水平摇床上室温温育 1小时。封闭后，用 IxPBST 洗膜≡次， 每次 5- 10分钟。  After the end of the electrophoresis, the protein was transferred to a nitrocellulose membrane (NC membrane) by: cutting two 2. 5 mm thick filter paper (Bio-Rad, Inc.) and a nitrocellulose membrane into gel size. Soak for 30 minutes in lx transfer buffer (39 mmol/L glycine, 8mraol/L Tris, 0.0037% SDS and 20% methanol). Subsequently, it was placed on the semi-dry transfer apparatus (Bio-Rad, Inc.) from top to bottom in the order of filter paper-gel-nitrocellulose membrane-filter paper, and the air bubbles in the interlayer were exhausted as much as possible. Transfer film voltage 15V, 2- 2. After 5 hours, remove the NC membrane, add 5 mL of blocking solution (IxPBST containing 5% skim milk powder), and incubate for 1 hour at room temperature on a gently shaken horizontal shaker. After blocking, the membrane was washed once with IxPBST for 5-10 minutes each time.
4、 免疫杂交  4, immune hybridization
用含有 0. 2%。叠氮钠的 PBST配制靶蛋白特异性的非标记抗磷酸化酪氨酸 抗体作为一抗 (anti-P-Tyr, 4G10, Upstate Biotechnology, Inc. ) ； 用含 5%脱脂奶粉和 0. 2%。叠氮钠的 PBST配制辣根过氧化物酶 （HRP) 标记羊抗鼠 抗体 (HRP -mouse Ig G) 作为二抗 (Amersham Pharmacia Biotech, Inc)。 将步骤 3封闭后的 NC膜与一抗在室温温育 1小时后， 用 lxPBST洗膜三次， 每次 5_10分钟； 随后加入酶标二抗， 同样在室温温育 1小时后， 用 lxPBST 洗膜三次， 每次 5-10分钟。 上述温育过程均在水平摇床上进行。 2%。 Contains 0.2%. Sodium azide in PBST formulated a target protein-specific non-labeled anti-phosphotyrosine antibody as a primary antibody (anti-P-Tyr, 4G10, Upstate Biotechnology, Inc.) 5% skim milk powder and 0.2%. Horseradish peroxidase (HRP)-labeled goat anti-mouse antibody (HRP-mouse Ig G) was prepared as a secondary antibody (Amersham Pharmacia Biotech, Inc) in PBST of sodium azide. After the NC membrane blocked in step 3 was incubated with primary antibody for 1 hour at room temperature, the membrane was washed three times with lxPBST for 5-10 minutes each time; then the enzyme-labeled secondary antibody was added, and after incubation for 1 hour at room temperature, the membrane was washed with lxPBST. Three times, 5-10 minutes each time. The above incubations were all carried out on a horizontal shaker.
5、 显色  5, color development
最后进行化学发光反应， 方法为： 将含有辣根过氧化物酶底物的显色液 WESTERN LIGHTNING (PerkinElmer Life Sciences, Inc. ) 均匀地滴在 NC 膜上， 反应 1分钟后将显色液吸干， 用 X光片进行曝光（X光片及显影液、 定 影液均购自 Kodak公司） 。  Finally, the chemiluminescence reaction is carried out by: uniformly dropping the sensitizing liquid WESTERN LIGHTNING (PerkinElmer Life Sciences, Inc.) containing the horseradish peroxidase substrate onto the NC film, and then reacting the coloring solution for 1 minute. Dry, exposed with X-ray film (X-ray film and developer, fixing solution were purchased from Kodak Company).
结果如图 1所示（ " + "表示转染， "-"表示未转染），与空载体 pCMV- Myc (图中以 "Myc- vector"表示） 转染结果相比， pcDNA 3-Flag- Gal3转染细胞 的表达产物 Flag-Gal3可以被 pCMV- Myc - c- Abl的表达产物 Myc-c-Abl磷酸化， 但是 pcDNA 3- Flag- Gal3转染细胞的表达产物 Flag- Gal3不能被  The results are shown in Figure 1 (" + " indicates transfection, "-" indicates no transfection), compared with the empty vector pCMV-Myc (indicated by "Myc- vector" in the figure), pcDNA 3-Flag The expression product Flag-Gal3 of Gal3 transfected cells can be phosphorylated by the expression product Myc-c-Abl of pCMV-Myc-c-Abl, but the expression product Flag-Gal3 of pcDNA 3-Flag-Gal3 transfected cells cannot be
pCMV-Myc-c-Abl (K290R) 的表达产物 Myc_c- Abl (K290R)磷酸化。 原因是 Abl的 K290位点是 C- Abl激酶结构域中 ATP结合位点的关键氨基酸， 被突变为精氨酸 后使 c-Abl丧失酪氨酸激酶活性， 上述检测结果表明 Gal3可特异性地被 c-Abl 磷酸化。 The expression product of pCMV-Myc-c-Abl (K290R) is phosphorylated by Myc_c-Abl (K290R). The reason is that the K290 site of Abl is a key amino acid of the ATP-binding site in the C-Abl kinase domain. When it is mutated to arginine, c-Abl loses tyrosine kinase activity. The above results indicate that Gal3 can specifically Phosphorylated by c-Abl.
四、检测非受体酪氨酸激酶 c-Abl特异性抑制剂 STI571对 c- Abl磷酸化 作用的抑制作用  4. Detection of non-receptor tyrosine kinase c-Abl specific inhibitor STI571 inhibits c-Abl phosphorylation
检测非受体酪氨酸激酶 C-Abl特异性抑制剂 STI571对 c- Abl磷酸化作用 的抑制作用， 检测方法为： 将 Flag-Gal3表达质粒 pcDNA 3_Flag-Gal3转染 293细胞， 24小时后用 10 mol/L STI571 (诺华公司产品， 商品名： 格列卫， 通用名： 甲磺酸伊马替尼）对转染细胞处理 12小时， 以用等量 DMS0 (STI571 的溶剂） 处理的转染细胞为对照， 裂解细胞， 用与步骤三相同的方法进行免 疫沉淀及免疫印迹检测。 Inhibition of c-Abl phosphorylation by non-receptor tyrosine kinase C -Abl specific inhibitor STI571 was detected by transfecting Flag-Gal3 expression plasmid pcDNA 3_Flag-Gal3 into 293 cells, 24 hours later. 10 mol/L STI571 (product of Novartis, trade name: Gleevec, generic name: imatinib mesylate) was transfected with transfected cells for 12 hours to be transfected with an equivalent amount of DMS0 (solvent of STI571) The cells were used as controls, and the cells were lysed, and immunoprecipitation and immunoblotting were carried out in the same manner as in the third step.
结果如图 2所示（ " + "表示经 STI571处理， "-"表示未经 STI571处 理， 即对照） ， 等量 DMS0处理的转染细胞中 Flag- Gal3的表达产物 Gal3可 以被酪氨酸磷酸化，但是经 10 μ raol/L STI571处理的转染细胞中表达的 Gal3 的酪氨酸磷酸化被抑制。 该结果证明 Gal3可以特异性的被细胞内源性 c- Abl 磷酸化， 同时 STI571对 c- Abl的磷酸化作用具有抑制作用 实施例 2、 检测经 STI571处理的 MCF- 7细胞中 Gal3蛋白的变化 The results are shown in Figure 2 ("+" indicates treatment with STI571, "-" indicates no STI571 treatment, ie control), and the expression product of Flag-Gal3 in the transfected cells treated with the same amount of DMS0 can be tyrosine phosphate. However, tyrosine phosphorylation of Gal3 expressed in transfected cells treated with 10 μ raol/L STI571 was inhibited. This result demonstrates that Gal3 can be specifically endogenous to the cell c-Abl Phosphorylation, while STI571 inhibits the phosphorylation of c-Abl. Example 2. Detection of changes in Gal3 protein in MCF-7 cells treated with STI571
一、 检测经 lOuM STI571处理的 MCF-7细胞中 Gal3蛋白的分布情况 用免疫染色法检测经 STI571处理的 MCF-7细胞中 Gal3蛋白的分布情况，具 体方法为： 将人乳腺癌细胞系 MCF- 7接种到 2个培养皿（Glass Bottom Culture Dishes, 购自军事医学科学院仪器分析测试中心）中， 接种量 102个 /皿， 细胞 贴壁 24h后进行下述处理： 第一组细胞不处理， 第二组细胞用 lOuM STI571处 理 18h， 然后对两组细胞进行人线粒体探针 Mito- Tracker ( Invitrogen公司） 标记： 将细胞培养皿中的 DMEM培养液 (Gibco公司）换成预热好的已加入 0. 2 uM mito-probe溶液的 DMEM培养液， 在 37°C培养箱中孵育 15min， 然后用新鲜的 DMEM培养液对细胞进行清洗， 用含 3. 7%多聚甲醛的 DMEM液固定细胞， 在 37°C 培养箱中孵育 15min， 用 PBS洗三次， 每次 5min。 加入 0. 1 % TritonX- 100， 室 温孵育 5min， 再用 PBS洗三次， 每次 5min。 然后用 1 %的 BSA封闭 lh， 再用 PBS 洗三次， 每次 5min。 Gal3蛋白染色和溶酶体染色一抗为分别为 I. Detection of Gal3 protein distribution in MCF-7 cells treated with lOuM STI571 The distribution of Gal3 protein in MCF-7 cells treated with STI571 was detected by immunostaining method. The specific method was as follows: Human breast cancer cell line MCF- 7 Inoculate 2 Petri dishes (Glass Bottom Culture Dishes, purchased from the Instrumental Analysis and Testing Center of the Academy of Military Medical Sciences), inoculate 10 2 / dish, and after the cells are attached for 24 hours, the following treatments are performed: The first group of cells is not treated, The second group of cells was treated with lOuM STI571 for 18 h, and then the two groups of cells were labeled with human mitochondrial probe Mito-Tracker (Invitrogen): DMEM medium (Gibco) in the cell culture dish was replaced with preheated and added. 0. 2 uM mito-probe solution in DMEM medium, incubate in a 37 ° C incubator for 15 min, then wash the cells with fresh DMEM medium, and fix the cells with DMEM containing 3.7% paraformaldehyde. Incubate for 15 min in a 37 ° C incubator and wash three times with PBS for 5 min each. Add 0.1% TritonX-100, incubate for 5 min at room temperature, and wash three times with PBS for 5 min each time. It was then blocked with 1% BSA for 1 h and washed three times with PBS for 5 min each. The Gal3 protein staining and lysosomal staining primary antibodies were
galectin- 3 (H-160) (santa cruz biotechnology, inc)抗体禾口 LAMP- 2 (LAMP- 2 为溶酶体膜标志蛋白）抗体 （santa cruz biotechnology, inc) ， 使用时按 1: 200比例进行稀释， 室温孵育 lh， 用 PBS洗三次， 每次 5min。 Gal3蛋白染色二 抗为 FITC-rabbit (北京中杉金桥公司） ， 使用时按 1: 200比例进行稀释， 室 温孵育 lh， 再用 PBS洗三次， 每次 5min。 溶酶体染色二抗为 TRITC- mouse，使用 方法同 Gal3染色。 最后进行细胞核染色， 核染料 Hochest33342 (购自鼎国生 物技术公司）的使用浓度为 lug/mL，室温孵育 30min，用 PBS洗三次，每次 5min。 置荧光显微镜下观察。 Galectin-3 (H-160) (santa cruz biotechnology, inc) antibody and LAMP-2 (LAMP-2 is a lysosomal membrane marker protein) antibody (santa cruz biotechnology, inc), used at a ratio of 1:200 Dilute, incubate for 1 h at room temperature, and wash three times with PBS for 5 min each time. The Gal3 protein stained secondary antibody was FITC-rabbit (Beijing Zhongshan Jinqiao Co., Ltd.), diluted at a ratio of 1:200 during use, incubated for 1 h at room temperature, and washed three times with PBS for 5 min each time. The lysosomal staining secondary antibody was TRITC-mouse and was stained with Gal3 using the method. Finally, nuclear staining was performed. The nuclear dye Hochest33342 (purchased from Dingguo Biotechnology Co., Ltd.) was used at a concentration of lu g /mL, incubated at room temperature for 30 min, and washed three times with PBS for 5 min each time. Observe under a fluorescence microscope.
未经 STI571处理的 MCF-7细胞的免疫染色结果见图 3中的图 A，红色为线粒 体， 绿色为 Gal3， 蓝色为细胞核， Gal3分布于全细胞， 并与线粒体有共定位； 经 10uM STI571处理 18h后的 MCF- 7细胞的免疫染色结果见图 3中的图 B (红色为 线粒体， 绿色为 Gal3， 蓝色为细胞核） ， Gal3蛋白发生点状聚集， 此时 Gal3 蛋白的聚集体与线粒体不再共定位， 而是如图 C所示（红色为 Gal3蛋白， 绿色 为溶酶体， 蓝色为细胞核） ， Gal3蛋白与溶酶体共定位， 即进入溶酶体。 上 述检测结果表明非受体酪氨酸激酶 c- Abl特异性抑制剂 STI571可通过抑制 c - Abl的激酶活性而导致 Gal3发生变性聚集并通过溶酶体降解。 图 3中， 图 A和 图 B中从左至右分别为绿色荧光标记的 Gal3蛋白，红色 Mito- Tracker标记的线 粒体， 核染料 Hochest33342标记的细胞核， 合并的图像。 图 C中从左至右分别 为绿色荧光标记的 Gal3蛋白， 红色荧光标记的溶酶体， 核染料 Hochest33342 标记的细胞核， 合并的图像。 The results of immunostaining of MCF-7 cells without STI571 are shown in Figure A in Figure 3, red is mitochondria, green is Gal3, blue is nucleus, Gal3 is distributed throughout cells, and colocalized with mitochondria; 10uM STI571 The immunostaining results of MCF-7 cells after 18h treatment are shown in Figure B in Figure 3 (red is mitochondria, green is Gal3, blue is nucleus), Gal3 protein is punctate, and Gal3 protein aggregates and mitochondria No longer co-localization, but as shown in Figure C (Gal3 protein in red, lysosome in green, nucleus in blue), Gal3 protein co-localizes with lysosomes, ie enters lysosomes. The above test results indicate that the non-receptor tyrosine kinase c-Abl specific inhibitor STI571 can be inhibited The kinase activity of c-Abl causes Gal3 to undergo degenerative aggregation and is degraded by lysosomes. In Fig. 3, from left to right in Fig. A and Fig. B, green fluorescently labeled Gal3 protein, red Mito-Tracker labeled mitochondria, nuclear dye Hochest33342 labeled nuclei, combined images. In Figure C, from left to right are the green fluorescently labeled Gal3 protein, the red fluorescently labeled lysosome, the nuclear dye Hochest33342 labeled nuclei, and the combined images.
二、 检测经 5uM STI571、 0. 5uM凋亡诱导剂 STS单独处理及两者共同处理 的 MCF-7细胞中 Gal3蛋白量的变化  2. Changes in the amount of Gal3 protein in MCF-7 cells treated with 5uM STI571, 0.5 uM apoptosis inducer STS alone and both.
1、 免疫染色检测  1, immunostaining detection
用免疫染色法检测经 5uM STI57U 0. 5uM凋亡诱导剂 STS单独处理及两者 共同处理的 MCF-7细胞中 Gal3蛋白量的变化， 实验分为四组： 第一组细胞不处 理； 第二组细胞经 5uM STI571处理 18h; 第三组细胞经 0. 5uM凋亡诱导剂 STS The amount of Gal3 protein in MCF-7 cells treated with 5uM STI57U 0.5 uM apoptosis inducer STS alone and treated together was detected by immunostaining. The experiment was divided into four groups: the first group of cells was not treated; 5uM apoptosis inducer STS was treated with 5uM STI571 for 18h;
( strurosporine) ( sigma公司， S4400产品）处理 150min， 第四组细胞经 5uM STI571处理 18h后加入 0. 5uM STS再处理 150min。 免疫染色方法与步骤一相同。 (strurosporine) (sigma, S4400) was treated for 150 min, and the fourth group was treated with 5 uM STI571 for 18 h and then added with 0.5 uu STS for 150 min. The immunostaining method is the same as step one.
结果如图 4所示 （左上图： 未经处理细胞， 右上图： 经 0. 5uM凋亡诱导剂 STS处理 150min细胞， 左下图： 经 5uM STI571处理 18h细胞， 右下图： 经 5uM STI571处理 18h后加入 0. 5uM STS处理 150min细胞） ， 红色为线粒体， 绿色为 Gal3, 蓝色为细胞核， 细胞单独用凋亡诱导剂 STS处理时， 细胞中 Gal3没有发 生明显变化； 当用 STI571处理时， 细胞中 Gal3发生聚集； 当用 STI和 STS共同 处理细胞时， Gal3蛋白量明显减少。  The results are shown in Figure 4 (top left panel: untreated cells, upper right panel: 150 min cells treated with 0.5 uM apoptosis inducer STS, left lower panel: 18 h cells treated with 5 uM STI571, bottom right panel: treated with 5 uM STI571 for 18 h After adding 0.5 μl of STS for 150 min, the red color is mitochondria, the green color is Gal3, and the blue color is the nucleus. When the cells are treated with the apoptosis inducer STS alone, there is no significant change in Gal3 cells; when treated with STI571, the cells are treated with STI571. The aggregation of Gal3 occurs; when the cells are treated together with STI and STS, the amount of Gal3 protein is significantly reduced.
2、 免疫印迹检测  2, immunoblotting detection
将 MCF- 7细胞接种到 4个 100皿细胞培养皿中， 与上述免疫染色实验相 同的处理分为四组， 用免疫印迹 Western blot法对细胞中 Gal3蛋白量的变 化作进一步检测， Gal3检测一抗为 galectin- 3 (B-2) (santa cruz  The MCF-7 cells were inoculated into four 100-well cell culture dishes. The same treatment as the above immunostaining experiments was divided into four groups. The change of Gal3 protein in the cells was further detected by Western blotting. Gal3 detection Resistance to galectin-3 (B-2) (santa cruz
biotechnology, inc) , 二抗为 HRP -mouse Ig G (santa cruz Biotechnology, inc) , secondary antibody for HRP -mouse Ig G (santa cruz
biotechnology, inc) , 以 β - actin作为内参 (一抗为 β - actin单克隆抗体 ( santa cruz biotechnology, inc) ， 二抗为 HRP -mouse Ig G (santa cruz biotechnology, inc) ， 以上抗体均购自友谊中联生物科技有限公司。 Biotechnology, inc) , using β-actin as an internal reference (the primary antibody is β-actin monoclonal antibody ( santa cruz biotechnology, inc), the secondary antibody is HRP -mouse Ig G (santa cruz biotechnology, inc) , the above antibodies are purchased from Friendship Zhonglian Biotechnology Co., Ltd.
结果如图 5所示 （ " + "表示添加， "-"表示未添加） 在 e -actin平 衡的情况下检测到，用 STI571处理后，细胞中 Gal3含量下降;用 STS和 STI571 共同处理后 Gal3蛋白几乎全部消失； 而单独使用 STS处理时， 细胞中 Gal3 含量几乎没有改变。 检测结果与步骤 1的免疫染色结果相符。 The results are shown in Fig. 5 (" + " indicates addition, "-" indicates no addition). In the case of e-actin equilibrium, Gal3 content in the cells was decreased after treatment with STI571; Gal3 was treated with STS and STI571. Almost all of the protein disappears; when treated with STS alone, Gal3 in the cell The content has hardly changed. The test results were consistent with the immunostaining results of step 1.
上述实验结果表明，非受体酪氨酸激酶 c- Abl特异性抑制剂 STI571通过 对 c- Abl激酶活性的抑制使 Gal3蛋白的 c- Abl激酶依赖性的稳定性丧失而发 生降解， 失去了 Gal3作为癌症相关蛋白的功能， 从而可以抑制肿瘤细胞的黏 附、 增殖、 分化、 血管生成、 恶化及转移。  The above experimental results indicate that the non-receptor tyrosine kinase c-Abl specific inhibitor STI571 degrades due to the inhibition of c-Abl kinase-dependent stability of the Gal3 protein by the inhibition of c-Abl kinase activity, and loses Gal3. As a function of a cancer-associated protein, it is possible to inhibit adhesion, proliferation, differentiation, angiogenesis, deterioration, and metastasis of tumor cells.
实施例 3、 检测经 ΙΟμΜ STI571、 0. 5μΜ STS单独处理及两者共同处理的 MCF- 7细胞的凋亡情况  Example 3: Detection of apoptosis of MCF-7 cells treated with ΙΟμΜ STI571, 0.5 μΜ STS alone and both
实验分为四组： 第一组 MCF-7细胞不处理； 第二组 MCF-7细胞经 ΙΟμΜ STI571处理 18h; 第三组 MCF- 7细胞经 0. 5μΜ凋亡诱导剂 STS处理 4- 5h， 第 四组 MCF- 7细胞经 ΙΟμΜ STI571处理 18h后加入 0. 5μΜ STS再处理 4_5h。  The experiment was divided into four groups: the first group of MCF-7 cells were not treated; the second group of MCF-7 cells were treated with ΙΟμΜ STI571 for 18 h; the third group of MCF-7 cells were treated with 0.5 μμ of apoptosis inducer STS for 4-5 h, The fourth group of MCF-7 cells were treated with ΙΟμΜ STI571 for 18 h and then added with 0.5 μM STS for 4-5 h.
一、 TUNEL法检测经 ΙΟμΜ STI571、 0. 5μΜ STS.单独处理及两者共同处理 的 MCF- 7细胞的凋亡情况  1. TUNEL method was used to detect the apoptosis of MCF-7 cells treated with ΙΟμΜ STI571, 0.5 μΜ STS.
用 TUNEL- FLU0S试剂盒（购自 Roche公司）并参照试剂盒说明书检测上述 经不同处理的四组 MCF-7细胞的凋亡情况， 具体方法为： 用新配制的 4%多聚 甲醛在室温下对细胞固定 30min， PBS洗片后与阻断剂（¾02甲醇溶液）在室温 下孵育 30min， PBS洗片， 加入 0. 1 %的 Triton X- 100在冰浴中孵育 2min。 PBS 洗两次， 滴加 50μ1 TUNEL反应混合液， 在湿盒中 37°C孵育 60min， PBS洗三次， 然后进行 Gal3蛋白免疫染色和细胞核染色， 免疫染色方法见实施例 2， 染色后 在荧光显微镜下观察。 The apoptosis of the four groups of MCF-7 cells treated with different treatments was detected by TUNEL-FLU0S kit (purchased from Roche) and with reference to the kit instructions. The specific method was as follows: using freshly prepared 4% paraformaldehyde at room temperature. The cells were fixed for 30 min, washed with PBS and incubated with blocking agent (3⁄40 2 methanol solution) for 30 min at room temperature, washed with PBS, and 0.1% Triton X-100 was added and incubated for 2 min in an ice bath. Wash twice with PBS, add 50μ1 TUNEL reaction mixture, incubate in a humid box for 60min at 37°C, wash three times with PBS, then perform Gal3 protein immunostaining and nuclear staining. The immunostaining method is shown in Example 2, after staining in fluorescence microscope. Under observation.
结果如图 6所示（红色为 Gal3蛋白， 绿色为 FITC标记酶标记抗体显示的细 胞凋亡情况， 蓝色为细胞核）， 如图 6中的图 1所示， 正常的 MCF-7细胞检测不 到细胞凋亡， 即细胞核中没有绿色荧光， 但可以看到红色的 Gal3蛋白分布于 全胞质； 如图 6中的图 2所示， 细胞经 lOuM STI571处理 18h后有细胞凋亡， 可 以看到细胞核中有绿色荧光， 红色的 Gal3蛋白呈点状聚集； 如图 6中的图 3所 示， 细胞经 0. 5uM STS处理 4-5h后也有细胞凋亡， 可以看到细胞核中有绿色荧 光， 红色的 Gal3蛋白依然分布于全胞质； 如图 6中的图 4所示， 细胞经 10uM STI571处理 18h后加入 0. 5uM STS再处理 4- 5h后， 可以看到很强的绿色荧光在 细胞核中表达， 而且细胞核变小皱缩， 此时红色的 Gal3蛋白表达极弱， 细胞 凋亡显著。 图 6中， 从左至右分别是由核染料 HocheSt33342标记的细胞核， Tunnel标记的绿色荧光染色， 红色荧光标记的 Gal3蛋白， 合并的图像。 上述检测结果表明非受体酪氨酸激酶 c- Abl特异性抑制剂 STI571可使 Gal3蛋白降解从而诱导肿瘤细胞发生凋亡。 The results are shown in Figure 6 (red is Gal3 protein, green is the apoptosis of the FITC-labeled antibody, blue is the nucleus), as shown in Figure 1 of Figure 6, normal MCF-7 cells are not detected. To apoptosis, there is no green fluorescence in the nucleus, but it can be seen that the red Gal3 protein is distributed in the whole cytoplasm; as shown in Figure 2 in Figure 6, the cells have apoptosis after treatment with lOuM STI571 for 18h, you can see There is green fluorescence in the nucleus, and the red Gal3 protein is punctate. As shown in Figure 3 in Figure 6, the cells also have apoptosis after being treated with 0.5 uM STS for 4-5 hours, and green fluorescence can be seen in the nucleus. The red Gal3 protein is still distributed in the whole cytoplasm; as shown in Figure 4 in Figure 6, the cells are treated with 10uM STI571 for 18h and then added to 0. 5uM STS for another 4-5h, a strong green fluorescence can be seen. It is expressed in the nucleus, and the nucleus becomes small and shrunken. At this time, the red Gal3 protein is extremely weakly expressed and the apoptosis is remarkable. In Fig. 6, from left to right are the nuclei labeled with the nuclear dye Hoch eS t33342, the green fluorescent staining of the tunnel marker, the red fluorescently labeled Gal3 protein, and the combined images. The above results indicate that the non-receptor tyrosine kinase c-Abl specific inhibitor STI571 can degrade Gal3 protein and induce apoptosis of tumor cells.
二、 Annexin- V- FLU0S法检测经 lOuM STI57 0. 5uM STS单独处理及两 者共同处理的 MCF- 7细胞的凋亡情况  2. Annexin-V-FLU0S method for detection of apoptosis of MCF-7 cells treated with lOuM STI57 0. 5uM STS alone and co-treated by both
再用 Annexin-V-FLUOS试剂盒（购自 Roche公司）并参照试剂盒说明书检 测上述经不同处理的四组 MCF-7细胞的凋亡情况， 具体方法为： 500g离心 5min 收集细胞， 用 PBS重悬细胞， 洗一次， 将细胞重悬于 200μ1的 binding buffer (试剂盒自带） 中， 加入 ΙΟμΙ Annexin- V-FITC和 5μ1 ΡΙ， 轻轻混匀， 避光室 温孵育 15min， 用流式细胞检测仪检测。  The apoptosis of the four groups of MCF-7 cells treated with different treatments was detected by using the Annexin-V-FLUOS kit (purchased from Roche) and the kit was as follows: The cells were collected by centrifugation at 500 g for 5 min, and the cells were harvested with PBS. Suspend the cells, wash once, resuspend the cells in a 200 μl binding buffer (provided with the kit), add ΙΟμΙ Annexin-V-FITC and 5μ1 ΡΙ, mix gently, incubate for 15 min at room temperature, and use flow cytometry. Instrument detection.
结果如图 7所示 （左上图： 正常的 MCF-7细胞， 右上图： 经 0. 5μΜ凋亡 诱导剂 STS处理 4-5h的 MCF- 7细胞， 左下图： 经 10μΜ STI571处理 18h的 MCF- 7细胞，右下图：经 10|oM STI571处理 18h后加入 0. 5 M STS再处理 4- 5h 的 MCF- 7细胞）， 用 STI571和 STS单独处理均可使 MCF-7细胞发生一定程度 的凋亡， 当用 STI571和 STS共同处理时细胞凋亡显著， 进一步证明非受体酪 氨酸激酶 c- Abl特异性抑制剂 STI571可诱导肿瘤细胞发生凋亡。  The results are shown in Figure 7 (top left panel: normal MCF-7 cells, upper right panel: MCF-7 cells treated with 0.5 μμΜ apoptosis inducer STS for 4-5 h, lower left panel: MCF treated with 10 μΜ STI571 for 18 h- 7 cells, right lower panel: after treatment with 10|oM STI571 for 18h, 0.5 M STS was added for 4-5h of MCF-7 cells, and treatment with STI571 and STS alone could cause MCF-7 cells to a certain extent. Apoptosis, when treated with STI571 and STS, showed significant apoptosis, further demonstrating that non-receptor tyrosine kinase c-Abl specific inhibitor STI571 can induce tumor cell apoptosis.
三、 Sub- G1法检测经 ΙΟμΜ STI57K 4μΜ CDDP单独处理及两者联合处理 的 MCF-7细胞的凋亡情况  3. Sub-G1 assay for apoptosis of MCF-7 cells treated with ΙΟμΜ STI57K 4μΜ CDDP alone or in combination
将 MCF- 7细胞分成 STI571 ( 10μΜ)处理和不处理两组， 18小时后， 将这 两组细胞再分成用化疗药物顺铂 CDDP (4MM)处理和不处理两组， 24小时后， 用流式细胞仪进行细胞凋亡分析。  MCF-7 cells were divided into STI571 (10 μΜ) treatment and no treatment. After 18 hours, the two groups of cells were subdivided into two groups treated with the chemotherapy drug cisplatin CDDP (4MM). After 24 hours, the flow was used. Apoptosis analysis was performed using a cytometer.
将细胞用胰蛋白酶消化后用 10- 12mL PBS收集细胞到 15ml的管中，500g， 3min离心沉淀细胞，用 5mL PBS洗细胞，再次离心沉淀细胞。逐滴加入 2mL70% 的乙醇（70%乙醇溶于 PBS中） ， 边加边摇晃。 - 20°C孵育过夜。 重复用 PBS 洗两次后离心沉淀， 加 2mL P-C Buffer, 孵育 30 min[0. 2 M NaH2P04 192mL; 0. 2M Sodium Citrate柠檬酸钠 8mL]。再用 PBS洗两次后离心沉淀，加入 0. 3mL RNase溶液 [20ug/mL， PBS溶解] , 30 min。 加 3uL pi溶液 [10mg/ml， PBS 溶解]染色 30min。 流式细胞仪检测 104细胞的 DNA含量， 通过 Modifit软件 分析凋亡细胞比例。 结果如图 8所示， 未处理组（图中以 MCF- 7表示） MCF- 7 细胞的凋亡率为 3%， STI571处理组（图中以 MCF- 7+STI表示）细胞的凋亡 率为 23. 7%, CDDP处理组（图中以 MCF - 7+CDDP表示）的细胞凋亡率为 5. 53%， 而 STI571和 CDDP双处理组（图中以 MCF- 7+STI+CDDP表示）的细胞凋亡率 为 48. 2%。上述结果说明， C- Abl/Arg特异性激酶抑制剂 STI571能够使细胞 发生凋亡， 当与低剂量的凋亡诱导剂共同使用时会出现协同诱导凋亡的效果。 实施例 4、 检测 STI571对裸鼠移植性肿瘤生长的抑制情况 The cells were trypsinized, and the cells were collected with 10-12 mL of PBS into a 15 ml tube, 500 g, and the cells were pelleted by centrifugation for 3 min, the cells were washed with 5 mL of PBS, and the cells were again pelleted by centrifugation. 2 mL of 70% ethanol (70% ethanol in PBS) was added dropwise while shaking. - Incubate overnight at 20 °C. After repeated washing with PBS twice, the pellet was centrifuged, and 2 mL of PC Buffer was added and incubated for 30 min [0. 2 M NaH 2 P0 4 192 mL; 0.2 M Sodium Citrate sodium citrate 8 mL]. After washing twice with PBS, the pellet was centrifuged, and 0.3 mL of RNase solution [20 ug/mL, PBS dissolved] was added for 30 min. Add 3uL pi solution [10mg/ml, PBS dissolved] for 30min. DNA content by flow cytometry 104 cells, the proportion of apoptotic cells analyzed by Modifit software. The results are shown in Fig. 8. The apoptotic rate of MCF-7 cells was 3% in the untreated group (indicated by MCF-7 in the figure), and apoptosis was observed in the STI571-treated group (indicated by MCF-7+STI in the figure). The rate was 23.7%, and the apoptotic rate of the CDDP-treated group (indicated by MCF-7+CDDP) was 5.33%, while the STI571 and CDDP double-treatment groups (MCF-7+STI+CDDP in the figure) 2%。 The apoptotic rate was 48.2%. These results indicate that the C-Abl/Arg-specific kinase inhibitor STI571 is capable of causing apoptosis in cells, and synergistically induces apoptosis when used in combination with a low dose of an inducer of apoptosis. Example 4: Detection of STI571 inhibition of transplanted tumor growth in nude mice
以 BABL/C裸鼠 （雄性， 18- 20g， 购自军事医学科学院实验动物中心） 为 实验动物。 取对数生长期的 MCF- 7细胞， 胰酶消化后按 5 X 10 6个细胞 /只以皮 下接种的方式接种裸鼠， 接种后将裸鼠随机分成 5组， 进行随机分组治疗， 每 组 7只。 第一组为生理盐水组（阴性对照组） ， 裸鼠在接种细胞后第二天以灌 胃方式给生理盐水，每天两次，每次 δμΐ/g体重；第二组为 STI571口服给药组， 裸鼠在接种细胞后第二天以灌胃方式给药， 每天两次， 每次 40mg/kg， 溶剂 是生理盐水， 每次灌胃体积是 δμΐ/g体重； 第三组为 1/4常规剂量 CTX (环磷酰 氨（江苏恒瑞医药股份有限公司产品） ， 腹腔注射组， 裸鼠在接种细胞后第 7 天进行腹腔注射给药， 两周一次， 每次 25mg/kg， 溶剂是生理盐水， 每次注射 体积是 δμΐ/g体重； 第四组为联合给药组， 按第二、 三组的给药量将 STI571和 CTX联合给药。 第五组， CTX全剂量腹腔注射组 （阳性对照组） ， 裸鼠在接种 细胞后第 7天进行腹腔注射给药，两周一次，每次 100mg/kg，溶剂是生理盐水， 每次注射体积是 δμΐ/g体重。 BABL/C nude mice (male, 18-20 g, purchased from the Experimental Animal Center of the Academy of Military Medical Sciences) were used as experimental animals. MCF-7 cells in logarithmic growth phase were inoculated into nude mice by pancreatic enzyme digestion at 5×10 6 cells/subcutaneously. After inoculation, nude mice were randomly divided into 5 groups and randomized into groups. 7 only. The first group was the saline group (negative control group), and the nude mice were given saline by intragastric administration twice a day, δμΐ/g body weight twice a day; the second group was STI571 oral administration group. The nude mice were administered by intragastric administration the next day after inoculation of cells, twice a day, 40 mg/kg each time, the solvent was normal saline, and the volume per administration was δμΐ/g body weight; the third group was 1/4 Conventional dose of CTX (cyclophosphamide (product of Jiangsu Hengrui Pharmaceutical Co., Ltd.), intraperitoneal injection group, nude mice were intraperitoneally administered on the 7th day after inoculation of cells, once every two weeks, 25 mg/kg each time, the solvent was Normal saline, each injection volume was δμΐ/g body weight; the fourth group was the combined administration group, and STI571 and CTX were administered in combination according to the second and third groups. Group 5, CTX full-dose intraperitoneal injection group (Positive control group), nude mice were intraperitoneally administered on the 7th day after inoculation of cells, once every two weeks, 100 mg/kg each time, the solvent was physiological saline, and the volume of each injection was δμΐ/g body weight.
在接瘤 24天后用脱颈法处死裸鼠， 解剖取瘤块， 称瘤重， 根据以下公式 计算肿瘤生长抑瘤率：  After 24 days of tumor attachment, the nude mice were sacrificed by neck dissection, and the tumor mass was dissected and weighed. The tumor growth inhibition rate was calculated according to the following formula:
IR= (1 -治疗组瘤重 /阴性对照组瘤重） X 100%。  IR = (1 - tumor weight of the treatment group / tumor weight of the negative control group) X 100%.
结果如图 9和 10所示， CTX全剂量给药组 （图中以 "CTX ( lOOmg/kg) "表 示） 的抑瘤率高达 85. 2%， 联合给药组 （图中以 "STI571+CTX (25mg/kg) " 表示） 的抑瘤率达 81. 4%左右， STI571给药组 （图中以 "STI571 "表示） 抑 瘤率达 45 %左右， 1/4剂量 CTX腹腔注射组 （图中以 "CTX (25rag/kg) "表示） 抑瘤率为 35%左右。 结果说明， STI571有一定的抑瘤效果， 与肿瘤化疗药物 CTX联合使用时可以起到协同抑瘤作用。 而且， 实验结束时这五组裸鼠体重统 计结果如图 10所示，第四组 STI571与 1/4常规剂量 CTX联合用药组 （图中以 "STI571 +CTX (25mg/kg) "表示）与第五组 CTX全剂量给药组（图中以 "CTX ( lOOmg/kg) "表示） 裸鼠平均体重存在显著差异， 而肿瘤治疗效果不存在 显著差异， 说明 STI571作为肿瘤化疗药物的联合用药能大大减少肿瘤化疗药 物的副作用。 图 9和 10中， Control为阴性对照组。 The results are shown in Figures 9 and 10. The inhibition rate of the CTX full-dose group (expressed as "CTX (100 mg/kg)" in the figure was as high as 85.2%, in the co-administration group ("STI571+" The inhibition rate of CTX (25mg/kg) "expressed" was about 81.4%, and the STI571 administration group (indicated by "STI571" in the figure) had a tumor inhibition rate of about 45%, and a quarter-dose CTX intraperitoneal injection group ( In the figure, "CTX (25rag/kg)" indicates that the tumor inhibition rate is about 35%. The results show that STI571 has a certain anti-tumor effect, and it can play a synergistic anti-tumor effect when combined with the tumor chemotherapy drug CTX. Moreover, at the end of the experiment, the weight results of the five groups of nude mice are shown in Figure 10. The fourth group of STI571 and 1/4 conventional dose CTX combination group (indicated by "STI571 + CTX (25mg/kg)") The fifth group of CTX full-dose administration group (indicated by "CTX (100 mg/kg)") showed significant differences in the average body weight of nude mice, but the tumor treatment effect did not exist. Significant differences indicate that STI571, as a combination of tumor chemotherapy drugs, can significantly reduce the side effects of cancer chemotherapy drugs. In Figures 9 and 10, Control is a negative control group.
对上述肿瘤进行肿瘤切片 HE染色。 如图 11所示， 表明阴性对照组（图 中以 "盐水组"表示）肿瘤中血管及腺体丰富， 1/4常规剂量 CTX腹腔注射组 (图中以 "CTX组"表示） 的肿瘤中血管及腺体受到一定的抑制， 而 STI571 口服给药组（图中以 "STI组"表示）和联合给药组（图中以 "STI+CTX组" 表示） 中看不到丰富的血管和腺体， 说明 STI有很好的抑制肿瘤血管生长及 肿瘤生长的作用。  Tumor sections were stained with HE for the above tumors. As shown in Fig. 11, it was shown that the negative control group (indicated by the "saline group" in the figure) was rich in blood vessels and glands in the tumor, and in the tumor of the 1/4 conventional dose CTX intraperitoneal injection group (indicated by "CTX group" in the figure). Vascular and glandularities were inhibited to a certain extent, while the STI571 oral administration group (indicated by the "STI group" in the figure) and the co-administration group (indicated by the "STI+CTX group" in the figure) did not have abundant blood vessels and Gland, indicating that STI has a good role in inhibiting tumor blood vessel growth and tumor growth.
工业应用 Industrial application
本发明提供了一种非受体酪氨酸激酶 c-Abl特异性抑制剂的新用途， 即 在制备抗肿瘤药物或肿瘤治疗辅助药物中的应用。 实验证明非受体酪氨酸激 酶 c- Abl特异性抑制剂通过抑制 c- Abl的激酶活性可导致 Gal3发生变性聚集 并通过溶酶体降解， 致使肿瘤细胞中 Gal3蛋白水平显著降低， 从而使肿瘤细 胞发生自噬性细胞凋亡而死亡。 同时， 非受体酪氨酸激酶 c- Abl特异性抑制 剂还可使肿瘤细胞对多种肿瘤治疗药物极为敏感， 短时间内低浓度的凋亡诱 导剂即可使细胞全部死亡， 因而非受体酪氨酸激酶 c-Abl特异性抑制剂可增 强现有细胞毒类化疗药物的治疗效果， 并可通过降低化疗药物的使用剂量， 使毒副作用大大减轻。 以非受体酪氨酸激酶 c- Abl特异性抑制剂为活性成分 制备抗肿瘤药物具有以下优点： 1 )疗效显著， 抑瘤率可达 81. 4% ; 2)安全 性高； 3)主要用药途径为口服， 用药方便； 4)该抑制剂价格低廉， 因而以 其制备抗肿瘤药物的成本较低， 从而可减轻病人的经济负担。 本发明为肿瘤 的防治提供了一条新途径， 在医药学领域的应用前景广阔。  The present invention provides a novel use of a non-receptor tyrosine kinase c-Abl specific inhibitor, i.e., in the preparation of an antitumor drug or a tumor therapeutic adjuvant. Experiments have shown that non-receptor tyrosine kinase c-Abl specific inhibitors can cause Gal3 to undergo degeneration and aggregation and lysosome degradation by inhibiting the kinase activity of c-Abl, resulting in a significant decrease in Gal3 protein levels in tumor cells, thereby allowing tumors to The cells die of autophagic cell apoptosis. At the same time, the non-receptor tyrosine kinase c-Abl specific inhibitor can also make tumor cells extremely sensitive to a variety of tumor therapeutic drugs, and a low concentration of apoptosis-inducing agents can cause all cells to die in a short period of time, thus The specific inhibitor of c-Abl, a tyrosine kinase, enhances the therapeutic effect of existing cytotoxic chemotherapeutic drugs, and can greatly reduce the side effects by reducing the dose of chemotherapeutic drugs. Preparation of anti-tumor drugs with non-receptor tyrosine kinase c-Abl specific inhibitor as active ingredient has the following advantages: 1) remarkable curative effect, tumor inhibition rate up to 81.4%; 2) high safety; 3) main The route of administration is oral, and the administration is convenient; 4) the inhibitor is inexpensive, and thus the cost of preparing the antitumor drug is low, thereby reducing the economic burden on the patient. The invention provides a new way for the prevention and treatment of tumors, and has broad application prospects in the field of medicine.

Claims

权利要求 Rights request
I、 非受体酪氨酸激酶 c- Abl特异性抑制剂在制备抗肿瘤药物和 /或肿瘤 治疗辅助药物中的应用。 I. Use of a non-receptor tyrosine kinase c-Abl specific inhibitor in the preparation of an antitumor drug and/or a tumor therapeutic adjuvant.
2、根据权利要求 1所述的应用，其特征在于：所述肿瘤为表达 Galectin - 2. The use according to claim 1, wherein said tumor is expressed by Galectin -
3的肿瘤。 3 tumors.
3、 根据权利要求 2所述的应用， 其特征在于： 所述肿瘤为乳腺癌、 前列 腺癌、 ***、 肺腺癌、 肝癌、 结肠癌、 甲状腺癌、 胰腺癌、 神经内分泌肿 瘤、 膀胱癌、 肾癌或胃癌。  3. The use according to claim 2, wherein the tumor is breast cancer, prostate cancer, cervical cancer, lung adenocarcinoma, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, neuroendocrine tumor, bladder cancer, Kidney cancer or stomach cancer.
4、 根据权利要求 1所述的应用， 其特征在于： 所述非受体酪氨酸激酶 c-Abl特异性抑制剂为 STI57K Sunitinib、 PKC412、 vandetanib, Sorafenib、 erlotinib-, gefitinib或 dasatinib。  4. Use according to claim 1, characterized in that the specific inhibitor of the non-receptor tyrosine kinase c-Abl is STI57K Sunitinib, PKC412, vandetanib, Sorafenib, erlotinib-, gefitinib or dasatinib.
5、 非受体酪氨酸激酶 c-Abl特异性抑制剂和顺铂在制备抗肿瘤药物和 / 或肿瘤治疗辅助药物中的应用。  5. Application of non-receptor tyrosine kinase c-Abl specific inhibitor and cisplatin in the preparation of antitumor drugs and/or tumor therapeutic aids.
6、根据权利要求 5所述的应用，其特征在于：所述肿瘤为表达 Galectin - 6. The use according to claim 5 wherein said tumor is expressed by Galectin -
3的肿瘤。 3 tumors.
7、 根据权利要求 6所述的应用， 其特征在于： 所述肿瘤为乳腺癌、 前列 腺癌、 ***、 肺腺癌、 肝癌、 结肠癌、 甲状腺癌、 胰腺癌、 神经内分泌肿 瘤、 膀胱癌、 肾癌或胃癌。  7. The use according to claim 6, wherein the tumor is breast cancer, prostate cancer, cervical cancer, lung adenocarcinoma, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, neuroendocrine tumor, bladder cancer, Kidney cancer or stomach cancer.
8、 根据权利要求 5所述的应用， 其特征在于： 所述非受体酪氨酸激酶 8. The use according to claim 5, wherein: said non-receptor tyrosine kinase
C- Abl特异性抑制剂为 STI571、 Sunitinib、 PKC412、 vandetanib、 Sorafenib, erlotinibs gefitinib或 dasatinib。 Specific inhibitors of C-Abl are STI571, Sunitinib, PKC412, vandetanib, Sorafenib, erlotinibs gefitinib or dasatinib.
9、非受体酪氨酸激酶 c- Abl特异性抑制剂和环磷酰氨在制备抗肿瘤药物 和 /或肿瘤治疗辅助药物中的应用。  9. Use of a non-receptor tyrosine kinase c-Abl specific inhibitor and cyclophosphamide in the preparation of an antitumor drug and/or a tumor therapeutic adjuvant.
10、根据权利要求 9所述的应用，其特征在于:所述肿瘤为表达 Galectin- 10. The use according to claim 9, wherein said tumor is expressed by Galectin-
3的肿瘤。 3 tumors.
I I、 根据权利要求 10所述的应用， 其特征在于： 所述肿瘤为乳腺癌、 前 列腺癌、 ***、 肺腺癌、 肝癌、 结肠癌、 甲状腺癌、 胰腺癌、 神经内分泌 肿瘤、 膀胱癌、 肾癌或胃癌。  II. The use according to claim 10, wherein the tumor is breast cancer, prostate cancer, cervical cancer, lung adenocarcinoma, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, neuroendocrine tumor, bladder cancer, Kidney cancer or stomach cancer.
12、 根据权利要求 9所述的应用， 其特征在于： 所述非受体酪氨酸激酶 c- Abl特异性抑制剂为 STI571、 Sunitinib、 PKC412、 vandetanib、 Sorafenib、 erlotinib、 gefitinib或 dasatinib。  The use according to claim 9, wherein the specific inhibitor of the non-receptor tyrosine kinase c-Abl is STI571, Sunitinib, PKC412, vandetanib, Sorafenib, erlotinib, gefitinib or dasatinib.
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