WO2012019432A1 - Reg4的用途及其药物组合物 - Google Patents
Reg4的用途及其药物组合物 Download PDFInfo
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- WO2012019432A1 WO2012019432A1 PCT/CN2011/001299 CN2011001299W WO2012019432A1 WO 2012019432 A1 WO2012019432 A1 WO 2012019432A1 CN 2011001299 W CN2011001299 W CN 2011001299W WO 2012019432 A1 WO2012019432 A1 WO 2012019432A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
Definitions
- the invention belongs to the field of biomedical technology, and particularly relates to the use of Reg4 and its pharmaceutical composition. Background
- Acute pancreatitis is one of the most common clinical diseases.
- the annual incidence rate in Western countries is about 30/100,000, and about 15-20% of patients suffer from severe acute pancreatitis (SAP).
- SAP severe acute pancreatitis
- Most patients with SAP have different levels of pancreatic endocrine and exocrine insufficiency, and very few develop chronic pancreatitis. Histologically, they show atrophy of acinus and interstitial fibrosis. Therefore, finding a drug that can protect acute pancreatitis, effectively reduce the mortality of SAP, and promote the recovery of pancreatic function is an urgent problem for clinicians.
- AP pathogenesis of AP has a variety of theories, including “trypsin self-digestion”, “leukocyte transitional activation”, “inflammatory factor cascade waterfall effect”, “pancreatic blood circulation disorder”, “intestinal bacterial translocation, endotoxin blood Symptoms and secondary infections, “apoptosis”, etc., eventually lead to systemic inflammatory response syndrome (SIRS) and even multiple organ failure (MOF).
- SIRS systemic inflammatory response syndrome
- MOF multiple organ failure
- AP is a common disease, especially SAP, and there is currently no ideal medical treatment [Frossard, J.L., MX. Steer, and CM. Pastor, Lancet, 2008. 371(9607): p. 143-52].
- the Regenerating gene (Reg ) family belongs to the calcium-dependent phytohemagglutinin superfamily member [Lasserre, C, et al, Eur J Biochem, 1994. 224(1): p. 29-38.; Chakraborty, C, Et al., Endocrinology, 1995. 136(5): p. 1843-9; Hartupee, JC, et al., Biochim Biophys Acta, 2001. 1518(3): p. 287-93].
- the Reg family is divided into four subtypes: Reg l, 2, 3, and 4, of which Reg2 is not expressed in humans.
- Reg4 is a new Reg family screened by Hartupee et al [Hartupee, JC, et a., family: Reg IV. Biochim Biophys Acta, 2001. 1518(3): p. 287-93] in a library of inflammatory bowel diseases. Members, expressed specifically in tissues, are mainly expressed in the gastrointestinal tract, and have low levels of expression in normal pancreatic tissues. Since the receptor for Reg4 has not yet been discovered, little is known about the biological function of Reg4 and its signaling pathways.
- REG4 is a special marker for pancreatic cancer, which can distinguish between pancreatic cancer patients and healthy controls by detecting serum REG4 (Takayama R, Nakagawa H, Sawaki A et al J Gastroenterol. 2010;45(l):52- 9 ).
- the technical problem to be solved by the present invention lies in the problem that the prior art means lacks effectiveness in the treatment of acute pancreatitis.
- the present invention discloses the use of a protein of (a) or (b) as follows in the preparation of a medicament for the treatment of acute pancreatitis:
- the acute pancreatitis is severe acute pancreatitis.
- the protein of (b) has the amino acid sequence set forth in SEQ ID NO.
- the present invention discloses a pharmaceutical composition
- a pharmaceutical composition comprising the following weight percentage components: 1% to 99% of the above protein as an active ingredient;
- a pharmaceutically acceptable carrier or excipient balance is provided.
- the formulation of the pharmaceutical composition is a parenteral formulation, which may be an injection or a sterile powder for injection.
- the invention discloses a method of treating acute pancreatitis by using the above protein as an active ingredient, the method comprising administering to the individual an effective amount of the above protein.
- the acute pancreatitis is severe acute pancreatitis.
- the individual is a mammal, preferably a human.
- the invention discloses a method for establishing an in vitro pancreatitis model, comprising the steps of:
- FIG. 1A Changes in serum amylase
- Figure 5A-D Semi-quantitative RT-PCR for detection of mRNA expression in inflammatory mediators of pancreatic tissue (* p ⁇ 0.05);
- Figure 8 Immunofluorescence identification of rat pancreatic acinar cells in primary culture amylase ( ⁇ 400 times);
- Figure 11 Hoechst33342/ PI double staining for necrosis and apoptosis of acinar cells ( ⁇ 400);
- FIG. 12 TUNEL detects apoptosis in acinar cells ( ⁇ 400 times);
- Figure 13A-B Reg4 inhibits arginine (Arg)-induced death of acinar cells
- Figure 14 Hoechst 33342 / PI double staining for necrosis and apoptosis of acinar cells. Planing method
- regenerating gene (Reg) family belongs to the calcium-dependent phytohemagglutinin superfamily. Currently, the Reg family is divided into four subtypes: Reg 1, 2, 3 and 4, where Reg2 is No expression in humans.
- Reg4 is a member of the new Reg family screened by Hartupee et al [Hartupee, JC, et al., Biochim Biophys Acta, 2001. 1518(3): p. 287-93] in a library of inflammatory bowel diseases. Specific expression, mainly expressed in the stomach In the intestine, there is a low level of table in normal pancreatic tissue. Reg4 protein promotes the proliferation and growth of several tumor cells, and is significantly elevated in colon cancer, gastric cancer, pancreatic cancer and prostate cancer, and inflammatory bowel disease.
- the human Reg4 gene (NM-032044.2) was 1285 bp and the CDS was 477 bp in length. The 66 bp of the CDS encodes a signal of 22 amino acids in length. The signal peptide is not contained in the secreted Reg4.
- the protein of the invention is a human Reg4 protein and a mutant thereof; a functionally active fragment thereof or an analog thereof; a homologue having a high degree of homology; and a protein encoding an amino acid sequence comprising SEQ ID NO:
- a vector such as a DNA vector (plasmid or virus).
- a functionally active fragment or analog can be formed by adding, inserting, modifying, substituting or deleting one or more amino acid residues in the amino acid sequences listed above.
- analog also includes chimeric proteins, fusion proteins, anti-idiotypic antibodies, precursors of the above compounds, and other functional equivalents or mimetics. Also included are synthetics that mimic the activity of Reg4 binding proteins.
- mutant refers to a mutant of the amino acid sequence of Reg4 as described in SEQ ID NO. Compared to the native Reg4 protein, this mutant has enhanced activity and/or altered stereospecificity compared to their wild type.
- the amino acid sequence mutant of the native protein can be prepared by introducing an appropriate nucleotide change into the nucleotide of the present invention, or by synthesizing the desired polypeptide in vitro. These mutants include, for example, deletions, insertions or substitutions of residues in the amino acid sequence.
- the final construct can be obtained by a combination of deletions, insertions and substitutions to provide the final protein product.
- GAP assay was tested on a region of at least 15 amino acids of the two sequences involved in the assay. More preferably, when the sequence being analyzed is at least 50 amino acids in length, the GAP assay is tested at a region of at least 50 amino acids of the two sequences involved in the assay. More preferably, when the sequence being analyzed is at least 100 amino acids in length, the GAP assay is tested at a region of at least 100 amino acids of the two sequences involved in the assay.
- the GAP assay is tested at a region of at least 250 amino acids of the two sequences involved in the assay. Even more preferably, when the sequence being analyzed is at least 500 amino acids in length, the GAP assay is tested in a region of at least 500 amino acids of the two sequences involved in the assay.
- aspects of the invention also include Reg4 protein analogs which are modified during or after their synthesis, for example, by biotinylation, benzylation, glycosylation, dormancy, phosphorylation, by known protection / Derivatization of blocking groups, cleavage by proteolysis, attachment to antibody molecules or other cellular ligands, etc. These modifications can be used to increase the stability and/or biological activity of the Reg4 protein of the present invention. Protein expression
- the present invention includes DNA encoding the Reg4 protein of the present invention, and vectors and transformants containing the DNA.
- the Reg4 protein can be prepared according to the Chinese patent application (Application No.: 200910056336.9) or according to the known human Reg4 gene (NM-032042) information, and can be prepared by a person skilled in the art without prior art modification by prior art.
- transformant is used to refer to a host cell with a heterologous DNA molecule.
- the invention also encompasses methods of producing the proteins of the invention by synthetic and recombinant techniques.
- Polynucleotides DNA or RNA
- vectors, transformants and organisms can be isolated and purified by methods known in the art.
- the vector used in the present invention may be, for example, a bacteriophage, a plasmid, a cosmid, a minichromosome, a virus or a retroviral vector.
- a vector useful for cloning and/or expressing a polynucleotide of the present invention is a vector capable of replicating and/or expressing a polynucleotide in a host cell in which a polynucleotide is to be replicated and/or expressed.
- polynucleotides and/or vectors can be used in any eukaryotic or prokaryotic cell, including mammalian cells (eg, human (eg, HeLa), monkey (eg, Cos), rabbit (eg, rabbit reticulocyte), rat, Hamsters (such as CHO, NSO, and baby hamster kidney cells) or mouse cells (such as L cells), plant cells, yeast cells, insect cells, or bacterial cells (such as E. coli). Suitable for appropriate carriers for multiple types of host cells For examples, see, for example, F. Ausubel et al "Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience (1992) and Sambrook et al. (1989). Host cells containing these polynucleotides can be used for large expression and can be used for Proteins such as drugs, diagnostics, vaccines and therapeutics.
- mammalian cells eg, human (eg, HeLa), monkey (eg, Cos), rabbit (eg,
- a variety of methods have been developed for operably linking polynucleotides to vectors via complementary cohesive ends. For example, a complementary homomeric sequence fragment can be added to the DNA segment to be inserted into the vector DNA. The vector and DNA segments are then joined by hydrogen bonding between the complementary homomeric tails to form a recombinant DNA molecule.
- a synthetic linker containing one or more restriction sites provides another means of joining the DNA segment to the vector.
- the DNA segment produced by endonuclease restriction digestion is treated with phage T4 DNA polymerase or E. coli DNA polymerase I, and the two polymerases are distinguished by their 3', 5'-nucleic acid exonuclease activity.
- the ⁇ -single-stranded end is used to fill the 3'-concave end with its polymerization activity. Thus, the combination of these activities produces a blunt-ended DNA segment.
- the blunt-ended segment is then incubated with a molar excess of the linker in the presence of an enzyme that catalyzes the ligation of the blunt-ended DNA molecule, such as phage ⁇ 4 DNA ligase.
- an enzyme that catalyzes the ligation of the blunt-ended DNA molecule such as phage ⁇ 4 DNA ligase.
- the reaction product is a DNA segment carrying a polymeric linker sequence at the end.
- These DNA segments are then cleaved with an appropriate restriction enzyme and ligated into an expression vector that has been cleaved with an enzyme that produces a terminus that is compatible with the DNA segment.
- Synthetic linkers containing multiple restriction endonuclease sites are available from a variety of merchants.
- the polynucleotide insert should be operably linked to a suitable promoter compatible with the host cell expressing the polynucleotide.
- the promoter may be a strong promoter and/or an inducible promoter. Examples of some of the promoters listed include the bacteriophage lambda PL promoter, E. coli lac, trP, phoA, tac promoter, SV40 early and late promoters, and the retroviral LTR promoter. Other suitable promoters are known to those skilled in the art.
- the expression recombinant vector further contains a transcription initiation and termination site, and contains a ribosome binding site for translation in the transcribed region.
- the coding portion of the transcript expressed by the recombinant vector can include a translation initiation codon at the start and a stop codon (UAA, UGA or UAG) suitably located at the end of the translated polypeptide.
- the expression vector can include at least one selectable marker.
- the marker includes dihydrofolate reductase, G418, glutamine synthase or neomycin resistance for eukaryotic cell culture; and tetracycline, kanamycin or for culture of E. coli and other bacteria Ampicillin resistance gene.
- suitable hosts include, but are not limited to, bacterial cells, such as E.
- yeast cells such as Saccharomyces cerevisiae or Pichia pastoris
- insect cells if flies S2 and Noctuida SF9 cells
- animal cells such as CHO, COS, NSO, 293 and Bowes melanoma cells
- plant cells Suitable media and culture conditions for the above host cells are known in the art.
- a tagged protein or a tag polypeptide which is convenient for isolation and purification.
- GST glutathione S-transferase
- His.Tag hexameric Histidine peptide
- protein A protein A
- cellulose binding domain a commonly used glutathione S-transferase
- the specific protein or polypeptide forms a fusion protein with the target protein, and the target protein can be isolated and purified by using the specific properties of the tagged protein or the tagged polypeptide.
- His.Tag specifically binds to the Ni-Chelating Sepharose column.
- the tagged protein or tag polypeptide can be purified by site-specific protease digestion to remove the fusion sequence, such as thrombin, enterokinase and factor Xa, to obtain the target protein.
- the invention also encompasses host cells comprising a nucleotide sequence of the invention operably linked to one or more heterologous control regions (such as promoters and/or enhancers) by techniques known in the art. Connected.
- a host strain capable of modulating the expression of the inserted gene sequence or modifying and processing the gene product in a specific manner as desired can be selected. In the presence of certain inducers, expression of certain promoters is increased; therefore, expression of the genetically engineered polypeptide can be controlled.
- different host cells have characteristic and specific mechanisms for translation, post-translational processing, and modification (eg, phosphorylation, cleavage) of proteins. Appropriate cell lines can be selected to ensure desirable modification and processing of the expressed foreign protein.
- the nucleic acid and nucleic acid recombinant vector of the present invention can be introduced into a host by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other methods. cell.
- the method is described in a number of standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986).
- a polynucleotide encoding the protein of the invention can be ligated to a vector containing a selectable marker for propagation in a host.
- a plasmid vector can be introduced into a precipitate, such as a calcium phosphate precipitate or a complex thereof with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into the host cell.
- Successfully transformed cells i.e., cells containing the DNA recombinant vector of the present invention
- cells obtained by expression of a recombinant vector can be cultured to produce a desired polypeptide.
- the cells are collected and lysed, and the presence of DNA in the DNA content is detected by the method described in Southern (1975) J. Mol. Biol. 95, 503 or Berent et al (1985) Biotech. 3, 208.
- antibodies are used to detect the presence of proteins in the supernatant.
- the protein of the invention can be purified using one or more of the chromatographic methods described above.
- the fusion protein of the invention may be purified using one or more of the following columns: Q sepharose FF column, SP sepharose FF column, Q sepharose High Performance column , Blue sepharose FF column, Blue column, Phenyl Sepharose FF column, DEAE Sepharose FF, Ni-Chelating Sepharose FF column or Methyl column.
- proteins of the invention can be purified using the methods described in International Publication No. WO 00/44772, which is incorporated herein by reference in its entirety.
- One of skill in the art can readily modify the methods described therein for purification of the proteins of the invention.
- the proteins of the invention can be recovered from products produced by recombinant techniques, including prokaryotic or eukaryotic hosts of, for example, bacteria, yeast, higher plant, insect and mammalian cells.
- a pharmaceutical composition for use in or containing a protein of the invention may be mixed with one or more pharmaceutically acceptable carriers or excipients to form pharmaceutical dosage forms of different administration routes, such as tablets and capsules. , powders, granules, syrups, solutions, oral liquids, elixirs, tinctures, aerosols, powders, injections, sterile powders for injection, suppositories, etc.
- a “pharmaceutically acceptable” ingredient is one which is suitable for use in humans and/or animals without excessive adverse side effects (e.g., toxicity, irritation, and allergies), i.e., having a reasonable benefit/risk ratio.
- a “pharmaceutically acceptable carrier” is a pharmaceutically or food acceptable solvent, suspending agent or excipient for delivering a fusion protein of the invention to an animal or human.
- the carrier can be a liquid or a solid.
- the proteins of the invention may be administered orally, intravenously, intramuscularly or subcutaneously.
- the dosage forms which can be orally administered in the above dosage forms are: tablets, capsules, powders, granules, syrups, solutions, elixirs.
- Solid carriers include: starch, lactose, calcium hydrogen phosphate, microcrystalline cellulose, sucrose, kaolin, micronized silica gel, talc, low-substituted hydroxypropylcellulose, sodium carboxymethyl starch, polyvinylpyrrolidone.
- Liquid carriers include: sterile water, ethanol, polyethylene glycol, nonionic surfactants, and edible oils such as corn oil, peanut oil, and sesame oil.
- Adjuvants commonly used in the preparation of pharmaceutical compositions include: flavoring agents, coloring agents, preservatives (such as hydroxybenzidine, sodium benzoate, sorbic acid) and antioxidants (such as vitamin E, vitamin C, coke) Sodium sulfite and dibutylhydroxytoluene).
- the dosage forms of the above dosage forms which can be administered by the injection route include: injections, sterile powder for injection, which are in the form of a mixture of the drug and one or more pharmaceutically acceptable excipients for administration by injection.
- Solvents include: sterile water, Ethanol, glycerin, propylene glycol, polyethylene glycol.
- bacteriostatic agents such as benzyl alcohol, hydroxybutyrate, thimerosal
- isotonic regulators such as sodium chloride, glucose
- suspending agents such as sodium carboxymethyl cellulose, methyl cellulose).
- solubilizers Teween-80, lecithin
- antioxidants such as vitamin E, vitamin C, sodium metabisulfite
- fillers such as lactose, mannitol).
- compositions of the present invention can be prepared according to well known and recognized methods of pharmaceutical production requirements.
- the pharmaceutical composition suitably comprises the protein of the invention together with a pharmaceutically acceptable carrier, and is suitably in unit dosage form.
- the pharmaceutical compositions of the present invention may comprise a protein of the invention in the form of a prodrug which is metabolically converted in the recipient host to the active form of the invention.
- compositions of the invention may also be used in combination with other therapies, such as simultaneous, sequential or separate application.
- the pharmaceutical composition of the present invention may contain other active agents.
- the protein of the present invention is effective for treating acute pancreatitis and may provide a new choice for the treatment of clinical acute pancreatitis in the future.
- a method of preventing or treating an acute pancreatitis reaction by using the protein as an active ingredient comprises administering to a patient an effective amount of the protein of (a) or (b) above.
- Effective dose refers to an amount sufficient to produce a therapeutic effect.
- An effective amount can be administered in one or more divided doses. Generally, an effective amount is sufficient to alleviate, improve, stabilize, slow or delay further progression of the disease.
- the effective dose of the active ingredient employed will vary with the mode of administration and the severity of the condition being treated. For most large mammals, the total daily dose of active ingredient is about 0.01-1000 mg. Generally, the clinical dose for adults ranges from 0.01 to 200 mg/H, preferably from 0.05 to 100 mg/ ⁇ .
- the invention will be further elucidated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually in accordance with conventional conditions or according to the conditions recommended by the manufacturer.
- the Reg4 protein can be prepared according to the Chinese patent application (Application No.: 200910056336.9) or according to the known human Reg4 gene (NM-032042) information, and can be prepared by a person skilled in the art without prior art modification by prior art.
- Example 1 Re g 4 protein attenuates the severity of pancreatitis in vivo
- mice (SPF grade, 25-30g, 8w-10w) were purchased from Shanghai Slack Laboratory Animal Co., Ltd. All mice were purchased in the animal house at 23 °C for one week after purchase. Free diet and drinking before the experiment. .
- DANase I TAKARA; Protease K: TAKARA; MPO Activity High Quality Sensitive Colorimetric Quantification Kit: GENMED; In Situ Cell Death Detection Kit POD: Roche; Mouse IL-1/IL-1F2 Immunoassay: R&D the company
- the level of serum amylase and lipase was measured by an automatic biochemical analyzer.
- MPO myeloperoxidase
- Pancreatic tissue MPO activity is detected by the MPO activity high-quality sensitive colorimetric quantitative detection kit (GENMED), and the specific procedure is carried out according to the instructions.
- GENEMED MPO activity high-quality sensitive colorimetric quantitative detection kit
- pancreatic tissue was fixed in 4% paraformaldehyde, routinely dehydrated, embedded, sectioned, stained, and sectioned.
- Semi-quantitative integration of pancreatic tissue reference [Toma ⁇ H., et al., Gastroenterology, 2000. 119(5): p. 1373-81], see Table 1 for details.
- Serum IL- ⁇ levels were obtained using the Mouse IL-1/IL-1F2 Immunoassay (R&D) kit. The specific procedures were followed according to the instructions.
- Apoptosis-positive cells were quantified: 10 fields were selected at 200-fold for each section, and apoptotic positive cells (nuclear showing green fluorescent cells) in each field were counted, and the average value was positive cells/200-fold field of view.
- the measurement data were expressed by mean SEM.
- the SAS 8.0 software package was used for statistical analysis.
- the survival curve was compared with log-rank test.
- the two groups were compared with Mann-Whitney non-parametric U test, with P ⁇ 0.05. The difference was statistically significant.
- Serum amylase activity in arginine-induced acute pancreatitis group was 5399 ⁇ 876, 13168 ⁇ 2604, 3473 ⁇ 286 ( U/ml) on days 2, 3, and 4, respectively;
- Reg4 treatment group was 44101 ⁇ 479, 8990 ⁇ 1526, 3502 ⁇ 317 (U/ml), respectively, which was significantly lower (? ⁇ 0.05) on days 2 and 3 than in the NS group, as shown in Figure 2A.
- Serum lipase activity in the NS group was 528 ⁇ 120, 1663 ⁇ 341, and 244 ⁇ 52 ( U/ml) on days 2, 3, and 4, respectively; 238 ⁇ 78, 758 ⁇ 96, and 181 ⁇ 48 in the Reg4 treatment group, respectively.
- U/ml was significantly lower (? ⁇ 0.01) than the NS group on days 2 and 3, as shown in Figure 2B.
- pancreatic interstitial edema pancreatic interstitial edema, acinar cell vacuolation, focal and flaky necrosis, and infiltration of a large number of inflammatory cells were observed in the NS group, while the Reg treatment group (Reg4 group) acinar cells. Necrosis and inflammatory cell infiltration were significantly alleviated, especially on days 3 and 4.
- the severity of pancreatitis was determined by pancreatic histopathological scoring.
- the pathological score of the Reg4 treatment group was significantly lower than that of the NS group, which was 6.8 ⁇ 1.5, 9.9 ⁇ 1.8, 8.5 ⁇ 1.9 to 5.4 ⁇ 1.4, 7.4 on days 2, 3, and 4, respectively. ⁇ 1.9, 5.5 ⁇ 1.0 ( p ⁇ 0.05 )»
- Reg4 protein reduces arginine-induced MPO activity in mouse pancreatic tissue
- MPO is present in the azurophilic granules of polynuclear leukocytes, especially in neutrophils and monocytes, and therefore, MPO activity can reflect the degree of infiltration of neutrophils in tissues.
- the activity of MPO in pancreatic tissue of NS group was 8.4 ⁇ 1.7, 63.3 ⁇ 7.6, 25.4 ⁇ 3.0 (activity unit / g protein) on days 2, 3, and 4, respectively, and 7.0 ⁇ 0.9 in Reg treatment group (Reg4 group). 40.4 ⁇ 4.0, 17.6 ⁇ 2.3 (activity unit / g protein), significantly lower than the NS group on days 3 and 4 (p ⁇ 0.05), as shown in Figure 4.
- the degree of death of acinar cells is directly proportional to the severity of acute pancreatitis, which includes both necrosis and apoptosis.
- acinar cell necrosis was significantly reduced in the Reg4 treatment group compared with the NS group.
- arginine-induced acute pancreatitis not only showed necrosis of acinar cells, but also showed apoptosis of acinar cells by TUNEL.
- the Reg4 treatment group (Reg4 group) was slightly reduced compared with the NS group, especially the third. 4 days is even worse, as shown in Figure 7A/B. This study found that the application of Reg4 protein significantly reduced arginine-induced mortality in acute pancreatitis in mice.
- urea Urea
- ALT alanine aminotransferase
- AST aspartate aminotransferase
- necrosis/apoptosis ratio is positively correlated with the severity of experimental pancreatitis [Gukovskaya, AS and S ⁇ Pandol, Pancreatology, 2004. 4(6): p. 567-86], inducing apoptosis in acinar cells to reduce pancreas Inflammation of inflammation.
- apoptotic cells are swallowed by surrounding macrophages and do not produce an inflammatory response.
- Miwa et al. [Miwa, K., et al., Nat Med, 1998. 4(11): p. 1287-92] found that the implantation of tumor cells expressing Fas ligand into wild mice can cause a large number of Leukocyte infiltration, but no leukocyte infiltration in IL- ⁇ / ⁇ knockout mice, suggesting that apoptosis can also induce inflammatory response, thus challenging the traditional view that apoptosis does not produce an inflammatory response.
- acinar cell apoptosis plays an important role in the atrophy of chronic pancreatitis acinar cells [Bateman, AC, et al., Gut, 2002. 50(4): p. 542-8]schreib
- acinar cell death including necrosis and apoptosis Two forms, but mainly necrosis.
- Reg4 not only inhibits arginine-induced necrosis of acinar cells, but also partially inhibits apoptosis of acinar cells, but it is not significant.
- necrosis includes accidental And programmed necrosis, and there may be a common signaling pathway between programmed necrosis and apoptosis [Proskuryakov, SY, AG Konoplyannikov, and VL Gabai, Exp Cell Res, 2003. 283(1): p. 1-16; Edinger, AL and CB Thompson, Curr Opin Cell Biol, 2004. 16(6): p. 663-9].
- FasL and TNFa not only induce cell apoptosis, but also induce cell necrosis when apoptosis is inhibited.
- Reg4 protein inhibits necrosis and inflammation of pancreatic acinar cells, thereby reducing the mortality and severity of experimental pancreatitis.
- Example 2 In vitro study of Reg4 inhibiting pancreatic acinar cell death
- Penicillin/streptomycin Gbico; DMEM/Ham F-12 medium: Gbico; fetal bovine serum: Gbico; bovine serum albumin: Sigma; trypsin/EDTA: Gbico; 200 mesh stainless steel cell screen : BD; type I collagenase: Sigma; type IV collagenase: Sigma; trypsin inhibitor: Ameresco; aprotinin: Ameresco; Cell Counting Kit-8 kit: Dojindo, Japan; : Invitrogen; PI: Sigma; Hoechest33342: Biyuntian; RIPA Lysis: Biyuntian; RevertAid Premium First Strand cDNA Synthesis Kit: Fermentas; PrimeScriptTM RT reagent Kit: TAKARA; PVDF Membrane: Millipore; Mouse ⁇ -actin monoclonal antibody: Santa cruz; mouse-derived amylase monoclonal antibody: Santa cruz; rabbit-derived Bcl-2 monoclonal antibody: Cell signaling; rabbit-
- Isolation and culture of acinar cells The collagenase digestion method is used, and is appropriately modified.
- Cell smear HE Prepare the cell suspension and dilute it appropriately. Apply the smear to the slide with a sputum machine. Fix 4% paraformaldehyde for 20 min at room temperature, and routinely stain with HE. The specific steps are the same as before.
- Trypan blue exclusion test (Trypan blue): Assessment of necrosis of acinar cells.
- a single cell suspension was prepared and appropriately diluted (10 6 /ml).
- the culture supernatant was collected.
- the cells were lysed with lysate and the lysate was collected.
- the LDH content in the supernatant and cell lysate was determined by conventional biochemical colorimetry.
- LDH release rate (%) supernatant LDH / (cell lysate LDH + supernatant LDH) ⁇ 100%
- CCK-8 solution ⁇ ⁇ /well was added and incubated at 37 ° C for 1 h to determine the OD450nm value.
- blank wells medium, CCK-8 solution
- control wells cells, drug dissolution medium, culture solution, CCK-8 solution
- 6 replicate wells were set for each group, and the results were averaged.
- Both PI and Hoechst 33342 bind to nuclear DNA (or RNA).
- PI cannot pass through the normal cell membrane, and Hoechst is a membrane-permeable fluorescent dye. Therefore, when the cell is necrotic or late, the cell membrane is destroyed, and the PI is red.
- Both normal cells and early-stage apoptotic cells can be stained by Hoechst, but the Hoechst staining of normal nuclei is circular, pale blue, with deep blue particles; and the nucleus of apoptotic cells is concentrated. Bright blue, or nuclear lobed, fragmented, edge set.
- the PI is colored as necrotic cells; the bright blue color, or the nucleus is lobulated, and the Hoechst staining of the edge set is apoptotic cells. Excited by ultraviolet light, Hoechst/PI double staining can be seen under fluorescence microscopy:
- Late apoptotic cells Also stained red, but significant chromatin condensation was observed.
- Non-apoptotic dead cells stained red, nuclear normal structure
- the cells were collected and made into a cell suspension. After the cells were sputum, the acetone was fixed for 5 mins, and the subsequent steps were the same as those in the third chapter.
- Measurement data were expressed by mean SEM, statistical analysis was performed using SAS 8.0 software package, Mann-Whitney non-parametric U test was used for comparison between the two groups, and one-way ANOVA was used for comparison between groups, with P ⁇ 0.05 as the difference. It is statistically significant.
- pancreatic acinar cells Under the phase contrast microscope, the pancreatic acinar cells showed non-adherent growth and clustered into clusters. The cell boundaries were clear and the refractive index was strong. The cells were densely enriched with zymogen. The expression of amylase in primary cultured cells was detected by immunofluorescence assay. The results showed that almost all cultured cells expressed amylase, which was determined to be pancreatic acinar cells, as shown in Fig. 8.
- the activity of freshly isolated rat pancreatic acinar cells was higher than that of trypan blue staining, which was more than 95%. With the prolongation of in vitro culture, the cell viability decreased to some extent, and at 24 o'clock, the cell viability was above 80%. After cultured with pancreatic acinar cells and different concentrations of arginine, cell viability decreased significantly in a time-dose-dependent manner. At 24 h in the 10 mg/ml arginine group, the cell viability was less than 15%, as shown in Table 3. Show.
- the lactate dehydrogenase release rate test showed that the release rate of lactate dehydrogenase was significantly increased after 6-10 mg/ml of arginine and acinar cells were treated for 6 h. Time and dose dependence. Further CCK-8 assay cell viability results showed that acinar cell survival rate decreased in a time- and dose-dependent manner after 6 h, 12 h and 24 h of different concentrations of arginine, as shown in Figure 10.
- Reg4 protein inhibits arginine-induced death of acinar cells
- Figure 13 (A: lactate dehydrogenase release rate to detect necrosis of acinar cells; B: CCK-8 to detect acinar cell survival rate, compared with the control group, *P ⁇ 0.05, **P ⁇ 0.01), and Arginine group (Arg+0 ⁇ rReg4) compared to Reg4 group
- Lactate dehydrogenase leakage rate was significantly reduced at each concentration (4 g/ml, 8 ⁇ ⁇ 0.01), as shown in Fig. 13A.
- the cell viability of CCK-8 assay showed that the concentration of 4-4 ⁇ / ⁇ Reg4 significantly increased the survival rate of acinar cells.
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MOTOO Y ET AL.: "Arginine induces apoptosis and gene expression of pancreatitis-associated protein in rat pancreatic acinar AR4-2J cells", PANCREAS, vol. 20, no. 1, 2000, pages 61 - 66 * |
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