CN109870583A - The relevant metabolin of acute pancreatitis and its application - Google Patents
The relevant metabolin of acute pancreatitis and its application Download PDFInfo
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- CN109870583A CN109870583A CN201910298887.XA CN201910298887A CN109870583A CN 109870583 A CN109870583 A CN 109870583A CN 201910298887 A CN201910298887 A CN 201910298887A CN 109870583 A CN109870583 A CN 109870583A
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- acute pancreatitis
- metabolin
- cinanserin
- hydrochloric acid
- cefazaflur
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Abstract
The present invention provides the relevant metabolin of acute pancreatitis and its applications, the metabolin is hydrochloric acid cinanserin or Cefazaflur, and the present invention is used for acute pancreatitis subject for diagnosis, the prediction of acute pancreatitis using metabolin hydrochloric acid cinanserin or Cefazaflur and reacts the response of therapeutic agent.Invention also provides the pharmaceutical composition for the treatment of pancreatitis, described pharmaceutical composition can cause the variation of metabolite level.
Description
Technical field
The invention belongs to field of biotechnology, it is related to metabolin relevant to acute pancreatitis and its application.
Background technique
Acute pancreatitis (Acute pancreatitis, AP) is to cause one of common disease of acute abdominalgia, it be by
Different types of etiopathogenises causes the pancreatin in pancreas to be activated, and causes pancreatic tissue autodigestion, oedema, bleeding, and inflammation locally occurs for pancreas
Disease reaction is the disease of main feature, and comprehensive sign (the Systemic inflammatory of systemic inflammatory response can occur for serious patient
Response syndrome, SIRS), and with organ dysfunction (Organ dsfunction, OD).Acute pancreatitis
Clinical manifestation is varied, and specificity is low, is lower than 40% (Steinberg for the prediction sensibility of poor prognosis
WM.Predictors of severity of acute pancreatitis[J].Gastroenterol C1inN Am,
1990,19 (4): 849-61.), and the pathological basis of AP generation systemic inflammatory response syndrome is not yet clear.Due to the AP cause of disease
Multiplicity, pathogenesis are complicated, the death rate is higher, and the overall case fatality rate of acute pancreatitis is up to 5%~10%, severe acute pancreatitis
(Severe acute pancreatitis, SAP) patient's case fatality rate be up to 36%~50% (Lankisch PG, Apte M,
Banks PA.Acute pancreatitis [J] .Lancet, 2015,386 (9988): 85-96), therefore judge as early as possible acute
The severity of Pancreatitis Patients identifies change of illness state, especially Severe Acute Pancreatitis SAP (Severe acute
Pancreatitis, SAP) patient, SAP patient is monitored closely in time and active treatment, to improve survival and
It is most important to mitigate economic pressures etc..Clinically there is the multiple points-scoring system of multiple assessment AP severity, such as APACHE
II, Ransom BISAP etc., but consuming time is long for these evaluation systems, variable is more, is difficult to apply to other than severe department
Clinical department (Pavlidis TE, Pavlidis ET, Sakantamis AK.Advances in prognostic
factors in acute pancreatitis:a mini-review[J].Hepatobiliary Pancreat Dis
Int.2010,9 (5): 482-6.), it is unfavorable for disease early diagnosis and treatment.
Metabolism group is after genomics, transcription group and proteomics, and the important composition of systems biology is led
Domain (Coodacre R.Marking sence of the metabolome using evolutionary computation:
seeing the wood with trees[J].J Exp Bot 2005,56(410):245-254.).It refers to nuclear-magnetism
The system researches means such as resonance, efficient liquid phase spectrum, gas-chromatography, mass spectrum, the stimulation of analysis Pathological Physiology and genetic modification cause
Biology body fluid, endogenous metabolites spectrum in tissue variation to study whole biological condition and gene function tune
Section.Metabolism group is dynamic, multi-parameter response a new technology and method, reflection be gene, environment, cause a disease because
The variant factors such as element, nutrition, drug, time act on total reaction after body, are to determine health, disease and treatment effect
The suitable elements collection of fruit (Holmes E, Tang H R, Wang Y L, et al.The assessment of plant
metabolite profiles by NMR-based methodologies[J].Plant Med,2006,72(9):771-
785.).It skips the intracorporal complicated regulation process of life, by the analysis to metabolin provide it is final, whole as a result,
Health evaluating, medical diagnosis on disease, therapeutic evaluation, drug development etc. have big advantage, can be not only used for some diseases
Diagnosis, discloses its pathologic process, and can indicate lapsing to for part disease, while in personalized treatment, health monitoring, disease
The various aspects such as diagnosis can provide technology platform (the Schnackenbrg LK, Beger for finding relevant biomarkers object
RD.Monitoring the health to disease continuum with global metabolic profiling
and systems biology[J].Pharmacogenomics,2006,7(7):1077.).Phase is studied with traditional metabolism
Than metabolism group detects the variation of the entire metabolite spectrum of body by the instrument analysis technology of modern chemistry, and by polynary
The whole biological function situation of statistical analysis technique research.Metabolism group research is occurred with disease and drug therapy is mutually tied
Close, the rule that changes over time of quantization biological metabolism, come recognize and parse the physiology of research object, pathological state and environment because
Son, the relationship of gene composition etc., and the biomarker for being possible to associated is found out, health status and disease are held to reach
The effect of sick remedy measures.
Summary of the invention
It is an object of the invention to find metabolite markers relevant to acute pancreatitis and its treatment response, pass through inspection
Survey the content of the metabolite markers, it can be determined that whether patients with acute pancreatitis suffers from acute pancreatitis and control drug
The response for the treatment of, so that the personalized treatment for patient provides guidance.
The first aspect of the present invention provides a kind of reagent, and the reagent can detecte metabolin hydrochloric acid cinanserin
The level of (Cinanserin hydrochloride) or Cefazaflur (Cefazaflur).
Further, the reagent includes the examination by chromatography, spectrum or mass spectrograph/spectroscopic methodology detection metabolin content
Agent.
Further, the chromatography is detected as the detection of ultra high efficiency liquid chromatography.
The second aspect of the present invention provides a kind of product of vitro detection metabolin, and the product includes the present invention first
Reagent described in aspect.
The third aspect of the present invention provides a kind of method of the drug candidate of screening treatment acute pancreatitis, including step
It is rapid:
The cultivating system containing hydrochloric acid cinanserin or Cefazaflur is added in substance to be screened, and
The content of hydrochloric acid cinanserin or Cefazaflur in the system is detected,
If substance to be screened can increase the content of hydrochloric acid cinanserin, Cefazaflur, show that the substance to be screened is to control
Treat the drug candidate of acute pancreatitis.
The fourth aspect of the present invention provides a kind of pharmaceutical composition, described pharmaceutical composition include hydrochloric acid cinanserin or
The promotor of Cefazaflur.
Further, the promotor is the reagent for increasing hydrochloric acid cinanserin or Cefazaflur.
The fifth aspect of the present invention provides following described in any item applications:
A. application of the reagent described in first aspect present invention in the product of preparation diagnosis acute pancreatitis；
B. reagent described in first aspect present invention is in the response product that preparation judges therapeutic agent treatment acute pancreatitis
Application
C. application of the product described in second aspect of the present invention in the tool of preparation diagnosis acute pancreatitis；
D. product described in second aspect of the present invention is in the response tool that preparation judges therapeutic agent treatment acute pancreatitis
Application；
E. application of the pharmaceutical composition described in fourth aspect present invention in the tool of preparation treatment acute pancreatitis；
F. the application of hydrochloric acid cinanserin, Cefazaflur in the computation model of building prediction acute pancreatitis；
G. the calculating mould that hydrochloric acid cinanserin, Cefazaflur are responded in the treatment of building prediction drug therapy acute pancreatitis
Application in type.
The advantages of the present invention:
Present invention firstly discovers that relevant to acute pancreatitis and medication effect metabolin, passes through detection metabolism
The content of object, it can be determined that it is anti-to the response of drug therapy whether subject suffers from acute pancreatitis and patients with acute pancreatitis
It answers, is conducive to the personalized treatment of patients with acute pancreatitis.
Detailed description of the invention
Fig. 1 is mice serum amylase content figure.
Fig. 2 is mice pancreatic organization chart.
Fig. 3 is mice pancreatic histo pathological change figure.
Fig. 4 is QC sample negative ions (ES+/-) Mode B PI and TIC figure, wherein figure A is cation (ES+) Mode B PI
Figure, figure B are cation (ES+) mode TIC figures, and figure C is anion (ES-) Mode B PI figure, and figure D is anion (ES-) mode
TIC figure.
Fig. 5 is OPLS-DA analysis chart.
Specific embodiment
In order to assess metabolin can as the marker of acute pancreatitis, the present invention is based on mass chromatography joint technology,
The metabolism group for the sample for using drug therapy acute pancreatitis subject is studied, is found normally and disease, Yi Jiji
Significant differences metabolism list is established in the variation of the pretherapy and post-treatment metabolin of disease, study its characterized it is related to acute pancreatitis
Biological information, and therefrom further screening key metabolites biomarker, thus make by it is a kind of it is improved in a manner of and
The early stage screening of disease and diagnosis acute pancreatitis are possibly realized, and are allowed more acurrate and reliably predicted with acute
Whether the patient of pancreatitis may have reaction to medicinal treatment.
Two or more biological aspects may be distinguished from each other generally, due to biomarker, therefore it is that one kind has
The tool of value, as normal biological processes, the indicant of pathogenic course or as the reaction of pharmaceutical intervention.Metabolin is low point
Sub- compound (< 1kDa) is less than most protein, DNA and other macromoleculars.The small variation of protein active causes biochemical anti-
It answers and its great variety of metabolin (=metabolism biological marker is conceived to the metabolism of body), concentration, flow and conveyance
It makes sensitive to disease and pharmaceutical intervention.This may obtain reflection science of heredity and environmental factor (such as: nutrition, body movement, intestines are micro-
Biology and drug) physiologically substance and pathologic, physiologic substance individual overview.Therefore, be such as used as biomarker rather than generation
The protein or hormone for thanking to biomarker compare, and metabolism biological marker provides more fully information.
The present invention is sequenced by metabolism group and bioinformatic analysis, has found hydrochloric acid cinanserin or cephalo for the first time
Significant difference is presented between different groups in nitrogen fluorine.Hydrochloric acid cinanserin (Cinanserin hydrochloride), chemical combination
Object ID is CSID4938327, molecular formula C20H24ClN2OS, content dramatically increases in acute pancreatitis, uses drug therapy
Afterwards, the horizontal of the metabolin significantly reduces, and there was no significant difference with normal control；Cefazaflur (Cefazaflur) compound ID
For CSID36777, molecular formula C13H13F3N6O4S3, content dramatically increases in acute pancreatitis, should after drug therapy
The horizontal of metabolin significantly reduces, and there was no significant difference with normal control.
Terms used herein " metabolin biomarker " or short " biomarker " are defined as being suitable as urgency
Property pancreatitis exist and the reference compound of state, this compound are the metabolism occurred in metabolic process in the mammalian body
Object or metabolic compounds.The present invention provides by comparing metabolite level detected and metabolin reference levels, provide pair
Whether patient suffers from the diagnosis of acute pancreatitis, or judges subject patient to the response condition for the treatment of acute pancreatitis agent.
" reference levels " of term metabolin mean to indicate disease specific state, its phenotype or shortage and morbid state
Combination, its phenotype or shortage metabolin level.In one embodiment, the disease reference levels of metabolin mean
The level of the metabolin of the positive diagnosis of acute pancreatitis can be indicated in patient.In another embodiment, metabolin is " strong
Health reference levels " mean the level that can indicate the metabolin of the positive diagnosis of health status in patients.
As a kind of enforceable scheme, " reference levels " of metabolin can be the exhausted of one or more metabolins below
Pair or relative quantity or concentration；The presence or shortage of metabolin；The amount of metabolin or the range of concentration；The minimum of metabolin and/or
Maximum or concentration；The average magnitude or concentration of metabolin；And/or the median amount or concentration of metabolin.Alternatively may be used
The scheme of implementation, " reference levels " for the combination of metabolin can also be the exhausted of two or more related mutual metabolins
Pair or relative quantity or concentration ratio.It can be by the level of detection required metabolin in one or more patients appropriate, really
The positive and negative reference of the fixed appropriate metabolin for particular disease states, its phenotype or shortage are horizontal, and such reference
Level can be made it suitable for special PATIENT POPULATION by modification, and (e.g., reference levels can match for the age, in order to derive from
Metabolite level in the Patient Sample A at some age and disease specific state, its phenotype or the shortage in some age group
It is compared between reference levels).Alternatively implementable solution, it is special that reference levels can be made it suitable for by modification
Technology, the technology can be used for detecting the metabolite level (such as LC-MS, GC-MS) in biological sample, there metabolin
Level can be different based on special technique used.
One aspect of the present invention is related to the diagnosis of acute pancreatitis development.In one embodiment, can disease into
The clinical sign of exhibition makes diagnosis before occurring.In one embodiment, the present invention provides diagnosis subject in patients and suffers from
The method of acute pancreatitis, wherein collecting the level of one or more metabolins in sample and test sample from patient.Based on institute
The level of the metabolin of detection, judges whether patient suffers from acute pancreatitis.It is detected in such embodiment
Metabolin includes hydrochloric acid cinanserin, Cefazaflur.The present invention can be used method well known in the art and detect, including not
It is limited to such as manual comparison method of simple comparison method, statistical analysis method such as t- is examined, Welch ' s T- inspection, the inspection of Wilcoxon ' s sum of ranks
It tests, random forests algorithm.
Response the present invention also relates to judgement or prediction patient to the therapeutic agent for using acute pancreatitis, as used in this article
, " response " means to show the patient of following situations: the decline of metabolite level associated with acute pancreatitis；With acute pancreas
The associated metabolite level of adenositis increases；The decline of metabolite level associated with health status；With the shape with health
The associated metabolite level of condition increases.In specific embodiments of the present invention, response does not refer to using after therapeutic agent, and not
The patients with acute pancreatitis for receiving treatment is compared, and hydrochloric acid cinanserin or the horizontal of Cefazaflur increase；Or it is tested with health
Person compares, and there was no significant difference for hydrochloric acid cinanserin or Cefazaflur.
In the present invention, any appropriate method known in the art for such metabolin can be used to analyze life
Specific metabolite in object sample.It the use of quantitative analysis method include but is not limited to chromatography, spectroscopic methodology and mass spectrography.Typically
Mass spectrograph by the source of the molecular ionization in sample and is used to detect the detector of ionized molecule or molecule fragment and formed by it.
The non-limiting example in common source includes electron bombardment, electronic spraying ionization (ESI), atmospheric pressure chemical ionization (APCI), big
Air pressure photo-ionisation (APPI), Matrix Assisted Laser Desorption ionize (MALDI), surface-enhanced laser desorption ionization (SELDI), and its
Development.Common mass spectrum separation and detection system may include level Four, level Four ion trap, linear ion hydrazine, flight time
(TOF), sectorial magnetic field, ion cyclotron (FTMS), orbit ion trap, and its development and combination.FTMS is better than other bases
It is its high resolution in the advantage of the platform of MS, allows to separate difference only in the metabolin of hundreds of dalton, many such generals
It is missed by the instrument of lower resolution.
Chromatography may include GC, LC, HPLC and UHPLC；Spectrum may include UV/Vis, IR and NMR；Mass spectrometer/light
Spectrum may include ESI-QqQ, ESI-QqTOF, MALDI-QqQ, MALDI-QqTOF and MALDI-TOF-TOF.It is highly preferred that matter
Contents analyzer/spectrum analysis includes quadrupole rod mass analyzer, ion trap mass spectrometry device, TOF (flight time) quality analysis
Device, orbitrap mass analyser, magnetic sector mass analyzer, electrostatic field sector mass analyzer (Electrostatic
Sector Mass Analyzer), ion cyclotron resonance (ICR) and the combination of mass analyzer (including single quadrupole rod (Q) and
Triple quadrupole bar (QqQ), QqTOF, TOF-TOF, Q orbit trap).Preferably use FLA- and HPLC- tandem mass spectrometry.
Wherein, GC=gas-chromatography, CE=Capillary Electrophoresis, LC=liquid chromatogram, HPLC=height liquid chromatogram,
UHPLC=ultra performance liquid chromatography, UV-Vis=UV, visible light, IR=is infrared, NIR=near-infrared, NMR=nuclear magnetic resonance, ESI
The ionization of=electronic spraying, MALDI=is substance assistant laser desorpted/ionization, the TOF=flight time, APCI=atmospheric pressure chemical electricity
From ((Q1 and Q3 quadrupole rod is mass filter to also referred to as Qlq2Q3 to the configuration of QqQ=triple quadrupole bar, and q2 is that massless differentiates four
Pole bar (no mass-resolving quadrupole))).
In the present invention, at least one metabolin can also be measured by specified chemical or biological test.Institute
Test is stated by the tool comprising allowing at least one metabolin described in specific detection sample.Preferably, the tool can
The chemical structure of specific recognition metabolin, or the ability reacted with other compounds based on it or its in biological read out system
The ability specificity identification metabolin of middle evoked response (such as induction of reporter).It is capable of the change of specific recognition metabolin
Learn structure tool be preferably antibody, or specifically with chemical structure such as other of receptor or enzyme interacting protein.Such as
Metabolin can be used as antigen, specific antibody is obtained by method known in the art.The antibody that such place refers to includes
Polyclonal antibody and monoclonal antibody and its segment, such as can be in conjunction with 2 segment of Fv, Fab and F (ab) of antigen or hapten.This
Invention also includes the hybrid antibody of humanization, wherein showing the amino acid sequence of the non-human donor antibody of desired antigentic specificity
With the combined sequence of human receptor antibody.In addition, also included is single-chain antibody.Donor sequences typically at least include the antigen of donor
It in conjunction with amino acid residue, but also may include the other structures and/or the relevant amino acid residue of function of donor antibody.Such heterozygosis
Body can be prepared by several method known in the art.The suitable protein for capableing of specific recognition metabolin is preferred
For enzyme, the metabolic conversion of the metabolin is participated in.The enzyme can be used metabolin as substrate or can with conversion of substrate at
For metabolin.In addition, the antibody may be used as generating the basis of the oligopeptides of specific recognition metabolin.These oligopeptides will for example
Binding structural domain comprising enzyme or the bag for receiving the metabolin.Test based on suitable antibody and/or enzyme can be RIA
(radiommunoassay), ELISA (enzyme linked immunosorbent assay (ELISA)), interlayer Dot Enzyme Immunoassay, electrochemical luminescence interlayer immunoassays
(ECLIA), dissociation enhancing lanthanide fluoro immuno measures (dissociation-enhanced lanthanide fluoro
Immuno assay, DELFIA) or solid phase immuno-assay.In addition, metabolin may be based on the energy that it is reacted with other compounds
Power (passing through special chemical reaction) is identified.Suitable reaction is known in the art, and it is anti-to preferably include enzymatic
It answers, enzymatic spectrophotometric determination method, fluorescence spectrophotometry and fluorescence；Chemiluminescence.Other detection methods can be used such as
Capillary Electrophoresis and colorimetric method.In addition, metabolin can be surveyed due to its ability of evoked response in biological read out system
It is fixed.The reading that biological response will test as the presence and/or amount that indicate the metabolin for including in sample.Biological response can be with
It is for example, the phenotype of the induction of gene expression or cell or organism responds.
In the present invention, biological sample is derived from mammal, preferably mouse, rat, cavy, dog, miniature pig or people, most
It is preferred that being people.Biological sample is preferably blood, however, allow to be measured according to the present invention is well known by persons skilled in the art
Any other biological sample is also suitable.Blood sample is usually whole blood, serum or blood plasma.
Although being sufficient using a kind of metabolin for diagnosis acute pancreatitis disease, use is one or more, two
Kind is a variety of, and metabolin as three kinds or a variety of or four kinds or a variety of is included in the present invention, and in many cases,
This is preferred.It can analyze metabolin, and in any combination for diagnosing.
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this
It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip
Part.
Treatment of 1 Chinese medicine of embodiment to acute pancreatitis
1, experimental material
1.1 experiment main materials
Table 1 uses the bill of materials
The 1.2 main equipment of experiment
Surgical instrument: sterile micro- curved tweezer 2, eye scissors 2, needle forceps 1, scalpel 1.
Other: dry cotton ball, medical cotton stick, suture, operating lamp, surgical plate, fixed rubber band, alcohol swab, sterile gauze,
Sterile petri dish, physiological saline, 10mL syringe, 1mL syringe, No. 4 sewing needles.
2, experimental method
2.1 animal packet
30 SPF grades of C57BL/6 mouse, male, 8 week old, 20g or so, adaptive feeding are randomly divided into five groups after 1 week,
Every group 6, grouping situation is as shown in table 2:
2 mice group situation of table
2.2 drugs are prepared
3 drug of table is prepared
The building of 2.3 animal models and drug-treated
Fasting 12h, can't help water before experiment mice molding.Control group injects isometric physiological saline, remaining each group receives 7 times
It is injected intraperitoneally caerulin (50 μ g/kg, every minor tick 1h), after having injected for the last time, the LPS of 30mg/kg dosage is injected intraperitoneally,
Prepare AP model.Rhein treatment group, honokiol treatment group, Rhein+honokiol treatment group in injecting caerulin for the first time
1h, 5h, 9h distinguish 0.2% Rhein 4mg/kg of tail vein injection afterwards, and honokiol 5mg/kg is injected intraperitoneally.
The collection of 2.4 serum and pancreas
After the completion of model construction, i.e., after injecting caerulin 7h for the first time, mouse orbit blood sampling.In caerulin is injected intraperitoneally for the first time
Eyeball of mouse is carried out after 12h under aseptic conditions and takes blood, after whole blood is placed at room temperature for 2h or 4 DEG C overnight, 6000r/min, centrifugation
5min collects serum, and -80 DEG C save backup.Pancreatic tissue is taken simultaneously, takes pictures, pancreatic tissue is divided into 2 in same position
Part, 1 part is fixed with formalin solution, put after 1 part of direct liquid nitrogen flash freezer -80 DEG C freeze it is spare.
The detection of 2.5 serum amylases
It is formed sediment using mouse amylase (AMS) kit (ELISA) the detection serum of the enzyme-linked Biotechnology Co., Ltd in Shanghai
Powder enzyme content.
1) lath needed for being taken out from the aluminium foil bag after equilibrium at room temperature 20min.
2) be loaded: standard items or sample to be tested 50 μ L, enzyme 50 μ L of labeling antibody working solution is added in every hole.Reaction plate is filled
Divide and mixes 37 DEG C of insulating boxs incubation 60min of postposition.
3) board-washing: sufficiently being washed reaction plate 4-6 times with cleaning solution, is printed on filter paper dry.
4) substrate A, each 50 μ L of B is added in every hole, and 37 DEG C are protected from light incubation 15min.
5) every hole is added 50 μ L terminate liquids, and in 15min, the OD value in each hole is measured at 450nm wavelength.
6) using standard items as abscissa, OD value is ordinate, draws standard curve.On the graph according to sample OD value
Find factor protein content to be measured in respective sample.
The HE of 2.6 pancreatic tissues is dyed
1) paraffin section makes
1. slice: after tissue block after embedding is trimmed, 5 μm of paraffin band is cut into slicer.
2. patch: tissue paraffin block being opened up piece in 50 DEG C of warm water, then fishes out piece, taeniae telarum with the clean glass slide of processing
It can stick on glass slide.
3. roasting piece: baking piece 2h in 60 DEG C of insulating boxs.
2) dewaxing and aquation
1. toasting 20min in 60 DEG C of insulating boxs.
2. impregnating 10min in dimethylbenzene, 10min is impregnated again after replacing dimethylbenzene；
3. impregnating 5min in dehydrated alcohol；
4. impregnating 5min in 95% ethyl alcohol；
5. impregnating 5min in 70% ethyl alcohol；
6. impregnating 5min in 50% ethyl alcohol；
7. impregnating 5min in pure water；
3) haematoxylin dyeing 5min；Acidic alcohol breaks up 5s；
4) tap water returns blue 15min.
5) eosin stains 5min.
6) 95% ethyl alcohol Rapid Cleaning 2 times.
7) dehydrated alcohol 2min fast dewatering, the transparent 5min*3 of dimethylbenzene.
8) gummy mounting, microscopy are taken pictures.
3, experimental result
3.1 mice serum amylase contents
7h, 12h mice serum amylase content as shown in Figure 1 (*: compared with the control group, p < 0.05, * *: with control group phase
Than p < 0.01, #: compared with model group, p < 0.05, ##: compared with model group, p < 0.01), caerulin 7h is injected intraperitoneally for the first time
Afterwards, it measures content of Diastase in blood serum in pancreatitis mouse model and is significantly higher than blank control group and drug treatment group, illustrate mouse
The success of pancreatitis model construction, and the significant effect of Rhein honokiol power drug combination group is better than the effect of independent medication.
3.2 mice pancreatic tissue pictures
After injecting caerulin 12h for the first time, pancreatic tissue is taken to take pictures, as a result as shown in Fig. 2, comparing blank control group, model
The pancreatic tissue of group animal has apparent oedema, and Rhein+honokiol drug combination is than being used alone both effect of drugs
It is good, the pancreatic tissue oedema phenomenon of pancreatitis model mice can be alleviated.
3.3 HE coloration results
For HE coloration result as shown in figure 3, compared with blank control group, the intercellular gap of pancreatitis model group is larger, and
Nucleus is more, illustrates that inflammatory model will lead to macrophage etc. and be gathered in around inflammation.Rhein treatment group, honokiol are controlled
Treatment group is reduced with respect to pancreatitis model group, intercellular gap, and cell nuclei decreases.Rhein+honokiol connection
Medicine group is shared with respect to pancreatitis model group, the number of nucleus can be significantly reduced, intercellular gap is also reduced, and illustrates to combine
Medication can alleviate pancreatitis inflammation.
Relevant metabolin embodiment 2 screening and influence acute pancreatitis
1, the collection of sample
It is small that blank control group, model group and Rhein+honokiol combination therapy group in embodiment are collected in this experiment
Each 10, rat tissue sample.
2, experiment information
2.1 instruments and reagent
4 reagent of table
5 instrument of table
2.2 experimental method
2.2.1 metabolin extracts
1) it takes 100mg to organize, the lysate (MeOH:H of 500 μ L is added2O=1:1)；
2) it smashes, 6,500MHz, 1min, vibrates 3 times in historrhexis's instrument；
3) it is vortexed after mixing, is stood overnight in -20 DEG C of refrigerators；
4) it is centrifuged: 4 DEG C, 13,000rpm, 20min；
5) same volume supernatant is taken to be lyophilized；
6) 100 μ L lysates are added to redissolve, ultrasonic 5min；
7) it is centrifuged: 4 DEG C, 13,000rpm, 20min；
8) machine on supernatant is taken.
2.2.2 instrument parameter
1) liquid-phase condition
Column temperature (DEG C): 45
Sample temperature (DEG C): 4
Liquid phase flow rate: 0.35ml/min
A phase :+0.1% formic acid of water
B phase :+0.1% formic acid of acetonitrile
6 liquid-phase condition of table
2) Mass Spectrometry Conditions
Under cation acquisition mode, Masslynx software is based on MSE function and carries out level-one, second order ms data to sample
Acquisition.Capillary voltage: 2.5kV, orifice potential 24V, remove solvent stream speed 800L/h, taper hole air-flow by 100 DEG C of ion source temperature
The ion that m/z is 50-1500Da is scanned in fast 50L/h, 14min, 0.2s/ circulation.
Under anion acquisition mode, Masslynx software is based on MSE function and carries out level-one, second order ms data to sample
Acquisition.Capillary voltage: 2.5kV, orifice potential 25V, remove solvent stream speed 600L/h, taper hole air-flow by 100 DEG C of ion source temperature
The ion that m/z is 50-1500Da is scanned in fast 10L/h, 14min, 0.2s/ circulation.
2.2.3 upper machine testing
In order to preferably acquire data, it is ensured that the optimum state of instrument needs to make of QC sample before machine on official sample
The same QC sample is generally repeated 10 needle of sample introduction or so by stability test, this sample is condition QC, to instrument stabilizer
It can sample introduction.A QC is inserted into every 10 this centre of needle-like, it is ensured that the stability during instrument sample introduction.
3, experimental result
3.1 data result
Cation has detected 15013 features, anions altogether and has detected 10431 altogether in this experiment
features.The wherein BPI (base peak intensity, base peak figure) and TIC (total ion of QC sample
Chromatogram, total ion chromatogram) as shown in figure 4, the figure upper right corner indicates peak intensity, sample ID is indicated in the upper left corner.
Peak type in BPI figure is narrow, quantity is more, shows that chromatographic isolation effect is good.
3.2 data Quality Controls
During instrument carries out data acquisition, the QC sample being inserted among sample is done into overlay, when determining reservation
Between and peak intensity be held essentially constant；PCA analysis is carried out to QC sample, Dimension Reduction Analysis, test experience mainly are carried out to data
The repeatability in otherness and group between group, analyzes the correlation of QC sample, and it is real to investigate the multiple biology of sample
The repeatability tested, the results show that time and peak intensity are held essentially constant in data acquisition；And the opposite collection of QC sample
In together, there is no changing at any time, any two groups Pearson for repeating to test quantitative polypeptide intensity value altogether are related
Coefficient is all larger than 0.9, has preferable consistency, illustrates that laboratory apparatus has preferable stability.
4, data are analyzed
4.1 PCA analysis
Initial data importing Progenesis QI (Waters) software of acquisition is for statistical analysis, first to data
The peak of the mass fraction correction and retention time that carry out Lockmass is aligned, and is then grouped to sample, it is soft to import EZinfo
The screening of part progress difference metabolin.PCA analysis is the first step of EZinfo software analysis, mainly carries out dimensionality reduction point to data
Analysis, can detecte the repeatability in the otherness and group between experimental group.In a two dimensional, the first two principal component PC1, PC2 is taken to come
Indicate sample, spatial distribution differences are smaller, indicate that the data of two samples are closer.It is repeated preferably to test, in same group
Different samples should be gathered in the range of a Relatively centralized, and can with other group data aggregation zone distinguish.
4.2 OPLS-DA (orthogonal ginsenoside)
For incoherent noise information of eliminating and classify, and also to screen the credible metabolism for the difference that causes to classify
Object chooses OPLS-DA analysis filtering and the incoherent signal, i.e. orthogonal signalling of classifying, OPLS-DA model is obtained, to model
Quality is tested with cross-validation method and (makes grouping model with a part of sample data, another part data are used to survey
Try the model being grouped), obtained R2X and Q2 respectively represents the interpretable variable of model and predictable degree, can be to the excellent of model
Bad to be differentiated and (be detailed in Fig. 5), the R2Y and Q2 of the OPLS-DA model in experiment are 99% and 98% respectively, and it is good to indicate that model has
Good interpretable ability and predictable packet capability.VIP marking screening is carried out to metabolin by model analysis, VIP score is got over
High metabolin, bigger to the contribution of grouping, VIP>1 is chosen in this experiment, the metabolin of value<0.05 P is difference metabolin.
5, result
By data analysis found that metabolin hydrochloric acid cinanserin or Cefazaflur in blank control group and model group, control
There are significant differences between treatment group and model group, and then there was no significant difference between blank control group and treatment group, compared to sky
The content of white control group, hydrochloric acid cinanserin or Cefazaflur in model group significantly reduces, and about reduces by 1.51 times and 2.13 times,
Compared to model group, the content of hydrochloric acid cinanserin or Cefazaflur in treatment group is dramatically increased, increase separately about 1.45 times and
2.07 times, illustrates that hydrochloric acid cinanserin or Cefazaflur can be used as the generation of marker instruction pancreatitis, can simultaneously serve as medicine
Object treats the monitoring index of validity, and the effect applied to drug therapy pancreatitis judges.
The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.
Claims (8)
1. a kind of reagent, which is characterized in that the reagent can detecte the level of metabolin hydrochloric acid cinanserin or Cefazaflur.
2. reagent according to claim 1, which is characterized in that the reagent includes passing through chromatography, spectrum or mass spectrograph/light
Spectrometry detects the reagent of the content of metabolin.
3. reagent according to claim 2, which is characterized in that the chromatography is detected as the detection of ultra high efficiency liquid chromatography.
4. a kind of product of vitro detection metabolin, which is characterized in that the product includes claim 1-3 described in any item
Reagent.
5. a kind of method of the drug candidate of screening treatment acute pancreatitis, which is characterized in that comprising steps of
The cultivating system containing hydrochloric acid cinanserin or Cefazaflur is added in substance to be screened, and
The content of hydrochloric acid cinanserin or Cefazaflur in the system is detected,
If substance to be screened can increase the content of hydrochloric acid cinanserin, Cefazaflur, show that the substance to be screened is that treatment is anxious
The drug candidate of property pancreatitis.
6. a kind of pharmaceutical composition, which is characterized in that described pharmaceutical composition includes the rush of hydrochloric acid cinanserin or Cefazaflur
Into agent.
7. pharmaceutical composition according to claim 6, which is characterized in that promotor is to increase hydrochloric acid cinanserin or cephalo
The reagent of nitrogen fluorine.
8. following described in any item applications:
A. application of the described in any item reagents of claim 1-3 in the product of preparation diagnosis acute pancreatitis；
B. the described in any item reagents of claim 1-3 are in the response product that preparation judges therapeutic agent treatment acute pancreatitis
Using；
C. application of the product as claimed in claim 4 in the tool of preparation diagnosis acute pancreatitis；
D. application of the product as claimed in claim 4 in the response tool that preparation judges therapeutic agent treatment acute pancreatitis；
E. application of the pharmaceutical composition described in claim 6 or 7 in the tool of preparation treatment acute pancreatitis；
F. the application of hydrochloric acid cinanserin, Cefazaflur in the computation model of building prediction acute pancreatitis；
G. hydrochloric acid cinanserin, Cefazaflur are in the computation model of the treatment response of building prediction drug therapy acute pancreatitis
Application.
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