CN107202810A

CN107202810A - One kind analysis IC50Influence method of the dosage methyltocol to RAW264.7 and K562 cells

Info

Publication number: CN107202810A
Application number: CN201710384527.2A
Authority: CN
Inventors: 耿越; 陆洋; 李慧
Original assignee: Shandong Normal University
Current assignee: Shandong Normal University
Priority date: 2017-05-26
Filing date: 2017-05-26
Publication date: 2017-09-26
Anticipated expiration: 2037-05-26
Also published as: CN107202810B

Abstract

It is based on the invention discloses one kind¹H NMR metabolism group method analyzes IC₅₀Influence method of the dosage methyltocol to RAW264.7 and K562 cells, including following methods：(1) IC of the methyltocol to RAW264.7 or K562 cells is determined₅₀Dosage；(2) preparation of cell extraction liquid；(3) nuclear magnetic resonance is pre-processed；(4)¹H magnetic resonance detections；(5) screening of otherness metabolin；(6) metabolic pathway is analyzed.Present invention application hydrogen spectrum nuclear magnetic resonance technique combination metabolism group method, have studied the influences of different Cell differentials metabolins and metabolic pathway, there is provided a kind of analysis method of research methyltocol to the inferior toxicity of RAW264.7 or K562 cells.

Description

One kind analysis IC50Influence of the dosage Delta-Tocopherol to RAW264.7 and K562 cells Method

Technical field

The present invention relates to based on¹H NMR metabolism group method analyzes IC₅₀Dosage Delta-Tocopherol is to RAW264.7 and K562 The influence method of cell, belongs to the metabonomic analysis of cellular level.

Background technology

The bioactivity of vitamin E：

Vitamin E is to maintain the metabolism of human normal and molecule necessary to every physiological function.Also, vitamin E human bodies itself can not be generated, it is necessary to be absorbed by food from the external world.Food rich in vitamin E includes almond, fibert, soybean And avocado etc..Vitamin E also known as tocopherol, according to methyl in the position of chroman ring and quantity, can be divided into following eight kinds of α- TOH, β-TOH, γ-TOH, four kinds of tocopherols of δ-TOH；α-TT, β-TT, γ-TT, four kinds of tocotrienols of δ-TT.Wherein α-TOH Bioactivity is maximum, and γ-TOH and δ-TOH activity only account for the 10% and 1% of α-type respectively；However, oxidation resistance δ-TOH It is most strong, it is 100 times of α-TOH oxidation resistances.Vitamin E has different physiological roles, including anti-oxidant, anti-aging, Frostbite is treated, blood vessel is protected, cell membrane, even signal transduction and gene expression etc. is protected.Vitamin E can also be by regulating and controlling to believe Number path alleviates obesity and its Metabolic complication, and the path of influence includes Wnt, JAK-Stat (the Janus Kinase-signal transducer and activator of transcription), PI3K (the - the kinase of phosphatidylinositol 3 ')/Akt/mTOR (mammalian target of rapamycin) signal lead to Road, and these signal paths are many all related to tumor promotion.There is researcher to find that γ-TOH can be by suppressing to have recently Oxygen glycolysis suppresses tumour.And it has been reported that, the different subtype of vitamin E has different metabolism in vivo and in vitro Approach.According to WHO (World Health Organization) as shown by data normal adults to vitamin E (δ- Tocopherol recommended intake (Recommended Nutrient Intakes, RNIs)) is daily 10mg, be can tolerate most High intake (Tolerable Upper Intake Level, UL) is 1000mg.Nano Silver is produced to Mouse Liver primary cell Toxicity can be protected by the vitamin Es of doses.In the toxicity reality to the different canceration period galactophore epithelial cells of mouse Test middle discovery, precancerous lesion period, the galactophore epithelial cell δ-TOH in tumour period and malignant tumour period IC₅₀Dosage is distinguished For 55 μM, 47 μM and 23 μM.There are some researches show vitamin E can be modified due to white thin caused by some chemotherapeutics very early Born of the same parents are reduced, so as to play booster action to the chemotherapeutic treatment of cancer.The tocopherol of different subtype is to macrophage RAW264.7's IC₅₀Concentration is also not quite similar, and as a result shows that cytoactive influences of the δ-TOH on mouse macrophage RAW264.7 is more than α-TOH Influence.Excessive vitamin E cell can be caused how the influence in terms of metabolism group, seldom have been reported that research.

The related application of metabolism group：

Current metabolism group has extensively and ripe application in every field, including treatment of cancer and Chinese medicine into Divide research etc..These are all based on metaboilic level Molecular characterization and determine quantifier elimination.Along with the development of metabolism group, cancer The otherness of cell and normal cell is also progressively determined.There are some researches show candidate stem cell shifts the mode for the treatment of cancer recently RAEB or acute myeloid leukemia may be caused, this process includes alanine and aspartic acid generation Thank, the metabolism of dicarboxyl acid metabolic, phenylalanine, the regulation and control of the metabolic pathway such as citrate cycle and aminoacyl tRNA biosynthesis.With This is relatively fewer than K562 cell lines simultaneously for the research in terms of RAW264.7 cell line metabolism group.Major part is to macrophage The research in cell metabolism group direction lays particular emphasis on the influence of δ-TOH and γ-TOH to its bioactivity.

Drenched therefore, it is necessary to study δ-TOH in mouse macrophage RAW264.7 and chronic myelogenous leukemia pleural effusion Bar cell K562 IC₅₀During concentration, the influence to different Cell differentials metabolites and metabolic pathway, this contributes into one Step understands δ-TOH inferior toxicity.

The content of the invention

For deficiency of the prior art, first purpose of the invention is to provide a kind of IC₅₀Dosage Delta-Tocopherol pair The analysis method of RAW264.7 and K562 cell inferior toxicities.

The technical solution adopted by the present invention is as described below：

A kind of IC₅₀Dosage Delta-Tocopherol comprises the following steps to the analysis method of RAW264.7 or K562 cell inferior toxicities：

(1) IC of the Delta-Tocopherol to RAW264.7 or K562 cells is determined₅₀Dosage；

(2) preparation of cell extraction liquid：

δ-TOH organize the preparation of cell extraction liquid：By RAW264.7 or K562 cells in corresponding Delta-Tocopherol IC₅₀Under dosage Cultivated, cold methanol is added after culture is quenched cell metabolism, then carries out ultrasonication using methanol chloroform water extraction method Extract, aqueous phase is collected by centrifugation；

The preparation of blank group cell extraction liquid：RAW264.7 or K562 cells are cultivated, cold methanol is added after culture Cell metabolism is quenched, ultrasonication extraction is then carried out using methanol chloroform water extraction method, aqueous phase is collected by centrifugation；

(3) nuclear magnetic resonance is pre-processed：

Used after the δ-TOH aqueous phase rotations organized with blank group are evaporated and contain (3- trimethylsilyls) -2,2,3,3- The D of four deuterated sodium propionates (TSP)₂O dissolves, and centrifuges, and freezes；Sample D after will be lyophilized₂The phosphate buffer that O is prepared is molten Solution, centrifugation, obtains supernatant；

(4)¹H magnetic resonance detections：The supernatant of the pretreated δ-TOH groups of nuclear magnetic resonance and blank group is subjected to nuclear-magnetism Resonance detection, obtains cell nmr spectrum, so as to obtain the signal value of metabolin in RAW264.7 or K562 cell samples；

(5) screening of otherness metabolin：The integral area of cell nmr spectrum is normalized, obtained Integral area data, differentiate that (PLS-DA) and orthogonal-offset minimum binary differentiate (OPLS-DA) analysis number using offset minimum binary According to, and checking screening otherness variable is carried out using arrangement experiment and CV-ANOVA, choose offset minimum binary and differentiate (PLS-DA) The variable weight importance ranking value of middle first principal component>0.1 metabolin obtains otherness generation as otherness metabolin Thank to the relative amount value of thing, the variation tendency of each quantitative otherness metabolin；

(6) metabolic pathway is analyzed：The otherness metabolin of determination is imported into capital of a country gene and the encyclopaedical data of genome In storehouse, belong to the metabolic pathway of correlation；By metabolism group DAS MetaboAnalyst to otherness metabolin Metabolic pathway enrichment analysis is carried out, the metabolic effect analysis path and metabolism sequence for obtaining being belonged to by otherness metabolin are rich Set analysis path, the path influence value of metabolic effect analysis path>0.1 metabolic pathway and metabolism sequence enrichment analysis path Path influence value<0.1 metabolic pathway is identified as based on the significantly affected path of Delta-Tocopherol group；It is last from Representative metabolic pathway is filtered out in the significantly affected path.

Based on above IC₅₀Analysis of the dosage Delta-Tocopherol to RAW264.7 or K562 cell inferior toxicities, of the invention second Individual purpose is to provide following any application：

(1) glycine betaine anti-metabolite agent is promoted to prepare treatment by IC₅₀Normal cell inferior toxicity caused by dosage Delta-Tocopherol Reagent in application；

(2) metabolism of pyruvate reagent is preparing treatment by IC in reduction anaerobic respiration₅₀Cancer caused by dosage Delta-Tocopherol is thin Application in the reagent of born of the same parents' inferior toxicity；Or be：Lactic acid antagonist/inhibitor is preparing treatment by IC₅₀Dosage Delta-Tocopherol draws Application in the reagent of the cancer cell inferior toxicity risen；

(3) Delta-Tocopherol antagonist/inhibitor is preparing the application in promotion mammal body in glycine betaine anti-metabolite agent；

(4) Delta-Tocopherol antagonist/inhibitor is in the breathing pattern for preparing promotion cancer cell is converted to aerobic respiration Using.

Wherein, it is that can promote the reagent that glycine betaine is metabolized in internal cell to promote glycine betaine anti-metabolite agent so that cell The content increase of internal glycine betaine.

Metabolism of pyruvate reagent is that can suppress the reagent of metabolism of pyruvate in anaerobic respiration in reduction anaerobic respiration；Lactic acid Antagonist/inhibitor is that can reduce the reagent of lactic acid concn/content in cell.Delta-Tocopherol antagonist/inhibitor is can Reduce IC in cell₅₀Dosage or more than IC₅₀The reagent of dosage Delta-Tocopherol concentration/content；To be small in the mammal body Mouse macrophage RAW264.7 or human leukemia lymphocyte K562；Described normal cell is mouse macrophage RAW264.7, the cancer cell behaviour Leukemic Lymphocytes K562.

Third object of the present invention is to provide a kind of IC₅₀Difference of the dosage Delta-Tocopherol to RAW264.7 or K562 cells The screening technique of property metabolin, comprises the following steps：

(2) preparation of cell extraction liquid：

(3) nuclear magnetic resonance is pre-processed：

δ-TOH are organized to use after the aqueous phase evaporation with blank group and contain the deuteriums of (3- trimethylsilyls) -2,2,3,3- four For sodium propionate (TSP) D₂O dissolves, and centrifuges, and freezes；Sample D after will be lyophilized₂The phosphate buffer dissolving that O is prepared, Centrifugation, obtains supernatant；

(5) screening of otherness metabolin：

The integral area of cell nmr spectrum is normalized, integral area data are obtained, using partially most A young waiter in a wineshop or an inn multiplies differentiation (PLS-DA) and orthogonal-offset minimum binary differentiates (OPLS-DA) analyze data, and using arrangement experiment and CV- ANOVA carries out checking screening otherness variable, chooses the variable weight that offset minimum binary differentiates first principal component in (PLS-DA) Importance ranking value>0.1 metabolin is used as otherness metabolin.

Fourth object of the present invention is to provide a kind of IC₅₀Difference of the dosage Delta-Tocopherol to RAW264.7 or K562 cells Property metabolin, including：

The otherness metabolin of RAW264.7 cells is acetic acid, lysine, choline, arginine, alanine, methanol, half Guang Amine, glycine betaine and isobutyric acid；

The otherness metabolin of K562 cells is hypotaurine, threonine, methanol, glucolactone, cysteamine, guanidine second Acid, lactic acid, choline, glycine betaine, butanedioic acid.

Compared with prior art, technical scheme has the advantages that：

(1) present invention application hydrogen spectrum nuclear magnetic resonance technique combination metabolism group method, have studied different Cell differentials generations Thanking to the influence of thing and metabolic pathway, there is provided a kind of point for studying Delta-Tocopherol to the inferior toxicity of RAW264.7 or K562 cells Analysis method.

(2) to IC₅₀Dosage Delta-Tocopherol act on after cell otherness metabolite carry out non-targeted classification, without right A certain or some targeting substances carry out qualitative, quantitative one by one and determined.

(3) nuclear magnetic spectrogram carries out multi-variate statistical analysis through MestReNova 6.11 and SIMCA-P+12.0 softwares and sets up PCA Model, PLS-DA models and OPLS-DA models, it is found that δ-TOH groups and blank group metabolin can be distinguished substantially, just can carry out difference Ownership and the path enrichment of different in nature metabolite.

(4)IC₅₀Dosage Delta-Tocopherol is to the metabolism of the otherness metabolins of RAW264.7 or K562 cells for two kinds of cells Path analysis provides basis.

Brief description of the drawings

The Figure of description for constituting the part of the present invention is used for providing a further understanding of the present invention, and of the invention shows Meaning property embodiment and its illustrate be used for explain the present invention, do not constitute inappropriate limitation of the present invention.

Fig. 1 is influences of the δ-TOH to RAW264.7 and K562 cytoactives, note：^{* *}P ＜ 0.005vs blank groups.

Fig. 2 is the regression equation that two kinds of cell line is obtained according to (Fig. 1).

Fig. 3 is influence of the ethanol to cytoactive of co-solvent concentration.

Fig. 4 is RAW264.7 cells¹H-NMR spectrum.

Fig. 5 is K562 cells¹H-NMR spectrum.

Fig. 6 is the PCA shot charts of RAW264.7 groups.

Fig. 7 is the PCA shot charts of K562 groups.

Fig. 8 is the PLS-DA shot charts of RAW264.7 groups.

Fig. 9 is the PLS-DA shot charts of K562 groups.

Figure 10 is the arrangement experimental model figure of RAW264.7 groups.

Figure 11 is the arrangement experimental model figure of K562 groups.

Figure 12 is the OPLS-DA shot charts of RAW264.7 groups.

Figure 13 is the OPLS-DA shot charts of K562 groups.

Figure 14 is the metabolic pathway analysis for being belonged in RAW264.7 cells by otherness metabolite；Wherein, A is The metabolic pathway gone out is belonged to by otherness metabolite in RAW264.7 cells；(1) biosynthesis of aminoacyl tRNA.(2) sweet ammonia Acid, serine and threonine metabolism.(3) L-lysine amino acid is synthesized.(4) biotin is metabolized.(5) taurine and hypotaurine generation Thank.(6) methane is metabolized.(7) it is metabolized containing selenoaminoacid.(8) lysine degradation.(9) metabolism of pyruvate.(10) alanine, asparagus fern Propylhomoserin and glutamic acid metabolism.(11) glycolysis and gluconeogenesis.(12) glycerophosphatide is metabolized.(13) arginine and proline Metabolism.B is RAW264.7 products of cellular metabolism group enrichment general introduction figure, is respectively from top to bottom：The biosynthesis of protein, beet Alkali metabolism, urea cycle, Methionine metabolism, biotin metabolism, alanine metabolism, taurine and hypotaurine metabolism, glucose sugar- Alanine cycle, lysine degradation, containing selenoaminoacid metabolism, phosphatide biosynthesis, metabolism of pyruvate, glycine serine and Soviet Union Propylhomoserin metabolism, arginine and Proline Metabolism.

Figure 15 is the metabolic pathway analysis for being belonged in K562 cells by otherness metabolite；A be in K562 cells by Otherness metabolite belongs to the metabolic pathway；(1) glycine, serine and threonine metabolism.(2) taurine and Ya Niu sulphurs Acid metabolic.(3) biosynthesis of valine, leucine and isoleucine.(4) glycolysis and gluconeogenesis.(5) phosphoric acid penta Sugared approach.(6) metabolism of pyruvate.(7) methane is metabolized.(8) propionic acid is metabolized.(9) glycerophosphatide is metabolized.(10) aminoacyl tRNA is given birth to Thing is synthesized.(11) arginine and Proline Metabolism.(12) porphyrin and chlorophyll metabolism.B is the enrichment of K562 products of cellular metabolism group General introduction figure；It is respectively from top to bottom：Glycine serine and threonine metabolism, taurine and hypotaurine metabolism, glycine betaine generation Thank, Methionine metabolism, pentose phosphate pathway, Protein synthesis, phospholipid metabolism, metabolism of pyruvate, arginine and proline Metabolism, gluconeogenesis.

Embodiment

It is noted that described further below is all exemplary, it is intended to provide further instruction to the present invention.Unless another Indicate, all technologies used herein and scientific terminology are with usual with general technical staff of the technical field of the invention The identical meanings of understanding.

It should be noted that term used herein above is merely to describe embodiment, and be not intended to restricted root According to the illustrative embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singulative It is also intended to include plural form, additionally, it should be understood that, when in this manual using term "comprising" and/or " bag Include " when, it indicates existing characteristics, step, operation and/or combinations thereof.

Explanation of the present invention to term：

Inferior toxicity：Refer to toxicity or slightly poisonous and be not included in State Council《Medical toxicant administrative law》It is defined Serious adverse reaction can be poisoned or be produced to toxic herb kind, Clinical practice excess or misapplication, using or pharmacy management on It need to be noted that.

IC₅₀(half maximal inhibitory concentration)：Refer to the semi-inhibit of measured antagonist Concentration

RAW264.7：Turnover of Mouse Peritoneal Macrophages cell line, is also one of conventional inflammatory cell model.

K562 is human chronic myeloblastic leukemia pleural effusion lymphocyte.

Topspin3.2 is a kind of nmr analysis software.

SIMCA-P softwares are multivariate analysis software.

PCA：Principal component analysis (Principal components analysis), is a kind of unsupervised mode statistical Method.The variable that one group there may be correlation is converted to by one group of linear incoherent variable by orthogonal transformation, after conversion This group of variable principal component.

PLS-DA：Offset minimum binary differentiates (Partial least squares projection to latent Structure-discriminant analysis), it is a kind of mode statistical method for having a supervision, utilizes PLS Projection Analysis is carried out to data structure.

OPLS-DA：Orthogonal-partial least squares discriminant analysis (orthogonal-PLS-DA), by Orthogonal Signal Correction The analysis that (orthogonal signal correction, OSC) is combined to be modified PLS-DA with PLS-DA Method.

Capital of a country gene and genome encyclopaedical (KEGG) are network analysis gene function, contact genomic information and function The database of information.

MetaboAnalyst：It is a kind of metabolism group data analysis website, can belongs to generation by otherness metabolite Thank to path.

As described in background technology, a kind of IC is lacked in the prior art₅₀Dosage Delta-Tocopherol to RAW264.7 and The impact analysis method of K562 cells, for above technical problem, it is an object of the present invention to use 503nhibiting concentration (IC₅₀) δ- TOH handles two kinds of cell lines of RAW264.7 cells and K562 cells, is existed using two kinds of cell lines of proton nmr spectra com-parison and analysis The change of metaboilic level.Pin is in this regard, the first aspect of the invention, proposes a kind of IC₅₀Dosage Delta-Tocopherol to RAW264.7 or The analysis method of K562 cell inferior toxicities, this method comprises the following steps：

IC of the Delta-Tocopherol to RAW264.7 or K562 cells is determined using mtt assay₅₀Dosage, wherein, RAW264.7's IC₅₀For 80 μM, K562 IC₅₀For 110 μM.

(2) preparation of cell extraction liquid：

δ-TOH organize the preparation of cell extraction liquid：By RAW264.7 or K562 cells in corresponding Delta-Tocopherol IC₅₀Under dosage Cultivated, cold methanol is added after culture is quenched cell metabolism, then carries out ultrasonication using methanol chloroform water extraction method Extract, collect aqueous phase；

During cell extraction liquid is prepared, institutional framework for RAW264.7 or K562 cells both cells and Composition, in order to ensure acquisition δ-TOH group cells as much as possible and blank group cell metabolite information, extracts reagent is preferentially selected Be methanol, chloroform and ultra-pure water mixed solvent, carry out that repeated ultrasonic is broken to be extracted to cell sample, can be carried out after extraction Nuclear magnetic resonance is pre-processed, and pre-treatment step is simple, it is easy to operate.

To reach the preferable effect for extracting metabolin, being more beneficial for improving follow-up otherness metabolin, metabolic pathway point The accuracy of analysis, the extractant that the present invention is used is the mixed solution of methanol, chloroform and ultra-pure water, and volume ratio is 4：4：2.8 ~2.9, in the preferred technical scheme of the present invention, the volume ratio of methanol, chloroform and ultra-pure water is 4：4：2.85.In this hair In bright preferred technical scheme, ultrasound 5 times altogether, ultrasound is stopped one minute for one minute.

(3) nuclear magnetic resonance is pre-processed：

Nuclear magnetic resonance testing sample preparatory phase, the preprocess method of sample can be to magnetic resonance detection result and principal component The accuracy of analysis produces influence, because metabolism group needs high-throughout test data, it is necessary to assure the stability of data and Comparability, this is based on influenceing magnetic resonance detection, and the present invention is established for RAW264.7 or K562 cell extraction liquid Preprocess method.Therefore in the preferred technical scheme of the present invention, the pre- specific processing method of nuclear magnetic resonance is：By δ-TOH groups Contain 0.1% (w/w) TSP D after being evaporated with the aqueous phase of blank group with 900 μ L₂O dissolves, and takes supernatant to freeze after centrifugation, will freeze Dry sample D₂The phosphate buffer that O is prepared is dissolved to 650 μ l, and supernatant is taken after centrifugation into nuclear magnetic tube, to be measured.

(4) magnetic resonance detection：The supernatant of the pretreated δ-TOH groups of nuclear magnetic resonance and blank group is carried out into nuclear-magnetism to be total to Shake detection, obtain cell nmr spectrum, so that the signal value of RAW264.7 or K562 cell sample metabolins is obtained, the letter Number value refers to the nuclear magnetic signal at different chemical shifts, and each signal peak had both contained the qualitative information of metabolin, and also included Quantitative information, qualitative information is exactly feature spectrogram and the chemical shift of the metabolin, quantitative information is exactly the core of the metabolin Magnetic response intensity, such as peak height, peak area；

In nuclear magnetic resonance sample detection-phase, the detection parameter of nuclear magnetic resonance produces influence to magnetic resonance detection result, Metabolism group is a kind of to need the technology of high throughput test data, it is necessary to assure the stability of test data, and present invention screening is excellent Change obtains a set of magnetic resonance detection condition for RAW264.7 or K562 cell extraction liquid.All proton nmr spectras are all Using being equipped with¹³C、¹The double optimization 5mm CPTCI tri- of H resonate the super shielding Fourier of III 600MHz superconductions of trans CryoProbeTM Leaf transformation nuclear magnetic resonance chemical analyser is detected.Wherein Proton Resonance Frequency is 600.104MHz, and pulse train is zg30, and spectrum width is 12019.230Hz, accumulative frequency is 256 times, and sky adopts number of times for 2 times, and experimental temperature is 298K, uses Topspin3.2 (BrukerBiospin, Germany) handles spectrogram.

(5) screening of otherness metabolin：Nmr spectrum uses the (Mestrelab of MestReNova 6.11 Research, Espain) processing, all spectrogram addition index window function (Exponential:0.5), to improve signal to noise ratio.Hand After dynamic phase correction, Baseline wander, calibrated with TSP, water peak cut off manually, selection 0.002ppm is separation standard subsection integral, This separation standard can improve the resolution of cell extraction liquid spectral peak, reduce error caused by nuclear magnetic resonance peak overlap, it Spectrogram integral area is normalized afterwards.Integral area data are preserved with Excel file, packet numbering.Afterwards, data are led Enter in SIMCA-P+12.0 softwares (Umetrics Inc., Umea, Sweden).Use partial least squares discriminant analysis (PLS- ) and orthogonal partial least squares discriminant analysis (OPLS-DA) analyze data DA.In order to ensure PLS-DA and OPLS-DA models are set up Credibility, with arrangement experiment and CV-ANOVA carry out checking screening otherness variable.Weighed according to the variable of first principal component Weight importance ranking value (VIP) and load weight (loading weights) determine otherness metabolite.It is chosen at partially minimum Two multiply the material that VIP values in discriminant analysis (PLS-DA) are more than 1, are determined as otherness metabolite.The positive and negative generation of load weight Upper downward relation of the table relevant difference opposite sex metabolite in blank group and δ-TOH groups.Finally by mankind's metabolism group database (HMDB) and biologic NMR database (BMRB) carry out qualitative analysis, obtain the species name of otherness metabolite.

(6) metabolic pathway is analyzed：The otherness metabolite of determination is imported into capital of a country gene and genome encyclopedia (KEGG) database obtains No. KEGG, and path enrichment analysis is then carried out in MetaboAnalyst (MetPA), is obtained by difference Property metabolin belong to the metabolic effect analysis path that and metabolism sequence enrichment analysis path, metabolic effect analysis path it is logical Road influence value>0.1 metabolic pathway and the path influence value for being metabolized sequence enrichment analysis path<0.1 metabolic pathway quilt It is identified as based on the significantly affected path of Delta-Tocopherol group；Last screened from the significantly affected path is provided Representational metabolic pathway.To obtain representative metabolic pathway, by Protein synthesis, various amino acid or ox The metabolism of sulfonic acid and hypotaurine is excluded, because when cell is by IC₅₀When dosage δ-TOH are acted on, above-mentioned metabolic pathway is Conventional metabolic pathway, that is to say, that when cell is in δ-TOH IC₅₀During concentration, Protein synthesis, various amino acid or The metabolism of taurine and hypotaurine belongs to the basic metabolism approach of cell, therefore, and cannot be used as representational metabolism way Footpath.

These representational metabolic pathways indicate specific biology mistakes of the δ-TOH to RAW264.7 and K562 impact cells Journey, such as upper downward of metabolism and the transformation of breathing pattern.

As a result find that δ-TOH can influence the Protein synthesis of two kinds of cell line, glycine betaine metabolism, urea cycle etc. A plurality of metabolic pathway.Cell metabolite level changes after being handled through δ-TOH, generates corresponding otherness metabolite.

Glycine betaine is significantly reduced in RAW264.7 cells, and lactic acid substantially increases in K562 cells, causes breathing pattern to nothing Oxygen breathing conversion.Experimental result helps to further appreciate that δ-TOH inferior toxicity.

Based on above-mentioned IC₅₀Analysis and research of the dosage Delta-Tocopherol to RAW264.7 or K562 cell inferior toxicities, it is of the invention Second aspect, proposes following any applications：

(4) Delta-Tocopherol antagonist/inhibitor is preparing the breathing pattern for promoting Leukemic Lymphocytes to aerobic respiration Application in conversion.

Metabolism of pyruvate reagent is that can suppress the reagent of metabolism of pyruvate in anaerobic respiration in reduction anaerobic respiration；Lactic acid Antagonist/inhibitor is that can reduce the reagent of lactic acid concn/content in cell.

Delta-Tocopherol antagonist/inhibitor is that can reduce IC in cell₅₀Dosage is more than IC₅₀Dosage Delta-Tocopherol concentration/ The reagent of content；It is mouse macrophage RAW264.7 or human leukemia lymphocyte K562 in the mammal body.

The proposition of above-mentioned application, to treat by IC₅₀Cell inferior toxicity caused by dosage Delta-Tocopherol provides certain base Plinth and reference.

There is provided a kind of IC for the third aspect of the invention₅₀Difference of the dosage Delta-Tocopherol to RAW264.7 or K562 cells The screening technique of property metabolin, it comprises the following steps：

(2) preparation of cell extraction liquid：

(3) nuclear magnetic resonance is pre-processed：

(5) screening of otherness metabolin：The integral area of cell nmr spectrum is normalized, obtained Integral area data are carried out ginsenoside by integral area data, choose variable weight importance ranking> 0.1 metabolin is used as otherness metabolin.

The concrete technical scheme of each step can refer to the first aspect of the invention.

There is provided a kind of IC for the fourth aspect of the invention₅₀Difference of the dosage Delta-Tocopherol to RAW264.7 or K562 cells Property metabolin, including：

IC₅₀Dosage Delta-Tocopherol is to the metabolic pathway of the otherness metabolins of RAW264.7 or K562 cells for two kinds of cells Analysis provides basis.

The major technique innovation of the present invention

(1) application metabolism group method is combined¹H NMR technologies analyze IC₅₀Dosage Delta-Tocopherol is to RAW264.7 and K562 The influence of cell, finds otherness metabolite.

Different from before to the study limitation of Delta-Tocopherol property in its influence to cellular level, the present invention combines metabolism Group learns multivariate analysis statistical method, passes through¹H NMR technologies set up model, complete the packet to Delta-Tocopherol sample before and after the processing, And belong to corresponding otherness metabolite and path.

(2) detection method, IC is quenched by cold methanol₅₀The cell of dosage Delta-Tocopherol processing, nuclear-magnetism pre-treatment is laggard OK¹H NMR are analyzed.Nmr spectrum is handled using MestReNova 6.11 (Mestrelab Research, Espain). Afterwards, import data in SIMCA-P+12.0 softwares (Umetrics Inc., Umea, Sweden).Use principal component analysis (PLS-DA) otherness metabolite and orthogonal partial least squares discriminant analysis (OPLS-DA) analyze data, is found.Pass through the mankind Metabolism group database (HMDB) and biologic NMR database (BMRB) carry out quantitative analysis.The otherness of determination is metabolized Product imports capital of a country gene with being led in encyclopaedical (KEGG) database of genome and MetaboAnalyst (MetPA) Road enrichment analysis.

(3) cell pre-treating method is simple to operate, and what extracts reagent was preferentially selected is the mixing of methanol, chloroform and ultra-pure water Solvent, the broken extraction of repeated ultrasonic is carried out to cell sample, and nuclear magnetic resonance pretreatment, pre-treatment step letter can be carried out after extraction It is single, it is easy to operate.

(4) nuclear magnetic resonance preprocess method is simple to operate, it was found from the analysis result of principal component analysis, common by the nuclear-magnetism Shake preprocess method, preferable, the forecasting accuracy height of model of principal component model fitting, smaller with group difference, no Sample separation with group is more obvious.

In order that technical scheme can clearly be understood by obtaining those skilled in the art, below with reference to tool The embodiment of body describes technical scheme in detail.

Embodiment 1

1st, basic skills principle

Determine IC of the Delta-Tocopherol to RAW264.7 and K562 cells₅₀Dosage, (be specifically using nuclear magnetic resonance chemical analyser AVANCE III 600MHz total digitalizations superconduction) detection sample, use Topspin3.2 (BrukerBiospin, Germany) Handle spectrogram.Nmr spectrum is handled using MestReNova 6.11 (Mestrelab Research, Espain).Afterwards, Import data in SIMCA-P+12.0 softwares (Umetrics Inc., Umea, Sweden).Use principal component analysis (PLS- DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) analyze data, otherness metabolite is found.It is metabolized by the mankind Group database (HMDB) and biologic NMR database (BMRB) carry out qualitative analysis, determine the species of otherness metabolin Title.By the otherness metabolite of determination import capital of a country gene and encyclopaedical (KEGG) database of genome and Path enrichment analysis is carried out in MetaboAnalyst (MetPA).

2nd, instrument and material

Instrument and equipment：

Spectrometer

Material and reagent：

3rd, cell culture

Macrophage RAW264.7 cells and K562 cells are purchased from Chinese Academy of Sciences's Shanghai cell bank.Culture medium is added using DMEM Plus 100U/mL penicillin, 100 μ g/mL streptomysins, 10 μ g/mL glutamine and 10% hyclone.K562 cells are used RPMI1640 medium cultures, the addition factor is identical with what is added in macrophage medium.Cell is all in 37 DEG C of constant temperature CO₂ Cultivate, be passed through in incubator containing 5%CO in incubator₂Wet air.δ-TOH group addition δ-TOH with 0.5% ethanol Dissolving is added in culture medium.

4th, cytoactive detection

Influences of the δ-TOH to cytoactive is detected using MTT experiment.Cell suspension adjusts concentration to every hole 1 × 10⁵It is individual thin Born of the same parents are plated in 96 orifice plates, 37 DEG C of culture 8h.Remove afterwards culture medium and add δ containing various concentrations-TOH culture medium (20, 40,50,80,100 μM), 37 DEG C of culture 48h.Continue 37 DEG C of culture 4h after the MTT that 20 μ L are added per hole.Afterwards, Kong Zhongpei is removed Base is supported, 150 μ L dimethyl sulfoxide (DMSO) (DMSO) is added per hole, room temperature shakes up culture 20 minutes.Because K562 is suspension cell, Therefore 96 orifice plates of the cell need 1,000rmp to centrifuge 10 minutes before removing culture medium addition DMSO.By the orifice plate of cell 96 The light absorption value at 492nm is detected with ELIASA.

5th, prepared by cell extraction liquid

According to MTT experiment result, δ-TOH act on RAW264.7 and K562 IC₅₀Respectively 80 μM and 110 μM.Cell Under corresponding δ-TOH concentration after 37 DEG C of culture 48h, original fluid is discarded, with PBS 3 times, adding 4ml cold methanols makes carefully Born of the same parents' metabolism quenching, is scraped using cell and cell directly is scraped off into culture dish.With 2.85:4:4 volume ratio adds ultra-pure water, methanol And chloroform.Mixed liquor is vortexed after concussion, and cell suspension carries out sonicated cells.Stop each 1min using super, 5 are carried out altogether Secondary, the common 5min of ultrasonic time, ultrasonic overall process need to be carried out in ice bath.12,000rpm, 4 DEG C afterwards, centrifuge 30 minutes, by water Mutually with organic phase separation, aqueous phase is collected.Organic phase supplements ultra-pure water in proportion and methanol is centrifuged again, and water is collected after being repeated 3 times Phase.Supernatant is stored in -80 DEG C.

6th, nuclear magnetic resonance (NMR) is pre-processed

Contain 0.1%TSP D after 60 DEG C of rotary evaporations of supernatant with 900 μ L₂O dissolves.12,000rpm, 4 DEG C of sample, from The heart is freezed after 15 minutes.The sample D freezed afterwards₂The phosphate buffer that O is prepared is dissolved to 650 μ l, 12,000rpm, 4 DEG C, centrifuge 15 minutes, take the μ l of supernatant 550 into 5mm nuclear magnetic tubes, it is to be measured.

7th, nuclear magnetic resoance spectrum map analysis

All proton nmr spectras are all used and are equipped with¹³C、¹The double optimization 5mm CPTCI tri- of the H trans ultralow temperature that resonates is visited The super shielding fourier transform NMR spectrometer detection of the III 600MHz superconductions of head.Wherein Proton Resonance Frequency is 600.104 MHz, pulse train is zg30, and spectrum width is 12019.230Hz, and accumulative frequency is 256 times, and sky adopts number of times for 2 times, real Temperature is tested for 298K, spectrogram is handled using Topspin3.2 (BrukerBiospin, Germany).

8th, achievements exhibition

(1) influences of the δ-TOH to cytoactive

In order to determine influences of the δ-TOH to RAW264.7 and K562 cytoactives, the present invention, which is used, contains various concentrations δ-TOH Medium treatment cell, the influence of cytoactive is as shown in Figure 1.MTT experiment result shows, after δ-TOH processing cells 48h There is obvious inhibiting effect, and two kinds of different IC of cell are determined₅₀。

According to growth curve Fig. 2, RAW264.7 IC is drawn₅₀For 80 μM, K562 IC₅₀For 110 μM.

Meanwhile, with the Ethanol Treatment cell of dissolving δ-TOH concentration, find to the cytoactive of two kinds of cell line without obvious shadow Ring, as shown in Figure 3.

Above test result indicates that macrophage RAW264.7 and K562 Leukaemia are to δ-TOH tolerance and sensitivity Property it is different.

(2) nuclear magnetic resoance spectrum map analysis

According to the proton nmr spectra spectrogram of two kinds of cell aqueous phase extracts of RAW264.7 and K562, it can determine corresponding Otherness metabolite simultaneously further carries out multi-variate statistical analysis.Two kinds of cell lines each δ-TOH group and blank group overlapping spectra Figure is as shown in Figures 4 and 5.From spectrogram, δ-TOH groups are compared with blank group, and both signal peaks have larger difference, this It imply that in δ-TOH groups and blank group that there is otherness metabolin, and the species of metabolin is very more.

(3) multi-variate statistical analysis

Nmr spectrum is handled using MestReNova 6.11 (Mestrelab Research, Espain), owned Spectrogram addition index window function (Exponential:0.5), to improve signal to noise ratio.After manual phase correction, Baseline wander, use TSP is calibrated, and water peak is cut off manually, and selection 0.002ppm is separation standard subsection integral, and spectrogram integral area is returned afterwards One changes.Integral area data are preserved with Excel file, packet numbering.Afterwards, SIMCA-P+12.0 softwares are imported data to In (Umetrics Inc., Umea, Sweden).Use principal component analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) analyze data.In order to ensure the credibility that PLS-DA and OPLS-DA models are set up, with arrangement experiment and CV- ANOVA carries out checking screening otherness variable.According to the variable weight importance ranking value (VIP) and load of first principal component Weight (loading weights) determines otherness metabolite.It is chosen in partial least squares discriminant analysis (PLS-DA) VIP values are more than 1 material, are determined as otherness metabolite.The positive and negative different in nature metabolite of relevant difference that represents of load weight exists Blank group and the upper downward relation in delta-TOH groups.Finally by mankind's metabolism group database (HMDB) and biological nuclear-magnetism Resonance data storehouse (BMRB) carries out qualitative analysis, obtains the species name of otherness metabolite, such as Tables 1 and 2.

By the descending arrangement of VIP values, the chemical shift of VIP >=1 is taken.Wherein bigger be grouped to otherness of VIP values is contributed It is bigger.The otherness metabolite that the chemical shift of two kinds of cell line is determined is shown in Table 1 and 2.For more preferable poor different in nature generation Thank to the upper downward degree of product, the integral area of otherness metabolite is normalized.Using integral area simultaneously The mode for being multiplied by 1000 obtains relative value (relative value), and the quantitative specific variation tendency of each metabolite is compared Compared with.RAW264.7 and K562 and the comparative result of respective blank group are shown in Table 3 and 4.

The otherness metabolite belonged in table 1.RAW264.7 cells and corresponding chemical shift

The otherness metabolite and corresponding chemical shift s- belonged in table 2.K562 cells is unimodal；D- is bimodal； The peaks of t- tri-

The relative value for the otherness metabolite that δ-TOH groups are compared with blank group and change become in table 3.RAW264.7 cells Gesture dd- double doublets；The peaks of q- tetra-

The relative value and variation tendency for the otherness metabolite that δ-TOH groups are compared with blank group in table 4.K562 cells

PCA the and PLS-DA analysis results that multi-variate statistical analysis is carried out in SIMCA-P softwares show, for RAW264.7 With K562 cells, δ-TOH groups and blank group can be separated.The shot chart (Fig. 6 and Fig. 7) and load diagram of two kinds of cells show, two Plant cell has respective otherness metabolite after δ-TOH processing.

First, dimension-reduction treatment is carried out to data using pca model, extracts the most information observation blank group in data set The natural packet conditions organized with δ-TOH.As a result show for RAW264.7 cells, δ-TOH groups and blank group can be from the (PC1 is substantially distinguished in one principal component and Second principal component,:88.38%；PC2:9.16%).Similarly, K562 δ-TOH groups Can also there is good differentiation (PC1 on its first principal component and Second principal component, with blank group:61.01%；PC2: 20.45%).PCA as the analysis method supervised without teacher, can only response data original condition.But, in the actual behaviour of experiment There is the accuracy of many Effects of Factors results, such as environment or some systemic mistakes in work.It is more accurate in order to obtain Packet situation, excludes these externality factors as far as possible, analysis method PLS-DA and OPLS- that we are supervised using teacher of the having DA processing datas.

The effect for evaluating PLS-DA models fittings generally uses R²X, R²Y and Q²These three indexs.R²X and R²Y can be represented The percentage of X and Y matrixes in model, and Q²Mean the predictive ability of model.Three above index value is said closer to 1 The confidence level of bright model is higher.Preferable (the R of the PLS-DA models fittings of RAW264.7 Establishment of Cell Line²X=0.975, R²Y= 0.981,Q²=0.972), as shown in Figure 8.

Equally, the PLS-DA models of K562 Establishment of Cell Line are also fitted good (R²X=0.813, R²Y=0.964, Q²= 0.964), as shown in Figure 9.

The validity of model can be verified by arranging experiment, as a result shows that the slope of two regression equations is larger, arbitrarily R²And Q²Value be both less than the point of the rightmost side, R²And Q²The regression equation rightmost side value difference away from smaller, this shows that model does not have Over-fitting.Two cell line arrangement experimental result pictures of observation, as shown in FIG. 10 and 11, illustrate what RAW264.7 and K562 were set up PLS-DA models are all without over-fitting.

The model for setting up RAW264.7 cells is analyzed using OPLS-DA, according to shot chart (Figure 12) it can be found that blank group It can farthest be distinguished with δ-TOH groups and group difference can be reduced.

K562 cells set up OPLS-DA models, and the observation of its shot chart (Figure 13) is found also to can be good at distinguishing.

Otherness metabolite can be obtained with reference to corresponding VIP values, wherein RAW264.7 has the metabolism production of 9 species diversity Thing, K562 has 10 kinds.Meanwhile, OPLS-DA can be able to verify that by CV-ANOVA (association's independent variable-variance analysis) P values The validity of model, P values, which are more than 0.05, proves that model checking is meaningful.The P values that two kinds of cells are obtained according to CV-ANOVA are all small (the P values of RAW264.7 cell lines are 0.00164 in 0.05；The P values of K562 cell lines are 0.02044), to show what is set up OPLS-DA models all have meaning.

(4) metabolic pathway is analyzed

In order to study the metabolic pathway that δ-TOH act on RAW264.7 and K562 cell associated biomarkers, the present invention Obtained otherness metabolin will be screened and determine No. KEGG by KEGG databases, then pass through MetaboAnalyst again (MetPA) (network address：http://www.metaboanalyst.ca/) belong to the metabolic pathways of these otherness metabolins, from And the interaction relationship probed between otherness metabolin and metabolic pathway.9 related otherness generations of RAW264.7 cells Thank to product and belong to 13 metabolic pathways, including：(1) biosynthesis of aminoacyl tRNA；(2) glycine, serine and threonine Metabolism；(3) L-lysine amino acid is synthesized；(4) biotin is metabolized；(5) taurine and hypotaurine metabolism；(6) methane is metabolized；(7) Containing selenoaminoacid metabolism；(8) lysine degradation；(9) metabolism of pyruvate；(10) alanine, aspartic acid and glutamic acid metabolism； (11) glycolysis and gluconeogenesis；(12) glycerophosphatide is metabolized；(13) arginine and Proline Metabolism.And K562 cells are related 11 biomarkers belong to 12 associated metabolic paths, including：(1) glycine, serine and threonine metabolism；(2) Taurine and hypotaurine metabolism；(3) biosynthesis of valine, leucine and isoleucine；(4) glycolysis and gluconeogenesis are made With；(5) pentose phosphate pathway；(6) metabolism of pyruvate；(7) methane is metabolized；(8) propionic acid is metabolized；(9) glycerophosphatide is metabolized；(10) Aminoacyl tRNA biosynthesis；(11) arginine and Proline Metabolism；(12) porphyrin and chlorophyll metabolism.

In order to further determine that size of these metabolic pathways to δ-TOH function influences, we are divided using MetPA Analysis.MetPA can obtain the KEGG sequence numbers (KEGG ID) of corresponding metabolin according to metabolic information database (KEGG), utilize The automatic Biochemical changes to different metabolic thing of computer are grouped, so as to search out degree of correlation highest metabolic pathway and disease Disease.Above-mentioned substance (RAW264.7 9 species diversity metabolins and K562 10 species diversity metabolins) is imported into MetPA soft In part, belong to metabolic pathway by two kinds of databases.One kind is path analysis, that is, integrates enrichment analysis and path topology analysis, The model can carry out visual analyzing to 21 kinds of model organisms.Acquired results are represented using path influencing characterisitic as abscissa - logP the values of metabolic pathway enrichment importance are ordinate, and the topological analysis for calculating metabolic pathway importance is mapped.Metabolism - logP the values of approach influence are bigger, and the metabolism correlation between different grouping is higher, and the bigger color of circle in figure is deeper to be located at Upper right comer region.The path analysis figure that two kinds of cells are obtained is shown in Figure 14 A and 15A.Another is the generation to the mankind and mammal Thank to thing group enrichment analysis (MSEA), the path attribution analysis carried out based on about 6300 groups of metabolomes in database.This reality Selection phenetic analysis (Over Representaion Analysis, ORA) is tested as enrichment parser, this method is only needed to The KEGG sequence numbers of correspondence compound, which are imported, can carry out subsequent analysis.The model by traditional feature selection approach or Clustering algorithm detects significant biological pattern.

Path analysis result shows that two kinds of cell lines have some paths all to be changed after being handled through δ-TOH, such as aminoacyl TRNA biosynthesis, glycine, serine and threonine metabolism, taurine and hypotaurine metabolism, metabolism of pyruvate, sugared ferment Solution and gluconeogenesis and glycerophosphatide are metabolized.However, the metabolic pathway for also having some different in two kinds of cells is changed. In RAW264.7 cells, L-lysine amino acid synthesis and degraded, biotin metabolism, containing selenoaminoacid metabolism and alanine, day Winter propylhomoserin and glutamic acid these paths are changed, but all do not occur significant change in K562 cells.

Equally, the biosynthesis of valine, leucine and isoleucine, pentose phosphate pathway, propionic acid metabolism, arginine and Proline Metabolism and porphyrin and chlorophyll metabolism all only there occurs significant change in K562 cells.The function of these paths is true The decline of mitochondria dysfunction and free radical (ROS) Scavenging activity can be can be shown that in fact.These are different in two kinds of cell line Metabolic pathway changes, and might mean that the different molecular mechanism side that δ-TOH are acted on produced by macrophage and leukaemia The influence in face.

It is enriched with and is analyzed by metabolome, we is it can be found that biomarker can belong to the metabolic pathway of determination. In RAW264.7 cells, protein synthesis, amino acid metabolism, metabolism of pyruvate, the exception of glycolysis and gluconeogenesis trigger The dysbolism (Figure 14 B) of cell.And K562 is analyzed, find similar amino acid metabolism, protein synthesis, pyruvic acid Metabolism and glycolysis and gluconeogenesis there occurs exception (Figure 15 B).

When analyzing the representational metabolic pathway after δ-TOH processing in cell, two kinds of figures of comprehensive analysis divide first Path analysis figure (such as Figure 14 A and Figure 15 A) is analysed, path of the path influencing characterisitic value more than 0.1 is identified as significantly affected Path, its is with a high credibility, can for analysis, such as Figure 15 A, at the same need analysis metabolome enrichment analysis chart (as scheme 14B and Figure 15 B), in Figure 14 B and Figure 15 B, P value are less than 0.1 interval scale, and it is significantly affected metabolic pathway (8e-04 represents 8 × 10 in note figure^-4).To obtain representative metabolic pathway, by Protein synthesis, various amino The metabolism of acid or taurine and hypotaurine is excluded, because when cell is by IC₅₀When dosage δ-TOH are acted on, above-mentioned metabolism way Footpath is conventional metabolic pathway, that is to say, that when cell faces the threat being killed, Protein synthesis, various amino The metabolism of acid or taurine and hypotaurine belongs to a kind of conventional metabolic pathway of cell, therefore, and cannot be used as representative The metabolic pathway of property.

Analysis chart 14B, for RAW264.7 cells, glycine betaine is metabolized as its representational metabolic pathway, that is to say, that δ- Influence of the tocopherol to the inferior toxicity of RAW264.7 cells is embodied in glycine betaine metabolic pathway.

Analysis chart 15A, for K562 cells, metabolism of pyruvate is its representational metabolic pathway (path influencing characterisitic value More than 0.1), that is to say, that influence of the Delta-Tocopherol to the inferior toxicity of K562 cells is embodied in metabolism of pyruvate approach.

To sum up, IC of the RAW264.7 cells in δ-TOH₅₀In the presence of dosage, glycine betaine metabolism reduction, this and macrophage Growth, which is suppressed, to be consistent.For K562 Leukaemia, through IC₅₀After the δ-TOH processing of dosage, lactic acid content increase Breathing pattern is caused to be converted to anaerobic respiration.In two kinds of cells, the content of cysteamine is all to reduce.The content of lactic acid exists Increase in K562 cells, but there is no significant change in RAW264.7 cells.

RAW264.7 and K562 is after δ-TOH processing, biosynthesis, glycine betaine metabolism in protein, urea cycle And all changed significantly on breathing pattern.This may illustrate that δ-TOH can be with different metabolic pathways to cell physiological work( Influence can be produced.Influence on δ-TOH to RAW264.7 and K562 cells by otherness metabolite and can be enriched to Path state.These metabolic pathways can explain that δ-TOH act on the influence to its physiological function after cell.However, by The cell acted in δ-TOH is different, and the path of metabolite level influence is also not quite similar.Test result indicates that glycine betaine is metabolized (table 3) is reduced in RAW264.7 cells, and (table 4) is raised in K562 cells.It is well known that glycine betaine is public affairs in biology The methyl donor recognized, it can promote DNA and RNA to synthesize.There are some researches show glycine betaine can suppress RAW264.7 cells To TNF (TNF-α), interleukin-6 (IL-6), nitric oxide synthase type (iNOS), cyclooxygenase-2 (COX-2) secretion of inflammatory factor such as.But also there are some researches show the oxidative stress and inflammation that glycine betaine can alleviate rat are anti- Should.Therefore, the IC that macrophage is acted in δ-TOH₅₀During concentration, cell DNA and RNA synthesis are reduced, but inflammatory factor is actually How change need further research.In chronic cerebral ischemia in rats model, glycine betaine may be by suppressing MDA (MDA) With active oxygen (ROS), while increasing superoxide dismutase (SOD) and glutathione (GSH), oxidative stress water is reduced with this It is flat.Because choline is the precursor of glycine betaine, so the trend that it changes in two kinds of cells is identical with glycine betaine.

All there is taurine and hypotaurine metabolism in the metabolic pathway of two kinds of cellular changes.Taurine is that one kind has The semi-dispensable amino acid of a variety of physiologically actives, it can influence the mitochondrial function of most mammalian cells, Apoptosis with And anti-oxidative defense.There are some researches show taurine can as neuroglial cytoma apoptosis mark, it means that The reduction of hypotaurine is possibly used for synthesizing more taurines in K562 cells, so as to trigger cell in IC₅₀The production of apoptosis It is raw.There are some researches show cystamine has many important metabolins, such as cysteamine, taurine, hypotaurine etc..It is thin at two kinds In born of the same parents, the content of cysteamine is all to reduce, and this might mean that δ-TOH regulate and control in a similar manner in terms of cysteamine metabolism Macrophage and leukaemia.

The content of lactic acid increases in K562 cells, but does not have significant change in RAW264.7 cells.This explanation δ-TOH Aerobic respiration may be converted into anaerobic respiration by promoting metabolism of pyruvate to change K562 cell energy-provision way, reduced thin Born of the same parents energize efficiency.We can also have found that urea cycle weakens in RAW264.7 cells and arginine increases, it means that The tricarboxylic acid cycle related to urea cycle may reduce cell energy supply.On the contrary, being used as urea synthesizing bottom in K562 cells The butanedioic acid of thing then has downward trend.In fact, the accumulation of butanedioic acid may cause cell to open anaerobic respiration pattern.However, The reinforcement of K562 cell anaerobic respirations means that cell more flocks together, and causes the tumor microenvironment of hypoxemia to produce, enters And strengthen the ability of K562 cell invasions and transfer.In summary, after cell is handled through δ-TOH, two kinds of cells are with different Metabolic pathway changes cellular respiration level, and wherein K562 aerobic respiration is converted into anaerobic respiration, causes cancer metastasis ability Enhancing.

To sum up, IC of the RAW264.7 cells in δ-TOH₅₀In the presence of dosage, glycine betaine metabolism reduction, this and macrophage Growth, which is suppressed, to be consistent；At the same time, in δ-TOH IC₅₀Secretion and oxidation of the glycine betaine to inflammatory factor under dosage Stress effect it is also to be studied.For Leukemic Lymphocytes K562, through IC₅₀After the δ-TOH processing of dosage, lactic acid contains Amount increase causes breathing pattern to be converted to anaerobic respiration.This change is probably to be led due to agglomerating flock together of K562 cells Cause, so as to strengthen the ability of cell invasion transfer.The difference of these metabolic pathways may confirm δ-TOH to cancer cell and normal The difference of impact cell.

It is follow-up to be verified by cell experiment, it was demonstrated that IC₅₀Influence of the dosage Delta-Tocopherol to the inferior toxicity of RAW264.7 cells It is embodied in glycine betaine metabolic pathway, IC₅₀Influence of the dosage Delta-Tocopherol to the inferior toxicity of K562 cells is embodied in pyruvic acid generation Thank in approach.

The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, for the skill of this area For art personnel, the present invention can have various modifications and variations.Within the spirit and principles of the invention, that is made any repaiies Change, equivalent substitution, improvement etc., should be included in the scope of the protection.

Claims

1. a kind of IC₅₀Dosage Delta-Tocopherol to the analysis methods of RAW264.7 or K562 cell inferior toxicities, it is characterized in that, including with Lower step：

(2) preparation of cell extraction liquid：

δ-TOH organize the preparation of cell extraction liquid：By RAW264.7 or K562 cells in corresponding Delta-Tocopherol IC₅₀Carried out under dosage Culture, cold methanol is added after culture is quenched cell metabolism, then carries out ultrasonication extraction using methanol chloroform water extraction method, Collect aqueous phase；

The preparation of blank group cell extraction liquid：RAW264.7 or K562 cells are cultivated, cold methanol is added after culture to be made carefully Born of the same parents' metabolism is quenched, and then carries out ultrasonication extraction using methanol chloroform water extraction method, aqueous phase is collected by centrifugation；

(3) nuclear magnetic resonance is pre-processed：

δ-TOH are organized to use after the aqueous phase evaporation with blank group and contain (3- trimethylsilyls) -2,2,3,3- tetra- deuterated third The D of sour sodium (TSP)₂O dissolves, and centrifuges, and freezes；Sample D after will be lyophilized₂The phosphate buffer dissolving that O is prepared, centrifugation, Obtain supernatant；

(4)¹H magnetic resonance detections：The supernatant of the pretreated δ-TOH groups of nuclear magnetic resonance and blank group is subjected to nuclear magnetic resonance Detection, obtains cell nmr spectrum, so as to obtain the signal value of metabolin in RAW264.7 or K562 cell samples；

(5) screening of otherness metabolin：The integral area of cell nmr spectrum is normalized, integrated Area data, differentiates that (PLS-DA) and orthogonal-offset minimum binary differentiate (OPLS-DA) analyze data using offset minimum binary, and Checking screening otherness variable is carried out using arrangement experiment and CV-ANOVA, offset minimum binary is chosen and differentiates first in (PLS-DA) The variable weight importance ranking value of principal component>0.1 metabolin obtains otherness metabolin as otherness metabolin Relative amount value, the variation tendency of each quantitative otherness metabolin；

(6) metabolic pathway is analyzed：The otherness metabolin of determination is imported in capital of a country gene and genome encyclopedia database, Belong to the metabolic pathway of correlation；Otherness metabolin is carried out by metabolism group DAS MetaboAnalyst Metabolic pathway enrichment analysis, obtains metabolic effect analysis path and the metabolism sequence enrichment point for being belonged to by otherness metabolin Analyse path, the path influence value of metabolic effect analysis path>0.1 metabolic pathway and metabolism sequence enrichment analysis path it is logical Road influence value<0.1 metabolic pathway is identified as based on the significantly affected path of Delta-Tocopherol group；It is last from it is described by Representative metabolic pathway is filtered out into the path significantly affected.

2. analysis method as claimed in claim 1, it is characterized in that, in step (1), Delta-Tocopherol pair is determined using mtt assay The IC of RAW264.7 or K562 cells₅₀Dosage, wherein, RAW264.7 IC₅₀For 80 μM, K562 IC₅₀For 110 μM.

3. analysis method as claimed in claim 1, it is characterized in that, in step (2), the volume ratio of methanol, chloroform and ultra-pure water Example is 4：4：2.8~2.9；It is preferred that, the volume ratio of methanol, chloroform and ultra-pure water is 4：4：2.85；

It is preferred that, ultrasound 5 times altogether, ultrasound is stopped one minute for one minute.

4. analysis method as claimed in claim 1, it is characterized in that, in step (3), the specific preprocess method of nuclear magnetic resonance It is：The D for containing 0.1% (w/w) TSP with 900 μ L after the aqueous phase of δ-TOH groups and blank group is evaporated₂O dissolves, and is taken after centrifugation Clear liquid is freezed, by lyophilized sample D₂The phosphate buffer that O is prepared is dissolved to 650 μ L, and supernatant is taken after centrifugation to nuclear-magnetism Guan Zhong, it is to be measured.

5. analysis method as claimed in claim 1, it is characterized in that, in step (4), using outfit¹³C、¹The double optimization 5mm of H CPTCI tri- resonates the super shielding fourier transform NMR spectrometer detection of III 600MHz superconductions of trans CryoProbeTM, Wherein Proton Resonance Frequency is 600.104MHz, and pulse train is zg30, and spectrum width is 12019.230Hz, and accumulative frequency is 256 Secondary, sky adopts number of times for 2 times, and experimental temperature is 298K, and spectrogram is handled using Topspin3.2.

6. analysis method as claimed in claim 1, it is characterized in that, in step (5), specific method is：

Nmr spectrum is handled using MestReNova 6.11, and all spectrograms add index window function；Manual phase correction, After Baseline wander, calibrated with TSP, water peak is cut off manually, selection 0.002ppm is separation standard subsection integral；Spectrogram is accumulated afterwards Facet product is normalized；Integral area data are preserved with Excel file, packet numbering；Afterwards, SIMCA-P+ is imported data to 12.0 in software；Use partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) point Analyse data；In order to ensure the credibility that PLS-DA and OPLS-DA models are set up, verified with arrangement experiment and CV-ANOVA Screen otherness variable；The material that first principal component VIP values in partial least squares discriminant analysis (PLS-DA) are more than 1 is chosen, It is determined as otherness metabolite；Finally by mankind's metabolism group database (HMDB) and biologic NMR database (BMRB) qualitative analysis is carried out, the species name of otherness metabolite is obtained.

7. analysis method as claimed in claim 1, it is characterized in that, in step (6), specific method is：By the difference of determination Property metabolite import encyclopaedical (KEGG) database of capital of a country gene and genome and obtain No. KEGG, then Path enrichment analysis is carried out in MetaboAnalyst (MetPA), obtain the metabolic pathway that is belonged to by otherness metabolin and Sequence enrichment analysis path is metabolized, representative metabolic pathway is obtained finally by the data of metabolism sequence enrichment.

8. following any applications：

(1) glycine betaine anti-metabolite agent is promoted to prepare treatment by IC₅₀The examination of normal cell inferior toxicity caused by dosage Delta-Tocopherol Application in agent；

(2) metabolism of pyruvate reagent is preparing treatment by IC in reduction anaerobic respiration₅₀Cancer cell caused by dosage Delta-Tocopherol is sub- Application in the reagent of toxicity；Or be：Lactic acid antagonist/inhibitor is preparing treatment by IC₅₀Caused by dosage Delta-Tocopherol Application in the reagent of cancer cell inferior toxicity；

(4) Delta-Tocopherol antagonist/inhibitor is converted in the breathing pattern for preparing promotion Leukemic Lymphocytes to aerobic respiration In application.

9. a kind of IC₅₀Dosage Delta-Tocopherol is to the screening technique of the otherness metabolin of RAW264.7 or K562 cells, its feature It is that it comprises the following steps：

(2) preparation of cell extraction liquid：

(3) nuclear magnetic resonance is pre-processed：

δ-TOH are organized to use after the aqueous phase evaporation with blank group and contain (3- trimethylsilyls) -2,2,3,3- four deuterated third The D of sour sodium (TSP)₂O dissolves, and centrifuges, and freezes；Sample D after will be lyophilized₂The phosphate buffer dissolving that O is prepared, centrifugation, Obtain supernatant；

(5) screening of otherness metabolin：The integral area of cell nmr spectrum is normalized, integrated Area data, differentiates that (PLS-DA) and orthogonal-offset minimum binary differentiate (OPLS-DA) analyze data using offset minimum binary, and Checking screening otherness variable is carried out using arrangement experiment and CV-ANOVA, offset minimum binary is chosen and differentiates first in (PLS-DA) The variable weight importance ranking value of principal component>0.1 metabolin is used as otherness metabolin.

10. the IC that screening technique according to claim 9 is obtained₅₀Dosage Delta-Tocopherol is to RAW264.7 or K562 cells Otherness metabolin, it is characterized in that：

The otherness metabolin of RAW264.7 cells is acetic acid, lysine, choline, arginine, alanine, methanol, cysteamine, sweet tea Dish alkali and isobutyric acid；

The otherness metabolin of K562 cells be hypotaurine, threonine, methanol, glucolactone, cysteamine, glucocyamine, Lactic acid, choline, glycine betaine, butanedioic acid.