CN107202810B - A kind of analysis IC50Influence method of the dosage Delta-Tocopherol to RAW264.7 and K562 cell - Google Patents

A kind of analysis IC50Influence method of the dosage Delta-Tocopherol to RAW264.7 and K562 cell Download PDF

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CN107202810B
CN107202810B CN201710384527.2A CN201710384527A CN107202810B CN 107202810 B CN107202810 B CN 107202810B CN 201710384527 A CN201710384527 A CN 201710384527A CN 107202810 B CN107202810 B CN 107202810B
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cell
analysis
metabolism
tocopherol
otherness
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CN107202810A (en
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耿越
陆洋
李慧
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山东师范大学
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Abstract

The invention discloses one kind to be based on1H NMR metabolism group method analyzes IC50Influence method of the dosage Delta-Tocopherol to RAW264.7 and K562 cell, including following methods: (1) determining Delta-Tocopherol to the IC of RAW264.7 or K562 cell50Dosage;(2) preparation of cell extraction liquid;(3) nuclear magnetic resonance pre-processes;(4)1H magnetic resonance detection;(5) screening of otherness metabolin;(6) metabolic pathway is analyzed.Present invention application hydrogen composes nuclear magnetic resonance technique combination metabolism group method, has studied the influence of different Cell differentials metabolins and metabolic pathway, provides a kind of Delta-Tocopherol of studying to the analysis method of the inferior toxicity of RAW264.7 or K562 cell.

Description

A kind of influence for analyzing IC50 dosage Delta-Tocopherol to RAW264.7 and K562 cell Method

Technical field

The present invention relates to be 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 technique

The bioactivity of vitamin E:

Vitamin E is molecule necessary to maintaining the metabolism and every physiological function of human normal.Also, vitamin E human body can not generate itself, need to absorb by food from the external world.Rich food containing vitamin E includes almond, fibert, big Beans and avocado etc..Vitamin E also known as tocopherol, position and quantity according to methyl in chroman ring can be divided into following eight kinds Four kinds of α-TOH, β-TOH, γ-TOH, δ-TOH tocopherols;Four kinds of α-TT, β-TT, γ-TT, δ-TT tocotrienols.Wherein α- TOH bioactivity is maximum, and the activity of γ-TOH and δ-TOH only account for the 10% and 1% of α-type respectively;However, oxidation resistance δ- TOH is most strong, is 100 times of α-TOH oxidation resistance.Vitamin E has different physiological roles, anti-ageing including anti-oxidant Always, frostbite is treated, blood vessel is protected, protects cell membrane, even signal transduction and gene expression etc..Vitamin E can also pass through tune It controls signal path alleviation obesity and its Metabolic complication, the access of influence includes Wnt, JAK-Stat (the Janus Kinase-signal transducer and activator of transcription), PI3K (the 3 '-kinase of phosphatidylinositol)/Akt/mTOR (mammalian target of rapamycin) signal is logical Road, and these signal path many are all related to tumor promotion.There is researcher to find that γ-TOH can be by inhibiting to have recently Oxygen glycolysis inhibits tumour.And it has been reported that the different subtype of vitamin E has different generations in vivo and in vitro Thank to approach.According to WHO (World Health Organization) statistics indicate that normal adult is to vitamin E (δ- Tocopherol recommended intake (Recommended Nutrient Intakes, RNIs)) is daily 10mg, be can tolerate Highest intake (Tolerable Upper Intake Level, UL) is 1000mg.Nano silver produces Mouse Liver primary cell Raw toxicity can be protected by the vitamin E of doses.In the toxicity to mouse difference canceration period galactophore epithelial cell It was found that, the IC of the galactophore epithelial cell δ-TOH in precancerous lesion period, tumour period and malignant tumour period50Dosage point Wei not be 55 μM, 47 μM and 23 μM.Some researches show that vitamin E can be modified white due to caused by certain chemotherapeutics very early Leukopenia, so that the chemotherapeutic treatment to cancer plays booster action.The tocopherol of different subtype is to macrophage RAW264.7 IC50Concentration is also not quite similar, and δ-TOH influences the cell activity of mouse macrophage RAW264.7 to be greater than α-as the result is shown The influence of TOH.Excessive vitamin E to cell will cause how the influence in terms of metabolism group, seldom have been reported that research.

The related application of metabolism group:

Metabolism group has extensively and mature application in every field at present, including treatment of cancer and Chinese medicine at Divide research etc..These are all based on Molecular characterization on metaboilic level and determine quantifier elimination.Along with the development of metabolism group, The otherness of cancer cell and normal cell also gradually determines.Some researches show that candidate stem cell shifts the side for the treatment of cancer recently Formula may cause myelodysplastic syndrome or acute myeloid leukemia, this process includes alanine and asparagus fern ammonia The tune of the metabolic pathways such as acid metabolic, dicarboxyl acid metabolic, phenylalanine metabolism, citrate cycle and aminoacyl tRNA biosynthesis Control.At the same time, relatively few for the research ratio K562 cell line in terms of RAW264.7 cell line metabolism group.It is most of The influence of δ-TOH and γ-TOH to its bioactivity is laid particular emphasis on to the research in macrophage metabolism group direction.

Therefore, it is necessary to study δ-TOH to drench in mouse macrophage RAW264.7 and chronic myelogenous leukemia pleural effusion The IC of bar cell K56250When concentration, influence to different Cell differentials metabolites and metabolic pathway, this facilitate into The inferior toxicity of one step understanding δ-TOH.

Summary of the invention

Aiming at the shortcomings in the prior art, the 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 toxicity.

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

A kind of IC50Analysis method of the dosage Delta-Tocopherol to RAW264.7 or K562 cell inferior toxicity, comprising the following steps:

(1) determine Delta-Tocopherol to the IC of RAW264.7 or K562 cell50Dosage;

(2) preparation of cell extraction liquid:

The preparation of δ-TOH group cell extraction liquid: by RAW264.7 or K562 cell in corresponding Delta-Tocopherol IC50Dosage Under cultivated, cold methanol is added after culture is quenched cell metabolism, and it is broken then to carry out ultrasound using methanol chloroform water extraction method Broken extraction, is collected by centrifugation water phase;

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

(3) nuclear magnetic resonance pre-processes:

It is used after the water phase rotation of δ-TOH group and blank group is evaporated and contains (3- trimethylsilyl) -2,2,3,3- The D of four deuterated sodium propionates (TSP)2O dissolution, is centrifuged, freeze-drying;By the sample D after freeze-drying2The phosphate buffer that O is prepared Dissolution, centrifugation, obtains supernatant;

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

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

(6) metabolic pathway is analyzed: determining otherness metabolin is imported capital of a country gene and genomic encyclopedia data In library, belong to relevant metabolic pathway out;Otherness is metabolized by metabolism group Data Analysis Software MetaboAnalyst Object carries out metabolic pathway enrichment analysis, obtains being belonged to by otherness metabolin metabolic effect analysis path and metabolism sequence out Column enrichment analysis path, the metabolic pathway and metabolism sequence enrichment point of access influence value > 0.1 of metabolic effect analysis path The metabolic pathway for analysing access influence value < 0.1 of access is identified as the access significantly affected based on Delta-Tocopherol group; Finally representative metabolic pathway is filtered out from the significantly affected access.

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

(1) promote glycine betaine anti-metabolite agent in preparation treatment by IC50Normal cell inferior toxicity caused by dosage Delta-Tocopherol Reagent in application;

(2) metabolism of pyruvate reagent in anaerobic respiration is reduced to treat in preparation by IC50Cancer caused by dosage Delta-Tocopherol is thin Application in the reagent of born of the same parents' inferior toxicity;Or are as follows: lactic acid antagonist/inhibitor is in preparation treatment by IC50Dosage Delta-Tocopherol draws Application in the reagent of the cancer cell inferior toxicity risen;

(3) Delta-Tocopherol antagonist/inhibitor promotes the application in glycine betaine anti-metabolite agent in the mammalian body in preparation;

(4) Delta-Tocopherol antagonist/inhibitor promotes the breathing pattern of cancer cell into aerobic respiration conversion in preparation Using.

Wherein, promoting glycine betaine anti-metabolite agent is that can promote the reagent that glycine betaine is metabolized in internal cell, so that cell The content of intracorporal glycine betaine increases.

Reducing metabolism of pyruvate reagent in anaerobic respiration is the reagent for being able to suppress metabolism of pyruvate in anaerobic respiration;Lactic acid Antagonist/inhibitor is that can reduce lactic acid concn/content reagent in cell.Delta-Tocopherol antagonist/inhibitor is can Reduce IC in cell50Dosage is greater than IC50Dosage Delta-Tocopherol concentration/content reagent;Described be small in the mammalian body Mouse macrophage RAW264.7 or human leukemia lymphocyte K562;The normal cell is mouse macrophage RAW264.7, the cancer cell are people's 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 cell The screening technique of property metabolin, comprising the following steps:

(1) determine Delta-Tocopherol to the IC of RAW264.7 or K562 cell50Dosage;

(2) preparation of cell extraction liquid:

The preparation of δ-TOH group cell extraction liquid: by RAW264.7 or K562 cell in corresponding Delta-Tocopherol IC50Dosage Under cultivated, cold methanol is added after culture is quenched cell metabolism, and it is broken then to carry out ultrasound using methanol chloroform water extraction method Broken extraction, is collected by centrifugation water phase;

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

(3) nuclear magnetic resonance pre-processes:

Contain four deuterium of (3- trimethylsilyl) -2,2,3,3- for using after the water phase evaporation of δ-TOH group and blank group For the D of sodium propionate (TSP)2O dissolution, is centrifuged, freeze-drying;By the sample D after freeze-drying2The phosphate buffer that O is prepared is molten Solution, centrifugation, obtains supernatant;

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

(5) screening of otherness metabolin:

The integral area of cell nmr spectrum is normalized, integral area data are obtained, using partially most Small two multiply differentiation (PLS-DA) and orthogonal-offset minimum binary differentiation (OPLS-DA) analysis data, and using arrangement experiment and CV- ANOVA carries out verifying screening otherness variable, chooses the variable weight that offset minimum binary differentiates first principal component in (PLS-DA) The metabolin of importance ranking value > 0.1 is 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 cell Property metabolin, comprising:

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

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

Compared with prior art, technical solution of the present invention has the following beneficial effects:

(1) present invention composes nuclear magnetic resonance technique combination metabolism group method using hydrogen, has studied different Cell differentials generations The influence for thanking object and metabolic pathway provides a kind of research Delta-Tocopherol to the inferior toxicity of RAW264.7 or K562 cell Analysis method.

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

(3) nuclear magnetic spectrogram carries out multi-variate statistical analysis through MestReNova 6.11 and SIMCA-P+12.0 software and establishes PCA Model, PLS-DA model and OPLS-DA model, discovery δ-TOH group and blank group metabolin can be distinguished obviously, can be carried out The ownership and access of otherness metabolite are enriched with.

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

Detailed description of the invention

The Figure of description for constituting a part of the invention is used to provide further understanding of the present invention, and of the invention shows Examples and descriptions thereof are used to explain the present invention for meaning property, does not constitute improper limitations of the present invention.

Fig. 1 is influence of the δ-TOH to RAW264.7 and K562 cell activity, note:* *P < 0.005vs blank group.

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

Fig. 3 is influence of the ethyl alcohol to cell activity of co-solvent concentration.

Fig. 4 is RAW264.7 cell1H-NMR spectrum.

Fig. 5 is K562 cell1H-NMR spectrum.

Fig. 6 is the PCA shot chart of RAW264.7 group.

Fig. 7 is the PCA shot chart of K562 group.

Fig. 8 is the PLS-DA shot chart of RAW264.7 group.

Fig. 9 is the PLS-DA shot chart of K562 group.

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

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

Figure 12 is the OPLS-DA shot chart of RAW264.7 group.

Figure 13 is the OPLS-DA shot chart of K562 group.

Figure 14 is to be analyzed in RAW264.7 cell by the metabolic pathway that otherness metabolite belongs to out;Wherein, A is Metabolic pathway out is belonged to by otherness metabolite in RAW264.7 cell;(1) biosynthesis of aminoacyl tRNA.(2) sweet ammonia Acid, serine and threonine metabolism.(3) L-lysine amino acid synthesizes.(4) biotin is metabolized.(5) taurine and hypotaurine generation It thanks.(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 from top to bottom respectively as follows: biosynthesis, the sweet tea of protein The metabolism of dish alkali, urea cycle, Methionine metabolism, biotin metabolism, alanine metabolism, taurine and hypotaurine metabolism, glucose Sugar-alanine cycle, contains selenoaminoacid metabolism, phosphatide biosynthesis, metabolism of pyruvate, glycine serine at lysine degradation With threonine metabolism, arginine and Proline Metabolism.

Figure 15 is to be analyzed in K562 cell by the metabolic pathway that otherness metabolite belongs to out;A be in K562 cell by Otherness metabolite belongs to metabolic pathway out;(1) glycine, serine and threonine metabolism.(2) taurine and Ya Niu Sulfonic acid metabolism.(3) biosynthesis of valine, leucine and isoleucine.(4) glycolysis and gluconeogenesis.(5) phosphoric acid Pentose 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.B is that K562 products of cellular metabolism group is rich Collection summarizes figure;From top to bottom it is respectively as follows: glycine serine and threonine metabolism, taurine and hypotaurine metabolism, glycine betaine Metabolism, Methionine metabolism, pentose phosphate pathway, Protein synthesis, phospholipid metabolism, metabolism of pyruvate, arginine and dried meat ammonia Acid metabolic, gluconeogenesis.

Specific embodiment

It is noted that described further below be all exemplary, it is intended to provide further instruction to the present invention.Unless another It indicates, all technical and scientific terms used herein has logical with general technical staff of the technical field of the invention The identical meanings understood.

It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root According to exemplary embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singular shape Formula be also intended to include plural form, additionally, it should be understood that, when in the present specification use term "comprising" and/or When " comprising ", existing characteristics, step, operation and/or their combination are indicated.