CN102175809A - New method for performing data correction by using cell metabolite relative content as cell number index - Google Patents
New method for performing data correction by using cell metabolite relative content as cell number index Download PDFInfo
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Abstract
The invention discloses a new method for performing data correction by using cell metabolite relative content as cell number index. The new method is characterized by comprising the steps of: realizing the fast quenching of biochemical metabolism in cells by using a simple sample pre-processing method; efficiently extracting small molecule metabolites by an optimized extraction solvent; and roundly and half quantitatively measuring the small molecule compounds in a biological sample by a metabonomics method based on measuring technology like mass spectrum, nuclear magnetic resonance and the like so as to identify a quantitative index of symbolic metabolites in cells to be as the cell number, and regarding the quantitative index as a weight factor to perform data correction. Compared with the quantitative index of the prior cell number, the method is simple, fast and with good repeatability; additional experimental working and the error caused by the additional working are reduced; the accuracy and reliability of experiment result are improved; the difficulty for relatively quantifying the number of in vitro cultured cells is solved; a new displacing method is supplied for cell metabonomics and relevant researches; and the method can be widely used in the relevant researches in the field of cell molecular biology.
Description
Technical field
The new method that the present invention relates to utilize cultured cell in vitro to carry out relevant metabolism group research such as disease, drug effect, drug toxicity, the interior micromolecular compound high efficiency extraction of quick quencher, cell, cell metabolite relative content that is specifically related to the cultured cell in vitro biochemical metabolism is as the cell number quantitative target and be used for the method for the metabolism group adjustment of data.
Background technology
At present, the method for cellular metabolism group research is still immature, and especially the metabolism group research of the attached cell of in vitro culture relatively lags behind, and has a series of problem, mainly shows following three aspects:
1.
The cell quantity quantitative problemThe difference of different cell proliferation rates, allogenic cell is subjected to the influence of initial inoculum density and different disposal method (environment or medicine irritation), the cell number that can cause different cell lines or allogenic cell strain to be grown under different conditions obtaining there are differences, and needs the pair cell quantity variance to proofread and correct when carrying out data processing.And be used for generally at present that methods such as common method that cell quantity proofreaies and correct such as cell count, wet cell weight weighing, protein content mensuration are not only time-consuming, (weigh as counting and cell not only needs to carry out additional experiments to effort, and attached cell also produces the skew that metabolism is composed inevitably under the digestion of pancreatin; The determining the protein quantity method need be carried out additional experiments, goes back quantity consumed simultaneously originally with regard to limited cell sample), cause the variation of various metabolite levels in the cell, also postponed the quencher of biochemical metabolism in the cell, cause the deviation of correction data.
2.
The extraction efficiency problemThe pre-treating method of biological specimen greatly influences the metabolism group result of study, particularly wherein the extracting method of micromolecular compound directly influences the extraction efficiency (recovery) and the precision (repeatability) of micromolecular compound in the cell, thereby the metabolism group result is exerted an influence.Screening suitable extraction solvent is to stablize and one of key issue that extracts the micromolecule metabolin in the cell fully.
3.
The problem of the quick quencher of biochemical metabolism in the cellIn order accurately to obtain micromolecular compound quantitative information in the cell, need ongoing biochemical metabolism in the quick quencher living cells, otherwise cause the skew of cellular metabolism spectrum easily, influence the reliability of experimental result.Because most cultured cell in vitro have the characteristic of adherent growth, how quick harvesting also carries out the metabolism quencher rapidly also is key issue for the metabolism group analysis.
In a word, the method limitation of existing cellular metabolism group research is bigger, on the one hand, existing cell quantity bearing calibration complex operation, cause endocellular metabolism thing content deviation easily, on the other hand the cell pre-treating method complexity of existing adhere-wall culture, be difficult to carry out metabolism quencher fast, easy drawing-in system error, press for and found new research method.
The metabolism quencher is fast more, and pre-treatment step is simple and easy more, and the result of study of metabolism group is also just reliable more.Consider that intracellular metabolite level should have positive correlation with cell quantity, seek micromolecule metabolin in the cell and substitute the possibility that weight that protein content is used for the metabolism group data as the index of cell quantity has reality.Studies show that, be determined as example with GC/TOFMS, it is relevant that the relative content that a lot of micromolecule metabolins comprise nearly all amino acid, glycitols compound, fatty acid, small molecular organic acid class, nucleosides and purine compound, ammoniac compounds in the cell and cell quantity/protein content are favorable linearity.Correlativity between some of them metabolite level and the cell quantity also demonstrates the characteristic that is independent of cell line, promptly all has linear dependence in different cell lines, as inositol, pantothenic acid, cholesterol etc.These potential significant metabolins may all have good universality to various kinds of cell, are the candidate markers of cell quantity, are expected to be used for to substitute protein content as the index of cell quantity and be used for the correction and the analysis of related experiment data.
Summary of the invention
Technical matters to be solved by this invention is: improve existing cell harvesting and metabolism method for quenching in the research of cellular metabolism group, optimize the scheme of solvent extraction, the micromolecule metabolin that utilization identifies is used for the weight of gained data as the index of cell number, solves a series of research difficult problems of existence in the research of cellular metabolism group.
For addressing the above problem, the invention provides following technical scheme, step comprises:
Sample collection and processing: cell is incubated in cell plates or the double dish according to standard practice instructions, treat that cell grows to suitable quantity, discard nutrient culture media and with the rapid drip washing cell surface of 0.5 ℃ of physiological saline of 2.5mL 2 times, in every duplicate samples, add the ultrapure water (size and cell quantity according to the cultivation vessel that adopted are taken all factors into consideration) of suitable volumes then, again sample is placed-70 ℃ refrigerator to preserve rapidly.The lysis suspension adds appropriate amount of organic and extracts behind multigelation 3 times, organic solvent comprises ethyl acetate, chloroform, ether, normal butyl alcohol, sherwood oil, methylene chloride, acetonitrile; Or through albumen precipitation, the method for albumen precipitation comprises that adding organic solvent independent or comprehensive modes such as (as methyl alcohol, ethanol, acetone, acetonitrile, isopropyl alcohols) handles; Sample can not carry out drying, or first drying is utilized all kinds of solvent dissolvings that contain organic solvent and water (saliferous or not saliferous separately or comprehensive) again; Sample does not carry out derivatization or utilizes reagent to carry out derivatization treatment.
Sample analysis and data processing: adopt based on mass spectroscopy and chromatograph joint used technical method or the assay method of nuclear magnetic resonance technique above-mentioned sample is detected, these methods can be liquid chromatography-mass spectrography, gas chromatography-mass spectrum, Capillary Electrophoresis chromatography-mass spectroscopy, nuclear magnetic resonance etc.; Through obtaining the quantitative data at each peak in the chromatogram after the data processing, these data can be peak area or peak height, or the quantitative data that is calculated by peak area or peak height.
An outstanding advantage of the present invention is can simple, convenient and rapidly make the cellular metabolism quencher by said method, obtain cell metabolism spectrum at once, the accuracy and the reliability of experimental result have been guaranteed, and sample once measured the correction/weight factor that can obtain the whole metabolism spectrum of cell and each sample, not only simple and efficient but also systematic error and the personal error of avoiding additional experiments (as cell count, determination of protein concentration) to be introduced of this method.
Beneficial effect of the present invention:
In the research of cultured cell in vitro metabolism group owing to can not carry out the metabolism quencher timely and effectively, tend to cause the change of cellular metabolism pattern, fast and convenient sample treatment comprises that effective cell harvesting and metabolism quencher fast are to guarantee that result of study is accurate, crucial reliably.The method complex operation that existing in addition cell count, protein quantification etc. are proofreaied and correct as cell quantity, cause endocellular metabolism thing content deviation easily, and the method for utilizing significant metabolin in the cell is easy, quick, its adjustment of data effect and traditional correction factor (as protein content, cell count) are suitable, demonstrate bigger superiority.
1. utilize improved cell pre-treating method can high efficiency extraction endocellular metabolism thing, effectively carry out the metabolism quencher, can improve the accuracy that metabolin detects on the one hand, prevent the deviation that the metabolism quencher not exclusively causes; Also simplify the process of complicated cell harvesting, cell metabolite extraction on the other hand, reduced operation and systematic error.
2. utilize significant metabolin in the cell to be used for the research of cellular metabolism group as the weight factor of cell quantity and can simplify experimental implementation, need not to carry out additional experiments and just can obtain weight factor, the while also reduces error at measurment and personal error.
3. significant metabolin is compared with protein content as weight factor in the employing cell, and the means that the micromolecule metabolite content is measured are more versatile and flexible, as LC-MS, and GC-MS etc., the sensitivity of its detection, reappearance and the range of linearity also improve greatly.
4. the interior significant metabolin of employing cell is weighed with cell count or cell and is compared, and quencher cellular biochemical metabolism rapidly reduces unnecessary experimental implementation, strengthens result's accuracy and reliability.
Description of drawings
Fig. 1 Martin reaches (the Madin-Darby canine kidney than dog renal epithelial cell system, MDCK) wild type (a, MDCK-WT) and the transfection type (b of transfection people Mdr1 gene, MDCK-MDR) GC/TOFMS measures total ions chromatogram, can find 252 chromatographic peaks in the chromatogram, compounds identified has 94.
Linear regression curve in the born of the same parents of Fig. 2 cell quantity between significant metabolin-malic acid, inositol, pantothenic acid and cholesterol and the protein content
A, the integral data in D:MDCK-WT and the MDCK-MDR cell
B, E: only comprise the data in the MDCK-WT cell
C, F: only comprise the data in the MDCK-MDR cell
Linear regression curve between born of the same parents' OM outer marker metabolin-inositol, serine and the sorbierite and the cell number/protein content of Fig. 3 cell quantity
A: the linear regression curve between born of the same parents' OM outer marker metabolin and the protein content
B: the linear regression curve between born of the same parents' OM outer marker metabolin and the cell number
The metabolism group data of Fig. 4 varying number cell sample are (A) and the sample distribution scatter diagram after the significant metabolin-pantothenic acid (C) of protein content (B) and the cell number that identifies and inositol (D) are proofreaied and correct before weight.The metabolism spectrum of MDCK-WT and MDCK-MDR cell is along with the different tracks that are dynamic change of cell quantity, and the difference of every kind of cell on the 1st major component and the 2nd major component all obviously reduces after inositol and the pantothenic acid weight, the point that looses in the group is assembled more, show that weight has reduced the metabolite level difference that cell quantity causes effectively, thereby increased the validity of model, and the weight effect can compare favourably with the protein content weight.
Embodiment
The present invention carries out detailed explanation by the following examples, but and does not mean that the present invention only limits to this.
Embodiment
Correction-Martin that cell metabolite is used for cellular metabolism group data as the index of cell quantity reaches that (Madin-Darby canine kidney, MDCK) the metabolism group example in transfection type (MDCK-MDR) cell of wild type (MDCK-WT) and transfection people Mdr1 gene is studied than dog renal epithelial cell system
1. experimental program and sample collection
The cell suspension of series volume (A-F:2.5,2.0,1.6,1.2,0.9,0.6mL, MDCK-WT 600, and 000/mL and MDCK-MDR 800 000/mL) are inoculated in (n=4) in six orifice plates, replenish blank nutrient culture media and make that every aerial volume is 2.5mL.Two cell lines place to grow about 3 days under the identical condition of culture does not change nutrient solution.Collect the media samples of every porocyte, cell then carries out pre-treatment according to the method described above, and is frozen to be measured in-70 ℃ refrigerator behind the ultrapure water of adding 300 μ L in every hole.
2. sample preparation
Cell sample: the water-splitting liquid of cell is behind multigelation 3 times, and the 20 μ L that take out wherein are used for determining the protein quantity, and all the other add 840 μ L and contain mark (IS: stable isotope in the 0.5 μ g/mL
13The meat bandit acid of C mark) methyl alcohol extracts and protein precipitation.Every part of cell fragment suspension was transferred to the shell pipe mesoscale eddies concussion of a 1.5mL after 5 minutes, 4 ℃ with 19600g centrifugal 5 minutes.The 400 μ L supernatants that shift equal portions are in the GC bottle, and decompression volatilizes.
Media samples: pipette the collected nutrient culture media of 50 μ L, add after 200 μ L contain the methanol extraction albumen of IS, place 4 ℃ refrigerator 1 hour.Then 4 ℃ with 19600g centrifugal 5 minutes, every duplicate samples shifts 100 μ L supernatants in the GC bottle, decompression volatilizes.
Add 30 μ L methoxamine pyridine solutions (10mg/mL) in the aforementioned GC bottle that contains cell/nutrient culture media extract, vortex vibration 3min leaves standstill 16h and carries out oximate under the room temperature.Add 30 μ L TMS trifluoroacetyl MSTFA (containing 1% TMCS) as catalyzer, vortex vibration 1min, room temperature leaves standstill 1h and carries out derivative reaction, adds the n-heptane solution (30 μ g/mL) of 30 μ L external standard methyl meat bandit acid esters at last again, carries out GC/TOFMS after the mixing and detects.
3.GC/TOFMS measure
Instrument: (GC:Angilent 6890N gas chromatograph is equipped with Angilent 7683B automatic sampler and 100 sample feeding dishes of G2614A type to gas chromatography-flight time mass spectrum (GC/TOFMS) monitoring system; TOF-MS:Pegasus III, Leco, USA).
Chromatographic condition: chromatographic column is DB-5 quartz capillary column (10m * 0.18mm i.d., J﹠amp; W Scientific, USA), sample size: 1 μ L, adopt the original mold formula that flows to that is regardless of; Carrier gas: helium; Constant current speed: 1mL/min; The temperature programme pattern: 70 ℃ keep 2.0min, with 35 ℃/min linearity linear temperature increase to 305 ℃ at the uniform velocity, keep 2.0min then.
Mass spectrum condition: injector temperature: 250 ℃; Scavenging period and flow velocity: 1min, 20mL/min; Transfer tube temperature: 250 ℃; Ion source temperature: 200 ℃; Ion gun voltage and current :-70eV, 3.0mA; MS adopts the full scan mode to carry out data acquisition, and MS adopts the full scan mode to carry out data acquisition; Sweep limit: m/z 50-800; Sweep velocity: 20spectra/s; Detecting voltage is-1690v.
Measurement result: the GC/TOFMS total ions chromatogram (Fig. 1) that under above-mentioned testing conditions, can obtain sample.The software (ChromaTOF 2.00) that utilizes instrument to carry can extract each chromatographic peak peak area in each working sample chromatogram, form a data matrix of forming by peak area, this data matrix comprises MDCK-WT (WA-WF) and MDCK-MDR (RA-RF) totally 72 samples (being first row) and 252 chromatographic peaks (being first row), is kept in the excel file.
Fig. 1 Martin reaches (the Madin-Darby canine kidney than dog renal epithelial cell system, MDCK) wild type (MDCK-WT) and transfection type (transfection people Mdr1 gene, MDCK-MDR) GC/TOFMS measures total ions chromatogram, can find 252 chromatographic peaks in the chromatogram, compounds identified has 94.
4. the significant metabolin of cell number is identified
By PLS correlation analysis and linear regression analysis, identify intracellular pantothenic acid, inositol, cholesterol and malic acid in two cell lines all with protein content high-positive correlation (r＞0.970), and this correlativity is independent of cell line, and correlation regression curve and parameter are seen Fig. 2 and table 1 respectively.Similarly, find in extracellular fluid, inositol and serine content and cell quantity negative correlation, and sorbierite and cell number are proportionate, its linear fit result is as shown in Figure 3.
5. the checking of weight factor validity
With above-mentioned data call in multivariate data analysis soft sim CA P-11 (Umetrics AB,
Sweden) in, utilize the PCA modeling, by of the influence of sample scatter diagram observation group cell quantity to its metabolism spectrum, assess the validity of weight by the loose validity of the gathering situation of point and model of sample between group between before and after the weight relatively, the result as shown in Figure 4, show significant born of the same parents' intracellular metabolite thing particularly pantothenic acid and inositol can effectively reduce the metabolite level difference that cell quantity causes, the effect of its weight effect and albumen weight is suitable.In addition, RSD value result's demonstration relatively in the group of each metabolin before and after the weight: the weight effect that is similar to protein, after significant metabolin especially pantothenic acid and inositol are proofreaied and correct in two cell lines in the group of most metabolite levels the RSD value all obviously reduced, thereby confirmed that further the cell metabolite relative content carries out the feasibility of the adjustment of data as the cell quantity index.
The significant metabolin of table 1. cell number and the linear regression parameter between the protein content
[notes] R﹠amp; Integral data in W:MDCK-WT and the MDCK-MDR cell; W: only comprise the data in the MDCK-WT cell; R: only comprise the data in the MDCK-MDR cell.
Claims (9)
1. the micromolecule metabolin is used for the weight of cellular metabolism group data as the cell number index in the application cell, and its feature comprises the following aspects:
A) adopt the cultured cell in vitro sample to detect;
B) the method smudge cells of employing multigelation, micromolecule extracts in the method pair cell of methanol extraction albumen;
C) adopt the method for TMS trifluoroacetyl MSTFA that sample is carried out derivatization;
D) adopt gas chromatography-flight time mass spectrum (GC/TOF-MS) to carry out sample analysis;
E) adopt the interior inositol of cell, pantothenic acid, cholesterol, malic acid that the metabolism group data are proofreaied and correct.
F) adopt cell culture fluid mysoinositol, pantothenic acid that the metabolism group data are proofreaied and correct.
2. the cultured cell in vitro sample in right requirement 1, also be applicable to other cultured cell, primary cell, original position histocyte etc.
3. require in 1 also to be applicable to the weight and the analysis of other molecular cell biological experimental data except that right as the metabolism group data weighting factor.
4. except that right requires the method smudge cells of multigelation in 1, also comprise the method for other method such as ultrasonication, hypertonic salt solution, organic solvent fragmentation; Except that the method for methanol extraction albumen is extracted the cell sample, also comprise through other albumen precipitation method, as add organic solvent (as ethanol, acetone, acetonitrile, isopropyl alcohol) or carry out liquid-liquid extraction through organic solvent, organic solvent comprises ethyl acetate, chloroform, ether, normal butyl alcohol, sherwood oil, methylene chloride, benzene, normal hexane, cyclohexane, acetonitrile.And the independent or comprehensive mode of method is handled in the claim 4.
5. except that right requires the derivatization method of TMS trifluoroacetyl (MSTFA) in 1, comprise that also other any commercial reagent carries out the method for derivatization treatment; Or do not carry out the method for any derivatization treatment.
6. require to adopt gas chromatography-flight time mass spectrum (GC/TOF-MS) to carry out the sample analysis in 1 except that right, also comprise other any analytical approach based on mass spectroscopy technology, nuclear magnetic resonance measuring technology and other metabolism group determination techniques.
7. resulting data can be the absolute quantitation data in the claim 1, also can be the sxemiquantitative data, as peak area, peak height, with the positive peak area of interior calibration, peak height, or carry out the data that mathematical computations obtains thus.
8. except that right requires compound that 1 mysoinositol, pantothenic acid, cholesterol, malic acid proofread and correct the metabolism group data, also comprise the detected intracellular matter that does not identify or identified of other any analytical approach, as Beta-alanine, hypoxanthine etc. based on mass spectroscopy technology, nuclear magnetic resonance measuring technology and other metabolism group determination techniques.
9. except that right requires compound that cell culture fluid mysoinositol in 1, pantothenic acid proofread and correct the metabolism group data, also comprise compound in the detected cell nutrient solution that does not identify or identified of other any analytical approach, as sorbierite, serine, inositol etc. based on mass spectroscopy technology, nuclear magnetic resonance measuring technology and other metabolism group determination techniques.
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Application publication date: 20110907 |