CN113390666B - Method for detecting performance index of chemical substance in cell - Google Patents
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Abstract
The invention discloses a method for detecting performance indexes of chemical substances in cells, which comprises the following steps: s1, preparing a sample slice; s2, sequentially carrying out MS detection, electrophysiology detection, western blot detection and immunofluorescence detection on the sample slice to obtain a target detection data set corresponding to the target sample slice, wherein the target detection data set comprises: MS data, electrophysiological data, western blot data, and immunofluorescence data; s3, analyzing the target detection data to obtain the performance index of the compound in the sample slice; the invention can carry out multiple detections based on the same sample slice, on one hand, the influence of other external factors on the detection can be reduced, so that different experimental results can be provided for the same experimental object, on the other hand, the requirements of different detections can be met, and the preliminary research direction of chemical substances in cells can be provided.
Description
Technical Field
The invention relates to the technical field of human biology, in particular to a method for detecting performance indexes of chemical substances in cells.
Background
With the development of science and technology, research on biological chemicals, especially intracellular chemicals, is becoming more and more important, and can contribute to human development in different fields, for example, many diseases can be overcome in the medical field, and research on intracellular chemicals cannot be separated from the detection process of intracellular chemicals, and only accurate detection can better understand the performance and application of chemicals.
At present, chemical substances in cells can be analyzed according to different experimental results to obtain different research results, and a set of system cannot be formed in the prior art for detecting the chemical substances in the cells so as to obtain a primary judgment direction for the chemical substances in cells with different components or contents and further research, so that misleading in the research on the chemical substances in the cells and waste of research and development resources and time are avoided.
Disclosure of Invention
In order to solve the problems in the prior art, multiple detections can be carried out based on the same sample slice, so that on one hand, the influence of other external factors on the detections can be reduced, different experimental results can be obtained for the same experimental object, on the other hand, different detection requirements can be met, and a preliminary research direction of chemical substances in cells can be provided; the embodiment of the invention provides a method for detecting performance indexes of chemical substances in cells, which comprises the following steps:
s1, preparing a sample slice;
s2, sequentially carrying out MS detection, electrophysiology detection, western blot detection and immunofluorescence detection on the sample slice to obtain a target detection data set corresponding to the target sample slice, wherein the target detection data set comprises: MS data, electrophysiology data, western blot data, and immunofluorescence data;
and S3, analyzing the target detection data to obtain the performance index of the compound in the sample slice.
Further, S1 further includes the steps of:
taking a brain tissue sample of a target subject;
placing the brain tissue sample in a slicing solution for culture treatment, wherein the slicing solution comprises the following components: sucrose, naCl, naHCO 3 、MgCl 2 Glucose, KCl and NaH 2 PO 4 ；
And slicing the cultured brain tissue sample to obtain the sample slice.
Further, sucrose, naCl, naHCO 3 、MgCl 2 Glucose, KCl and NaH 2 PO 4 Comprises the following chemical components in percentage by weight: 200mM, 30mM, 26mM, 1mM, 10mM, 4mM5mM and 1.2mM, the contents being in order of chemical composition.
Further, the sample slice thickness is 300 μm.
Further, the method further comprises analyzing the MS data by: and extracting the ion intensity of the metabolite from the MS data to obtain a change value between the isotope-labeled metabolite and the non-isotope-labeled metabolite according to the ion intensity of the metabolite.
Further, the method further comprises analyzing the electrophysiology detection data by: and (4) obtaining the variation degree of the excitatory postsynaptic current by the average value of 60-90 light excitatory postsynaptic currents in the electrophysiology detection data.
Further, the method further comprises analyzing the western blot data by: extracting protein bands in the western blot data, and calculating a target gray value according to the protein bands;
and comparing the target gray value with a preset gray threshold value, and adjusting the protein band.
Further, the method further comprises analyzing the immunofluorescence data by: and obtaining the quantity of the fluorescently-labeled NeuN protein according to the immunofluorescence data.
The method for detecting the performance index of the chemical substance in the cell has the following technical effects:
the method can prepare a sample slice, detect the sample slice to obtain the detection results of chemical substances and chemical substances in the sample slice, and analyze the detection results of the chemical substances to obtain the performance index value of the sample slice; wherein the detection result of the chemical substance comprises: MS data, electrophysiological data, western blot data and immunofluorescence data can realize multiple detections, do not need to change detection samples, guarantee the accuracy of detection, and adopt corresponding detection methods to detect according to different chemical substances, avoid the inaccuracy of detection results and then influence the judgment of the chemical substances in the cell, and enable researchers to predict the metabolic state of the cell according to the chemical substances in the cell.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a flow chart of a method for detecting performance indicators of chemicals in a cell, according to an embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in other sequences than those illustrated or described herein. Moreover, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or server that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
As shown in fig. 1, the present embodiment provides a method for detecting performance indicators of chemical substances in cells, the method comprising the following steps:
s1, preparing a sample slice;
s2, sequentially carrying out MS detection, electrophysiology detection, western blot detection and immunofluorescence detection on the sample slice to obtain a target detection data set corresponding to the target sample slice, wherein the target detection data set comprises: MS data, electrophysiology data, western blot data, and immunofluorescence data;
and S3, analyzing the target detection data to obtain the performance index of the compound in the sample slice.
Further, S1 further includes the steps of:
taking a brain tissue sample of a target subject;
placing the brain tissue sample in a slicing solution for culture treatment, wherein the slicing solution comprises the following components: sucrose, naCl, naHCO 3 、MgCl 2 Glucose, KCl and NaH 2 PO 4 ；
And slicing the cultured brain tissue sample to obtain the sample slice.
Further, sucrose, naCl, naHCO 3 、MgCl 2 Glucose, KCl and NaH 2 PO 4 Comprises the following chemical components in percentage by weight: 200mM, 30mM, 26mM, 1mM, 10mM, 4.5mM and 1.2mM, the contents being in order of chemical composition.
Further, the sample slice thickness is 300 μm.
Further, the method further comprises preparing a target sample slice from the sample slice, preparing a pre-prepared solution as follows, wherein the pre-prepared solution refers to a diluted solution of the labeled isotope histidine;
under the condition of room temperature, the sample slice is incubated in the prepared solution for a preset time;
cleaning the incubated sample slice, and obtaining a single neuron sample on the cleaned sample slice;
and injecting a preset isotope solution into the single neuron sample to obtain the target sample slice.
Further, the isotope may be used 13 C。
Further, before analyzing the MS data in S3, the method further includes: carrying out calibration processing on a UCA numerical curve in MS data, wherein the calibration processing comprises the following steps:
preparing a plurality of calibration solutions with different concentrations according to a standard sample, wherein each calibration solution is an intracellular solution;
and sequentially calibrating the UCA numerical curve according to the concentration based on each calibration solution.
Further, the chemical composition of the intracellular solution is: potassium gluconate, naCl, EGTA, HEPES, caCl 2 、CsOH、MgCl 2 Na-ATP and Na-GTP.
Further, potassium gluconate, naCl, EGTA, HEPES, caCl 2 、CsOH、MgCl 2 The contents of Na-ATP and Na-GTP are respectively as follows: 130mM, 6mM, 10mM, 1mM, 4mM, 1mM, 2mM and 0.2mM, wherein the order of contents is in the order of chemical components.
Further, the diluted solution of isotopic histidine is subjected to dilution and washing treatment by cerebrospinal fluid.
Further, in S3, obtaining a metabolic pathway of the chemical substance in the sample slice according to the analysis result of the MS data, further comprising the steps of:
comparing the increment corresponding to the metabolite ion strength of the labeled isotope with a preset threshold, wherein the increment is an increment value of the metabolite ion strength in mass number percentage of the substance;
and when the corresponding increment of the ion intensity of the metabolite of the labeled isotope is larger than the threshold value, tracking the metabolic path formed by the chemical substance of the labeled isotope.
Further, the method further comprises analyzing the MS data by: and extracting the ion intensity of the metabolite in the MS data to obtain a change value between the isotope-labeled metabolite and the non-isotope-labeled metabolite according to the ion intensity of the metabolite.
Preferably, the following formula is specifically adopted:
wherein the content of the first and second substances,
is a mark 13 The ratio of the metabolites of C is such that,
refers to the ratio of unlabeled 13C metabolites.
Therefore, when the MS data are preprocessed by the method, the metabolite ion intensity is divided by the total ion current (I/TIC) for standardization, so that the difference of the total ion current of different sample mass spectrum detection signals in the original data can be avoided, the comparability among samples is poor, and the comparability and the result of the signals among the samples are improved and more accurate. Further, the method further comprises analyzing the electrophysiological detection data by: and obtaining the variation degree of the excitatory postsynaptic current by the average value of 60-90 light excitatory postsynaptic currents in the electrophysiology detection data.
Specifically, the following formula is adopted for processing:
ρ2＝IQ(1-P)，
wherein N is the number of synapses, i.e., the number of neurosynaptic areas on the brain tissue slice for electrical stimulation in performing electrophysiological analysis;
q is quantum size, i.e. the postsynaptic response resulting from the release of a single neurotransmitter vesicle;
rho 2 is the degree of variation, and is a statistical index, namely the sample dispersion degree;
i is current, i.e., electrical physiology monitors post-synaptic excitatory current or post-synaptic inhibitory current, which is produced by the flow of charged ions due to the release of presynaptic transmitter acting on the post-synaptic ion channel;
p is the probability of release, i.e., the probability of an event that the presynaptic membrane releases a neurotransmitter.
Further, the method further comprises analyzing the western blot data by:
extracting protein bands in the western blot data, and calculating a target gray value according to the protein bands;
and adjusting the protein band according to the comparison between the target gray value and a preset gray threshold value.
Specifically, the following formula is adopted:
Furthermore, the reference protein band can be selected from NAPDH or beta-actin, but in some special tissue areas, the more specific reference protein can be selected, so that more accurate results can be obtained in corresponding tissues, and the experimental results have tissue specificity and targeting. If in the synapse area sample analysis, PSD-95 is selected as a reference protein; further, the method can avoid the situation that the absolute quantity of the sample is inevitably different, and the protein strip bandwidth of the sample with large absolute quantity is wide, so that the analysis result is possibly inaccurate.
Further, the method further comprises analyzing the immunofluorescence data by: and obtaining the quantity of the fluorescently-labeled NeuN protein according to the immunofluorescence data.
Specifically, the following formula is adopted:
Further, both a and B are extracted by immunofluorescence data, and the in-view refers to a view observed by a microscopic instrument, and those skilled in the art can select according to actual needs, and details are not described herein.
Therefore, the method can improve the accuracy of experimental results and eliminate errors caused by differences of shooting, slicing and tissue sizes. The image analysis uses the average gray value to calculate the fluorescence intensity, thereby effectively eliminating the difference of calculation results caused by the cell size and the cell number in each field.
In one specific example, data from blood and cerebrospinal fluid with and without isotope are compared as shown in Table 1.
TABLE 1
In a specific example, the results of the MS analysis data analysis corresponding to the target sample sections incubated with and without the isotope are compared, as shown in table 2.
TABLE 2
As can be seen from tables 1 and 2, metabolite and metabolic pathway tracing by tail vein injection of stable isotopes can be effectively demonstrated: the ability of metabolites to cross the blood brain barrier, the specificity and accuracy of the conversion between metabolites, and the accurate confirmation of metabolic pathways. In addition, the stable isotope has obvious advantages compared with the traditional radioactive isotope tracing, and the safety and the stability are obviously improved.
In summary, the table can be used for preparing serum and cerebrospinal fluid samples, preparing sample slices based on the serum and the cerebrospinal fluid, detecting the sample slices to obtain detection results of chemical substances and chemical substances in the sample slices, and analyzing the detection results of the chemical substances to obtain performance index values of the sample slices; wherein the detection result of the chemical substance comprises: one or more combinations of MS data, MR data, electrophysiological data, western blot data and immunofluorescence data can realize multiple detections without replacing detection samples, ensure the detection accuracy, and adopt corresponding detection methods to detect according to different chemical substances, so as to avoid the inaccuracy of detection results from influencing the judgment of the chemical substances in the cells, and enable researchers to predict the biological health according to the chemical substances in the cells.
Claims (2)
1. A method for detecting a performance indicator of a chemical in a cell, the method comprising the steps of:
s1, preparing a sample slice;
s2, sequentially carrying out MS detection, electrophysiology detection, western blot detection and immunofluorescence detection on the sample slice to obtain a target detection data set corresponding to the target sample slice, wherein the target detection data set comprises: MS data, electrophysiology detection data, western blot data, and immunofluorescence data;
s3, analyzing the target detection data set to obtain a performance index of a compound in the sample slice;
the step S1 further includes the steps of:
taking a brain tissue sample of a target subject; placing the brain tissue sample in a slicing solution for culture treatment, and then slicing to obtain a sample slice;
in step S3, the MS data is analyzed: extracting metabolite ion intensities in the MS data to obtain a variation value between the isotope-labeled metabolite and the non-isotope-labeled metabolite according to the metabolite ion intensities; the following formula is specifically adopted:
wherein, the first and the second end of the pipe are connected with each other,
is a mark 13 The ratio of the metabolites of C is such that,
means not marked 13 The ratio of metabolites of C;
I/TIC is the ratio of metabolite ion intensity to total ion current;
the method further comprises analyzing the electrophysiological measurements by: obtaining the variation degree of the excitatory postsynaptic current by the average value of 60-90 light-excited excitatory postsynaptic currents in the electrophysiology detection; the following formula is adopted for processing:
ρ2＝IQ(1-P)，
wherein Q is quantum size, i.e. a postsynaptic reaction caused by the release of a single neurotransmitter vesicle;
rho 2 is the degree of variation, and is a statistical index, namely the sample dispersion degree;
i is current, i.e., electrical physiology monitors post-synaptic excitatory current or post-synaptic inhibitory current, which is produced by the flow of charged ions due to the release of presynaptic transmitter acting on the post-synaptic ion channel;
p is the release probability, namely the probability of the event that the presynaptic membrane releases the neurotransmitter;
the method further comprises analyzing the western blot data by: extracting protein bands in the western blot data, and calculating a target gray value according to the protein bands;
comparing the target gray value with a preset gray threshold value, and adjusting the protein band; the following formula is adopted:
the method further comprises analyzing the immunofluorescence data by: obtaining the quantity of the fluorescently-labeled NeuN protein according to immunofluorescence data; the following formula is adopted:
s1 also comprises the following steps:
taking a brain tissue sample of a target subject;
placing the brain tissue sample in a slicing solution for culture treatment, wherein the slicing solution comprises the following components: sucrose, naCl, naHCO 3 、MgCl 2 Glucose, KCl and NaH 2 PO 4 ；
Slicing the cultured brain tissue sample to obtain a sample slice;
sucrose, naCl, naHCO 3 、MgCl 2 Glucose, KCl and NaH 2 PO 4 Comprises the following chemical components in percentage by weight: 200mM, 30mM, 26mM, 1mM, 10mM, 4.5mM and 1.2mM, the contents being in order of chemical composition.
2. The method of claim 1, wherein the section of the sample has a thickness of 300 μm.
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