CN113390666B - Method for detecting performance index of chemical substance in cell - Google Patents

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

A performance index method for detecting intracellular chemical substances

technical field

The invention relates to the technical field of human biology, in particular to a performance index method for detecting intracellular chemical substances.

Background technique

With the development of science and technology, people's research on biological chemical substances is becoming more and more important, especially the chemical substances in cells, which can contribute to the development of human beings in different fields. For example, in the field of medicine, many diseases can be overcome. The study of chemical substances in cells is inseparable from the detection process of chemical substances in cells. Only accurate detection can better understand the properties and uses of chemical substances.

At present, the chemical substances in cells are analyzed according to different experimental results, and different research results are obtained. In the prior art, it is impossible to form a system for the detection of chemical substances in cells, which can be used to target different components or contents of intracellular chemicals. Substances get a preliminary judgment direction for further research, avoiding misleading research on chemical substances in cells and wasting R&D resources and time.

Contents of the invention

In order to solve the problems of the existing technology, multiple detections can be performed based on the same sample slice. On the one hand, it can reduce the influence of other external factors on the detection, resulting in different experimental results for the same experimental object. On the other hand, it can meet different detection requirements. To meet the demand, provide the preliminary research direction of the chemical substances in the cells; the embodiment of the present invention provides a method for detecting the performance indicators of the chemical substances in the cells, and the method includes the following steps:

S1, preparing sample slices;

S2. Perform MS detection, electrophysiological detection, Western blot detection, and immunofluorescence detection on the sample slice in sequence to obtain a target detection data set corresponding to the target sample slice. The target detection data set includes: 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.

Further, S1 also includes the following steps:

Remove a sample of brain tissue from the subject;

placing the brain tissue sample in a slicing solution for culturing, wherein the components of the slicing solution include: sucrose, NaCl, NaHCO ₃ , MgCl ₂ , glucose, KCl and NaH ₂ PO ₄ ;

Slicing the cultured brain tissue samples to obtain the sample slices.

Further, the chemical composition contents of sucrose, NaCl, NaHCO ₃ , MgCl ₂ , glucose, KCl and NaH ₂ PO ₄ are: 200mM, 30mM, 26mM, 1mM, 10mM, 4.5mM and 1.2mM, the order of contents is according to the chemical composition sorted in order.

Further, the sample slice thickness is 300 μm.

Further, the method further includes analyzing the MS data as follows: extracting the ion intensity of the metabolites in the MS data, so as to obtain the change value between the isotope-labeled metabolite and the non-isotope-labeled metabolite according to the metabolite ion intensity .

Further, the method also includes the following method for analyzing the electrophysiological detection data: the average value of 60-90 light-excited excitatory postsynaptic currents in the electrophysiological detection data to obtain the variability of the excitatory postsynaptic current .

Further, the method also includes analyzing the western blot data as follows: extracting protein bands in the western blot data, and calculating the target gray value according to the protein bands;

Adjust the protein bands according to the comparison between the target gray value and the preset gray threshold.

Further, the method further includes analyzing the immunofluorescence data as follows: obtaining the quantity of fluorescently labeled NeuN protein according to the immunofluorescence data.

A performance index method for detecting intracellular chemical substances provided by the present invention has the following technical effects:

The present invention can prepare a sample slice, detect the sample slice, obtain the chemical substance in the sample slice and the detection result of the chemical substance, analyze the detection result of the chemical substance, and obtain the performance index value of the sample slice ; Wherein, the detection results of the chemical substances include: MS data, electrophysiological data, Western blot data and immunofluorescence data, which can realize multiple detections and do not need to replace the detection samples to ensure the accuracy of detection, and according to different chemical Substances are detected by corresponding detection methods to avoid inaccurate detection results and affect the judgment of the chemical substances in the cell, so that researchers can predict the metabolic state of the cell based on the intracellular chemical substances.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings that need to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those skilled in the art, other drawings can also be obtained based on these drawings without creative effort.

FIG. 1 is a flowchart of a method for detecting performance indicators of intracellular chemical substances according to an embodiment of the present invention.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

It should be noted that the terms "first" and "second" in the description and claims of the present invention and the above drawings are used to distinguish similar objects, but not necessarily used to describe a specific sequence or sequence. It is to be understood that the data so used are interchangeable under appropriate circumstances such that the embodiments of the invention described herein can be practiced in sequences other than those illustrated or described herein. Furthermore, the terms "comprising" and "having", as well as any variations thereof, are intended to cover a non-exclusive inclusion, for example, a process, method, system, product or server comprising a series of steps or elements is not necessarily limited to the expressly listed instead, may include other steps or elements not explicitly listed or inherent to the process, method, product or apparatus.

As shown in Figure 1, the present embodiment provides a method for detecting performance indicators of intracellular chemical substances, the method comprising the following steps:

S1, preparing sample slices;

S2. Perform MS detection, electrophysiological detection, Western blot detection, and immunofluorescence detection on the sample slice in sequence to obtain a target detection data set corresponding to the target sample slice. The target detection data set includes: 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.

Further, S1 also includes the following steps:

Remove a sample of brain tissue from the subject;

placing the brain tissue sample in a slicing solution for culturing, wherein the components of the slicing solution include: sucrose, NaCl, NaHCO ₃ , MgCl ₂ , glucose, KCl and NaH ₂ PO ₄ ;

Slicing the cultured brain tissue samples to obtain the sample slices.

Further, the chemical composition contents of sucrose, NaCl, NaHCO ₃ , MgCl ₂ , glucose, KCl and NaH ₂ PO ₄ are: 200mM, 30mM, 26mM, 1mM, 10mM, 4.5mM and 1.2mM, the order of contents is according to the chemical composition sorted in order.

Further, the sample slice thickness is 300 μm.

Further, the method also includes preparing target sample slices according to the sample slices, as follows to prepare a prefabricated solution, wherein the prefabricated solution refers to a diluted solution of the labeled isotope histidine;

Under room temperature conditions, incubating the sample slices in the prefabricated solution for a preset time;

Washing the sample slice after incubation, and obtaining a single neuron sample on the sample slice after washing;

A preset isotope solution is injected into the single neuron sample to obtain the target sample slice.

Further, the isotope may be ¹³ C.

Further, before analyzing the MS data in S3, it also includes: performing calibration processing on the UCA numerical curve in the MS data, and the calibration processing includes the following steps:

Prepare several calibration solutions with different concentrations according to the standard sample, wherein each calibration solution is an intracellular solution;

Based on each of the calibration solutions, the UCA value curve is calibrated sequentially according to the concentration.

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, the contents of potassium gluconate, NaCl, EGTA, HEPES, CaCl ₂ , CsOH, MgCl ₂ , Na-ATP and Na-GTP are respectively: 130mM, 6mM, 10mM, 10mM, 1mM, 4mM, 1mM, 2mM and 0.2mM, wherein, the order of content is sorted according to the order of chemical composition.

Further, the dilute solution of the isotope histidine is diluted and washed through the cerebrospinal fluid.

Further, in S3, according to the analysis result of the MS data, obtaining the metabolic pathway of the chemical substance in the sample slice also includes the following steps:

Comparing the increment value corresponding to the metabolite ion intensity of the labeled isotope with the preset threshold, the increment refers to the increment value of the metabolite ion intensity as a percentage of the mass number of the substance;

When the increase value corresponding to the ion intensity of the metabolite of the labeled isotope is greater than the threshold value, the formation of the chemical substance of the labeled isotope is tracked on the metabolic pathway.

Further, the method further includes analyzing the MS data as follows: extracting the ion intensity of the metabolites in the MS data, so as to obtain the change value between the isotope-labeled metabolite and the non-isotope-labeled metabolite according to the metabolite ion intensity .

Preferably, the following formula is specifically adopted:

为标记¹³C的代谢物的比值，

是指未标记13C的代谢物的比值。in,

is the ratio of ¹³ C-labeled metabolites,

Refers to the ratio of unlabeled 13C metabolites.

It can be seen that when the MS data is preprocessed by the above method, the metabolite ion intensity is divided by the total ion current (I/TIC) for normalization, which can avoid the difference in the total ion current of different sample mass spectrometry detection signals in the original data, As a result, the comparability between samples is poor, thereby improving the comparability of signals between samples and making the results more accurate. Further, the method also includes the following method for analyzing the electrophysiological detection data: the average value of 60-90 light-excited excitatory postsynaptic currents in the electrophysiological detection data to obtain the variability of the excitatory postsynaptic current .

Specifically, the following formula is used for processing:

ρ2=IQ(1-P),

Among them, N is the number of synapses, that is, the number of synapses in the area of electrical stimulation on the brain tissue slice in the electrophysiological analysis;

Q is the quantum size, which is the post-synaptic response caused by the release of a single neurotransmitter vesicle;

ρ2 is the degree of variability, which is a statistical indicator, that is, the degree of dispersion of the sample;

I is the current, which is the electrophysiological monitoring of post-synaptic excitatory current or post-synaptic inhibitory current, which is formed by the flow of charged ions caused by the release of pre-synaptic transmitters and acting on post-synaptic ion channels;

P is the release probability, which is the probability of the event that the presynaptic membrane releases the neurotransmitter.

Further, the method also includes analyzing the western blot data as follows:

Extract the protein bands in the western blot data, and calculate the target gray value according to the protein bands;

Adjust the protein bands according to the comparison between the target gray value and the preset gray threshold.

Specifically, the following formula is used:

其中，F为标准化后的蛋白条带的灰度值，D为目标灰度值，

为预设的灰度阈值，即可以参照蛋白条带的灰度值。

Among them, F is the gray value of the normalized protein band, D is the target gray value,

is the preset gray threshold, that is, the gray value of the protein band can be referred to.

Furthermore, NAPDH or β-actin can be selected as the reference protein band, but in some special tissue regions, a more specific reference protein can be selected to obtain more accurate results in the corresponding tissue, making the experimental results tissue-specific and targeting. For example, in the analysis of synaptic region samples, PSD-95 is selected as the reference protein; thus, it can avoid the unavoidable difference in the absolute amount of the sample, and the protein band width of the sample with a large absolute amount may lead to inaccurate analysis results.

Further, the method further includes analyzing the immunofluorescence data as follows: obtaining the quantity of fluorescently labeled NeuN protein according to the immunofluorescence data.

Specifically, the formula is as follows:

其中，S为平均荧光度，A为视野内整合光密度，B为荧光像素面积。

Among them, S is the average fluorescence intensity, A is the integrated optical density in the field of view, and B is the area of fluorescent pixels.

Further, both A and B are extracted through immunofluorescence data, and the field of view refers to the field of view observed by a microscopic instrument, which can be selected by those skilled in the art according to actual needs, and will not be repeated here.

It can be seen that the accuracy of the experimental results can be improved through the above method, and errors caused by differences in photographing, slicing, and tissue size can be eliminated. Image analysis uses the average gray value to calculate the fluorescence intensity, which effectively excludes the differences in the calculation results caused by the cell size and the number of cells in each field of view.

In a specific embodiment, the data of blood and cerebrospinal fluid with and without isotope are compared, as shown in Table 1.

Table 1

In a specific embodiment, the analysis results of MS analysis data corresponding to the slices of target samples incubated with and without isotopes were compared, as shown in Table 2.

Table 2

According to Table 1 and Table 2, it can be seen that traces of metabolites and metabolic pathways by injecting stable isotopes into the tail vein can effectively explain: the ability of metabolites to cross the blood-brain barrier, the specificity and accuracy of metabolite conversion, and the metabolic pathways. Accurate identification of pathways. In addition, stable isotopes have obvious advantages over traditional radioisotope tracers, and their safety and stability have been significantly improved.

In summary, the table can prepare samples of serum and cerebrospinal fluid, prepare sample slices based on the serum and cerebrospinal fluid, and test the sample slices to obtain the detection results of chemical substances and chemical substances in the sample slices. The detection results of the chemical substances are analyzed to obtain the performance index values of the sample slices; wherein the detection results of the chemical substances include: one of MS data, MR data, electrophysiological data, Western blot data and immunofluorescence data or A variety of combinations can achieve a variety of detection without changing the detection sample, to ensure the accuracy of detection, and to use corresponding detection methods for detection according to different chemical substances, so as to avoid inaccurate detection results and affect the intracellular chemistry. The judgment of substances allows researchers to predict the health of organisms based on intracellular chemicals.

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,

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:

wherein F is the gray scale value of the normalized protein band, D is the target gray scale value,

the gray value is a preset gray threshold value, namely the gray value of the protein strip can be referred to;

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:

wherein S is the average fluorescence, A is the integrated optical density in the visual field, and B is the area of the fluorescence pixel;

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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