CN113049475B - Identification method and application of M1 type macrophage - Google Patents
Identification method and application of M1 type macrophage Download PDFInfo
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Abstract
The invention relates to the technical field of cell detection and typing, and particularly discloses an identification method of M1 type macrophages. The method adopts the microfluidic technology to detect the electrical characteristics of the macrophages from the single cell layer, judges whether the sample to be detected is the M1 type macrophages according to the cell membrane specific capacitance of the detection result, or calculates the content or the proportion of the M1 type macrophages in the sample to be detected according to the detection result, and can be applied to high-flux drug screening of the targeted M1 type macrophages. The invention creatively utilizes the difference of the cell in the aspects of surface appearance, cell membrane structure, membrane protein expression, cytoplasmic components and the like to cause the difference of the cell electrical characteristics (such as cell membrane unit area capacitance, cytoplasm conductivity) characteristics, achieves the aim of cell identification and typing, and has higher sensitivity and specificity.
Description
Technical Field
The invention relates to the technical field of cell detection and typing, in particular to detection and typing of macrophages.
Background
Macrophages are mainly derived from bone marrow precursor cells, play an extremely important role in the innate immune response and the adaptive immune response of the body, and are closely related to inflammatory responses, immune defenses, angiogenesis, tumor formation, homeostasis, and the like.
In different microenvironments, macrophages can differentiate into different cell subsets: type M1 and type M2, the former promote inflammation and inhibit cell proliferation, the latter promote proliferation and tissue repair. The macrophage phenotype conversion is closely related to the occurrence, development, outcome and the like of a plurality of diseases including atherosclerosis, myocardial infarction, cerebral infarction, tumor, asthma, infectious diseases and the like. For example, patients with dementia AD have a reduced M2b macrophage subpopulation and an increased M1 macrophage subpopulation, while a reduced PM2K + CD14+ M2b macrophage subpopulation is manifested by a decreased cognitive function [ PMID:32170772]. The M1 type of tumor-associated macrophages in non-small cell lung cancer is positively correlated with survival time [ PMID:20338029]. The ratio of M2/M1 of macrophages can be used as a sensitive index for judging esophageal adenocarcinoma lymph node metastasis and poor prognosis, and M2d macrophages possibly play an important role in esophageal adenocarcinoma metastasis [ PMID:26263481]. In multivariate analysis, M1/M2 is a positive independent predictor of survival. Multivariate analysis demonstrated that M1 macrophage density and M1/M2 ratio could help predict survival time for patients after radical gastric carcinoma [ PMID:24283947]. The tissue-infiltrated M1 macrophage component has significant correlation with the tumor stage, stage and histology grade of the tumor of renal clear cell carcinoma [ PMID:33181696]. Osteoclast-like giant cells of invasive breast cancer have predominantly an M2 macrophage phenotype [ PMID:29129494]. Osteosarcoma is transferred to lung, and M2 macrophage mainly promoting immunosuppression in the metastatic tumor has a certain relation with low survival rate of patients. In contrast, the superiority of M1 macrophages is associated with a longer survival [ PMID:32313728]. The relative predominance of helper T-cytokine 1 and M1 macrophages in the blood and tumors of patients with chronic obstructive pulmonary disease means that these patients have a more potent pro-inflammatory pattern. Inflammation should not be targeted systematically in all lung cancer patients. Lung cancer patients should be screened for potential respiratory disease and the identification of specific inflammatory patterns at least in the early stages of the disease [ PMID:27793775]. It has also been shown that M1 macrophage-induced endothelial cell to mesenchymal cell transformation promotes regression of infantile hemangiomas and may provide a novel therapeutic approach for the treatment of such hemangiomas [ PMID:28710904]. Therefore, the number and the proportion of M1-type macrophages in the peripheral blood of a patient are detected, and the prognosis of clinically relevant diseases can be judged. The pathological state is improved by regulating the ratio of M1 to M2, and the method can become a new idea for treating the diseases.
In the prior art, macrophage typing is performed by detecting a group of cell surface markers and their differential expression levels. Markers for M1-type macrophages can be identified from many aspects, such as expression of membrane molecules, cytokine secretion, and arginine metabolic pathways. In terms of membrane molecules, CD86 may serve as an ideal marker for M1-type macrophages; the macrophage of the type highly expresses TNF, IL-12 and IL-18 under the condition of cytokine secretion, so the cytokines can be used as markers of the macrophage of the M1 type; in the aspect of arginine metabolic pathway, the synthesis of nitric oxide by taking arginine as a raw material is an important characteristic of M1 type macrophages, and inducible nitric oxide synthase iNOS can be used as a marker of the M1 type macrophages. However, the indexes to be detected are numerous, much time and expense are needed, and the indexes are controversial.
At present, molecular biological methods including flow cytometry, western blot, RT-PCR, ELISA, immunofluorescence and the like are mainly adopted for identifying M1 and M2 type macrophages to detect molecular markers of the M1 and M2 type macrophages. The method has the defects of multiple detection indexes, insufficient index specificity and time consumption, and is not easy to convert into clinic.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a convenient and quick macrophage typing method with high specificity.
The invention firstly provides an identification method of M1 type macrophages, which adopts a microfluidic technology to detect the electrical characteristics of the macrophages from the whole layer of single cells, takes the specific capacitance of cell membranes as a detection index to detect a sample to be detected, the sample to be detected is single cells or cell groups, judges whether the sample to be detected is the M1 type macrophages or calculates the content or the proportion of the M1 type macrophages in the sample to be detected according to the detection result.
When the cell membrane specific capacitance of the detected macrophage single cell meets the cell membrane specific capacitance range of the M1 type macrophage in the single cell electrical characteristic calibration system, the macrophage single cell is judged to be the M1 type macrophage.
The specific capacitance range of the cell membrane of the M1 type macrophage in the single-cell electrical characteristic calibration system is 2.700 +/-0.527 mu F/cm 2 。
Further, when the cell population is used as a sample to be detected, the specific capacitance of the cell membrane of the single cell in the cell population is detected, the number of M1-type macrophages is counted according to the same identification method, the proportion of the M1-type macrophages in the cell population is further calculated, and the quantitative detection of the M1-type macrophages in the cell population to be detected is realized.
When more than 50% of single cells in the cell population conform to the specific capacitance range of the cell membrane of M1 type macrophages, the cell population can be judged to be M1 type macrophages.
The detection of the specific capacitance of the cell membrane involved in the present invention can be performed by conventional detection methods for detecting the electrical characteristics of cells in the art, such as a patch clamp system or the cell membrane specific capacitance detection method described in chinese patent publication No. CN 106959391A.
The invention also provides application of the cell electrical characteristic detection method in identification and typing of M1 macrophages.
The invention also provides application of the specific capacitance of the cell membrane as a specificity index for identifying and typing M1 macrophage.
Based on the technical scheme, the invention also provides a macrophage fingerprint map, which is a scattered point distribution heat map of the electrical characteristics of single cells drawn by adopting a microfluidic technology, taking the cytoplasm conductivity as a horizontal coordinate, taking the cell membrane specific capacitance as a vertical coordinate and taking each cell as a point.
Further, on the basis that the aforementioned scheme can realize M1 type macrophage typing identification, the invention also provides application of the aforementioned method in predicting the progression of chronic diseases and the prognosis of acute diseases related to M1 type macrophages; and the use of the aforementioned method for the quality control of the in vivo reinfusion of M1-type macrophages for cell therapy; and the application of the method in screening related medicaments for targeted regulation of M1 type macrophages.
The operations involved in the present invention are conventional in the art unless otherwise specified.
The preferred conditions described above may be combined with each other to arrive at a specific embodiment, based on general knowledge in the art.
The invention has the beneficial effects that:
the invention creatively adopts the microfluidic technology, utilizes the difference of the cell in the aspects of cell membrane surface appearance, cell membrane structure, membrane protein expression and the like to cause the difference of the cell electrical characteristics (such as cell membrane specific capacitance and cytoplasm conductivity), realizes the capture of the single cell, and detects the cell membrane specific capacitance of the single cell, thereby achieving the purpose of cell identification and typing and having higher sensitivity and specificity.
The invention adopts the non-labeling detection technology of the multidimensional cells based on the size and the electrical characteristics, can realize the detection of the electrical characteristics 'fingerprint spectrum' of the macrophage, and identifies the macrophage M1 phenotype from the aspect of electrical characterization.
The method for detecting the specific capacitance of the cell membrane by adopting the microfluidic technology single-cell electrical flow cytometry can rapidly, specifically and highly discriminatively identify the M1 type macrophage without chemical markers, and on one hand, can help to evaluate the occurrence, development and regression of related diseases such as tumors, infectious diseases, asthma, atherosclerosis and the like; on the other hand, the method can also evaluate the efficacy of the drug for targeting and interfering M1 type macrophage.
From the aspect of index specificity for identifying M1 type macrophages, at present, no single index can identify M1 type macrophages, and multiple molecular markers are used for identifying M1 type macrophages together. The invention adopts the microfluidic technology to detect the electrical characteristics (cell membrane specific capacitance and cytoplasm conductivity) of the macrophages from the whole cell layer, finds that the cell membrane specific capacitance of the M1 type macrophages is obviously higher than that of the M0 type macrophages and the M2 type macrophages, and greatly improves the discrimination of the M1 type macrophages and the M0 and M2 type macrophages; and intervening M1 type macrophages by using an inhibitor Rosiglitazone maleate of M1 type macrophages and an activator GW9662 of M1 type macrophages, finding that the specific capacitance of cell membranes is remarkably reduced and increased respectively, and the influence of the two on the specific capacitance of the cell membranes presents opposite trends, thereby verifying that the specific capacitance of the cell membranes can be used as a specific marker of the M1 type macrophages.
From the aspect of identifying the level of M1 type macrophages, the microfluidic technology is a detection method of single cell level, and as with flow cytometry, the proportion of M1 type macrophages in a cell group can be analyzed, and the mark area is high; while the three methods of western blot, ELISA and RT-PCR are not single cell level analysis, but semi-quantitative methods for the total expression amount of markers of a cell population, and the analysis methods are rough.
From the aspect of testing the speed of M1 type macrophages, the detection speed of the microfluidic method adopted by the invention reaches the speed of 3-6 cells/second, and compared with flow cytometry, the method is also used for detecting the single cell level, but the step of marking cells is omitted; compared with western blot (1-2 days), ELISA (shortest about 2 hours) and RT-PCR (about 4 hours), the detection period is much shorter, the invention adopts the cross channel to measure the specific capacitance of the cell membrane of the cell, does not need to obtain the cell length through optical measurement, gets rid of expensive microscopes and high-speed cameras, and simplifies the measurement steps and the operation difficulty. The detection result does not need to carry out complex data processing processes such as image processing and the like, and can realize automation and real-time processing. Therefore, whether acute diseases or chronic diseases exist, the expression of M1 can be rapidly identified, the proportion of M1 type macrophages can be rapidly identified, and the method can be used for diagnosis and prognosis of diseases in the future and can also be used for screening drugs for targeted regulation of M1 type macrophage phenotypes.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious for those skilled in the art to obtain other drawings without inventive labor.
FIG. 1 is a photograph of macrophages in example 1 of the present invention;
FIG. 2 is a graph showing the cell membrane specific capacitance/cytoplasmic conductivity of M0, M1, M2-type macrophages in example 1 of the present invention.
FIG. 3 is an electrical diagram and a statistical diagram showing the effect of M0, M1, M2-type macrophages, and Rosiglitazone malonate, an inhibitor of M1-type macrophages, and GW9662, an activator of M1-type macrophages, on the cell membrane specific capacitance of M1-type macrophages in example 2 of the present invention.
Detailed Description
In order that the above objects, features and advantages of the present invention may be more clearly understood, a solution of the present invention will be further described below. It should be noted that the embodiments and features of the embodiments of the present invention may be combined with each other without conflict.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those described herein; it is to be understood that the embodiments described in this specification are only some embodiments of the invention, and not all embodiments.
The preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
This example illustrates the typing of macrophages, in particular M1-type macrophages, by the identification method according to the invention.
1. Inducing, culturing and collecting M0, M1 and M2 macrophage cells
Day0: (1) Two six-well plates were prepared and 6-well cells were cultured, two wells for each of M0, M1 and M2. To each1mL of cell culture solution is added into the hole; (2) The cells were taken from a flask that had been cultured for three days for passaging at a cell concentration of about 10 6 cells/mL, add 1mL cell suspension to each well; (3) Recording the picture in the biological safety cabinet (20 x, phase difference mode); (4) Add 2. Mu.L of 100. Mu.g/mL PMA (final concentration 100 ng/mL) to each well; induction was carried out for 2 days.
Day2: (1) Recording the picture in the biological safety cabinet (20 x, phase difference mode); (2) replacing 2mL of culture solution in each hole; (3) 2 wells of M1+ 2. Mu.L of 100. Mu.g/mL LPS (final concentration 100 ng/mL) and 2. Mu.L of 20. Mu.g/mL IFN-. Gamma. (final concentration 20 ng/mL); (4) 2 wells of M2 + 2. Mu.L of 20. Mu.g/mL IL-4and 4. Mu.L of 10. Mu.g/mL IL-13 (final concentration 20 ng/mL);
day3: record of taking a picture in the biological safety cabinet (20 x, difference mode)
Day4: (1) Recording the picture in the biological safety cabinet (20 x, phase contrast mode, fig. 1); (2) M0, M1 and M2 cells for electrical test, before electrical test, sucking out the culture solution, adding 1mL of trypsin, digesting for 5min, adding 2mL of culture solution to stop digestion, photographing and recording, sucking out the cells, loading the cells into a 5mL centrifuge tube, centrifuging (900 r/min,5 min), removing 2mL of supernatant, blowing the cells into a 1.5mL centrifuge tube, centrifuging (900 r/min,5 min), removing the supernatant, and adding 300 μ L of cell culture solution for resuspension.
2. Electrical measurement
The invention adopts a cell membrane specific capacitance detection system disclosed in Chinese patent with publication No. CN106959391A, which comprises a pair of cross channels, wherein a first channel of the cross channels is positioned in a first direction, and a second channel of the cross channels is positioned in a second direction crossed with the first direction. When the cell is driven by the driver and passes through the cell inflow channel from the cell passing through the compression channel, the impedance measurement module records the impedance change between the electrodes, and the specific capacitance of the cell membrane of the cell is calculated by combining with the cell electrical equivalent model.
In the detection process, the frequency of the electric signal is set to be 1kHz-10MHz, and the pressure of the driver is set to be 3kPa-20kPa. Each test period is 5min, 1000-2000 cells can be detected, and the specific capacitance of the cell membrane and the cytoplasm conductivity can be calculated. A thermogram was drawn with the cytoplasm conductivity as the abscissa and the cell membrane specific capacitance as the ordinate, as shown in fig. 2.
3. The result of the detection
As can be seen in fig. 1, M1 and M2 macrophages are larger in size from the cell size compared to M0 macrophages; from the cellular morphology, M1 macrophages are fusiform, and M1 and M2 macrophages are rounded. Indicating successful macrophage induction by M1 and M2 types.
As can be seen from fig. 2, the ordinate is the cell membrane specific capacitance and the abscissa is the cytoplasmic conductivity. As can be seen from the electrical diagram and the statistical analysis result, the specific capacitance of the cell membrane of the M1 type macrophage is obviously higher than that of the M0 type macrophage and the M2 type macrophage, and the specific capacitance can be used as an index for distinguishing M1 from M0 and M2.
Example 2
This example illustrates the typing of macrophages, in particular M1-type macrophages, by the identification method according to the invention.
1. Inducing M0, M1 and M2 type macrophage cells, interfering with M1 type macrophage inhibitor and M1 type macrophage agonist, culturing and collecting.
Studies show that the agonist Rosiglitazone of PPAR gamma can inhibit M1 type macrophage [ PMID:32345753] [ PMID:32622513], and the inhibitor GW9662 of PPAR gamma can promote the conversion of M1 type macrophage to M2 type [ PMID:32805581]. Therefore, intervention was performed with these two drugs as a validation of methodology.
Day0: (1) Two six-well plates were used to prepare 10-well cells, two wells for M0, M1+ Rosiglitazone malonate, M1+ GW9662, and M2. Add 1mL of cell culture medium to each well; (2) The cells were taken from the flasks that had been cultured for three days and were ready for passage at a cell concentration of about 10 6 cells/mL, add 1mL cell suspension to each well; (3) Recording the picture in the biological safety cabinet (20 x, phase difference mode); (4) Add 2. Mu.L of 100. Mu.g/mL PMA (final concentration 100 ng/mL) to each well; induction was carried out for 2 days.
Day2: (1) Taking a picture in the biological safety cabinet for recording (20 x, phase difference mode); (2) replacing 2mL of culture solution in each hole; (3) 2 wells of M1, 2 wells of M1+ Rosglitazone maleate, 2 wells of M1+ GW9662, 2. Mu.L of 100. Mu.g/mL LPS (final concentration 100 ng/mL) and 2. Mu.L of 20. Mu.g/mL IFN-. Gamma. (final concentration 20 ng/mL) were added to each of the above 6 wells; (4) 2 μ L of 20 μ g/mL IL-4and 4 μ L of 10 μ g/mL IL-13 (final concentration 20 ng/mL) were added to 2 wells of M2;
day3: (1) Recording the picture in the biological safety cabinet (20 x, phase difference mode); (2) adding a drug which can intervene in M1: m1+ 40. Mu.M Rosiglitazone maleate, M1+ 10. Mu.M GW9662;
day4: for the electrical test, M0, M1, M2, M1+ 40. Mu.M Rosiglitazone maleate, M1+ 10. Mu.M GW9662 cells were aspirated from the culture before electrical testing, 1mL of trypsin was added, digestion was performed for 5min, 2mL of culture was added to terminate digestion, recording was performed by photographing, aspirated cells were loaded into a 5mL centrifuge tube, centrifuged (900 r/min,5 min), 2mL of supernatant was removed, cells were blown out and loaded into a 1.5mL centrifuge tube, centrifuged (900 r/min,5 min), supernatant was removed, and 300. Mu.L of cell culture was added for resuspension.
2. Electrical measurement
Measurement methods as in example 1, the violin chart of the specific capacitance (expressed as Csm) and changes in each cell membrane after intervention of M0, M1, M2 type macrophages and M1 type macrophages with the M1 type macrophage inhibitor Rosiglitazone and the M1 type macrophage activator GW9662 is shown in FIG. 3.
3. The result of the detection
As can be seen from fig. 3, rosiglitazone, an inhibitor of M1-type macrophages, can reduce the specific capacitance of the cell membrane, and GW9662, an activator of M1-type macrophages, can significantly increase the specific capacitance of the cell membrane. The electrical detection rate of macrophages is about 3-6 cells/sec. And the data can be analyzed in real time by adopting the computing software, so that the high-efficiency data analysis is realized.
The foregoing are merely exemplary embodiments of the present invention, which enable those skilled in the art to understand or practice the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (4)
1. The identification method of M1 type macrophages is characterized in that the microfluidic technology is adopted to detect the electrical characteristics of the macrophages from the whole layer of single cells, the cell membrane specific capacitance is used as a detection index to detect a sample to be detected, the sample to be detected is single cells or cell groups, and whether the sample to be detected is the M1 type macrophages or not is judged according to the detection result; when in detection: the range of the frequency of an electric signal in the cell membrane specific capacitance detection system is 1kHz-10MHz, and the range of the pressure of a driver is 3kPa-20 kPa; when the specific capacitance of the cell membrane of the detected macrophage single cell meets the specific capacitance range of the cell membrane of the M1 type macrophage in the single cell electrical characteristic calibration system, the specific capacitance range is 2.700 +/-0.527 mu F/cm 2 When the temperature of the water is higher than the set temperature,
it was judged to be M1 type macrophage.
2. Use of the specific capacitance of cell membrane determined by the method for identifying M1-type macrophages according to claim 1 for identifying and typing M1-type macrophages.
3. The use of the specific capacitance of cell membranes measured by the method for identifying M1-type macrophages according to claim 1 as an index for identifying and typing M1-type macrophages.
4. Use of the identification method of claim 1 for screening for a related drug that targetedly modulates M1-type macrophages.
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