阅读说明：本技术 一种中性粒细胞的吞噬能力的检测方法 (Method for detecting phagocytic capacity of neutrophil ) 是由 王明义 刘鹏 丛海燕 于 2020-05-29 设计创作，主要内容包括：本发明公开了一种中性粒细胞的吞噬能力的检测方法,其解决了现有吞噬检测方法不符合实际,存在吞噬性能检测成本较高,不适合普及,抗生素使用较多导致与实际临床数据结果相比差异比较大,检测数据结果不准确,实验结果不稳定的技术问题。本发明提供一种中性粒细胞的吞噬能力的检测方法,中性粒细胞的吞噬能力为中性粒细胞经第一感染信号刺激后对第二种病原菌的吞噬能力,第一感染信号为被微需氧的细菌感染,第二种病原菌为活的异养兼性厌氧菌,采用细菌涂布接种培养,计数菌落数目,可广泛应用于医学检测技术领域。(The invention discloses a method for detecting phagocytosis capability of neutrophils, which solves the technical problems that the existing phagocytosis detection method is not practical, the phagocytosis performance detection cost is high, the existing phagocytosis detection method is not suitable for popularization, the difference of the actual clinical data result is large due to more antibiotics, the detection data result is inaccurate, and the experimental result is unstable. The invention provides a method for detecting phagocytic capacity of neutrophils, which is characterized in that the phagocytic capacity of the neutrophils is the phagocytic capacity of the neutrophils on second pathogenic bacteria after the neutrophils are stimulated by a first infection signal, the first infection signal is infected by microaerophilic bacteria, the second pathogenic bacteria are live heterotrophic facultative anaerobes, bacterial coating inoculation culture is adopted, the number of colonies is counted, and the method can be widely applied to the technical field of medical detection.)

1. A method for detecting the phagocytic capacity of neutrophils, which is characterized in that the phagocytic capacity of neutrophils is the phagocytic capacity of neutrophils on a second pathogenic bacteria after the neutrophils are stimulated by a first infection signal, the first infection signal is infected by microaerophilic bacteria, and the second pathogenic bacteria is live heterotrophic aerobic bacteria.

2. The method of claim 1, wherein the first infection signal is infection by helicobacter pylori and the second pathogen is live escherichia coli, staphylococcus, or streptococcus.

3. The method for detecting the phagocytic capacity of neutrophils according to claim 2, wherein the method comprises the following steps:

step (1) stimulating the neutrophils by a first infection signal by adopting an in-vitro co-culture method: under microaerophilic conditions, co-culturing helicobacter pylori and neutrophilic granulocytes by adopting an in-vitro co-culture technology, and stimulating the neutrophilic granulocytes by a first infection signal after the co-culture of the neutrophilic granulocytes and the helicobacter pylori mutually acts to obtain an in-vitro co-culture system after the neutrophilic granulocytes are infected and stimulated by the helicobacter pylori by the first infection signal;

and (2) adding bacteria for secondary in-vitro co-culture, wherein the neutrophil phagocytosis on a second pathogenic bacterium after being stimulated by the first infection signal in the step (1): culturing under normal aerobic condition, co-culturing the second pathogenic bacteria live bacteria and the in-vitro co-culture system in the step (1) for 1h, and then exposing the helicobacter pylori in the in-vitro co-culture system in the step (1) to the aerobic condition for 1h to lose activity, so that the second pathogenic bacteria phagocytosis and culture analysis of the neutral granulocytes infected and stimulated by the helicobacter pylori are not interfered when the helicobacter pylori is continuously cultured under the aerobic condition for 1 h;

the method for evaluating the phagocytosis capacity of the second pathogenic bacteria of the neutrophils after the stimulation of the first infection signal, namely the method for detecting the phagocytosis capacity of the second pathogenic bacteria of the neutrophils after the infection of the helicobacter pylori by the first infection signal, comprises the following steps: and counting the number of colonies after two groups of neutrophils are inoculated on the plate by adopting a bacterial coating inoculation culture method.

4. The method for detecting phagocytic activity of neutrophils according to claim 3, wherein in the step (1), the in vitro co-culture method comprises the following specific steps: inoculating helicobacter pylori into a well plate with live neutrophils; the helicobacter pylori concentration is 1.0 × 10E 6-2.0 × 10E7CFU/mL, and the neutrophil concentration is 1.0 × 10E 5-2.0 × 10E 6/mL.

5. The method according to claim 4, wherein the second pathogenic bacterium is Escherichia coli; the concentration of neutrophils and helicobacter pylori is in the range of 1: 10. the concentration of neutrophils and E.coli was co-cultured at 1: 10.

6. The method for detecting phagocytic activity of neutrophils according to claim 5, wherein in the step (2), the secondary in vitro co-culture comprises the following specific steps: inoculating live Escherichia coli of a second pathogenic bacterium into the pore plate with helicobacter pylori in the step (1), wherein the Escherichia coli concentration is as follows: 1.0 × 10E 6-2.0 × 10E7 CFU/mL.

7. The method for detecting phagocytic activity of neutrophils according to claim 5, wherein in the step (3), said bacterial coating inoculation culture method is used for counting the colony number of two groups of neutrophils after plating the plate, and the specific steps are as follows:

adding antibiotics with a certain concentration into the co-culture group cells which are added with the second pathogenic bacterium escherichia coli and co-cultured in vitro in the step (2) for the second time, killing extracellular bacteria in the culture medium, collecting the cells, washing the cells once by using sterile PBS, adding sterile water, shaking, uniformly mixing and cracking the neutral granulocytes, coating the lysate on an inoculated blood plate, culturing overnight, and counting the number of colonies.

8. The method of claim 7, wherein the antibiotic is antibiotic penicillin bisanti (penicillin streptomycin).

9. The method for detecting phagocytic activity of neutrophils according to claim 7, wherein in the step (3), said counting colony count method comprises: statistical analysis is carried out by using Microsoft Excel and GraphPad Prism 5.0 software, the phagocytic coefficient is the colony number x 5 ÷ cell count, two groups of groups are compared and accord with normal distribution, and LSD-t test is carried out.

Technical Field

The invention belongs to the technical field of medical detection, and particularly relates to a method for detecting phagocytic capacity of neutrophils.

Background

In human body, about 70% of leukocytes in blood circulation are neutrophils, normal human bone marrow can generate a large amount of fresh and mature neutrophils every day, the cells contain a plurality of neutral granules and are "precedent" of the innate immune system, and the neutrophils play an important role in the body to resist the invasion of pathogenic microorganisms. After the body is infected by pathogens, a plurality of chemotactic factors act on the neutrophils, roll, adhere to and cross an endothelial barrier, gradually migrate to an infected part along the gradient of a chemotactic agent and enter an infected focus, and kill pathogenic organisms through a series of bactericidal functions to maintain the health of the body, for example, pathogens are trapped and killed through respiratory outbreak, degranulation, NETs and other ways, so that the immunoregulation effect of the neutrophil is very important. Among them, phagocytosis is one of the important functions of neutrophils in the development of immune function, and neutrophils generate reactive oxygen species and various lysosomal enzymes to effectively kill phagocytosed microorganisms by phagocytosing bacteria and foreign substances and triggering respiratory burst or oxygen burst.

The main function of the neutrophil is to phagocytize foreign matters entering the human body, and under certain conditions, the neutrophil and bacteria are put together, so that the neutrophil can phagocytize the bacteria and can reflect whether the function of the neutrophil is normal or not. The phagocytosis rate and index of neutrophils are two common indicators for evaluating the phagocytosis function of neutrophils at present. The common method is Wirhgt-Gemsa composite staining method, blood smear is observed by a low power lens, and then the condition that 100 neutrophils phagocytize pathogenic bacteria is observed under an oil lens. And calculating the phagocytosis rate and the phagocytosis index according to a formula. The phagocytosis rate is 100% of the number of bacteria phagocytosed cells out of 100 neutrophils. The phagocytic index is the total number of phagocytic bacteria per 100 neutrophils. However, this method requires staining, and also requires identifying cell structure, phagocytizing bacteria, and counting cells and bacteria under a microscope, and the accuracy of the experimental result is highly correlated with the recognition level of the operator, and the experimental result is unstable. In the prior art, phagocytosis experiments generally use single common pathogenic bacteria (such as escherichia coli) or fluorescent microspheres (particles) as phagocytosed substances. However, the clinical situation of concurrent infection of multiple bacteria (two or three) is common, so the difference of the detection data result of the existing phagocytosis experiment is larger than the actual clinical data result, and the phagocytosis ability needs to be detected during concurrent infection, so that the phagocytosis ability of the detected neutrophils is inaccurate due to the fact that multiple antibiotics need to be used for multiple bacteria. Then, the existing methods for observing the phagocytosis of the neutrophil under a fluorescence microscope and detecting the phagocytosis function of the neutrophil by a flow cytometer need fluorescent dyes and special instruments, have higher cost and are not suitable for popularization. The phagocytosis rate and index of neutrophils are two common indicators for evaluating the phagocytosis function of neutrophils at present. However, this method requires special staining, and also requires cell structure identification, bacterial phagocytosis, and cell and bacterial counting under a microscope, and the accuracy of the experimental results is highly correlated with the recognition level of the operator, and the experimental results are unstable.

Disclosure of Invention

Aiming at the defects and shortcomings in the prior art, the invention provides the method for detecting the phagocytosis capability of the neutrophil, which can accurately evaluate the phagocytosis capability of the neutrophil, has the advantages of low detection cost, simple and convenient operation, simplicity and easiness, and good repeatability, and the experimental result is closer to clinical data, and provides a new experimental method and thought for the phagocytosis experiment.

Therefore, the invention provides a method for detecting the phagocytic capacity of neutrophils, wherein the phagocytic capacity of the neutrophils is the phagocytic capacity of the neutrophils on a second pathogenic bacterium after the neutrophils are stimulated by a first infection signal, the first infection signal is infected by microaerophilic bacteria, and the second pathogenic bacterium is live heterotrophic aerobic bacteria.

Preferably, the first infection signal is an infection by helicobacter pylori and the second pathogen is live escherichia coli, or staphylococcus, or streptococcus.

Preferably, the specific steps comprise:

step (1) stimulating the neutrophils by a first infection signal by adopting an in-vitro co-culture method: under microaerophilic conditions, co-culturing helicobacter pylori and neutrophilic granulocytes by adopting an in-vitro co-culture technology, and stimulating the neutrophilic granulocytes by a first infection signal after the co-culture of the neutrophilic granulocytes and the helicobacter pylori mutually acts to obtain an in-vitro co-culture system after the neutrophilic granulocytes are infected and stimulated by the helicobacter pylori by the first infection signal;

and (2) adding bacteria for secondary in-vitro co-culture, wherein the neutrophil phagocytosis on a second pathogenic bacterium after being stimulated by the first infection signal in the step (1): culturing under normal aerobic condition, co-culturing the second pathogenic bacteria live bacteria and the in-vitro co-culture system in the step (1) for 1h, and then exposing the helicobacter pylori in the in-vitro co-culture system in the step (1) to the aerobic condition for 1h to lose activity, so that the second pathogenic bacteria phagocytosis and culture analysis of the neutral granulocytes infected and stimulated by the helicobacter pylori are not interfered when the helicobacter pylori is continuously cultured under the aerobic condition for 1 h;

the method for evaluating the phagocytosis capacity of the second pathogenic bacteria of the neutrophils after the stimulation of the first infection signal, namely the method for detecting the phagocytosis capacity of the second pathogenic bacteria of the neutrophils after the infection of the helicobacter pylori by the first infection signal, comprises the following steps: and counting the number of colonies after two groups of neutrophils are inoculated on the plate by adopting a bacterial coating inoculation culture method.

Preferably, in step (1), the in vitro co-culture method comprises the following specific steps: inoculating helicobacter pylori into a well plate with live neutrophils; the helicobacter pylori concentration is 1.0 × 10E 6-2.0 × 10E7CFU/mL, and the neutrophil concentration is 1.0 × 10E 5-2.0 × 10E 6/mL.

Preferably, the second pathogenic bacterium is escherichia coli; the concentrations of neutrophils and helicobacter pylori were measured at 1: 10. the concentration of neutrophils and E.coli was co-cultured at 1: 10.

Preferably, in the step (2), the secondary in vitro co-culture comprises the following specific steps: inoculating live Escherichia coli of a second pathogenic bacterium into the pore plate with helicobacter pylori in the step (1), wherein the Escherichia coli concentration is as follows: 1.0 × 10E 6-2.0 × 10E7 CFU/mL.

Preferably, in step (3), the bacterial coating inoculation culture method is adopted to count the number of colonies after two groups of neutrophils are inoculated on the plate, and the specific steps are as follows:

adding antibiotics with a certain concentration into the co-culture group cells which are added with the second pathogenic bacterium escherichia coli and co-cultured in vitro in the step (2) for the second time, killing extracellular bacteria in the culture medium, collecting the cells, washing the cells once by using sterile PBS, adding sterile water, shaking, uniformly mixing and cracking the neutral granulocytes, coating the lysate on an inoculated blood plate, culturing overnight, and counting the number of colonies.

Preferably, the antibiotic is the antibiotic penicillin streptomycin (penicillin streptomycin).

Preferably, in step (3), the method for counting colonies comprises: statistical analysis is carried out by using Microsoft Excel and GraphPadprism 5.0 software, the phagocytic coefficient is the colony number x 5 ÷ cell count, two groups of groups are compared and accord with normal distribution, and LSD-t test is carried out.

The invention has the beneficial effects that:

(1) the method for detecting the phagocytic capacity of the neutrophil of the invention adopts the co-culture mode established in vitro between the human peripheral blood neutrophil and a first infection signal (helicobacter pylori), adds a second pathogenic bacterium (live escherichia coli) in an incubation system, the helicobacter pylori is microaerophilic bacterium and loses activity after being exposed in the air for about 1h, so that the phagocytosis and culture analysis of the second pathogenic bacterium by cells can not be interfered under aerobic condition, no extra antibiotic is needed to kill the helicobacter pylori, the influence of the added antibiotic on the survival of the escherichia coli of the second pathogenic bacterium is avoided, the phagocytic capacity of the neutrophil on the second pathogenic bacterium (live escherichia coli) after the stimulation of the first infection signal (helicobacter pylori infection) can be accurately evaluated, the phagocytic capacity of the neutrophil can be accurately evaluated, and the detection cost is low, the method is simple and easy to operate, the experimental result is closer to clinical data, the repeatability is good, a new experimental method and thought are provided for phagocytosis experiments, and the method has obvious progress.

(2) The existing phagocytosis experiment generally uses single common condition pathogenic bacteria (such as escherichia coli) or fluorescent microspheres (particles) as phagocytes; however, the infection conditions of multiple bacteria (two or three) are common in clinic, and the method for detecting the phagocytic capacity of the neutrophil provided by the invention adopts two pathogens, aims to simulate the infection conditions of the multiple bacteria, and does not need to kill the first infection signal bacteria by using antibiotics, so that the phagocytic performance of the neutrophil detected by the method provided by the invention is closer to the actual clinical condition, the detection data result is more accurate, and the repeatability is good.

(3) In the detection method, the phagocytosis condition of the neutrophil on the second bacterium can be evaluated by utilizing the microaerophilic survival characteristic of the microaerophilic bacterium helicobacter pylori only by changing microaerophilic conditions and aerobic conditions without adding antibiotics for killing the first infection signal bacterium, and the detection method has the advantages of low detection cost, simple and convenient operation, simplicity and easiness, and the experimental result is closer to clinical data.

Drawings

FIG. 1 is a graph of the growth of Escherichia coli colonies in one set of experimental groups according to an embodiment of the present invention;

FIG. 2 is a statistical table of data results (statistical mean of test data of multiple parallel experimental groups) in the example of the present invention.

Detailed Description

The following specific examples further illustrate the invention to aid in understanding the contents of the invention. The method used in the invention is a conventional method if no special provisions are made; the raw materials and the apparatus used are, unless otherwise specified, conventional commercially available products.

The phagocytic capacity of the neutrophil is the phagocytic capacity of the neutrophil on a second pathogenic bacterium after the neutrophil is stimulated by a first infection signal, the first infection signal is infected by microaerophilic bacteria, and the second pathogenic bacterium is live heterotrophic facultative anaerobe. The first infection signal is infection by H.pylori and the second pathogen is live Escherichia coli.

The method comprises the following specific steps:

step (1), taking peripheral blood of a human body, and separating neutral granulocytes from the peripheral blood respectively by adopting a density gradient centrifugation method;

step (2), the neutrophils separated in the step (1) are inoculated into a 24-well plate according to the density of 4.0 × 10E 5/well, and the obtained product is incubated in an incubator (37 ℃, 5% CO)₂) Culturing for 2 h;

and (3) stimulating the neutrophils by a first infection signal by adopting an in-vitro co-culture method: under microaerophilic conditions (5% O)₂、10％CO₂And 85% N₂) Adding H.pylori (H.pyrori) and serum into the 24-well plate inoculated with the neutrophils in the step (2) for co-incubation for 30min, co-culturing the H.pylori and the neutrophils by adopting an in-vitro co-culture technology, and continuing to culture for 1 h; after the co-culture and interaction of the neutrophils and the helicobacter pylori, the neutrophils are stimulated by a first infection signal, and an in-vitro co-culture system after the neutrophils are infected and stimulated by the helicobacter pylori with the first infection signal is obtained;

the experimental groups were: inoculating helicobacter pylori into 24-pore plate with neutrophilic granulocyte, and culturing under microaerophilic condition for 1 h; helicobacter pylori 4.0 × 10E6CFU/mL, neutrophil 4.0 × 10E 5/mL, 500 μ L/well;

the control group was set as: the 24-well plate of neutrophils was not inoculated with H.pylori, i.e., only with neutrophils, which were 4.0X 10E5 cells/mL, 500. mu.L/well.

And (4) carrying out secondary in-vitro co-culture, wherein the neutrophil phagocytosis on a second pathogenic bacterium after being stimulated by the first infection signal in the step (3):

taking a newly cultured quality control strain Escherichia coli (E.coli) (ATCC 25922), preparing into suspension, measuring an OD600 value, calculating the thallus concentration of the Escherichia coli, and inoculating thallus into a cell culture system according to the ratio of 1:10 of neutrophile to Escherichia coli, wherein the specific inoculation step is as follows;

incubate under normal aerobic conditions (37 ℃, 5% CO)₂) After co-culturing the second pathogenic bacterium escherichia coli and the in-vitro co-culture system in the step (3) for 1h, the helicobacter pylori in the in-vitro co-culture system in the step (3) loses activity after being exposed to the aerobic condition for 1h, so that the phagocytosis and culture analysis of the second pathogenic bacterium escherichia coli by the neutrophils after being infected and stimulated by the helicobacter pylori can not be interfered after the culture for 1h is continued under the aerobic condition;

the experimental groups were: inoculating live Escherichia coli of a second pathogen into the 24-hole plate with helicobacter pylori in the step (3), wherein the Escherichia coli is 4.0 × 10E6 CFU/mL;

the control group was: inoculating live Escherichia coli (4.0 × 10E6 CFU/mL) as the second pathogenic bacterium into the 24-well helicobacter pylori-free plate in step (3).

Step (5), evaluating the phagocytic ability of the second pathogenic bacterium of the neutrophil after being stimulated by the first infection signal, namely detecting the phagocytic ability of the second pathogenic bacterium of Escherichia coli of the neutrophil after being infected by the helicobacter pylori of the first infection signal: and counting the number of colonies after two groups of neutrophils are inoculated on the plate by adopting a bacterial coating inoculation culture method.

In the step (5), antibiotic green chain double antibody (penicillin streptomycin) with certain concentration is respectively added into the cultured cells of the experimental group neutrophile granulocyte and helicobacter pylori and the Escherichia coli co-cultured group and the cultured cells of the control group neutrophile and the Escherichia coli for 30min to kill extracellular bacteria (Escherichia coli) in the culture medium, the collected cells are washed once by 1mL of sterile PBS, then 0.5mL of sterile water is added, the neutrophile granulocytes are uniformly cracked and uniformly shaken for 10min, 0.1mL of lysate is taken to coat an inoculated blood plate, the overnight culture is carried out, and the colony count is counted.

The method for counting the number of colonies comprises the following steps: statistical analysis is carried out by using Microsoft Excel and GraphPad Prism 5.0 software, the phagocytic coefficient is the colony number x 5 ÷ cell count, two groups of groups are compared and accord with normal distribution, and LSD-t test is carried out.

The bacteria colony number is counted and inoculated by using sterile water to crack the cells, the error is small, the phagocytosis capacity of the neutrophils on a second pathogenic bacterium (live Escherichia coli) after the stimulation of a first infection signal (helicobacter pylori infection) can be accurately evaluated, and a new experimental method and a new thought can be provided for a phagocytosis experiment.

The above are only examples of the invention, for example, the second pathogenic bacterium is a live heterotrophic aerobic bacterium, such as a live Escherichia coli, or a staphylococcal bacterium, or a streptococcal bacterium; in the step (1), the in vitro co-culture method comprises the following specific steps: inoculating helicobacter pylori into a well plate with live neutrophils; the concentration of helicobacter pylori is 1.0 × 10E 6-2.0 × 10E7CFU/mL, and the concentration of neutrophils is 1.0 × 10E 5-2.0 × 10E 6/mL; in the step (2), the secondary in vitro co-culture comprises the following specific steps: inoculating live Escherichia coli of a second pathogenic bacterium into the pore plate with helicobacter pylori in the step (1), wherein the Escherichia coli concentration is as follows: 1.0 × 10E 6-2.0 × 10E7CFU/mL, experimental groups: the concentrations of neutrophils and helicobacter pylori were measured at 1: 10. the concentrations of neutrophils and escherichia coli were measured at 1:10 co-culture group; the control group was: neutrophils and escherichia coli were measured at a 1:10 co-culture group; the method for measuring the phagocytic ability of neutrophils according to the present invention can be realized.

The application of the principles of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

1 materials and methods

1.1RPMI 1640 medium is a product of Hyclone company; the fetal bovine serum is a product of biological science and technology limited company in Hangzhou in Zhejiang; PBS powder was purchased from wuxi aorui east source biotechnology limited; antibiotics were purchased from solibao corporation; blood plates were purchased from zhengzhou angtu bioengineering, inc;

1.2 human peripheral blood neutrophils were collected from healthy volunteers and cultured in a 37 ° incubator with RPMI 1640 containing 10% fetal bovine serum; the cells with good growth and cell survival rate (trypan blue exclusion method) > 95% are taken for experiment.

1.3 methods

Experiment set 2:

experimental groups: neutrophils and helicobacter pylori expressed as 1: 10. neutrophils and escherichia coli were measured at a 1:10 concentration co-cultured group.

Control group: neutrophils and escherichia coli were measured at a 1:10 co-culture group;

1.3.1 inoculating 4.0 × 10E5 neutrophils in a 6-well plate, 500 μ L/well, setting 3 wells, inoculating only neutrophils in a control group, inoculating neutrophils and helicobacter pylori in an experimental group, and culturing for 1h under microaerobic conditions; helicobacter pylori 4.0 × 10E6CFU/mL, neutrophil 4.0 × 10E 5/mL, 500 μ L/well;

1.3.2 Co-culture of the above-mentioned co-culture system with Escherichia coli in vitro by inoculating Escherichia coli in a 24-well plate with or without helicobacter pylori.

1.3.3 bacterial spread inoculation culture method

Culturing the group cells in the neutrophils and the Escherichia coli; in the cultured cells of the neutrophilic granulocyte, the helicobacter pylori and the Escherichia coli,

respectively adding antibiotics with a certain concentration to act for 30min, killing bacteria outside the neutrophils in the culture medium, collecting the neutrophils, washing the neutrophils once with sterile PBS, adding 0.5mL of sterile water, shaking, uniformly mixing and cracking the neutrophils for 10min, coating 0.1mL of lysate on an inoculated blood plate, culturing overnight, and counting the number of colonies;

1.3.4 statistical analysis is carried out by using Microsoft Excel and GraphPad Prism 5.0 software, the phagocytic coefficient is the colony number x 5 ÷ cell count, two groups are compared, if the normal distribution is met, the LSD-t test is carried out, and the difference is p <0.05, so that the statistical significance is achieved.

2 results

2.1 growth of E.coli colonies in each group, the results show (as shown in FIG. 1):

the number of the Escherichia coli phagocytosed by the neutrophils in the experimental group of the co-culture system of the neutrophils, the helicobacter pylori and the Escherichia coli is 12, the number of the Escherichia coli phagocytosed by the neutrophils in the system without the helicobacter pylori in the control group is 10, and the difference of the number of the Escherichia coli phagocytosed by the neutrophils in the experimental group and the control group has no statistical significance. Compared with the conventional method for evaluating the phagocytic capacity of neutrophils, which adopts a Wirhgt-Gemsa composite staining method as a common index, and requires identifying water of an operator to cause unstable experimental results, the method disclosed by the invention has the advantages that the accuracy of the experimental results is remarkably improved, special staining is not required in the detection method disclosed by the invention, antibiotics for killing the first infection signal thallus is not added, cells and bacteria are not required to be identified under a microscope, only cell coating inoculation is required to be cracked, and the bacterial colony number is counted, so that the time and labor are saved, the error is small, the accuracy is high, and the method has great influence on the technical field of medical detection.

3. Discussion of the related Art

After the pathogen invades the human body, the neutrophil is activated under the action of various factors, rolls, adheres and crosses an endothelial barrier, migrates to an infected part and enters an infected focus, and kills the pathogenic organism through a series of sterilization functions to maintain the health of the body. Among them, phagocytosis is one of the important functions of neutrophils in the development of immune function, and neutrophils generate reactive oxygen species and various lysosomal enzymes to effectively kill phagocytosed microorganisms by phagocytosing bacteria and foreign substances and triggering respiratory burst or oxygen burst.

The phagocytic capacity of neutrophils is changed into the phagocytic capacity of neutrophils on a second pathogenic bacterium after the neutrophils are stimulated by a first infection signal, the first infection signal is infected by microaerophilic bacteria, and the second pathogenic bacterium is a live heterotrophic facultative anaerobe. The first infection signal is infection by H.pylori and the second pathogen is live Escherichia coli.

In the detection method of the present invention, infection with the microaerophilic bacterium helicobacter pylori is used as a first infection signal, and the growth conditions thereof are microaerophilic; heterotrophic facultative anaerobe Escherichia coli is used as an infection pathogenic bacterium, is heterotrophic facultative anaerobe, and can perform corresponding living activities under the condition of oxygen or oxygen deficiency; helicobacter pylori is a microaerophilic bacterium and loses activity after being exposed in the air for 1 hour, so that during secondary bacterium-adding culture, phagocytosis and culture analysis of the second pathogenic bacterium (Escherichia coli) by the neutrophils cannot be interfered under aerobic conditions, and therefore, compared with other pathogenic bacteria, the helicobacter pylori is used for simulating the first infection signal, antibiotics for killing the thallus of the first infection signal are not needed to be added, survival of the second pathogenic bacterium (live Escherichia coli) cannot be influenced, and phagocytosis capability of the second pathogenic bacterium (live Escherichia coli) by the neutrophils after stimulation of the first infection signal (helicobacter pylori infection) can be accurately evaluated. In the prior art, the phagocytic capacity of neutrophils is evaluated by a phagocytic rate and a phagocytic index, which are commonly detected by staining with a Wirhgt-Gemsa complex staining method, observing a blood smear with a low power microscope, observing the condition that 100 neutrophils phagocytize pathogenic bacteria under an oil microscope, and calculating the phagocytic rate and the phagocytic index according to a formula (the phagocytic rate is 100% of the number of phagocytic bacteria in 100 neutrophils/100%, and the phagocytic index is 100% of the total number of phagocytic bacteria in 100 neutrophils/100). However, this method requires staining, the degree of staining quality is highly related to the professional skill level of the operator, and it is necessary to identify the cell structure, phagocytize bacteria, count cells and bacteria under a microscope, so the accuracy of the experimental result is highly related to the identification level of the operator, which often results in unstable experimental result. However, the detection method does not need special dyeing, does not need to identify cells and bacteria under a microscope, only needs to crack the cells, coat and inoculate the cells and count bacterial colonies, and has small error and high accuracy.

In addition, in the prior art, phagocytosis experiments generally use single common pathogenic pathogen (such as escherichia coli) or fluorescent microspheres (particles) as phagocytosed substances. However, the infection condition of multiple bacteria (two or three) is common in clinic, and the detection method of the invention adopts two pathogens and aims to simulate the infection condition of the multiple bacteria. Therefore, compared with the phagocytosis experimental result in the prior art, the detection scheme provided by the invention is closer to the actual clinical situation, the phagocytosis result of the neutrophil is closer to the actual clinical situation, the detection data result is more accurate, and the error is small.

In addition, the detection method of the invention utilizes the microaerophilic survival characteristic of the bacteria helicobacter pylori, only needs the transformation of microaerophilic conditions and aerobic conditions, and does not need to add antibiotics for killing the first infection signal, so that the phagocytosis condition of the neutrophils to the second bacteria can be evaluated, the whole operation is simple, the time and the labor are saved, the result statistics is more intuitive, and the universality is strong.

In conclusion, the method for detecting the phagocytic capacity of the neutrophils has the advantages of low detection cost, mild conditions, simplicity and convenience in operation, time and labor conservation, high repeatability, more accurate detection data result, small error and high popularization, is more approximate to the actual clinical situation, and provides a new experimental method and thought for phagocytic experiments.

The above are merely examples of the invention and are not intended to limit the scope of the invention, for example, the second pathogenic bacterium is a live heterotrophic aerobic bacterium, such as a live Escherichia coli, or a Staphylococcus, or a Streptococcus; in the step (1), the in vitro co-culture method comprises the following specific steps: inoculating helicobacter pylori into a well plate with live neutrophils; the concentration of helicobacter pylori is 1.0 × 10E 6-2.0 × 10E7CFU/mL, and the concentration of neutrophils is 1.0 × 10E 5-2.0 × 10E 6/mL; in the step (2), the secondary in vitro co-culture comprises the following specific steps: inoculating live Escherichia coli of a second pathogenic bacterium into the pore plate with helicobacter pylori in the step (1), wherein the Escherichia coli concentration is as follows: 1.0 × 10E 6-2.0 × 10E7CFU/mL, experimental groups: the concentrations of neutrophils and helicobacter pylori were measured at 1: 10. the concentrations of neutrophils and escherichia coli were measured at 1:10 co-culture group; the control group was: neutrophils and escherichia coli were measured at a 1:10 co-culture group; the method for measuring the phagocytic ability of neutrophils according to the present invention can be realized.

However, the above description is only exemplary of the present invention, and the scope of the present invention should not be limited thereby, and the replacement of the equivalent components or the equivalent changes and modifications made according to the protection scope of the present invention should be covered by the claims of the present invention.