阅读说明：本技术 一种体外评价卡介苗对中性粒细胞活性影响的方法 (Method for evaluating influence of BCG (bacillus calmette guerin) on neutrophil activity in vitro ) 是由 陈炳地 黄鑫 乐文俊 崔征 于 2019-10-29 设计创作，主要内容包括：本发明公开了一种体外评价卡介苗对中性粒细胞活性影响的方法,至少包括如下步骤：提供培养板；在所述培养板上接种靶细胞；制备卡介苗悬液；将中性粒细胞和所述卡介苗悬液共同作用于所述靶细胞；向所述培养板中添加检测试剂。本发明在体外就可以确定中性粒细胞对卡介苗有无应答反应。(The invention discloses a method for evaluating influence of BCG on neutrophil activity in vitro, which at least comprises the following steps: providing a culture plate; seeding the target cells on the culture plate; preparing BCG vaccine suspension; allowing the neutrophil and the BCG suspension to act on the target cells together; adding a detection reagent to the culture plate. The invention can determine whether the neutral particle cell has response reaction to the BCG vaccine in vitro.)

1. A method for evaluating the influence of BCG on the activity of neutrophils in vitro is characterized by at least comprising the following steps:

providing a culture plate;

seeding the target cells on the culture plate;

preparing BCG vaccine suspension;

allowing the neutrophil and the BCG suspension to act on the target cells together;

adding a detection reagent to the culture plate;

detecting a change in absorbance in the culture plate;

and evaluating the influence of the BCG on the activity of the neutrophils according to the change of the absorbance.

2. The method of claim 1, wherein the step of preparing the bcg suspension comprises:

inoculating the BCG vaccine into a liquid culture medium for culture;

measuring the OD600 value in the liquid medium;

when the OD600 value reaches 2.0-2.1 value or above, the BCG vaccine in the liquid culture medium is collected and centrifuged to obtain supernatant and a precipitate layer;

and (4) sucking the supernatant, and suspending the precipitation layer through cell culture medium to obtain the BCG suspension.

3. The method according to claim 2, wherein the culture conditions are 5-14 days at 35-37 ℃ after inoculating the bcg vaccine in the liquid medium.

4. The method of claim 2, wherein the cell culture medium is the same type of medium as the medium in which the target cells are seeded, and the medium in which the target cells are seeded is located within the culture plate.

5. The method of claim 1, wherein the step of obtaining the neutrophils comprises:

preparing a plurality of groups of lymphocyte separation solutions with concentration gradients;

adding the plurality of groups of lymphocyte separation solutions with concentration gradients into a centrifuge tube according to the sequence of the concentration from large to small, so that the lymphocyte separation solution with the minimum concentration is positioned on the uppermost layer of the centrifuge tube;

collecting 1-2ml of peripheral blood and diluting by 1-3 times to obtain diluted peripheral blood;

adding 1-4ml of the diluted peripheral blood to the lymphocyte separation solution with the minimum concentration;

and (3) obtaining the neutrophils by using the lymphocyte separation liquid with the plurality of groups of concentration gradients through a separation and extraction step.

6. The method of claim 5, wherein the number of groups of lymphocyte separation fluid is 5-8 groups.

7. The method as claimed in claim 1, wherein the ratio of the effect between BCG, the neutrophils and the target cells in the BCG suspension is (500- & 2000): (3-20): 1.

8. the method of claim 1, wherein the culture plate is a 96-well cell culture plate.

9. The method of claim 1, wherein the density of the target cells in the step of seeding the target cells on the culture plate is 8000-12000 cells/well.

10. The method of claim 1, wherein the detection reagent is one of a cell proliferation/toxicity detection reagent, a thiazole blue reagent, or a trypan blue reagent.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for evaluating influence of BCG (bacillus calmette guerin) on activity of neutrophils in vitro.

Background

At present, if the effect of BCG on cancer cells is to be determined, the BCG is generally injected into a human body by adopting a perfusion method, so that the risk rate is high and the human body is injured. The invention provides a method for evaluating the influence of BCG on the activity of neutrophils in vitro, which can determine whether the neutrophils have response to the BCG in vitro, thereby reasonably selecting whether to use the BCG according to individual conditions.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention provides a method for in vitro evaluation of the effect of BCG on neutrophil activity, which can determine in vitro the presence or absence of response of neutrophils to BCG based on the activity of neutrophils on target cells, thereby identifying the effect of BCG on individuals in vitro.

In order to achieve the above objects and other objects, the present invention provides a method for in vitro evaluation of the effect of BCG on neutrophil activity, comprising at least the following steps:

providing a culture plate;

seeding the target cells on the culture plate;

preparing BCG vaccine suspension;

allowing the neutrophil and the BCG suspension to act on the target cells together;

adding a detection reagent to the culture plate;

detecting a change in absorbance in the culture plate;

and evaluating the influence of the BCG on the activity of the neutrophils according to the change of the absorbance.

In one embodiment, the step of preparing the bcg suspension comprises:

inoculating the BCG vaccine into a liquid culture medium for culture;

measuring the OD600 value in the liquid medium;

when the OD600 value reaches 2.0-2.1 value or above, the BCG vaccine in the liquid culture medium is collected and centrifuged to obtain supernatant and a precipitate layer;

and (4) sucking the supernatant, and suspending the precipitation layer through cell culture medium to obtain the BCG suspension.

In one embodiment, the culture conditions are culture at 35-37 ℃ for 5-14 days after inoculating the BCG vaccine in the liquid medium.

In one embodiment, the cell culture medium is the same type of culture medium as the medium in which the target cells are seeded, and the medium in which the target cells are seeded is located within the culture plate.

In one embodiment, the step of obtaining the neutrophils comprises:

preparing a plurality of groups of lymphocyte separation solutions with concentration gradients;

adding the plurality of groups of lymphocyte separation solutions with concentration gradients into a centrifuge tube according to the sequence of the concentration from large to small, so that the lymphocyte separation solution with the minimum concentration is positioned on the uppermost layer of the centrifuge tube;

collecting 1-2ml of peripheral blood and diluting by 1-3 times to obtain diluted peripheral blood;

adding 1-4ml of the diluted peripheral blood to the lymphocyte separation solution with the minimum concentration;

and (3) obtaining the neutrophils by using the lymphocyte separation liquid with the plurality of groups of concentration gradients through a separation and extraction step.

In one embodiment, the number of sets of lymphocyte separation fluid is 5-8 sets.

In one embodiment, the ratio of the effect between BCG, the neutrophils and the target cells in the BCG suspension is (500-: (3-20): 1.

in one embodiment, the culture plate is a 96-well cell culture plate.

In one embodiment, in the step of seeding the target cells on the culture plate, the density of the target cells is 8000-12000 cells/well.

In one embodiment, the detection reagent is one of a cell proliferation/toxicity detection reagent, a thiazole blue reagent, or a trypan blue reagent.

In the present invention, the change of the survival rate of cancer cells after the stimulation of the neutrophils by the BCG can be detected in vitro in different subjects. The method can simply, conveniently and quickly screen out the person who has a response to the BCG in vitro, thereby reasonably selecting whether to use the BCG according to individual conditions, greatly reducing the use risk of the BCG in a human body and playing a good precaution role. The method has the advantages of small wound on a subject, small blood consumption, simple operation, high repeatability, batch operation and the like. The method fills the blank of the technical field, and is used for evaluating the response reaction of the neutrophils to the BCG in vitro for the first time, thereby providing a reference basis for whether an individual needs to use the BCG.

Drawings

FIG. 1 is a schematic diagram of an experimental process according to an embodiment of the present invention;

FIG. 2 is a graph showing the survival rate of cancer cells after co-incubation of BCG, neutrophils and cancer cells for the first neutrophil in accordance with one embodiment of the present invention;

FIG. 3 is a graph showing the survival rate of cancer cells after co-incubation of BCG, neutrophils and cancer cells for a second neutrophil according to one embodiment of the present invention;

FIG. 4 is a graph showing the survival rate of cancer cells after co-incubation of BCG, neutrophils and cancer cells with a third neutrophil in accordance with an embodiment of the present invention.

Detailed Description

The following description of the embodiments of the present invention is provided by way of specific examples, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure herein. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention. It is to be noted that the features in the following embodiments and examples may be combined with each other without conflict. It is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the description of the present invention, and any methods, apparatuses, and materials similar or equivalent to those described in the examples of the present invention may be used to practice the present invention.

According to the method, the response result of the neutrophils to the BCG is determined in vitro, so that whether the BCG is used or not is reasonably selected according to individual conditions, the use risk of the BCG is greatly reduced, and a good precaution effect is achieved.

As shown in FIG. 1, the present invention provides a method for in vitro evaluation of the effect of BCG on neutrophil activity, comprising at least the following steps:

s1, providing a culture plate;

s2, inoculating target cells on the culture plate;

s3, preparing BCG suspension;

s4, allowing the neutrophil and the BCG suspension to act on the target cell together;

s5, adding a detection reagent into the culture plate;

s6, detecting the change of absorbance in the culture plate;

s7, evaluating the influence of the BCG on the activity of the neutrophils according to the change of the absorbance.

Specifically, in step S1, the culture plate is, for example, a 96-well cell culture plate, and the culture plate contains a culture medium corresponding to the inoculated target cells.

Specifically, in step S2, the target cells are seeded at a density of 8000-.

Specifically, in step S3, the step of preparing bcg suspension comprises: inoculating BCG vaccine into 20-30ml liquid culture medium, such as 7H9 culture medium, and culturing at 35-37 deg.C for 5-14 days; measuring the OD600 value in the liquid medium; when the OD600 value reaches 2.0-2.1 value or above, the BCG in the liquid culture medium is collected and centrifuged under the condition of centrifugation, such as centrifugation at 3000-3500rpm for 5-10 minutes, so as to obtain a supernatant and a precipitate layer; the supernatant is aspirated off and the pellet layer is suspended by cell culture medium, for example in a volume of 10-20ml, to obtain the bcg suspension.

Specifically, in the step S3, the bacterial concentration of the bcg suspension needs to be detected while preparing the bcg suspension, so as to facilitate the subsequent calculation of the action ratio among bcg, neutrophils and cancer cells. The method comprises the following specific steps:

(1) diluting BCG vaccine suspension with high-sugar improved eagle culture medium containing 10-20% fetal calf serum by certain times of 5-1000 times, and detecting OD600 value;

(2) respectively taking 150ul of 100-fold bacteria liquid from some dilutions in 5-1000 folds, inoculating to Middlebrook 7H11 agar medium, and culturing at 35-37 deg.C for 2-3 weeks;

(3) counting the number of BCG colonies on the agar medium, and drawing a standard curve by taking OD600 values as abscissa and bacterial liquid concentrations of different dilution times as ordinate under different dilution times;

(4) in the subsequent experiment, the optical density value of the BCG suspension with the concentration to be measured is brought into the obtained standard curve, and the corresponding bacterial concentration can be calculated.

Specifically, in the step of S4, the step of obtaining the neutrophil includes: preparing a plurality of groups of lymphocyte separation solutions with concentration gradients; adding the plurality of groups of lymphocyte separation solutions with concentration gradients into a centrifuge tube according to the sequence of the concentration from large to small, so that the lymphocyte separation solution with the minimum concentration is positioned on the uppermost layer of the centrifuge tube; collecting 1-2ml of peripheral blood and diluting by 1-3 times to obtain diluted peripheral blood; adding 1-4ml of the diluted peripheral blood to the lymphocyte separation solution with the minimum concentration; and (3) obtaining the neutrophils by using the lymphocyte separation liquid with the plurality of groups of concentration gradients through a separation and extraction step. The number of the lymphocyte separating medium groups is 5-8 groups. The action ratio among the BCG, the neutrophil and the target cell in the BCG suspension is (500- & 2000): (3-20): 1.

specifically, in step S5, the detection reagent is one of a cell proliferation/toxicity detection reagent, a thiazole blue reagent, and a trypan blue reagent.

In one embodiment, the specific process of extracting neutrophils in, for example, human peripheral blood is: preparing 5-8 groups of lymphocyte separating medium with different densities, such as Percoll lymphocyte separating medium, taking 5 groups as an example, respectively: the density of the first group is greater than that of the second group, the density of the third group is greater than that of the fourth group, the density of the first group is 70% -75% of Percoll lymphocyte separation liquid, the density of the second group is 60% -63% of Percoll lymphocyte separation liquid, the density of the third group is 45% -55% of Percoll lymphocyte separation liquid, the density of the fourth group is 36% -40% of Percoll lymphocyte separation liquid, the density of the fifth group is 10% -25% of Percoll lymphocyte separation liquid, then 1-2mL of each of the first group, the second group, the third group, the fourth group and the fifth group is respectively sucked, and then the first group, the second group, the third group, the fourth group and the fifth group are sequentially added into a centrifuge tube; collecting 1-2mL of human peripheral blood, diluting 1-3 times with PBS, sucking 1-4mL of diluted peripheral blood, adding to the fifth group, centrifuging at 500-1000rpm for 30-60 min, centrifuging for 5-10 min to separate the Percoll lymphocyte separation liquid from the neutrophils to obtain a neutrophilic granulocyte layer, sucking the neutrophilic granulocyte layer, adding 10-20 times of cell culture medium such as high-sugar modified eagle culture medium containing 10-20% of fetal calf serum, centrifuging for 5-10 min, removing the upper layer solution after centrifugation, adding 0.5-50mL of cell culture medium such as high-sugar modified eagle culture medium containing 10-20% of fetal calf serum in volume fraction to the lower layer precipitate to keep the neutrophils in suspension, suspending, the neutrophils were counted using an automated cell counter for the purpose of subsequent calculation of the interaction ratio between bcg, neutrophils and cancer cells. The method has the advantages of small wound on a subject, small blood consumption, simple operation, good repeatability, batch operation and the like.

The concrete steps for preparing the BCG vaccine suspension are as follows: inoculating BCG strain in 20-25mL of culture medium such as 7H9 culture medium, culturing at 37 deg.C for 5-14 days, measuring OD600 every 3-5 days until OD600 reaches 2.0-2.1 value or reaches 2.0-2.1 above (wherein OD600 value refers to optical density value at 600nm wavelength); then, the BCG suspension is diluted by phosphate buffer PBS by different times, and the OD600 value after dilution is measured and recorded, and is inoculated in Middlebrook 7H11 agar culture medium, and is cultured for 2-4 weeks at 37 ℃, and the colony number of the BCG on the agar culture medium is counted, and a standard curve is drawn. Collecting bacteria in the culture medium, centrifuging at the room temperature at 3000-3500rpm for 5-10 minutes to obtain supernatant and lower-layer sediment, sucking the supernatant, suspending the lower-layer sediment, namely the BCG, by using 10-20mL of high-sugar modified eagle culture medium containing 10-20% of fetal calf serum to obtain BCG suspension, and measuring OD600 at the moment to calculate the concentration of the BCG according to a standard curve, wherein the calculation of the concentration of the BCG is used for subsequently calculating the action ratio among the BCG, the neutrophils and the cancer cells. The culture process of the BCG is very difficult, and the method for preparing the BCG suspension ensures that the survival rate of the cultured BCG is higher, and can better provide convenience for the in vitro evaluation method.

The specific process of in vitro co-incubation of neutrophils, BCG and target cells such as cancer cells is as follows: cancer cells such as human cervical cancer cells HeLa, human bladder cancer cells 5637 or human bladder cancer cells J82 are seeded at a density of 8000-; removing the cell culture medium, washing with phosphate buffer PBS for 2-3 times, adding BCG suspension and obtained human neutrophils into cancer cells, and culturing at 35-37 deg.C for 12-24 hr, wherein the action ratio of BCG, neutrophils and cancer cells is (500-) -2000): (3-20): 1. the invention adopts the co-incubation of the neutrophils, the BCG vaccine and the target cells to fully simulate the in vivo environment and avoid the result deviation caused by other factors, and utilizes a 96-hole cell culture plate to artificially simulate the bladder environment of a human body, so that the experimental result is close to the in vivo real result, and the credibility of the result is high.

The specific process for detecting the survival rate of the cancer cells comprises the following steps: removing the cell culture medium, human neutrophil granulocytes and BCG vaccine in a culture plate such as a 96-well cell culture plate, washing with Phosphate Buffered Saline (PBS) for 2-3 times, adding a detection reagent such as a WST-1 cell proliferation/toxicity detection reagent into a high-sugar modified eagle culture medium containing 10-20% fetal calf serum according to the proportion of 1 (10-15), and uniformly blowing to obtain a diluted detection solution; the diluted detection solution is added into a 96-well cell culture plate, wherein each well is 110-. WST-1 is an upgrading substitute product of MTT, and has the advantages that MTT is not dissolved in water and needs to be subjected to a dissolution step, so that the problem is solved; formazan reduced by dehydrogenases in mitochondria of WST-1, XTT, and MTS is water-soluble, and a subsequent solubilization step can be omitted; however, formazan produced by WST-1 was more soluble than both XTT and MTS; secondly, WST-1 is more stable than XTT and MTS; finally, WST-1 has wider linear range and higher sensitivity than XTT and MTT, wherein the WST-1, the MTT, the XTT and the MTS are cell proliferation and toxicity detection reagents.

The specific process of calculating the survival rate of the cancer cells is as follows: the absorbance of experimental wells inoculated with cancer cells, neutrophils, with or without bcg is indicated by letter a; the absorbance of experimental wells inoculated with cancer cells only is indicated by the letter B; the absorbance of experimental wells inoculated with neutrophils only, with or without bcg, is indicated by the letter C; the absorbance of the blank wells not inoculated with any cells is represented by the letter D, and the survival rate of cancer cells is (a-C)/(B-D) × 100%.

By applying the method, the survival rates of normal cancer cells, cancer cells incubated by the neutrophils and cancer cells incubated by the BCG and the neutrophils are detected and compared. The results are shown in FIGS. 2 to 4.

Referring to fig. 2, the experimental results show that, for the first neutrophil, the subject from which the neutrophil is derived is a bladder cancer patient, wherein the neutrophil can significantly inhibit the survival rates of human cervical cancer cells HeLa, human bladder cancer cells 5637 and human bladder cancer cells J82. After the BCG is added, the inhibition effect of the sex granulocyte on the survival rate of human cervical cancer cells HeLa and 5 human bladder cancer cells 5637 is more obvious, but the inhibition effect on the survival rate of human bladder cancer cells J82 is not obviously improved. Taken together with the results for the 3 target cells, the first neutrophil showed a positive result for 67% of the target cells after stimulation with bcg. Therefore, the test result in vitro judges that the subjects providing the first kind of neutrophils can use the BCG and the effect is obvious. The clinical treatment result is consistent with the in vitro detection judgment result aiming at the patient displayed by the method, and the accuracy and the reliability of the method are further verified.

Referring to fig. 3, the experimental results show that, regarding the second neutrophil, the subject derived from the neutrophil is also a bladder cancer patient, wherein the neutrophil can significantly inhibit the survival rates of human cervical cancer cell HeLa, human bladder cancer cell 5637 and human bladder cancer cell J82. After the BCG is added, the inhibition effect of the sex granulocyte on the survival rate of 3 target cells is not obviously improved. In vitro assays the subjects providing the second neutrophil need not be considered for bcg, as judged by the results of the in vitro assays. The clinical treatment result is consistent with the in vitro detection judgment result aiming at the patient displayed by the method, and the accuracy and the reliability of the method are further verified.

Referring to fig. 4, the experimental results show that, regarding the third kind of neutrophils, the subject derived from the neutrophils is also a bladder cancer patient, wherein the neutrophils can significantly inhibit the survival rates of the human cervical cancer cell HeLa and the human bladder cancer cell J82, but have no significant inhibition effect on the survival rate of the human bladder cancer cell 5637. After the BCG is added, the inhibition effect of the sex granulocyte on the survival rate of the human cervical cancer cells HeLa is more obvious, but the inhibition effect on the survival rate of the human bladder cancer cells 5637 is not obviously improved. Combining the results of 3 target cells, the third neutrophil showed a positive result for 67% of the target cells after stimulation with bcg. Therefore, the third neutrophil was judged to respond well to BCG as a result of in vitro assay evaluation. The clinical treatment results are also consistent with the in vitro test and evaluation results shown by the method of the invention for the patient, which further verifies the accuracy and reliability of the method of the invention.

The target cell of the invention comprises one of bladder cancer cells, cervical cancer cells, lung cancer cells, liver cancer cells, breast cancer cells, prostate cancer cells, thyroid cancer cells, ovarian cancer cells, lymph cancer cells, kidney cancer cells, stomach cancer cells, intestinal cancer cells, pancreatic cancer cells, bone cancer cells, melanoma cells or leukemia cancer cells.

The invention can detect the change of the survival rate of cancer cells of different subjects after the stimulation of the neutrophils by the BCG vaccine in vitro. The method can simply, conveniently and quickly screen out the person who has or does not have a response to the BCG in vitro, thereby reasonably selecting whether to use the BCG according to individual conditions, providing reference basis for whether a receptor is suitable for using the BCG, reducing the risk of the BCG in a human body and playing a good precaution role. The method fills the blank of the technical field, and is used for evaluating the response reaction of the neutrophils to the BCG vaccine in vitro for the first time.

Furthermore, it is to be understood that one or more method steps mentioned in the present invention does not exclude that other method steps may also be present before or after the combined steps or that other method steps may also be inserted between these explicitly mentioned steps, unless otherwise indicated; it should also be understood that unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention which may be practiced, nor is it intended to limit the relative changes or modifications to the scope of the invention which may be practiced without materially changing the technical details.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.