Helicobacter pylori-containing transplantation tumor as well as preparation method and application thereof

Abstract

The invention discloses a transplanted tumor containing helicobacter pylori and a preparation method and application thereof, belongs to the technical field of biology, and further discloses a reagent for preparing a transplanted tumor tissue containing helicobacter pylori, and the reagent comprises tumor cells and helicobacter pylori and further comprises at least one of matrigel and defibrated sheep blood. The helicobacter pylori-containing transplanted tumor obtained by utilizing the reagent and the preparation method disclosed by the invention can be used for preparing a quality control substance or a reference substance in immunological detection, the HP thallus positive staining is stronger, and besides being used as positive quality control, the helicobacter pylori-containing transplanted tumor also has internal control of negative cell hematoxylin staining, so that the helicobacter pylori-containing transplanted tumor can be used for preparing the quality control substance or the reference substance. The same quality control product has both thallus positive staining and histocyte negative staining, has consistency with conventional case tissue detection, and can play a monitoring role in HP immunohistochemical quality control.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a transplanted tumor containing Helicobacter pylori and a preparation method and application thereof. Background Art

[0002] Helicobacter pylori (HP) is a Gram-negative Campylobacter. The pathological report of H. pylori gastritis can identify the degree of inflammation (mild, moderate, severe). If there is active inflammation, the degree of activity (mild, moderate, severe) can be reported. If there is intestinal metaplasia, the degree of intestinal metaplasia (mild, moderate, severe) can be reported. If there is dysplasia, the grade of dysplasia (low or high) should be reported. Long-term infection with H. pylori is an important cause of the occurrence and development of chronic digestive diseases such as type B (antral) gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue (MALT) gastric cancer, etc. The World Health Organization (WHO) has classified it as a Class I carcinogen.

[0003] HP immunohistochemical staining can be automated using a fully automatic immunohistochemical staining instrument. Various special guidelines have proposed the need to set up reasonable quality control for immunohistochemistry to ensure the correctness of the experimental results, so as to obtain accurate diagnostic results. The laboratory's quality management system requirements also require the addition of controls during the implementation of immunohistochemistry to ensure that all tests conducted by the laboratory have traceable standards. Therefore, convenient, stable and reliable quality control products have become a new requirement for pathology laboratories to achieve immunohistochemical quality control.

[0004] There is a big gap between immunohistochemistry and other medical testing methodologies in terms of quality control. Currently, the most common quality control method for Helicobacter pylori immunohistochemistry is to use the remaining tissue of the patient as a control sample in subsequent experiments. This method is limited by the number of laboratory samples and requires a lot of manpower during implementation. Therefore, this method has not been widely used in domestic pathology laboratories. Therefore, it is very necessary to develop a quality control product for Helicobacter pylori immunohistochemistry. Summary of the invention

[0005] In order to solve one of the above problems, the present invention provides a transplanted tumor containing Helicobacter pylori and a preparation method and application thereof.

[0006] In order to achieve the above objectives, the present invention adopts the following technical means: The first aspect of the present invention provides a reagent for preparing transplanted tumor tissue containing Helicobacter pylori, the reagent comprising tumor cells and Helicobacter pylori, and also comprising at least one of matrix gel and defibrinated sheep blood.

[0007] In some embodiments of the present invention, the concentration of tumor cells in the reagent is 5-6.5 M / mL, and the density of Helicobacter pylori is 1.5×108 -3×10 8 As a preferred solution in the present invention, a tumor cell suspension with a concentration of 8 M / mL and a density of 8×10 8 cfu / mL of Helicobacter pylori culture solution were mixed in a volume ratio of (2-4):1.

[0008] In some embodiments of the present invention, in the reagent, the volume fraction of the matrix gel is 30%-40%, and the volume fraction of the defibrinated sheep blood is 2%-3.5%.

[0009] In some embodiments of the present invention, preferably, the tumor cells are HeLa cells.

[0010] In the present invention, the inventors found that simply injecting Helicobacter pylori liquid subcutaneously in nude mice was difficult to cause tumors to form in nude mice; it was also difficult to obtain cell and HP transplant tumor samples by first injecting cells and then injecting Helicobacter pylori after tumor formation. Later, it was found that by injecting nude mice with a reagent containing tumor cells and Helicobacter pylori, and at least one of the auxiliary reagents matrix gel and defibrinated sheep blood as an injection reagent, transplant tumor tissue containing Helicobacter pylori could be obtained in 1-2 weeks.

[0011] The second aspect of the present invention provides the use of the first aspect in preparing transplanted tumor tissue containing Helicobacter pylori.

[0012] The third aspect of the present invention provides a method for preparing a transplanted tumor containing Helicobacter pylori, comprising the following steps: (1) culturing tumor cells to obtain a tumor cell suspension; the tumor cell suspension is prepared by suspending the cells in a culture medium to a concentration of 16 M / mL; (2) Cultivating Helicobacter pylori to obtain a Helicobacter pylori bacterial solution; the Helicobacter pylori bacterial solution is mixed with 10 mg / mL tryptone broth medium to a density of 16×10 8 cfu / mL; (3) preparing the reagent described in the first aspect as an injection reagent; (4) Injecting the injection reagent prepared in step (3) into nude mice to obtain Helicobacter pylori-containing xenograft tumors.

[0013] In some embodiments of the present invention, four stock solutions are used to prepare the injection reagent: A. Mix the cell suspension in step (1) with an equal volume of PBS solution to make the final cell concentration 8M / mL; B. Mix the bacterial solution in step (2) with 10 mg / mL tryptone broth medium in equal volumes to make the final bacterial density 8×10 8 cfu / mL; C. A mixture of equal volumes of the tumor cell suspension obtained in step (1) and matrix gel, wherein the final concentration of the tumor cells is 8-10 M / mL; D. A mixture of the Helicobacter pylori solution obtained in step (2): defibrinated sheep blood: tryptone broth medium in a volume ratio of 5:1:4, with a final density of 8×10 8 -10×10 8 cfu / mL; The reagents described in the first aspect are prepared using the aforementioned four mother solutions.

[0014] In some embodiments of the present invention, the mother liquor of step C is mixed with the mother liquor of step D at a ratio of 4:1, or the mother liquor of step C is mixed with the mother liquor of step B at a ratio of 2:1, or the mother liquor of step A is mixed with the mother liquor of step D at a ratio of 2:1; preferably, the mother liquor of step C is mixed with the mother liquor of step D at a ratio of 2:1.

[0015] In some embodiments of the present invention, the concentration of the tryptone broth medium is 1-10 mg / mL; preferably, the tumor cells are Hela cells.

[0016] The fourth aspect of the present invention provides a transplanted tumor containing Helicobacter pylori prepared according to the method described in the third aspect.

[0017] The fifth aspect of the present invention provides a use of the Helicobacter pylori-containing transplanted tumor described in the fourth aspect in immunological detection or analysis; in some embodiments, it is used to prepare quality control products and reference products in immunological detection or analysis.

[0018] In some embodiments of the present invention, there is also provided the use of a transplanted tumor containing Helicobacter pylori as a drug research model.

[0019] When the transplanted tumor containing Helicobacter pylori is used for immunohistochemistry (IHC) detection, it can be clearly seen that the HP transplanted tumor has tissue characteristics. In addition to the aggregated HP bacterial staining, it can also be seen that other tissue cells are obviously negative and hematoxylin staining is blue. Compared with simple bacterial staining, using it as a quality control product has stronger staining. In addition to being a positive quality control, there is also an internal control of negative cell hematoxylin staining, so that the same quality control product has both bacterial positive staining and tissue cell negative staining, which is consistent with routine case tissue testing.

[0020] In some embodiments of the present invention, the immunological detection or analysis is selected from one of immunohistochemistry, immunofluorescence, and immunoblotting.

[0021] In some embodiments of the present invention, it is used to monitor the HP immunohistochemical staining process.

[0022] In some embodiments of the invention, the monitoring includes monitoring changes in primary antibody concentration and repair conditions.

[0023] The sixth aspect of the present invention provides a Helicobacter pylori detection kit, wherein the kit comprises the quality control product prepared from the transplanted tumor containing Helicobacter pylori as described in the fourth aspect.

[0024] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a reagent for preparing transplanted tumor tissue containing Helicobacter pylori. By injecting nude mice with the reagent, transplanted tumor tissue containing Helicobacter pylori can be obtained in 1-2 weeks. The Helicobacter pylori transplanted tumor quality control product prepared by using the transplanted tumor tissue has more advantages. Compared with simple bacterial staining, the Helicobacter pylori transplanted tumor quality control product has stronger staining in addition to being used as a positive quality control; at the same time, there is also an internal control of negative cell hematoxylin staining, so that the same quality control product has both bacterial positive staining and tissue cell negative staining, which is consistent with conventional case tissue detection, can be used in quality control products and reference products in tissue cell immune detection or analysis methods, and plays a monitoring role in HP immunohistochemistry quality control: when the HP primary antibody concentration and repair conditions change, the HP transplanted tumor staining also changes accordingly, and the quality control product can be used for conventional immunohistochemistry HP quality control control. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The growth diagram of Helicobacter pylori in Example 1 of the present invention is shown; Figure 2 The results of mixed staining of HP bacteria and HeLa cells in different proportions in Example 3 of the present invention are shown; Figure 3 The application of the lysate prepared from HeLa-HP transplanted tumor in the present invention in Western blot is shown; Figure 4 The results of immunohistochemical staining of the HP pure bacterial liquid quality control product of the present invention are shown; Figure 5 The results of immunohistochemical staining of HP transplanted tumor quality control products in the present invention are shown; Figure 6 The staining change diagram of the HP transplant tumor quality control product in the present invention under different primary antibody dilution conditions is shown. DETAILED DESCRIPTION

[0026] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0028] The technical solution of the present application is further described in detail below in conjunction with specific implementation methods.

[0029] All immunohistochemical experiments in the present invention used the LYNX480 immunohistochemical staining machine platform and supporting reagents of Tuling (Hangzhou) Biopharmaceutical Co., Ltd., and the HP primary antibody used Abcam's HP antibody (clone number: EPR10353, product number: ab172611) for conventional immunohistochemical staining experiments.

[0030] Example 1 Cultivation of Helicobacter pylori (1) Take out 1 mL of Helicobacter pylori culture stored at -80℃, quickly resuscitate it in 38℃ water, open the tube in the sterile biological safety cabinet, and add it evenly to 2 Columbia blood agar culture plates, 500μL per plate. Use a sterilized and clean spreading stick to spread the HP culture on 2 plates. Add 2 mL of 10 mg / mL tryptic soy broth medium to the culture plate.

[0031] (2) Place the coated HP culture plate in a three-gas incubator and culture it under the following conditions: 37°C, 5% O2, 10% CO2, 85% N2. Culture it statically for 3 to 5 days. Generally, the HP bacteria can be subcultured or harvested after about 4 days of culture.

[0032] (3) During the HP culture process, the typical morphological characteristics of bacterial growth are: colony diameter is 0.5-1mm, round, with neat edges, opaque, grayish white on the front, raised in the middle, smooth and moist on the surface, easy to pick up, no pigment, Gram-negative (red), bacillus. HP growth reference pictures are as follows: Figure 1 shown.

[0033] (4) Add 5 mL of DPBS to each HP plate (grown to 70%-90% of the entire plate), gently rinse the HP, and use a 1 mL pipette to aspirate the bacterial solution into a clean 50 mL centrifuge tube to collect the bacterial solution. Be careful to avoid aspirating solid culture medium. Follow this process to collect bacterial solution for each plate. Use a pipette to pipette several times to mix the collected HP bacterial solution for subsequent experiments.

[0034] Example 2 Preparation of Helicobacter pylori suspension (1) Set the speed of the collected Helicobacter pylori culture to 8000 rpm and centrifuge at room temperature for 5 min. Discard the supernatant and add 50 mL of DPBS to continue washing for 2 more times.

[0035] (2) Add 2 mL of neutral formalin fixative, blow evenly, and fix for 22 hours.

[0036] (3) Set the speed to 8000 rpm, centrifuge at room temperature for 5 min, discard the supernatant, add 50 mL of DPBS and continue washing for 2 more times.

[0037] (4) Discard the supernatant and resuspend the cells in the semisolid buffer described in the patent "CN112611614B-A method for preparing a semisolid cell suspension and its application". Adjust the density to 1×10 8 cfu / mL to form the final Helicobacter pylori suspension, which was stored at -20°C for later use.

[0038] Example 3 Study on immunohistochemical staining of Helicobacter pylori suspension The Helicobacter pylori suspension prepared in Example 2 and the Hela cell suspension (the Hela cell suspension was prepared by the relevant method described in the patent "CN112611614B-A method for preparing a semi-solid cell suspension and its application", and Hela cells are HP staining negative cells, and other negative cells can also be used instead) were mixed at a volume ratio of 1:0, 1:3, 1:2, 1:1, 2:1, 3:1, and 0:1, and after being fully mixed, they were spotted on a blank glass slide, with 2 μL spotted on each spot. After the slide was dried, conventional immunohistochemical staining was performed on the machine using a primary antibody dilution of 1:500; the experimental results are as follows Figure 2 shown.

[0039] The results show that: Figure 2It can be seen that after mixing the Helicobacter pylori suspension and the Hela cell suspension, the bacteria are small and the cells are large. After mixing the two in different volume ratios, compared with the staining of only the Helicobacter pylori suspension, the positive staining of the HP bacteria is not obvious, and the HP bacteria and Hela cells are separated. Compared with tissue staining (HP bacteria adhere to the gastric mucosal epithelium), the mixed suspension staining is quite different, which is not conducive to the judgment of the control. The pure Helicobacter pylori suspension staining is observed under a microscope, and the phenomenon is brown bacteria positive staining. Since there is no negative staining, it is also not conducive to the consistency judgment of tissue and liquid staining.

[0040] Example 4 Establishment of Helicobacter pylori transplant tumor model 1. Inoculation of nude mice with simple Helicobacter pylori suspension (1) The collected Helicobacter pylori culture was centrifuged at 8000 rpm for 5 min at room temperature. The supernatant was discarded and washed once with 50 mL of DPBS. The concentration was 4.28 × 10 8 cfu / mL, 20mL in total.

[0041] (2) After centrifuging the HP bacterial solution, add 10 mg / mL tryptone broth medium to adjust the HP density to 10×10 8 cfu / mL, inoculated six BALB / C female nude mice. Each nude mouse was inoculated at four points under the armpits of the limbs, and 100uL was inoculated at each site. The same inoculation method was used in subsequent experiments. After 1 to 2 weeks, the growth of Helicobacter pylori transplanted tumors was observed. It can be seen that none of the six female nude mice grew transplanted tumors under the skin. The parts of the six mice injected with Helicobacter pylori liquid showed varying degrees of skin purple or ulceration, causing an inflammatory reaction. This shows that it is difficult for nude mice to form tumors subcutaneously by simply injecting Helicobacter pylori liquid subcutaneously.

[0042] 2. Inject cells first, then inject Helicobacter pylori after tumor formation HeLa cells were cultured according to conventional methods, with the culture medium being RPMI1640 + 10% FBS, cultured at 37°C, 5% CO2, and cultured and subcultured until the cells were collected. The cells were suspended in the culture medium to 16 M / mL (i.e., there were approximately 16×10 6 cells), mixed with equal volumes of matrix gel to a final cell concentration of 8 M / mL, and inoculated subcutaneously into 5 female nude mice, with the number of cells inoculated being approximately 3.2 M / mouse. After approximately 1-2 weeks, subcutaneous tumor formation was observed in 3 of the nude mice.

[0043] The cultured Helicobacter pylori culture was washed with DPBS and then 10 mg / mL tryptone broth was added to adjust the density to 8 × 10 8 cfu / mL(8×10 8cfu / mL-10×10 8 cfu / mL), and selected three female nude mice with HeLa cell tumors. HP bacterial solution was inoculated into the grown cell tumors, and the tumor growth of the nude mice was observed after 1 to 2 weeks. It can be seen that the tumors of the three nude mice that were subcutaneously injected with HeLa cells and grew tumors disappeared or were extremely small after the injection of HP bacterial solution, making it difficult to use them for subsequent experiments. This is probably because the body's immune system was activated after the injection of HP, resulting in tumor ablation. It is also difficult to obtain cell and HP transplant tumor samples according to this method.

[0044] 3. Mixed injection of cells and Helicobacter pylori into nude mice (1) Culture HeLa cells according to conventional methods, using RPMI1640 + 10% FBS as the culture medium, at 37°C, 5% CO2, and culture and subculture until the cells are collected. Suspend the cells in the culture medium to 16M / mL.

[0045] (2) After the Helicobacter pylori culture collected from the laboratory was washed with DPBS, the HP culture was mixed with 10 mg / mL tryptone broth medium to a density of 16×10 8 cfu / mL.

[0046] (3) Prepare four kinds of stock solutions: The first mother solution is pure Hela cells, that is, the cell suspension in step (1) is mixed with equal volumes of commercially available conventional PBS solution to make the final cell concentration 8M / mL; The second mother solution is pure HP bacteria, that is, the bacterial solution in step (2) is mixed with 10 mg / mL tryptone broth medium in equal volumes to make the final bacterial density 8×10 8 cfu / mL; The third mother solution is Hela cells with added matrix gel, that is, the cell suspension in step (1) is mixed with equal volumes of matrix gel to make the final cell concentration 8M / mL; Mother solution 4 is HP bacteria with defibrinated sheep blood added, that is, based on the total volume of 100%, 50% by volume of the bacterial solution in step (2), 10% by volume of defibrinated sheep blood, and 40% by volume of 10 mg / mL tryptone broth medium are mixed to make the final bacterial density 8×10 8 cfu / mL.

[0047] (4) The stock solutions were mixed according to the volume ratio in Table 1. Five BALB / C female nude mice were injected into each group. The growth of the transplanted tumors in the nude mice was observed after 1-2 weeks. The statistical results are shown in Table 1.

[0048] Table 1 Injection formula and transplanted tumor growth statistics

[0049] The results showed that nude mice in groups 4, 6, 7, and 8 all had transplanted tumors, among which the mice in group 8 had the most transplanted tumors, and the other groups were the same. Therefore, the optimal injection formula for forming transplanted tumors was mother solution 3 (HeLa-matrix gel complex, the final cell concentration was about 8M / mL): mother solution 4 (HP-defibrotized sheep blood complex, the final bacterial density was about 8×10 8 The volume ratio of cfu / mL was 2:1.

[0050] (5) The nude mice that successfully grew tumors were killed, and the tumors were removed subcutaneously for subsequent experiments.

[0051] Example 5 Preparation of Helicobacter pylori transplant tumor quality control The HeLa-HP transplanted tumor was cut into small pieces and fixed in neutral formalin fixative for 24 hours. After fixation, the HeLa-HP transplanted tumor tissue was dehydrated. The dehydration process was 70% ethanol dehydration for 1.5 hours, 95% ethanol dehydration for 1.5 hours, and anhydrous ethanol dehydration for 1 hour. The HP transplanted tumor was then placed in xylene for transparency for 40 minutes. After transparency, the transplanted tumor was soaked in paraffin in a paraffin tissue embedding box and embedded in paraffin for 3 hours. After sufficient wax immersion, the entire embedding box was perfused and sealed and cooled to room temperature until completely solidified. After the wax solidifies, it is placed in a -20℃ freezer to cool overnight. The next day, the wax block is removed from the metal embedding base and placed in the wax block warehouse for use.

[0052] Example 6 Application of Western blot in Helicobacter pylori transplanted tumors (1) Cut the HeLa-HP transplanted tumor removed from the subcutaneous tissue of the nude mouse into small pieces, freeze them with liquid nitrogen, grind them into powder in a mortar, and then add lysis buffer to lyse them. After the HP transplanted tumor sample is fully lysed, centrifuge and take the supernatant to quantify the concentration of the lysate (BCA quantitative method).

[0053] (2) After the concentration of HP transplant tumor lysate was quantified, the lysate was used for polyacrylamide gel electrophoresis. The loading amount of HeLa-HP transplant tumor lysate was 20 μg / lane. The electrophoresis conditions were: 80V, 40min; 120V, 80min.

[0054] (3) Transfer: Remove the gel from the glass plate after electrophoresis in step (2), soak it in transfer buffer for 30 min, and then perform transfer experiment. The transfer conditions are: 0.4A lateral flow transfer for 20 min.

[0055] (4) After the transfer is completed, remove the PVDF membrane, rinse the PVDF membrane with ddH2O for 5 minutes, add 10mL of Ponceau red for staining, wash the membrane with methanol for 5 minutes, dry it and set it aside. At this time, the red area of ​​the cell protein lysate on the PVDF membrane indicates that the protein lysate on the cells has been transferred to the PVDF.

[0056] (5) Blocking: Take out the PVDF membrane prepared in step (3), activate it with methanol for 30 seconds, and then wash the membrane with ddH2O for 5 minutes. Then add 10 mL of 5% skim milk powder to block the nonspecific binding sites on the membrane, and incubate at 60 rpm for 1 hour at room temperature.

[0057] (6) Primary antibody incubation: After step (4) is completed, add a 1:1000 diluted anti-HP monoclonal antibody (developed by Hangzhou Bailing Biotechnology, the cloned immunogen is HP catalase with a molecular weight of 59 kD) and incubate at room temperature and 60 rpm for 1 h.

[0058] (7) Secondary antibody incubation: After step (5) is completed, add 1:4000 diluted HRP-conjugated goat anti-rabbit IgG polyclonal antibody and incubate at room temperature at 60 rpm for 1 h.

[0059] (8) Development and photography: After step (6), add the prepared ECL developer to the membrane. Place the PVDF membrane in a UV exposure instrument for photography.

[0060] (9) The image is inverted and the result is as follows: Figure 3 shown.

[0061] The results in the figure show that the anti-HP rabbit monoclonal antibody can recognize the HP protein in the HP transplant tumor lysate, with an apparent molecular weight of 59kD, and no obvious mixed bands appear, which is in line with theoretical expectations. This shows that the HP transplant tumor lysate can be used for Western blot detection experiments in the development of HP antibodies.

[0062] Example 7 Immunohistochemistry (IHC) Verification of Helicobacter pylori Transplant Tumor Quality Control (1) Use a pipette to spot 2 μL of the pure HP bacterial solution prepared in Example 2 on a clean glass slide and dry it for later use; slice the transplanted tumor quality control sample prepared in Example 5 to a thickness of 1-3 μm, pick up the slices and adhere them to a clean glass slide, and dry them for later use.

[0063] (2) Use HP primary antibody at a dilution of 1:500 for routine immunohistochemical staining experiments.

[0064] (3) After immunohistochemical staining is completed, the sections are placed under a microscope for reading and photographing.

[0065] (4) Immunohistochemical staining images of HP pure bacterial liquid quality control products taken under a microscope after immunohistochemical staining are as follows: Figure 4 As shown; the immunohistochemical staining picture of HP transplanted tumor liquid quality control product after immunohistochemical staining and photographed under the microscope is as follows Figure 5 shown.

[0066] The results show that: Figure 4 It can be clearly seen that due to the small diameter of the HP bacterial suspension, only brown dots stained with DAB can be seen under a 40x microscope for immunohistochemical staining, confirming that they are positive HP bacteria. Figure 5 It can be clearly seen that the HP transplant tumor has tissue characteristics. In addition to the aggregated HP bacterial staining, other tissue cells can be clearly seen to be negative, and the hematoxylin staining is blue. This quality control product has more advantages. Compared with simple bacterial staining, the HP transplant tumor liquid quality control product has stronger staining in addition to being a positive quality control; at the same time, there is also an internal control for negative cell hematoxylin staining, so that the same liquid quality control product has both bacterial positive staining and tissue cell negative staining, which is consistent with routine case tissue testing.

[0067] Example 8 Monitoring effect of Helicobacter pylori transplant tumor quality control products on HP immunohistochemistry quality control 1. Monitoring of HP primary antibody concentration changes by HP immunohistochemistry quality control products On the LYNX480 immunohistochemical staining machine platform, HP primary antibody (clone number: EPR10353, catalog number: ab172611) was set at 1:50, 1:200, 1:800, and 1:3200 to perform routine immunohistochemical staining on positive tissue stomach and HP transplant tumor quality control products. The staining results are shown in Table 2 below. The staining changes of HP transplant tumor quality control products under different primary antibody dilution conditions are shown in Table 2. Figure 6 shown.

[0068] Table 2 Statistics on the effect of HP primary antibody concentration changes on staining results

[0069] The results showed that under normal staining conditions, the dilution of the HP primary antibody was 1:200, and the positive gastric tissue was strongly stained with a clean background. When the antibody concentration was increased by 4 times, the background staining of the tissue and HP transplant tumor quality control products was darker. When the antibody concentration was gradually reduced, the staining intensity of the tissue and HP transplant tumor quality control products decreased, maintaining good consistency. Therefore, the HP transplant tumor liquid quality control product can monitor the staining process of improper use of HP primary antibodies.

[0070] 2. Monitoring of changes in HP primary antibody repair conditions by HP transplant tumor quality control products The HP primary antibody target was set to 1:200 dilution on the LYNX480 immunohistochemical staining machine platform, and the repair conditions were set to no repair, citric acid repair for 20 minutes, Tris-EDTA repair for 5 minutes, Tris-EDTA repair for 10 minutes, Tris-EDTA repair for 20 minutes, and Tris-EDTA repair for 40 minutes, a total of 6 repair conditions, and the rest were performed as usual for immunohistochemical staining. The staining results are shown in Table 3.

[0071] Table 3 Statistics on the impact of changes in HP repair conditions on staining results

[0072] The results showed that the normal staining repair condition was Tris-EDTA repair for 20 minutes, and the result was strong staining with a clean background. When the repair condition was no repair, there was no staining for both tissue and HP transplant tumor quality control products; when the repair conditions were citric acid repair for 20 minutes, Tris-EDTA repair for 5 minutes, and Tris-EDTA repair for 10 minutes, compared with the conventional staining conditions, the positive staining rate and staining intensity of tissue and HP transplant tumor quality control products decreased; when the repair condition was Tris-EDTA repair for 40 minutes, compared with the conventional staining conditions, the positive staining rate of tissue and HP transplant tumor quality control products did not change significantly, but the staining background deepened, affecting the result judgment.

[0073] It can be seen that the quality control product of the transplanted tumor maintains good consistency with the tissue staining results, and can monitor the staining process for improper use of repair conditions.

[0074] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.

Claims

1. A reagent for preparing transplanted tumor tissue containing Helicobacter pylori, characterized in that: The reagents include tumor cells and Helicobacter pylori, and also include at least one of matrix gel and defibrinated sheep blood.

2. The reagent according to claim 1, characterized in that: The tumor cell concentration in the reagent is 5-6.5 M / mL, and the Helicobacter pylori density is 1.5×10 8 -3×10 8 cfu / mL.

3. The reagent according to claim 1, characterized in that: In the reagent, the volume fraction of the matrix gel is 30%-40%, and the volume fraction of the defibrotized sheep blood is 2%-3.5%.

4. Use of the reagent according to any one of claims 1 to 3 in the preparation of transplanted tumor tissue containing Helicobacter pylori.

5. A method for preparing a transplanted tumor containing Helicobacter pylori, characterized in that: The steps include: (1) Cultivating tumor cells to obtain tumor cell suspension; (2) culturing Helicobacter pylori to obtain Helicobacter pylori bacterial liquid; (3) Preparing the reagent according to any one of claims 1 to 3 as an injection reagent; (4) Injecting the injection reagent prepared in step (3) into nude mice to obtain Helicobacter pylori-containing xenograft tumors.

6. The Helicobacter pylori-containing transplanted tumor prepared according to the method of claim 5.

7. Use of the Helicobacter pylori-containing transplanted tumor according to claim 6 in the preparation of quality control products and reference products for immunological detection or analysis.

8. The use according to claim 7, characterized in that: The immunological detection or analysis is selected from one of immunohistochemistry, immunofluorescence and immunoblotting.

9. Use of the Helicobacter pylori-containing transplanted tumor according to claim 6 as a drug research model.

10. A Helicobacter pylori detection kit, characterized in that: A quality control product prepared from the Helicobacter pylori-containing transplanted tumor as described in claim 6.

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