Cell infection model for efficient proliferation of chlamydia trachomatis and application

Abstract

The invention provides a chlamydia trachomatis efficient proliferation cell infection model and an establishment method thereof, and the establishment method specifically comprises the following steps: taking immortalized human placental trophoblast cells HTR-8 / SVneo as hosts, and infecting the host cells with chlamydia trachomatis. The chlamydia trachomatis infection model based on healthy human source cells is established for the first time, so that the chlamydia trachomatis can present infection, survival and proliferation states in normal cells. According to the cell infection model, rapid growth of the chlamydia trachomatis can be guaranteed under the condition that actinomycete ketone is not added, the step that the chlamydia trachomatis is infected and amplified through mode hosts such as Hela cells is simplified, and compared with the mode hosts such as the Hela cells, the infection speed and proliferation efficiency of the chlamydia trachomatis in the cell infection model are remarkably improved. The invention provides an efficient infection model for developing pathogenic characteristic research of the chlamydia trachomatis and an interaction mechanism of the chlamydia trachomatis and a host.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a cell infection model for efficient proliferation of Chlamydia trachomatis and an application thereof. Background Art

[0002] Chlamydia is a small prokaryotic organism that lives as an obligate parasite in eukaryotic cells. Its growth and development depend on the host. According to the classification of the International Commission on Prokaryotes in 2022, Chlamydia is classified into Chlamydiae, Chlamydiae, Chlamydiales, Chlamydiaceae, and Chlamydia. The life cycle of Chlamydia has two forms: elementary body (EB) and reticulate body (RB). EB is a small spherical cell that is infectious and capable of infection. Its cell wall is thick and dense, unable to move and grow, and the ratio of RNA to DNA is about 1:1; RB is a large spherical cell that is non-infectious and non-infectious. Its cell wall is thin and fragile, easily deformed, and grows faster. The ratio of RNA to DNA is about 3:1. EB infects the host and transforms into RB. RB reproduces by binary fission in the host cell to form inclusion bodies and re-transforms into EB at the same time. EB is released by host cells and waits for the opportunity to infect new host cells.

[0003] Chlamydia trachomatis (Ct) can cause diseases such as trachoma, venereal lymphogranuloma and urogenital tract infection, and is one of the most common pathogens of sexually transmitted diseases. In 1956, Chinese scholar Tang Feifan first used the chicken embryo yolk sac inoculation method to culture and isolate Ct, solving the long-disputed etiology of trachoma and setting off a climax in chlamydia research. Since the chicken embryo yolk sac inoculation method is cumbersome, Ct culture and isolation have been improved to use Hela cells or L929 cells as model hosts. However, since Hela cells are cancer cells and L929 cells are mouse cells, they cannot directly reflect the interaction between Ct and target cells, especially healthy human cells, when infecting the body. In addition, in the process of modeling based on the two, the inclusion body grows slowly, and cycloheximide needs to be added to promote the growth of chlamydia. Therefore, the establishment of a cell infection model with efficient Ct proliferation will effectively promote the study of Ct etiology and the exploration of the interaction mechanism between Ct and the host, which is of great value for the development of public health affairs and the maintenance of people's health and safety. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a cell infection model for efficient proliferation of Chlamydia trachomatis and its application in view of the deficiencies of the above-mentioned prior art. The present invention establishes for the first time a cell infection model based on immortalized human placental trophoblast cells (HTR-8 / SVneo cells) that can efficiently proliferate Ct. The cell model is used to amplify Ct, which simplifies the modeling steps, improves the Ct amplification efficiency, and can present the infection, survival and proliferation status of Chlamydia trachomatis in normal somatic cells, providing an efficient infection model for studying the pathogenic characteristics of Chlamydia trachomatis and the interaction mechanism between Chlamydia trachomatis and the host.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] The invention discloses a cell infection model for efficient proliferation of Chlamydia trachomatis. The cell infection model uses immortalized human placental trophoblast HTR-8 / SVneo cells as hosts and infects the host cells with Chlamydia trachomatis.

[0007] Preferably, the placental trophoblast HTR-8 / SVneo cells are isolated from human placental tissue in early pregnancy, and are immortalized by introducing the simian virus 40 large T antigen (SV40Tag) gene.

[0008] The present invention also provides a method for establishing a cell infection model for efficient proliferation of Chlamydia trachomatis, the method comprising the following steps:

[0009] S1. Immortalized human placental trophoblast HTR-8 / SVneo cells were collected and inoculated into 6-well plates, and DMEM / F-12 cell culture medium containing 10% fetal bovine serum was added, and the cells were cultured in an incubator until the HTR-8 / SVneo cell confluence reached 60%-80%, and the cell culture medium was discarded, and then the cells were treated with DEAE-dextran solution in an incubator for 20-30 minutes, and the supernatant was discarded to obtain treated HTR-8 / SVneo cells;

[0010] S2, adding Chlamydia trachomatis infection solution to the treated HTR-8 / SVneo cells obtained in S1, incubating in an incubator for 30 minutes and then centrifuging, and continuing to incubate in the incubator for 30 minutes after the centrifugation, discarding the supernatant, and obtaining Chlamydia trachomatis-infected HTR-8 / SVneo cells;

[0011] S3. Add DMEM / F-12 cell culture medium containing 10% fetal bovine serum to the HTR-8 / SVneo cells infected with Chlamydia trachomatis obtained in S2, and culture them in an incubator for 12-14 hours to obtain a Chlamydia trachomatis HTR-8 / SVneo cell infection model.

[0012] Preferably, the culture conditions of the incubator are: temperature of 37° C. and 5% CO 2 .

[0013] Preferably, the concentration of the DEAE-dextran solution in S1 is 30 μg / mL.

[0014] The present invention also provides an application of the cell infection model in the study of the pathogenic characteristics of Chlamydia trachomatis and the study of the interaction mechanism between Chlamydia trachomatis and the host.

[0015] The present invention has significant technical effects due to the adoption of the above technical solution:

[0016] 1. The present invention provides a cell infection model for efficient proliferation of Chlamydia trachomatis, which uses immortalized human placental trophoblast HTR-8 / SVneo cells as hosts and infects the host cells with Chlamydia trachomatis. Compared with Hela cells or mouse L929 cells, the model does not require the addition of cycloheximide during establishment, which can simplify the Ct amplification step, and the Ct proliferation rate in HTR-8 / SVneo cells is faster; in addition, the present invention can present the infection, survival and proliferation status of Chlamydia trachomatis in normal somatic cells, and can directly reflect the interaction between Ct and target cells, especially healthy human somatic cells when infecting the body, and provide an efficient and convenient cell infection model and modeling technology for studying the pathogenic characteristics of Chlamydia trachomatis and the interaction mechanism between Chlamydia trachomatis and the host.

[0017] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a flow chart of establishing the HTR-8 / SVneo cell infection model and the Hela cell infection model of Chlamydia trachomatis (Ct) in the present invention;

[0019] Figure 2 The inclusion body formation at each time point of the HTR-8 / SVneo cell infection model of Ct in Example 2 of the present invention;

[0020] Figure 3 This is the inclusion body formation at various times in the Hela cell infection model of Ct in Example 2 of the present invention. DETAILED DESCRIPTION

[0021] Example 1

[0022] This example is the establishment of an HTR-8 / SVneo cell infection model of Chlamydia trachomatis, and the method is as follows:

[0023] S1. Collect immortalized human placental trophoblast HTR-8 / SVneo cells and inoculate them in 6-well plates. Add 2 mL of DMEM / F-12 cell culture medium containing 10% fetal bovine serum to each well of cells. Incubate in a 37°C, 5% CO2 incubator until the cell confluence reaches 60%, and then discard the cell culture medium. Add 2 mL of 30 μg / mL DEAE-dextran solution to each well of cells, treat in a 37°C, 5% CO2 environment for 30 minutes, and then discard the DEAE-dextran solution to obtain treated HTR-8 / SVneo cells.

[0024] S2. Add Chlamydia trachomatis infection solution to each well of HTR-8 / SVneo cells treated with S1, incubate in a 37°C, 5% CO2 incubator for 30 minutes, and centrifuge at 1500 rpm and 30°C for 60 minutes. After centrifugation, continue to incubate in a 37°C, 5% CO2 incubator for 30 minutes, discard the remaining Chlamydia trachomatis infection solution, and obtain HTR-8 / SVneo cells infected with Chlamydia trachomatis.

[0025] S3. Add 2 mL of DMEM / F-12 cell culture medium containing 10% fetal bovine serum to each well of HTR-8 / SVneo cells infected with Chlamydia trachomatis obtained in S2, and culture in a 37°C, 5% CO2 incubator for 12 h to obtain the Ct HTR-8 / SVneo cell infection model.

[0026] Example 2

[0027] This example is a comparison of the infection rate and proliferation rate of Chlamydia trachomatis on HTR-8 / SVneo cells and Hela cells.

[0028] 1. Establishment of HTR-8 / SVneo cell infection model of Chlamydia trachomatis (Ct): According to the steps described in Example 1, an HTR-8 / SVneo cell infection model of Chlamydia trachomatis was established, and the inclusion body formation was observed under an optical microscope at 0 h, 12 h, 24 h, 36 h and 48 h of infection.

[0029] 2. Establishment of Hela cell infection model of Chlamydia trachomatis (Ct): Hela cells were collected and inoculated into 6-well plates, 2 mL of DMEM high-glucose cell culture medium containing 10% fetal bovine serum was added to each well of cells, and the cells were cultured in a 37°C, 5% CO2 incubator until the cell confluence reached 60%, and the cell culture medium was discarded. 2 mL of 30 μg / mL DEAE-dextran solution was added to each well of cells, and the DEAE-dextran solution was discarded after being treated in a 37°C, 5% CO2 environment for 30 minutes. Chlamydia trachomatis infection solution was added to each well of cells, and after incubation in a 37°C, 5% CO2 incubator for 30 minutes, centrifuged at 1500 rpm and 30°C for 60 minutes. After centrifugation, the cells were continued to be incubated in a 37°C, 5% CO2 incubator for 30 minutes, and the remaining Chlamydia trachomatis infection solution was discarded. Add 2 mL of DMEM high-glucose cell culture medium containing 10% fetal bovine serum and 5 μg / mL cycloheximide to the cells in each well, and culture in a 37°C, 5% CO2 incubator for 12 h to obtain the Hela cell infection model of Chlamydia trachomatis. Observe the inclusion body formation under an optical microscope at 0 h, 12 h, 24 h, 36 h and 48 h of infection.

[0030] Figure 1 This is a comparison chart of the establishment process of the HTR-8 / SVneo cell infection model and the Hela cell infection model of Chlamydia trachomatis (Ct).

[0031] Figure 2 The figures show the inclusion body formation at different time points in the HTR-8 / SVneo cell infection model of Ct. It was found that initial inclusion bodies, which were white, translucent, and punctate vesicles, appeared in the cells 12 hours after Ct infection. As the infection time increased, the volume of the inclusion bodies in the cells gradually increased. At 48 hours after Ct infection, the size of the inclusion bodies accounted for about 2 / 3 of the cell volume and even filled the entire cell. Figure 3 The figures show the inclusion body formation at different time points in the Hela cell infection model of Ct. It was found that early inclusion bodies with white, translucent, punctate vesicles appeared in the cells 24 hours after Ct infection, and only a few inclusion bodies reached 2 / 3 of the cell volume 48 hours after Ct infection, indicating that compared with Hela cells, Ct inclusion bodies were formed earlier and grew faster in HTR-8 / SVneo cells, and there was no need to treat HTR-8 / SVneo cells with cycloheximide during the infection process.

[0032] Example 3

[0033] This example is to collect Chlamydia trachomatis progeny virus from HTR-8 / SVneo cells, the method is as follows:

[0034] According to the steps described in Example 1, the HTR-8 / SVneo cell infection model of Chlamydia trachomatis was established. When the average size of each inclusion body was about 2 / 3 of the cell size, and the Ct in the inclusion body was observed to move violently, the cell culture medium was discarded, and 1 mL of 4°C pre-cooled DMEM / F-12 basal medium was added to each well cell, and the cells were scraped off with a cell scraper under ice bath conditions and mixed evenly. The cell suspension was transferred to a 15 mL centrifuge tube pre-cooled at 4°C, and the cells were ultrasonically broken 6 times under ice bath conditions with a power of 300W and a program of opening 5s, stopping 5s, and 30s / times, and the ultrasonic sequence was stopped by 30s, 60s, 90s, 120s, and 150s. After the ultrasonic lysis is completed, mix and dispense into small volume centrifuge tubes, and store Ct progeny virus seeds in an environment of -80°C.

[0035] The present invention establishes for the first time a cell infection model based on immortalized human placental trophoblast cells (HTR-8 / SVneo cells) that can efficiently proliferate Ct. Compared with Hela cells or mouse L929 cells, the model establishment process does not require the addition of cycloheximide. The use of the cell model to amplify Ct simplifies the modeling steps, improves the Ct amplification efficiency, and can present the infection, survival and proliferation status of Chlamydia trachomatis in normal somatic cells, and can directly reflect the interaction between Ct and target cells, especially healthy human somatic cells when infecting the body, providing an efficient and convenient cell infection model and modeling technology for studying the pathogenic characteristics of Chlamydia trachomatis and the interaction mechanism between Chlamydia trachomatis and the host.

[0036] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A cell infection model for efficient proliferation of Chlamydia trachomatis, characterized in that: The cell infection model uses immortalized human placental trophoblast HTR-8 / SVneo cells as hosts, and the host cells are infected with Chlamydia trachomatis.

2. A cell infection model for efficient proliferation of Chlamydia trachomatis according to claim 1, characterized in that: The placental trophoblast HTR-8 / SVneo cells are separated from human placental tissues in early pregnancy, and are immortalized by introducing simian virus 40 large T antigen genes.

3. A method for establishing a cell infection model as claimed in claim 1, characterized in that: The steps include: S1. Immortalized human placental trophoblast HTR-8 / SVneo cells were collected and inoculated into 6-well plates, and DMEM / F-12 cell culture medium containing 10% fetal bovine serum was added, and the cells were cultured in an incubator until the HTR-8 / SVneo cell confluence reached 60%-80%, and the cell culture medium was discarded, and then the cells were treated with DEAE-dextran solution in an incubator for 20-30 minutes, and the supernatant was discarded to obtain treated HTR-8 / SVneo cells; S2, adding Chlamydia trachomatis infection solution to the treated HTR-8 / SVneo cells obtained in S1, incubating in an incubator for 30 minutes and then centrifuging, and continuing to incubate in the incubator for 30 minutes after the centrifugation, discarding the supernatant, and obtaining Chlamydia trachomatis-infected HTR-8 / SVneo cells; S3. Add DMEM / F-12 cell culture medium containing 10% fetal bovine serum to the HTR-8 / SVneo cells infected with Chlamydia trachomatis obtained in S2, and culture them in an incubator for 12-14 hours to obtain a Chlamydia trachomatis HTR-8 / SVneo cell infection model.

4. The method according to claim 3, characterized in that The culture conditions of the incubator are: temperature of 37° C. and 5% CO 2 .

5. The method according to claim 3, characterized in that: The concentration of the DEAE-dextran solution in S1 was 30 μg / mL.

6. An application of the cell infection model as claimed in claim 1 in the study of the pathogenic characteristics of Chlamydia trachomatis and the study of the interaction mechanism between Chlamydia trachomatis and the host.

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