Construction method and application of cervical normal organoid virus infection model

Abstract

The invention relates to the technical field of biological medicine and cell engineering, in particular to a construction method and application of a cervical normal organoid virus infection model. According to the invention, the invasion and infection process of the natural human papilloma virus in normal human cervical organs is simulated and represented to the maximum extent in vitro for the first time, and a new strategy is provided for preventing HPV infection.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine and cell engineering technology, and in particular to a construction method and application of a normal cervical organoid virus infection model. Background Art

[0002] Human papillomavirus (HPV) is a small, non-enveloped virus with a spherical shape and a diameter of about 55 nm, and its genetic material consists of double-stranded DNA. Persistent infection with high-risk HPV is the main cause of cervical cancer.

[0003] In the traditional virus research process, cell lines such as African green monkey kidney cells (Vero, AGMK, BGM) and human embryonic lung fibroblasts are used to inoculate viruses, amplify and culture the viruses, isolate the viruses, and then inoculate them into animal models for virological research. However, due to the non-lethal nature of HPV infection, most of the current research on HPV etiology relies on the VLP model. Artificially synthesized VLPs cannot truly reflect the infection characteristics of natural HPV to a certain extent, especially in related research fields such as studying virus-host interactions. The characteristics of natural HPV severely limit the universality of model cell and animal experimental results.

[0004] Organoids are microscopic three-dimensional structures that can self-assemble when pluripotent stem cells (PSC) and adult stem cells (ASC) are cultured in vitro. Organoids can simulate human organs in structure and function, have genomic stability during long-term in vitro expansion, and have good application potential in regenerative medicine, gene editing, precision medicine, organ development, disease modeling, etc. Organoid models retain the original characteristics of tissues and can more accurately reflect the human body's response to pathogens and drugs. These advantages highlight the great potential of organoids in personalized treatment, especially in predicting drug responses and simulating the interaction between the human body and pathogenic microorganisms.

[0005] Based on the characteristics of the organoid model, this paper proposes for the first time to use normal human cervical organoids to construct a natural human papillomavirus invasion and infection model. The establishment of this model can simulate and characterize the invasion and infection process of natural human papillomavirus in normal human cervical organoids to the greatest extent in vitro, and can be applied to the mechanism research and drug development of HPV vaccines, as well as the study of the pathogenic mechanism of viral diseases, which has important social, economic and scientific significance. In view of this, we propose a method for constructing and applying a normal cervical organoid virus infection model. Summary of the invention

[0006] The purpose of the present invention is to provide a method for constructing an organoid viral infection model. The establishment of this model can be applied to the mechanism research and drug development of HPV vaccines, as well as the study of the pathogenic mechanism of viral diseases, providing a new strategy for preventing HPV infection.

[0007] To achieve the above object, the present invention provides the following technical solutions: A method for constructing an organoid virus infection model, comprising: (1) Normal cervical organoids are mixed with the virus suspension and then incubated on a flat surface for a certain period of time to complete the infection process; (2) After infection, the organoids are collected and resuspended in Matrigel to continue culturing and growing.

[0008] (3) After 3D culture, the 3D organoids isolated and cultured after virus infection in step (2) are broken into cells, and the viruses are detected and captured by electron microscopy; Preferably, the normal cervical organoids co-cultured with the virus suspension in step (1) need to be treated with digestive fluid to remove the matrix gel, and have an appropriate size, preferably a cell sphere formed by 5-7 layers of cells; Preferably, in step (1), the components of the organoid digestion solution are: Advanced DMEM / F12 culture medium solution containing collagenase IV at a final concentration of 5 mg / ml; Preferably, in step (1), the virus infection liquid is a natural HPV infection liquid, and the virus infection liquid is a solution obtained by diluting the natural HPV infection liquid with a culture medium for organoid culture; the dilution ratio is 1:10-1:5, which is appropriately adjusted according to the virus infection ability; Preferably, in step (1), the planar co-incubation is to first place a thin layer of matrix gel mixture at the bottom of the cell culture dish, place it in a 37°C incubator and incubate it for 30 minutes until the matrix gel solidifies; then co-incubate the organoids without matrix gel wrapping with the virus suspension in the culture dish to complete the infection process; the composition of the matrix gel mixture is: the ratio of organoid culture medium to matrix gel is 9:1, and then add appropriate virus suspension.

[0009] Preferably, the mixture of organoids and virus suspension collected in step (2) is placed in a 15 ml sterile centrifuge tube pre-rinsed with 1% BSA, centrifuged at 500G for 5 min to collect the organoids, and after removing the virus supernatant, the organoids are resuspended in PBS, and the organoids are re-centrifuged and rinsed twice; the organoids are resuspended in matrix gel and cultured; Preferably, the crushing in step (3) is mechanical crushing, lysis solution crushing or ultrasonic crushing; Preferably, after the cell disruption, the virus is further precipitated and concentrated; more preferably, the virus is precipitated and concentrated by a method including PEG / NaCl precipitation, ammonium sulfate precipitation or sucrose gradient differential centrifugation; preferably, the PEG is PEG-8000; More preferably, in step (3), the isolated and cultured 3D organoids are cultured in 3D for 0 to 72 hours; preferably, after 3 hours, 6 hours, 9 hours, and 48 hours of 3D culture, the virus-infected organoids are ground and repeatedly frozen and thawed to break the cells, and then centrifuged to obtain the supernatant; Preferably, in step (3), the virus detection includes detecting the virus infection efficiency, which includes detecting the copy number of HPV in the cell through qPCR, combining sampling at different time points, and mapping the infection and replication process of the virus in the organoid; preferably, the nucleic acid is detected by specific primers and probes; more preferably, the specific probes and primers are selected from the primers described in the table.

[0010] Preferably, the 3D organoids separated and cultured in step (3) are cultured in 3D for 0 to 72 hours; preferably, after 3 hours, 6 hours, 9 hours, and 48 hours of 3D culture, the encapsulated matrix gel is removed with organoid digestion solution, and the centrifuged organoids are resuspended with electron microscopy fixative, and transmission electron microscopy scanning is performed to capture HPV.

[0011] Through the above technical solution, the present invention provides a method for constructing an organoid virus infection model. It has at least the following beneficial effects: (1) This invention is the first to achieve the purpose of natural HPV infection of normal human cervix in vitro by infecting normal human cervical organoids. The human organoid model can eliminate species differences and more realistically simulate the actual situation of human organ tissues. The mechanism research and drug development of HPV vaccines, as well as the research on the pathogenic mechanism of viral diseases, provide a new strategy for preventing HPV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application: Figure 1 This is a white light image of a normal cervical organoid; Figure 2 Schematic diagram of normal human cervical organoids infected with natural HPV; Figure 3 Transmission electron microscopy images of normal cervical organoids infected with natural HPV at different time points in the present invention; Figure 4 This is a radio electron micrograph of HPV particles appearing in the nucleus of a normal cervical organoid infected with natural HPV in the present invention; Figure 5This is a diagram showing the effect of virus infection after natural HPV infects normal cervical organoids in the present invention; DETAILED DESCRIPTION

[0013] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0014] Example 1: Natural HPV infection of normal human cervical organoids (P3 laboratory) Schematic diagram of normal human cervical organoids infected with natural HPV Figure 2 shown.

[0015] 1. Prepare human normal cervical organoids in 3D culture from surgical specimens ( Figure 1 ) were separated from the well plate together with the matrix gel and collected into a 15 ml sterile centrifuge tube pre-rinsed with 1% BSA.

[0016] 2. Centrifuge the mixture containing organoids and Matrigel obtained in step 1 at 500G for 5 min to collect the organoids, remove the supernatant, and obtain a mixture containing organoids and Matrigel.

[0017] 3. Add an appropriate amount of organoid digestion solution (Advanced DMEM / F12 culture medium solution containing collagenase IV at a final concentration of 5 mg / ml) to the mixture containing organoids and Matrigel obtained after centrifugation in step 2, and place the organoids resuspended in the digestion solution in a 37°C incubator for an appropriate digestion time to remove the matrix gel.

[0018] 4. Terminate the digestion of the organoid digestion solution in step 3 with complete culture medium, centrifuge at 400G for 5 minutes, remove the supernatant, and obtain the organoid precipitate.

[0019] 5. Add 2 mL of trypsin to the organoid precipitate obtained in step 4 and blow thoroughly until the organoid is roughly a cell ball formed by 5-7 layers of cells. Stop digestion and centrifuge at 400G for 5 minutes to obtain the organoid precipitate.

[0020] 6. Take the pre-cooled Matrigel matrix gel and Advanced DMEM / F12, 5% FBS solution mixture (1:1) and evenly spread it on a 6 cm dish. Place it in a 37°C incubator for 20 minutes to allow the matrix gel to completely solidify, and obtain a 6 cm dish covered with Matrigel matrix gel.

[0021] 7. Dilute the natural HPV virus concentrate with 6 mL of normal cervical organoid culture medium at a dilution ratio of 1:10 to obtain natural HPV virus infection fluid.

[0022] 8. Resuspend the organoid precipitate obtained in step 5 with the natural HPV infection fluid obtained in step 7 to obtain a mixture of the natural HPV infection fluid and the organoid precipitate, and evenly drop the mixture into a 6 cm dish covered with solidified Matrigel obtained in step 6. Incubate in a CO2 incubator at 37°C for a specific time to complete the infection process and obtain organoids infected with natural HPV.

[0023] 9. Collect the organoids infected with natural HPV obtained in step 8 together with the virus solution in a 15 mL centrifuge tube, gently pipette and centrifuge at 400G for 5 min, remove the supernatant containing the virus, and obtain the precipitate of organoids infected with natural HPV.

[0024] 10. The organoid precipitate obtained in step 9 was resuspended on ice with Advanced DMEM / F12 and 5% FBS solution, and the ratio of solution to matrix gel was 1:4; after the matrix gel was spread on the well plate, the well plate was placed in a 37°C incubator for 30 minutes. After the matrix gel solidified, culture medium was added for subsequent culture.

[0025] Example 2: Transmission electron microscopy scanning of normal cervical organoids infected with natural HPV The human normal cervical organoids infected with natural HPV obtained in Example 1 were cultured and collected into 15 mL centrifuge tubes after infection was completed (i.e., cultured for 0 h), 3 h, 6 h, 9 h, and 48 h, respectively. The organoids were centrifuged at 500 G for 5 min to collect the organoids, and the supernatant was removed to obtain a mixture containing the organoids and Matrigel matrix glue.

[0026] Add an appropriate amount of organoid digestion solution (Advanced DMEM / F12 culture medium solution containing collagenase IV at a final concentration of 5 mg / ml) to the centrifuged mixture containing organoids and Matrigel obtained in step 1, and place the organoids resuspended in the digestion solution in a 37°C incubator for an appropriate digestion time to remove the matrix gel.

[0027] The organoid digestion solution in step 2 was terminated with complete culture medium, centrifuged at 400G for 5 minutes, and the supernatant was removed to obtain the organoid precipitate.

[0028] Resuspend the organoids obtained in step 3 at different time points after infection with natural HPV in the electron microscopy fixative, centrifuge at 400G for 5 minutes, fully fix the organoids at room temperature in the dark, and transport them to the transmission electron microscopy laboratory at 4°C for preparation and photography. Figure 3 , 4 ) Example 3: Detection of viral copy number in normal cervical organoids 1. Cultivate the normal cervical organoids infected with natural HPV obtained in Example 1, collect them into 15 mL centrifuge tubes after the infection is completed (i.e., culture for 0 h), culture for 3 h, 6 h, 9 h and 48 h, respectively, and centrifuge at 500 G for 5 min to collect the organoids, remove the supernatant, and obtain a mixture containing organoids and Matrigel matrix glue.

[0029] 2. Add an appropriate amount of organoid digestion solution (Advanced DMEM / F12 culture medium solution containing collagenase IV at a final concentration of 5 mg / ml) to the centrifuged mixture containing organoids and Matrigel obtained in step 1, and place the organoids resuspended in the digestion solution in a 37°C incubator for an appropriate digestion time to remove the matrix gel.

[0030] 3. Terminate the digestion of the organoid digestion solution in step 2 with complete culture medium, centrifuge at 400G for 5 minutes, remove the supernatant, and obtain the organoid precipitate.

[0031] 4. The FastPure Cell / TissueTotal RNA Isolation Kit V2 and its HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) provided by Novagen were used to extract RNA at the corresponding time points of 0h, 3h, 6h, 9h and 48h from the organoid precipitate obtained in step 3. The RNA was used as a template to detect the viral copy number of the organoid using the primers provided in Table 1 ( Figure 5 ).

[0032] Table 1: It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0033] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for constructing a normal cervical organoid virus infection model, characterized in that: (1) Normal cervical organoids are mixed with the virus suspension and then incubated on a flat surface for a certain period of time to complete the infection process; (2) After infection, the organoids were collected and resuspended in Matrigel to continue culturing and growth; (3) After 3D culture, the 3D organoids isolated and cultured after virus infection in step (2) are broken into cells, and the viruses are detected and captured by electron microscopy.

2. The method for constructing a normal cervical organoid virus infection model according to claim 1, characterized in that: The normal cervical organoids co-cultured with the virus suspension in step (1) need to be treated with digestive fluid to remove the matrix gel, and the size should be appropriate, preferably a cell sphere formed by 5-7 layers of cells; In step (1), the components of the organoid digestion solution are: Advanced DMEM / F12 medium solution containing collagenase IV at a final concentration of 5 mg / ml; In step (1), the virus infection liquid is a natural HPV infection liquid, which is a solution obtained by diluting the natural HPV infection liquid with a culture medium for organoid culture; the dilution ratio is 1:10-1:5, which is appropriately adjusted according to the virus infection ability; In step (1), the planar co-incubation is to first place a thin layer of matrix gel mixture at the bottom of the cell culture dish, incubate in a 37°C incubator for 30 minutes, and wait for the matrix gel to solidify; then co-incubate the organoids without matrix gel wrapping with the virus suspension in the culture dish to complete the infection process; the composition of the matrix gel mixture is: the ratio of organoid culture medium to matrix gel is 9:1, and then add appropriate virus suspension.

3. The method for constructing a normal cervical organoid virus infection model according to claim 1, characterized in that: In step (2), the mixture of organoids and virus suspension is collected in a 15 ml sterile centrifuge tube pre-rinsed with 1% BSA, and centrifuged at 500G for 5 min to collect the organoids. After removing the virus supernatant, the organoids are resuspended in PBS and re-centrifuged and rinsed twice; the organoids are resuspended in matrix gel and cultured.

4. The method for constructing a normal cervical organoid virus infection model according to claim 1, characterized in that: The crushing in step (3) is mechanical crushing, lysis solution crushing or ultrasonic crushing; After the cells are broken, the virus is precipitated and concentrated; the virus is precipitated and concentrated by PEG / NaCl precipitation, ammonium sulfate precipitation or sucrose gradient differential centrifugation; the PEG is PEG-8000; In step (3), the isolated and cultured 3D organoids are cultured in 3D for 0 to 72 hours; after 3 hours, 6 hours, 9 hours, and 48 hours of 3D culture, the virus-infected organoids are ground and repeatedly frozen and thawed to break the cells, and then centrifuged to obtain the supernatant; In step (3), the virus detection includes detecting the virus infection efficiency, which includes detecting the number of HPV copies in the cell through a nucleic acid detection method (Taqman method), combining sampling at different time points, and mapping the infection and replication process of the virus in the organoid; The nucleic acid is detected by specific primers and probes; the specific probes and primers are selected from the primers described in Table.

5. The method for constructing a normal cervical organoid virus infection model according to claim 1, characterized in that: The 3D organoids isolated and cultured in step (3) are cultured in 3D for 0 to 72 hours; after 3 hours, 6 hours, 9 hours, and 48 hours of 3D culture, the matrix gel is removed with organoid digestion solution, and the centrifuged organoids are resuspended with electron microscopy fixative, and transmission electron microscopy scanning is performed to capture HPV.

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