CN113832103A

CN113832103A - Preparation and application of exosome of toxoplasma DC (dendritic cell) infected

Info

Publication number: CN113832103A
Application number: CN202110932048.6A
Authority: CN
Inventors: 蔺智兵; 陈兆国; 陆金苗
Original assignee: Shanghai Veteromaru Research Institute Caas China Animal Health And Epidemiology Center Shanghan Branch Center
Current assignee: Shanghai Veteromaru Research Institute Caas China Animal Health And Epidemiology Center Shanghan Branch Center
Priority date: 2021-08-13
Filing date: 2021-08-13
Publication date: 2021-12-24
Anticipated expiration: 2041-08-13
Also published as: CN113832103B

Abstract

The invention discloses preparation and application of exosome of toxoplasma infected Dendritic Cells (DC); in particular to a strategy for treating tumors by infecting DC with Toxoplasma gondii and obtaining exosomes from DC. Toxoplasma gondii infects exosomes of DC source, treats colorectal cancer mouse through intratumoral injection, and its tumor volume reduces, and mouse survival rate is showing and is rising, and the result shows that the exosomes that toxoplasma gondii infects DC produces has obvious tumor suppression effect. The toxoplasma gondii is used for infecting DC, and exosome with controllable quality and capable of being prepared in a large scale is prepared by an ultracentrifugation method for treating tumors, so that a novel method is provided for treating tumors.

Description

Preparation and application of exosome of toxoplasma DC (dendritic cell) infected

Technical Field

The invention belongs to the technical field of medical biology, and particularly relates to a preparation method of an exosome for toxoplasma gondii infected DC and application of the exosome in tumor treatment.

Background

Tumors are a disease in which the cells of cancer patients are affected by genetic and environmental factors and their proliferation is uncontrolled (cancer, A., et al., Parasites and immunology: immunological effect of leishmania spp. In cancer treatment (vol 110, s33,2019. European Journal of cancer,2019.119: p.199-199). Canceration can be caused by a variety of factors, among which infectious parasites play an important role in the development of tumors. The Toxoplasma gondii, Echinococcus granulosus and Trypanosoma cruzi can relieve immunosuppression caused by tumor through Th1 type immune response and inhibit tumor growth; the taenia adiposa can generate anti-inflammatory factors through macrophages to inhibit inflammatory reaction generated by tumors; trypanosoma cruzi can activate caspase of host and promote tumor cell apoptosis. Echinococcus granulosus and Trichinella spiralis can arrest cell cycle and inhibit the continuous proliferation of tumor cells. The ilexoplasma gondii, plasmodium falciparum, trypanosoma cruzi can inhibit tumor angiogenesis by inhibiting VEGF expression (Callejas, b.e., et al, Parasites as negative regulators of cancer Reports, 2018.38.). During the parasitic infection, the host shows Th1 type immune response, and achieves the anti-tumor effect. However, no safe and reliable parasitic insect strain is currently available for tumor therapy.

Dendritic Cells (DCs) are the most efficient antigen presenting cells in humans. DCs can capture antigens produced during tumorigenesis and process them for presentation to T cells, thereby generating an anti-tumor T cell immune response. Mature DC has high expression of MHC and costimulatory molecules, and the secreted exosome surface of DC carries functional MHC I/II and T cell costimulatory molecules, the exosome from DC cell maintains the basic immunostimulation ability of DC, and the stable characteristic of the exosome membrane enables the exosome membrane to be stored in a frozen state for at least 6 months. DC-derived exosomes are therefore of potential interest for research in Tumor therapy (Ugel, S., et al, Tumor-induced myeloid depletion: When myeloid-derived supressor cells for Tumor therapy, 2015.125(9): p.3365-3376.Tian, H.and W.Li, discrete cell-derived exosomes for cancer immunotherapy: hose and catalysts. Annals of Translational Medicine,2017.5 (10)).

Exosomes function as "messengers" of intercellular communication, carrying proteins and nucleic acids to target cells, performing biological regulatory functions (Ferguson, s.w.and j.nguyen, Exosomes as therapeutics: The antigens of molecular composition and exosomal homology. journal of controlled release,2016.228: p.179-190.). Can regulate the transcription and translation of gene, and plays a key role in the physiological and pathological processes of cell differentiation, development, inflammation, angiogenesis and the like. Toxoplasma infected mouse myoblast (L6) exosomes were found to inhibit tumor cell proliferation, altering cell cycle (Baird, J.R., et al, Averunt toxoplasma grandis genes therapeutic anti-cancer or immunological recovery, 2013.73(13): p.3842-3851.). Serum exosomes from plasmodium-infected mice can inhibit angiogenesis at the tumor site (Sanders, k.l., et al, infected toxoplasma gondii vaccines immunity to genetic cancer therapy of cancer Immunology Research,2015.3(8): p.891-901). Plasmodium infected macrophage exosomes undergo antigen presentation by surface MHC molecules, completing the activation of specific T lymphocytes (Sanders, k.l., et al, attentuated toxoplasma grandis therapy of differentiated systemic cancer cells long-lasting immunity to systemic cancer cells, oncoimmumology, 2016.5(4): p.e. 1104447.).

When a host is infected with a parasite, the host cells can transmit "infected" information to immune cells (DCs, macrophages, neutrophils, monocytes) by releasing exosomes and activate NK cells, macrophages, monocytes, T cells and B cells, exerting an anti-infective effect (Wu, z.y., et al, Extracellular vector-mediated communication with host-parasite interactions.

The exosome prepared by using the toxoplasma infected single cell subset has simple components as an activating molecule for activating immunosuppression of tumor-bearing mice. And cells from exosomes can be edited subsequently, so that exosomes generated by the cells have targeting property. Not only solves the potential safety hazard of tumor treatment by using live insects, but also can achieve the effect of tumor treatment.

Disclosure of Invention

The invention aims to overcome the safety risk of parasites in tumor treatment, and provides a preparation method of Toxoplasma gondii infected DC exosomes and application thereof in tumor treatment.

The purpose of the invention is realized by the following technical scheme:

the invention relates to a preparation method of exosome of Toxoplasma gondii infected DC, which comprises the following steps:

s1, carrying out separation culture on the mouse bone marrow-derived DC;

s2, adding an exosome-removing culture medium into the DC to resuspend the toxoplasma gondii (insect strain Me49), inoculating the toxoplasma gondii and host DC cells onto DC monolayer cells according to the ratio of 1:5-5:1, and inoculating 5% CO₂And then the static culture is carried out for 12 hours in an incubator at 37 ℃;

s3, collecting cell culture supernatant, centrifuging for 10min at 500g, and taking the supernatant; then 16500g is centrifuged for 30 min; filtering the supernatant with 0.22 μm filter membrane, centrifuging 120000g of the filtered liquid for 90min, and discarding the supernatant;

s4, washing the precipitate with PBS (2 times; 120000g, 90min), and resuspending the precipitate with an appropriate amount of PBS to obtain the exosome.

As an embodiment of the present invention, in step S1, the isolation culture includes the steps of: separating femur and tibia of mouse, washing bone marrow cells with a culture medium injector filled with 1640, washing with PBS for 2 times, and centrifuging at 250g for 8 min; lysing erythrocyte lysate for 8min, counting by using a hemocytometer, adding 1640 complete medium containing GM-CSF 40ng/ml and IL-420ng/ul, and adding 5% CO₂And culturing in a 37 ℃ cell culture box for 9 days to obtain the bone marrow-derived DC.

As an embodiment of the invention, the toxoplasma strain comprises Me49 and RH.

As an embodiment of the present invention, step S2 is preceded by a step of recovering and collecting toxoplasma gondii. Isolation culture frozen Toxoplasma gondii strains were removed from liquid nitrogen, recovered in Vera T25 cell culture flasks containing 70% and cultured in a 37 ℃ incubator containing 5% CO 2.

As an embodiment of the present invention, the collecting comprises: in the recovery process, when 70 +/-5% of toxoplasma gondii is released by Vera cells, scraping Vera cells infected by the toxoplasma gondii by using cells, then filtering and centrifuging the cell suspension, and removing the supernatant to obtain the toxoplasma gondii.

As an embodiment of the present invention, the step S4 further comprises an identification step after resuspending the pellet; and detecting exosome surface markers CD9, CD63 and TSG101 through nanoparticle tracking analysis, electron microscope observation and Western Blot.

The invention also relates to an exosome obtained by the preparation method of the toxoplasma gondii infected DC exosome.

The invention also relates to application of the exosome in tumor treatment.

As one embodiment of the present invention, the formulation is injected intratumorally or intravenously.

Compared with the prior art, the invention has the following beneficial effects:

1) the exosome is used for replacing toxoplasma gondii to treat the tumor, so that the potential safety hazard of live insect treatment on the tumor is solved;

2) by adopting a single insect strain to infect the exosome from a single cell strain, the problem of controllability of the composition and function of the exosome is solved, and industrial scale production and clinical drug approval are facilitated;

3) the exosome prepared by the single cell subgroup can be edited for cells from exosome sources subsequently, so that the exosome generated by the cells has targeting property;

4) the toxoplasma gondii is used for infecting the DC, the cell source is single, the composition of the exosome is relatively simple, and the cells cultured in vitro are beneficial to large-scale preparation and industrial production of the exosome;

5) the exosome is derived from host cells, has good cell compatibility, can directly regulate cellular immune response and reduce toxic and side effects, and is used as a natural nano carrier for tumor treatment;

6) the Toxoplasma gondii infected DC-derived exosome has the immunostimulation capacity, can achieve the tumor inhibition effect, and avoids the risk caused by live insect treatment.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is electron microscopy of exosomes; wherein, (a) toxoplasma infects vesicle TeM images derived from DC cells, showing exosome morphology (scale bar 100 nm); (B) western blot analysis of the vesicles derived from Toxoplasma gondii-infected DC cells with anti-CD 63, CD9, TSG 101; (C) analyzing the size distribution and the particle number of the toxoplasma gondii infected

DC

12h, 24h and 36h by using nanoparticle tracking;

FIG. 2 is a schematic diagram showing the imaging, fluorescence intensity analysis and survival curve of a toxoplasma infection mouse bone marrow-derived DC exosome for treating tumor-bearing mice in vivo; wherein, (A) exosome treatment tumor-bearing mice live imaging day 12; 1: PBS treatment of tumor-bearing mouse groups; 2: treating the tumor-bearing mouse group with the DC-derived exosomes; 3: treating a tumor-bearing mouse group with RH-infected DC-derived exosomes; 4: me49 infected DC-derived exosomes to treat tumor-bearing mouse exosome groups; (B) in vivo imaging mean fluorescence intensity analysis; (C) tumor-bearing mice survival curves after exosome treatment.

Detailed Description

The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention.

Example 1

Separation culture of mouse bone marrow-derived dendritic cells

(1) The femur and tibia of the mouse were separated, and the bone marrow cells were washed with a medium syringe filled with 1640, then washed 2 times with PBS, and centrifuged at 250g for 8 min. The erythrocyte lysate is lysed for 8min and counted on a blood counting plate.

(2) 1640 complete medium (10% fetal bovine serum, 1% streptomycin penicillin) containing GM-CSF 40ng/ml and IL-420ng/ul was added and cultured in a carbon dioxide cell incubator for three days.

(3) After 72h, culture was continued for 72h with fresh 1640 medium (containing 10% fetal calf serum, 1% streptomycin penicillin, 40ng/ml GM-CSF and 20ng/ul IL-4).

Example 2

Culture of Toxoplasma gondii

The frozen toxoplasma strains Me49 and RH were removed from liquid nitrogen, recovered to Vera T25 cell culture flasks containing 70% and cultured in a 37 ℃ incubator containing 5% CO 2. When about 70% of the toxoplasma was released, the Vera cells infected with toxoplasma were scraped off with a cell scraper, then the cell suspension was transferred to a 15ml centrifuge tube, the cell suspension was filtered 3 times with a syringe with a 25-gauge needle, after centrifugation, the supernatant was discarded, and the pellet was resuspended in 10ml PBS and counted by a microscope.

Example 3

Toxoplasma infected mouse DC cell

(1) Complete medium 1640 (10% fetal bovine serum, 1% streptomycin penicillin solution) with exosomes removed was added to mouse DC cells to resuspend Me49 worm strain, and the parasites were mixed with host cells according to the following ratio of 1:5 was inoculated onto DC monolayer cells and incubated at 37 ℃ in a 5% CO2 incubator for 24 h.

(3) After 12h, 24h and 36h of culture, cell culture supernatant is collected.

Example 4

Exosome isolation identification

(1) The collected cell supernatant was centrifuged at 500g for 10min to obtain the supernatant. Then, the mixture was centrifuged at 16500g for 30 min. The precipitate was discarded and the supernatant was filtered through a 0.22 μm filter.

(2) 120000g of the filtered supernatant was centrifuged for 90min, and the precipitate was washed twice with pbs. Finally the pellet was resuspended in the appropriate amount of PBS.

(3) Exosome surface markers CD9, CD63, TSG101 were detected by Nanoparticle Tracking Analysis (NTA), electron microscopy, Western Blot.

Example 5

Establishment of colorectal cancer tumor-bearing mouse model

CT26-luc tumor cells were revived, and then each mouse was injected 2X 10 tumor cells by axillary injection⁶cell。

Example 6

Tumor-bearing mouse for treating colorectal cancer by exosome

Exosomes were injected intratumorally into each mouse at 50 μ g per treatment 3 times per mouse (once every other day).

Example 7

In vivo imaging: luciferase was injected intraperitoneally into mice and imaged in a dark box using a chemiluminescent fluorescence imaging system (IVISTM).

Results of the experiment

1 exosome isolation identification

As shown in FIG. 1, exosomes of Toxoplasma infected DC cells were successfully obtained by ultracentrifugation. The membrane structure of exosome under transmission electron microscope is cup stand shape, and the diameter range is 100-200nm (figure 1A). The exosome membrane surface proteins CD63, CD9 and transmembrane protein TSG101 were detected by Westernblotting experiments, and the results are shown in fig. 1B. Nanoparticle tracking analysis found the most exosome secreted at 12h in toxoplasma infected DC cells, as shown in figure 1C.

2 exosome treatment of tumor bearing mice

After a colorectal cancer solid tumor mouse is injected into a toxoplasma gondii infected mouse DC-derived exosome tumor, in-vivo imaging is carried out 12 times later to detect the tumor volume, and the result shows that the tumor volume of the mouse treated by Me49 infected DC cell-derived exosome is obviously reduced and can not be seen by naked eyes (figure 2A). Mean fluorescence intensity of mice treated with Me 49-infected DC cell-derived exosomes was significantly different from that of PBS group (fig. 2B). Infection of DC cell-derived exosomes with Me49 significantly increased survival of mice (fig. 2C). The results indicate that Me49 infected mouse bone marrow-derived DC cell-derived exosomes can significantly inhibit tumor growth.

It can be seen that when toxoplasma Me49 infects DC cell-derived exosomes and is injected into colorectal cancer mice via tail vein, the tumor volume is reduced and the survival rate of the mice is significantly increased. The result shows that the exosome generated by the Toxoplasma gondii infected DC has obvious tumor treatment effect.

In conclusion, the present invention addresses the risks associated with live insect tumor therapy by "editing" the DC exosomes with the pathogen (toxoplasma gondii). The method can change the secretion level and the function of exosome of immune cell. And the exosome can be treated by intratumoral or tail vein injection, so that the formation of tumor metastasis is quickly and efficiently inhibited. Therefore, the toxoplasma infects antigen presenting cells, the preparation quality of the exosome is controllable, and the exosome can be prepared in a large scale, so that a new method is provided for treating tumors.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Claims

1. A method for preparing exosomes of Toxoplasma infected DC, comprising the steps of:

s1, carrying out separation culture on the mouse DC;

s2, adding an exosome-removing culture medium into the DC to resuspend the toxoplasma gondii, inoculating the toxoplasma gondii and the DC cells onto DC monolayer cells according to the ratio of 1:5-5:1, and inoculating 5% CO₂And then the static culture is carried out for 12 hours in an incubator at 37 ℃;

s3, collecting cell culture supernatant, centrifuging for 10min at 500g, and taking the supernatant; then 16500g is centrifuged for 30 min; filtering the supernatant with 0.22um filter membrane, centrifuging 120000g of the filtered liquid for 90min, and discarding the supernatant;

and S4, washing the precipitate with PBS, and resuspending the precipitate with a proper amount of PBS to obtain the exosome.

2. The method for preparing exosomes of toxoplasma gondii infected DCs according to claim 1, wherein in step S1, the isolation culture comprises the steps of: separating femur and tibia of mouse, washing bone marrow cells with a culture medium injector filled with 1640, washing with PBS for 2 times, and centrifuging at 250g for 8 min; lysing erythrocyte lysate for 8min, counting by using blood count plateAdding 1640 complete medium containing GM-CSF 40ng/ml and IL-420ng/ul, 5% CO₂And culturing in a 37 ℃ cell culture box for 9 days to obtain the bone marrow-derived DC.

3. The method of preparing an exosome according to claim 1, wherein the toxoplasma strain comprises Me49 and RH.

4. The method for preparing exosomes of toxoplasma infected DC according to claim 1, wherein step S2 is preceded by the steps of toxoplasma resuscitation and collection.

5. The method for preparing exosomes of Toxoplasma gondii-infected DC cells according to claim 4, wherein said recovery comprises taking out frozen Toxoplasma gondii strains from liquid nitrogen, recovering to Vera T25 cell culture bottles containing 70% and culturing in 37 ℃ incubator containing 5% CO 2.

6. The method of preparing exosomes of toxoplasma infected DCs according to claim 4, wherein the collecting comprises: in the recovery process, when 70 +/-5% of toxoplasma gondii is released by the Vera cells, scraping the Vera cells infected by the toxoplasma gondii by the cells, then filtering and centrifuging the cell suspension, and removing the supernatant to obtain the toxoplasma gondii.

7. The method of preparing exosomes of toxoplasma infected DC according to claim 1, wherein step S4 further comprises an identification step after resuspending the pellet; and detecting exosome surface markers CD9, CD63 and TSG101 through nanoparticle tracking analysis, electron microscope observation and Western Blot.

8. An exosome obtained by the method for producing an exosome of toxoplasma infected DC according to any one of claims 1 to 7.

9. Use of an exosome according to claim 8 in a tumour therapy formulation.

10. Use according to claim 9, wherein the formulation is administered by intratumoral or intravenous injection.