CN113144222B

CN113144222B - Experimental method for preventing cerebral malaria by exosomes

Info

Publication number: CN113144222B
Application number: CN202110401910.0A
Authority: CN
Inventors: 陈�光; 杜云婷
Original assignee: Taizhou University
Current assignee: Taizhou University
Priority date: 2021-04-14
Filing date: 2021-04-14
Publication date: 2023-05-23
Anticipated expiration: 2041-04-14
Also published as: CN113144222A

Abstract

The invention relates to an experimental method for preventing cerebral malaria, which comprises the steps of utilizing a P.y17XNL mouse malaria model, extracting peripheral blood, and separating and purifying the exosomes by an ultracentrifugation method or a magnetic bead immunocapture method; normal C57BL/6J mice were immunized with exosomes or exosomes in combination with CpGODN, challenged with p.b. aka at fixed time points, and the immune effects of the exosomes were determined by dynamic observation of the course of infection, neurological symptoms and Blood Brain Barrier (BBB) integrity. By extracting exosomes in peripheral blood of a host after infection of a heterologous plasmodium, the C57BL/6J mice are immunized twice on 0 day and 20 days respectively by adopting two methods of singly immunizing exosomes by tail vein injection or subcutaneously injecting exosomes and combining CpGODN, and 1X 10 mice are immunized after 20 days of the second immunization ⁶ bANKA-parasitic erythrocytes (pRBC) challenge immunized mice. Experimental results show that compared with a model group, the insect blood disease of mice in an immunized group is obviously reduced, and the survival rate is as high as 90-100%. Neurological symptoms did not appear and the mouse BBB remained intact; the exosomes are proved to be effective in protecting C57BL/6J mice from cerebral malaria.

Description

Experimental method for preventing cerebral malaria by exosomes

Technical Field

The invention relates to the technical field of malaria prevention, in particular to an experimental method for preventing cerebral malaria by using exosomes.

Background

Malaria is a global infectious disease that severely jeopardizes human health. Malaria is reported by 2020WHO, 89 countries and regions worldwide in 2019, with about 2.29 million people infected and up to 40.9 million deaths due to malaria. Malaria remains a major cause of death in children under 5 years of age, particularly in developing countries such as africa. At least 20 tens of thousands of people die annually from severe malaria only in saharan africa, with Cerebral Malaria (CM) caused by plasmodium falciparum (Pf) being the main cause of mortality in severe patients. CM mortality is as high as 15-25%, mortality accounts for 90% of malaria total mortality, and one-fourth surviving patients suffer from long-term neurological and cognitive deficits.

Thus, a thorough understanding of the pathogenic mechanisms involved in CM development and its interactions with the host are fundamental and preconditions for controlling and eradicating malaria worldwide and developing effective malaria vaccines.

Both parasite and parasitic site tissues are capable of secreting exosomes, but insect-derived exosomes are indistinguishable from host-derived exosomes. At present, the extraction of tissue exosomes is still in a starting stage, so that the selection of the optimal extraction method is the key point of future work. The mechanism by which insect-derived exosomes act on the host is still unknown, and their control of the bioactive substances of the host cells has yet to be studied.

Disclosure of Invention

The experimental method for preventing cerebral malaria by using exosomes provided by the invention can solve the technical problems.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

an experimental method for exosomes for preventing the occurrence of cerebral malaria, comprising:

s1, extracting peripheral blood by using a P.y17XNL mouse malaria model, and separating and purifying exosomes by using an ultracentrifugation method or a magnetic bead immunocapture method;

s2, immunizing a normal C57BL/6J mouse by utilizing exosomes or exosomes combined with CpGODN, dynamically observing the infection process, nervous system symptoms and Blood Brain Barrier (BBB) integrity at a fixed time point of P.bANKA attack, and determining the immune effect of the exosomes.

Further, the S1 specifically comprises exosome extraction and exosome identification;

wherein the exosome extraction comprises intraperitoneal injection of BALB/c mice by 1×10 ⁶ P.y17xnl parasitic erythrocytes (pRBC), extracting exosomes after 20% -30% of the insect disease is determined;

extracting exosomes in peripheral blood of P.y17XNL infected BALB/c mice by adopting an ultracentrifugation method or a magnetic bead immunocapture method, measuring protein concentration, and placing in a refrigerator at-80 ℃ for later use;

the exosome identification comprises analysis of the extracted exosome morphological structure, particle size and expression of surface specific molecules by adopting a transmission electron microscope and a WB method.

Further, the step of S2 specifically includes the following step of exosome immunization:

c57BL/6J mice were injected 5 μg/mouse tail intravenously and immunized once on

days

0 and 20, respectively; c57BL/6J mice were intraperitoneally injected 1X 10 20 days after the second immunization ⁶ bANKA-parasitic erythrocytes pRBC;

exosomes in combination with CpGODN immunization: c57BL/6J mice were subcutaneously injected with 10. Mu.g of exosomes/10. Mu.g/only in combination with CpG ODN, and after 20 days the exosomes were subcutaneously injected again with 5. Mu.g/only alone; c57BL/6J mice were intraperitoneally injected 1X 10 20 days after the second immunization ⁶ bANKA-parasitic erythrocytes (pRBC).

On the other hand, the invention also discloses the exosomes extracted in the steps.

According to the experimental method for preventing cerebral malaria by extracting exosomes in peripheral blood of a host after infection of a heterologous plasmodium, the separate immunization of exosomes by tail vein injection or the combination of CpGODN and C57BL/6J mice are immunized twice on

days

0 and 20 respectively by subcutaneous injection, and 1×10 is obtained after 20 days of secondary immunization ⁶ The experimental result shows that compared with a model group, the worm blood disease of mice in the immunized group is obviously reduced, and the survival rate is as high as 90-100%. Nervous system symptoms did not appear and the BBB of the mice remained intact. Proved by the verification, the exosome can effectively protect the C57BL/6J mice from cerebral malaria.

Drawings

FIG. 1 is a flow chart of the method of the present invention;

FIG. 2 is a projection electron microscope and WB identification of peripheral blood derived exosomes of malaria-infected hosts;

FIG. 3 is protozoal blood levels and survival rates of exogenously immunized mice;

fig. 4 is an exogenously immunized mouse Blood Brain Barrier (BBB)) integrity test.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention.

As shown in fig. 1, the experimental method for preventing cerebral malaria by using exosomes according to this embodiment includes the following steps:

s1, extracting peripheral blood by using a P.y17XNL mouse malaria model, separating and purifying exosomes by using an ultracentrifugation method or a magnetic bead immunocapture method, and purifying exosomes and detecting endotoxin;

s2, immunizing a normal C57BL/6J mouse by utilizing tail vein injection exosome or subcutaneous injection exosome combined with CPGODN, carrying out P.bANKA attack at a fixed time point, dynamically observing the infection process, nervous system symptoms and Blood Brain Barrier (BBB) integrity, and determining the immune effect of the exosome;

the following detailed description:

(1) Exosome extraction: BALB/c mice were intraperitoneally injected 1X 10 ⁶ P.y17xnl parasitic erythrocytes (pRBC), extracting exosomes after determination of worm disease (20% -30%); extracting exosomes in peripheral blood of P.y17XNL infected BALB/c mice by adopting an ultracentrifugation method or a magnetic bead immunocapture method, measuring protein concentration, and placing in a refrigerator at-80 ℃ for later use;

(2) Exosome identification: analyzing the extracted exosome shape structure, particle size and expression of surface specific molecules by adopting a transmission electron microscope and WB method;

(3) Exosome immunization method:

exosome immunity: c57BL/6J mice were injected with exosomes (5 μg/mouse) intravenously, and immunized once each of 0 and 20 days; c57BL/6J mice were intraperitoneally injected 1X 10 20 days after the second immunization ⁶ bANKA-parasitic erythrocytes (pRBC).

Exosomes in combination with CpGODN immunization: c57BL/6J mice were subcutaneously injected with exosomes (10 μg/dose) in combination with CpGODN (10 μg/dose), and after 20 days the exosomes were separately subcutaneously injected again(5. Mu.g/min); c57BL/6J mice were intraperitoneally injected 1X 10 20 days after the second immunization ⁶ bANKA-parasitic erythrocytes (pRBC).

(4) Exosome immune effect:

infection process observations of plasmodium challenged immunized mice: the worm blood disease and survival rate were recorded. Tail vein blood sampling, preparing a thin blood film, dyeing by Giemsa, and dynamically monitoring the level and survival rate of protozoal hematoma;

neurological symptom assessment: monitoring was performed twice daily from day 5 post infection to assess ECM occurrence.

Scoring criteria: fur erection, bow back, gait instability, quadriplegia, convulsion and coma. A score of 1, 4 or more was given per symptom, and considered severe ECM.

Blood Brain Barrier (BBB) integrity assay: on day 5 after mice were infected, 200 μl of Evansblue dye (2% Evansblue, EB, sigma) was intravenously injected, 1 hour later, normal saline was perfused through the heart until clear liquid was drained, the brain was taken and placed in a medium-sized tube with 1mL formamide (sigma), incubated at 37℃for 48h with an incubator, centrifuged at 3000r/min for 15min, and the supernatant was measured for dye extravasation on an ultraviolet spectrophotometer (wavelength 630 nm) to detect BBB permeability.

The following examples are given:

1. extraction and identification of exosomes

Transmission electron microscopy showed that exosomes in peripheral blood of p.y17xnl-infected BALB/c mice were all round or oval vesicle structures with diameters between 50-150nm (fig. 2a,2 b); CD9 and TSG101 are characteristic proteins of exosomes which are commonly used, and the result shows that the protein expression amounts of CD9 and TSG101 in the exosomes and serum which are isolated by WB are positive, but the protein expression amounts of CD9 and TSG101 in the exosomes are obviously higher than that of the supernatant (figure 2C), which indicates that the protein concentration of the exosomes which are isolated by the WB is higher, and the exosomes can be used for subsequent experiments.

2. Malaria host derived exosome immune mice worm blood disease and survival rate

C57BL/6J mice were immunized twice at fixed time points alone or in combination with CpGODN tail vein (IV) or Subcutaneously (SC) by extracting exosomes in peripheral blood of P.y17XNL-infected BALB/C mice, followed by P.bANKA challenge. The experimental results found that the protozoa in the IV and SC groups increased slowly compared to the model group mice, peaked at day 9 post-infection, only 14% and 18%, and there was no difference between the IV and SC groups. Model mice all die from cerebral malaria 6-10 days post infection. The survival time of exosomally immunized mice was significantly prolonged, and death began to occur at day 10 after infection, but survival rates were as high as 90-100% (see fig. 3a,3 b).

3. Malaria host-derived exosome immune mice Blood Brain Barrier (BBB) integrity

The invention adopts an in-vivo Evan blue blood brain barrier permeability test to detect the protection effect of exosome immunity on the integrity of the BBB of the ECM mice. As a result, it was found that the amount of Evans blue dye leaked from the BBB of C57BL/6J mice immunized by the IV and SC routes was significantly reduced (P < 0.05) as compared with the model group mice (see FIGS. 4A, 4B).

Conclusion of the experiment

1. The IV (tail vein injection) and SC (subcutaneous injection) groups of mice had a slow rise in protozoa compared to the model group of mice, peaking at 9 days post infection, only 14% and 18% with no difference between the IV and SC groups.

2. Model mice all die from cerebral malaria 6-10 days post infection. The survival time of the exosomally immunized mice is obviously prolonged, death starts to occur at the 10 th day after infection, but the survival rate is as high as 90-100%.

3. In vivo evans blue Blood Brain Barrier (BBB) permeability assay: the leakage of Evan blue dye from the BBB of C57BL/6J mice immunized by the IV and SC routes was significantly reduced (P < 0.05) compared to model mice, demonstrating that exosome immunization protects the integrity of the BBB of ECM mice.

The exosomes extracted in the steps can be prepared into corresponding preparations, including pharmaceutically acceptable auxiliary agents, wherein the preparations can be powder, granules, capsules, solutions, tablets or injections, and the exosomes can be used for preventing cerebral malaria pathological damage.

In summary, the invention utilizes the exosome and CpGODN to immunize under skin and normally utilizes the P.y17XNL mouse malaria model, extracts peripheral blood and then separates and purifies the exosome by an ultracentrifugation method or a magnetic bead immunocapture method, utilizes the exosome or the exosome and CpGODN to immunize a normal C57BL/6J mouse, utilizes the P.bANKA attack at a fixed time point, dynamically observes the infection process, the nervous system symptoms and the Blood Brain Barrier (BBB) integrity, and determines the immune effect of the exosome.

The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An experimental method for preventing cerebral malaria by exosomes, characterized by:

s2, immunizing a normal C57BL/6J mouse by utilizing exosomes or exosomes combined with CpGODN different ways, dynamically observing the infection process, nervous system symptoms and blood brain barrier BBB integrity at a fixed time point by P.bANKA attack, and determining the immune effect of the exosomes;

the S1 specifically comprises exosome extraction and exosome identification;

wherein the exosome extraction comprises intraperitoneal injection of BALB/c mice by 1×10 ⁶ P.y17XNL parasitized erythrocytes pRBC, extracting exosomes after 20% -30% of the insect diseases are determined;

the exosome identification comprises analysis of the extracted exosome form structure, particle size and expression of surface specific molecules by adopting a transmission electron microscope and a WB method;

the step of S2 specifically comprises the following steps of exosome immunization:

c57BL/6J mice were injected 5 μg/mouse tail intravenously and immunized once on days 0 and 20, respectively; c57BL/6J mice were intraperitoneally injected 1X 10 20 days after the second immunization ⁶ bANKA-parasitic erythrocytes pRBC;

exosomes in combination with CpGODN immunization: c57BL/6J mice were subcutaneously injected with 10. Mu.g of exosomes/10. Mu.g/only in combination with CpG ODN, and after 20 days the exosomes were subcutaneously injected again with 5. Mu.g/only alone; c57BL/6J mice were intraperitoneally injected 1X 10 20 days after the second immunization ⁶ bANKA parasitic erythrocytes pRBC.