CN117904031A - Extraction method and application of intestinal organoid outer vesicle - Google Patents
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Abstract
The invention relates to the technical field of biological medicines, and particularly provides an extraction method of an external vesicle of an intestinal organoid and application of the external vesicle in preparation of a medicament for treating osteoporosis complicated with inflammatory bowel disease. According to the invention, the intestinal organoid and the outer vesicle thereof are adopted for treating inflammatory bowel diseases, a new approach is provided for treating inflammatory bowel diseases and improving the treatment of concurrent osteoporosis, and an innovative method is provided for treating inflammatory bowel diseases based on the extracellular vesicles of the intestinal organoid. The research of the invention discovers that the extracellular vesicles derived from the intestinal organoids have the function of immunoregulation, thereby reducing colon injury caused by dextran sodium sulfate, improving symptoms of inflammatory bowel diseases, saving bone loss, improving bone mass and improving osteoporosis.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to an extraction method of an external vesicle of an intestinal organoid and application thereof in treating inflammatory bowel diseases and improving associated osteoporosis.
Background
Inflammatory bowel disease (inflammatorybowel disease, IBD) is a chronic inflammatory disease of the intestinal tract, including ulcerative colitis (Ulcerative colitis, UC) and Crohn's Disease (CD), clinically manifested as chronic diarrhea, abdominal pain, anemia and malnutrition, and is a series of chronic, recurrent and debilitating inflammatory diseases with the concomitant occurrence of osteoporosis in patients. IBD morbidity is rising rapidly and becomes a major social problem seriously affecting public health, with the number of patients in the united states and europe currently reaching 100 tens of thousands and 250 tens of thousands, respectively; the number of patients in 2025 of China can reach 150 ten thousand, which brings heavy burden to global medical care and affects millions of people. In IBD, increased inflammatory factors such as tumor necrosis factor alpha and interleukin-1 beta impair bone formation and result in decreased bone mass. Most of the therapies currently used for preventing or treating osteoporosis focus on anti-malabsorption drugs that inhibit the activity of osteoclasts, while neglecting the function of immune cells in the progression of osteoporosis, resulting in a greatly compromised therapeutic effect. With the advancement of osteoporosis research, osteoporosis is also classified as a chronic inflammatory bone disease. Regulatory T cells (Regulatory T cells, treg) and helper T cells 17 (Th 17) are critical for maintaining bone homeostasis, especially the regulation of Th17 cells in osteoclast differentiation. Overactivation of Th17 cells, in which a large amount of the cytokine RANKL is secreted, promotes the osteoclast phenotype. Restoring immune homeostasis of Treg/Th17 cells can inhibit bone loss.
The research shows that the extracellular vesicles (extracellular vesicles, EVs) are a membranous carrier with the thickness of 30-100 nm, have unique nano structure, no cell system, stable drug carrying capacity and good biocompatibility, are novel drug delivery carriers with great clinical application potential, and can regulate body functions by regulating signal paths of hosts or delivering bioactive substances into host cells. Intestinal epithelial cell derived EVs play an important role in the induction of Treg cells, and are capable of mediating interactions between intestinal epithelial cells and the immune system. The intestinal organoids are three-dimensional in vitro models, contain most of physiological related characteristics of intestinal tissues in vivo, and have physiological conditions which are closer to those of natural tissues. Thus, the extra-intestinal organoid vesicles (INTESTINAL ORGANOID EXTRACELLULAR VESICLES, IOEVs) secreted by the intestinal organoids have a greater number and better physiological effects. The existing medicines for treating osteoporosis complicated with inflammatory bowel disease have unsatisfactory effects, and the traditional IBD treatment schemes, such as anti-inflammatory medicines or immunosuppressant medicines, often cannot fully control IBD symptoms, so that the situation of bone loss cannot be improved. In addition, existing intravenous strategies are generally systemic, with some limitations that prevent accurate access to the site of the disease. Therefore, there is a need to develop a novel safe drug that can be orally administered.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides an extraction method of an external vesicle of an intestinal organoid and application of the external vesicle in preparation of a medicament for treating osteoporosis complicated with inflammatory bowel disease. According to the invention, the intestinal organoids and the vesicles thereof are used for treating inflammatory bowel diseases, a new way is provided for treating inflammatory bowel diseases and improving the treatment of concurrent osteoporosis, and an innovative method is provided for treating inflammatory bowel diseases based on the EVs of the intestinal organoids. The research of the invention discovers that EVs derived from intestinal organoids have the function of immunoregulation, thereby reducing colon injury caused by dextran sodium sulfate, improving symptoms of inflammatory bowel disease, saving bone loss, improving bone mass and improving osteoporosis.
The aim of the invention can be achieved by the following technical scheme:
In one aspect, the present invention provides a method for extracting an external vesicle of an intestinal organoid, the method comprising the steps of:
(1) Organoid culture: culturing intestinal organoids with organoid medium, changing and collecting the medium every three days;
(3) Organoid outer vesicle extraction: centrifuging the culture medium at a low speed, filtering supernatant obtained by centrifuging at a low speed by using a sterile filter, centrifuging the collected concentrated solution by using an ultrafiltration tube, centrifuging by using a density gradient, re-suspending the centrifuged sediment by using PBS, and performing ultracentrifugation, and collecting the sediment to obtain the external intestinal organoid vesicle;
further, the intestinal organoid outer vesicle is a mouse small intestine and a mouse colon-derived intestinal organoid outer vesicle.
Further, the low-speed centrifugation condition in the step (2) is 10000-12000 g of low-speed centrifugation for 10-30 minutes at 4 ℃; preferably 10000g,30 minutes, 4 ℃.
Further, the supernatant after low-speed centrifugation described in step (2) was filtered using a sterile filter membrane having a pore size of 0.22. Mu.m.
Further, the ultrafiltration tube used in the concentration process in the step (2) is 80-150 kDa; preferably 100kDa.
Further, the medium used in the density gradient centrifugation in the step (2) is iodixanol.
Further, the density gradient centrifugation condition in the step (2) is 120000-200000 g, 1-18 hours, 4 ℃; preferably 150000g for 3 hours at 4 ℃.
Further, the ultracentrifugation condition in the step (2) is 100000-150000 g, 1-3 hours, 4 ℃; preferably 100000g,1 hour, 4 ℃.
In another aspect, the invention provides an application of an external vesicle of an intestinal organoid in preparing a medicament for treating inflammatory bowel disease complicated with osteoporosis.
Further, the extra-intestinal organoid vesicles may be used to reduce colonic shortening caused by inflammatory bowel disease.
Further, the external vesicles of the intestinal organoids can reduce bone loss caused by inflammatory bowel disease and increase bone mass.
Further, the inflammatory bowel disease includes inflammatory bowel disease caused by dextran sodium sulfate.
Further, the outer vesicles are administered by oral gavage or intravenous injection.
Further, the oral gavage administration doses (1 x10 9-1x1011) are particles at a time; preferably 1x10 10 parts.
Compared with the prior art, the invention has the following effects:
The invention provides an extraction method of an organoid outer vesicle and application thereof in preparing a medicament for treating inflammatory bowel disease, and improves the complicated osteoporosis, in particular to a novel application of the organoid outer vesicle in treating inflammatory bowel disease complicated with osteoporosis caused by dextran sodium sulfate. Animal experiments prove that the external vesicle of the intestinal organoid is very effective for reducing colon shortening in inflammatory bowel disease caused by dextran sodium sulfate, and simultaneously reducing osteoporosis in the inflammatory bowel disease. The external vesicles of organoids are used for treatment, the effect is remarkable, the preservation is facilitated, and the occurrence of systemic side effects can be reduced by oral gastric lavage administration. Therefore, the external vesicle of the intestinal organoid provides a novel and effective treatment means for treating inflammatory bowel disease caused by dextran sodium sulfate and improves osteoporosis caused by inflammatory bowel disease.
Drawings
Fig. 1 shows an open field image of an intestinal organoid prepared according to the invention.
Fig. 2 TEM images (panel a) and NTA images (panel b) of the extra-intestinal organoid vesicles prepared according to the invention.
FIG. 3 fluorescence images of the arrival of the mice after oral gavage 5-labeled extra-intestinal organoid vesicles for 4 h.
Fig. 4. Colon length comparison after oral administration of the mice perfused with the external vesicles of the gastrointestinal organoids.
Figure 5 comparison of trabecular numbers after oral administration of the organoid vesicles to the gastrointestinal tract in mice.
Figure 6 comparison of bone volume fraction after oral administration of the organoid vesicles to the gastrointestinal tract in mice.
FIG. 7 is a graph showing the ratio of bone surface area to bone volume after oral administration of the organoid vesicles to the gastrointestinal tract in mice
Fig. 8. Three-dimensional image of distal femur after oral administration of the organoid vesicles to the gastrointestinal tract in mice.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications could be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.
The various reagents and materials used in the present invention are commercially available or may be prepared by known methods unless otherwise specified.
Example 1 external vesicle extraction
(1) Intestinal organoids are cultured:
Intestinal organoid medium provided by Boehmeria nivea biotechnology; then, the crypt cells from the small intestine of the mouse are planted in matrigel, added with intestinal organoid culture medium and placed in a carbon dioxide constant temperature incubator at 37 ℃; the medium was changed every three days.
(2) Extraction of intestinal organoid outer vesicles:
Centrifuging the collected culture medium at a low speed for 10,000g for 30 minutes at 4 ℃; then, the supernatant obtained by low-speed centrifugation is filtered by a sterile filter membrane with the pore diameter of 0.22 μm; then concentrating the filtered supernatant by using a 100kDa ultrafiltration tube, and carrying out density gradient centrifugation on the collected concentrated solution by using iodixanol for 150,000g for 3 hours at 4 ℃; the pellet obtained after centrifugation was resuspended in PBS and ultracentrifuged for 100,000g,1 hour, at 4℃and the pellet was collected with PBS to obtain the extra-intestinal organoid vesicles. 100 μl was pipetted for counting by nanoparticle tracking analysis technique and the remaining samples were stored at-80deg.C.
Example 2 animal model experiments
Mouse model: c57BL/6 male SPF grade mice of 9 weeks old were selected and had a body weight of 18-22 g, and were purchased from Slak laboratory animal company. The food water is fed freely, and the circadian rhythm is maintained.
Grouping experiment: the C57BL/6SPF mice were randomly divided into 3 blank groups (Sham group), model group (IBD group) and dosing group (IOEVs group), 7-10 of each group, and experiments were performed.
Sham group: each lavage with 0.3ml of PBS was repeated 7 times per week at weekly intervals.
IBD group: each time, 0.3ml of PBS was infused into the stomach, 7 times a week, and repeated at intervals of one week; the drinking water was added with 3% dextran sodium sulfate, changed every 2 days for 7 days, then normal water was changed, and repeated at intervals of one week.
IOEVs groups: IOEVs, 0.3ml of each gastric lavage, was performed 7 times per week alternately at intervals of one week; the drinking water was added with 3% dextran sodium sulfate, changed every 2 days for 7 days, then normal water was changed, and repeated at intervals of one week.
The drinking water for the IBD, IOEVs group was added with 3% dextran sodium sulfate, changed every 2 days for 7 days, and the Sham group was only gavaged. Normal water was then changed and administered as described above, alternately for one month. Mice are killed after dislocation, specimens such as colon tissues, thighbones and the like are collected, the length change of the colon is detected, and the thighbones are analyzed by Micro-CT.
Fig. 1 is a prepared bright field image of an intestinal organoid, and fig. 2 is a TEM (a) and NTA (b) image of an external intestinal organoid vesicle; in the NTA image of the right graph, the abscissa indicates the size, and the ordinate indicates the number of nanoparticles. FIG. 3 is a graph showing the fluorescence profile in the intestinal tract after oral gavage CY5 labeled engineered bacterial outer vesicles in mice for 4 h; wherein, group CY5 represents the dye group that is not bound to the extra-intestinal organoid vesicles, and group IOEVs represents the extra-intestinal organoid vesicles labeled with CY 5. From the graph, the method for administering the external vesicle of the intestinal organoid successfully reaches the intestinal tract and has higher accumulation in the intestinal tract. Fig. 4 is a comparison of colon length after oral administration of the organoid vesicles to the gastrointestinal tract in mice. From the figure, it can be seen that the extra-intestinal organoid vesicles can alleviate colon shortening by dextran sodium sulfate. Fig. 5 is a graph of comparison of trabecular numbers after oral administration of the organoid vesicles to the gastrointestinal tract in mice. As can be seen from the figure, the number of bone trabeculae is significantly increased compared with the control group after oral administration of the external intestinal organoid vesicles. Fig. 6 is a graph of bone volume fraction comparison after oral administration of the organoid vesicles to the gastrointestinal tract in mice. As can be seen from the figure, the bone volume fraction is significantly improved over the control group after oral administration of the organoid vesicles. FIG. 7 is a graph showing the ratio of bone surface area to bone volume after oral administration of the organoid vesicles to the gastrointestinal tract in mice. As can be seen from the figure, after the external vesicle of intestinal organoids is orally taken, the ratio of the surface area to the volume of the bone is obviously improved compared with that of a control group, which indicates that the bone mass is improved. Fig. 8 is a three-dimensional image of the distal femur of a mouse after oral administration of the organoid vesicles to the gastrointestinal tract. From the figure, it can be seen that the bone mass is significantly increased after oral administration of the external vesicles of the intestinal organoids, indicating an improvement in osteoporosis.
As can be seen from fig. 1 to 8, the extraction method can successfully extract the external vesicles of the intestinal organoids, and the oral administration method can successfully reach the intestinal tract and increase the accumulation amount in the intestinal tract. The IOEVs groups of colon length is increased, the number of bone trabeculae at the distal end of femur is increased, the bone volume fraction is increased, and the bone surface area and bone volume ratio are increased, which shows that IOEVs has remarkable protection effect, can be used for treating inflammatory bowel diseases, simultaneously reduces bone mass loss, and improves osteoporosis caused by the inflammatory bowel diseases.
Claims (10)
1. A method for extracting an external vesicle of an intestinal organoid, comprising the steps of:
(1) Organoid culture: culturing intestinal organoids with organoid medium, changing and collecting the medium every three days;
(2) Organoid outer vesicle extraction: and (3) centrifuging the culture medium at a low speed, filtering supernatant obtained by centrifuging at the low speed by using a sterile filter, concentrating the collected concentrated solution by using an ultrafiltration tube, centrifuging by using a density gradient centrifugation, re-suspending the centrifuged sediment by using PBS, and performing ultracentrifugation, and collecting the sediment to obtain the organoid outer vesicle.
2. The method of claim 1, wherein the external intestinal organoid vesicles are external intestinal organoid vesicles derived from the small intestine of mice and the colon of mice.
3. The method according to claim 1, wherein the low-speed centrifugation conditions in the step (2) are 10000 to 12000g for 10 to 30 minutes at 4 ℃.
4. The method according to claim 1, wherein the supernatant after low-speed centrifugation in step (2) is filtered using a sterile filter membrane having a pore size of 0.22. Mu.m.
5. The extraction method according to claim 1, wherein the ultrafiltration tube used in the concentration process in step (2) is 80-150 kDa.
6. The method according to claim 1, wherein the medium used in the density gradient centrifugation in the step (2) is iodixanol; the density gradient centrifugation condition in the step (2) is 120000-200000 g, 1-18 hours, 4 ℃; the ultracentrifugation conditions described in step (2) are 100000 to 150000g,1 to 3 hours, 4 ℃.
7. An application of external vesicle of intestinal organoid in preparing medicine for treating osteoporosis complicated with inflammatory bowel disease is provided.
8. The use according to claim 7, wherein the extra-intestinal organoid vesicles are useful for reducing colonic shortening caused by inflammatory bowel disease.
9. The use according to claim 7, wherein said extra-intestinal organoid vesicles are useful for reducing bone loss and increasing bone mass due to inflammatory bowel disease.
10. The use according to claim 7, wherein the inflammatory bowel disease comprises inflammatory bowel disease caused by dextran sodium sulfate.
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| CN202410170972.9A CN117904031A (en) | 2024-02-06 | 2024-02-06 | Extraction method and application of intestinal organoid outer vesicle |
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