CN117757885A - Intestinal flora metabolite screening method based on high content - Google Patents
Intestinal flora metabolite screening method based on high content Download PDFInfo
- Publication number
- CN117757885A CN117757885A CN202311805673.XA CN202311805673A CN117757885A CN 117757885 A CN117757885 A CN 117757885A CN 202311805673 A CN202311805673 A CN 202311805673A CN 117757885 A CN117757885 A CN 117757885A
- Authority
- CN
- China
- Prior art keywords
- intestinal
- intestinal flora
- damage
- metabolites
- screening
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000000968 intestinal effect Effects 0.000 title claims abstract description 88
- 239000002207 metabolite Substances 0.000 title claims abstract description 57
- 238000012216 screening Methods 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 23
- 210000002220 organoid Anatomy 0.000 claims abstract description 49
- 230000006378 damage Effects 0.000 claims abstract description 44
- 238000012258 culturing Methods 0.000 claims abstract description 9
- 238000005516 engineering process Methods 0.000 claims abstract description 5
- 210000004027 cell Anatomy 0.000 claims description 27
- 210000002472 endoplasmic reticulum Anatomy 0.000 claims description 23
- 230000003833 cell viability Effects 0.000 claims description 19
- 210000003712 lysosome Anatomy 0.000 claims description 18
- 230000001868 lysosomic effect Effects 0.000 claims description 18
- 208000027418 Wounds and injury Diseases 0.000 claims description 16
- 230000000694 effects Effects 0.000 claims description 16
- 208000014674 injury Diseases 0.000 claims description 16
- 231100000167 toxic agent Toxicity 0.000 claims description 14
- 239000003440 toxic substance Substances 0.000 claims description 14
- 239000000523 sample Substances 0.000 claims description 13
- 238000001514 detection method Methods 0.000 claims description 10
- 210000003470 mitochondria Anatomy 0.000 claims description 10
- 230000002438 mitochondrial effect Effects 0.000 claims description 10
- GRTOGORTSDXSFK-XJTZBENFSA-N ajmalicine Chemical compound C1=CC=C2C(CCN3C[C@@H]4[C@H](C)OC=C([C@H]4C[C@H]33)C(=O)OC)=C3NC2=C1 GRTOGORTSDXSFK-XJTZBENFSA-N 0.000 claims description 8
- 238000011109 contamination Methods 0.000 claims description 8
- 238000010186 staining Methods 0.000 claims description 6
- 230000002132 lysosomal effect Effects 0.000 claims description 5
- 230000006676 mitochondrial damage Effects 0.000 claims description 4
- 230000003013 cytotoxicity Effects 0.000 claims description 3
- 231100000135 cytotoxicity Toxicity 0.000 claims description 3
- 208000015181 infectious disease Diseases 0.000 claims description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 abstract description 8
- 238000011156 evaluation Methods 0.000 abstract description 6
- 208000037817 intestinal injury Diseases 0.000 abstract description 6
- 231100000419 toxicity Toxicity 0.000 abstract description 6
- 230000001988 toxicity Effects 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract description 3
- 230000007246 mechanism Effects 0.000 abstract description 3
- 230000009456 molecular mechanism Effects 0.000 abstract description 3
- 238000002560 therapeutic procedure Methods 0.000 abstract description 3
- 108010082117 matrigel Proteins 0.000 description 12
- 210000001519 tissue Anatomy 0.000 description 11
- 238000010790 dilution Methods 0.000 description 9
- 239000012895 dilution Substances 0.000 description 9
- 239000006228 supernatant Substances 0.000 description 9
- 239000003085 diluting agent Substances 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000012634 fragment Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000007792 addition Methods 0.000 description 3
- 238000007664 blowing Methods 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 210000002288 golgi apparatus Anatomy 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 210000000936 intestine Anatomy 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 239000007640 basal medium Substances 0.000 description 2
- DEGAKNSWVGKMLS-UHFFFAOYSA-N calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000023011 digestive tract development Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 238000002952 image-based readout Methods 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 229960002378 oftasceine Drugs 0.000 description 2
- 238000009736 wetting Methods 0.000 description 2
- 241001052560 Thallis Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000007366 host health Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 208000028774 intestinal disease Diseases 0.000 description 1
- 230000003871 intestinal function Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 231100000683 possible toxicity Toxicity 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a high-content intestinal flora metabolite screening method, and belongs to the technical field of cell biology. Co-culturing the intestinal flora metabolite and the intestinal organoids, detecting physiological indexes of the intestinal organoids by combining a high content technology, and screening the intestinal flora metabolite. The invention provides an intestinal organoid toxicity evaluation system based on high content analysis, and constructs a rapid, effective and reliable screening and evaluation system for finding early intestinal injury and clarifying intestinal injury mechanism, medicine and therapy. The screening method established by the invention can realize rapid screening of the intestinal flora metabolites by using the intestinal organoids, can rapidly discover toxicity of the intestinal flora metabolites in an early stage and discover early damage indexes, and provides a basis for elucidating molecular mechanisms of the intestinal flora metabolites on intestinal tracts.
Description
Technical Field
The invention relates to the technical field of cell biology, in particular to a method for screening intestinal flora metabolites based on high content.
Background
The gastrointestinal tract is both the main organ of digestive absorption in the human body and an important barrier to protect the body from potentially harmful molecules and pathogenic organisms. In addition, the human intestinal tract contains tens of thousands of intestinal flora. The intestinal flora is a microbial flora living in human gastrointestinal tract, and has a huge and various quantity, about 1000 bacteria in human gastrointestinal tract, and a quantity as high as 10 14 The total number of bacteria is 10 times more than the total number of human cells, the number of carried genes is 100 times more than the total number of human cells, and the total amount of thalli accounts for 1/60-1/50 of the mass of human body. Intestinal flora plays an important role in the metabolic regulation process, and this mode of action is mainly the metabolite produced by the intestinal flora. Intestinal flora metabolites are closely related to host health and disease, even in a single metabolite. Influencing factors mainly comprise genetic factors, life style factors, environment factors and the like. These intestinal flora metabolites directly or indirectly affect the physiological function of the host.
In recent years, intestinal organoids are increasingly becoming in vitro surrogate models for studying intestinal development, function and disease. Compared with the prior in vitro animal model, the organoid has the advantages of high success rate of culture, short culture period, small tissue difference and the like. In contrast to single species 2D cultures, 3D organoids contain multiple cell types, which can establish physiological cell-cell and cell-extracellular matrix interactions that mimic the specificity of natural tissues.
The high content cell imaging system is used for simultaneously detecting the influence of different conditions on the aspects of cell morphology, growth, differentiation, migration, apoptosis, metabolic pathways, signal transduction and the like under the condition of maintaining the cell activity, structure and functional integrity, and acquiring a large amount of relevant information from a single experiment to determine the biological activity and potential toxicity of the cell. The existing intestinal organoids are mostly used for researching intestinal development and intestinal diseases, and are mostly used for screening medicines and small molecular compounds in screening, and no related report on screening of the intestinal organoids for the metabolites of the flora is seen yet.
Disclosure of Invention
The invention aims to provide a method for screening intestinal flora metabolites based on high connotation so as to solve the problems in the prior art.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a method for screening intestinal flora metabolites based on high connotation, which comprises the steps of co-culturing the intestinal flora metabolites and intestinal organoids, detecting physiological indexes of the intestinal organoids by combining a high connotation technology, and screening the intestinal flora metabolites.
Preferably, the physiological index includes cell viability, golgi damage, mitochondrial damage, lysosomal damage, and endoplasmic reticulum damage.
Preferably, the cell viability is detected by using a cell viability detection kit.
Preferably, the detection method of golgi damage, mitochondrial damage, lysosomal damage and endoplasmic reticulum damage comprises the following steps: detecting cell viability, golgi, mitochondria, lysosome and endoplasmic reticulum with high connotation, staining golgi, mitochondria, lysosome and endoplasmic reticulum with golgi, mitochondrial, lysosome and endoplasmic reticulum staining probes, detecting changes in golgi, mitochondria, lysosome and endoplasmic reticulum; the cell activity is detected by using a cell activity and cytotoxicity detection kit.
Preferably, the intestinal organoids are colorectal organoids of 200 μm or less.
Preferably, the method comprises the following steps:
(1) Co-culturing intestinal flora metabolites and intestinal organoids, detecting cell viability by using a high content technology after culturing, and screening out a toxic substance I for reducing the cell viability;
(2) The toxic substance I screened in the step (1) is utilized to infect the intestinal organoid, and after the infection is finished, the Golgi body injury, the mitochondrial injury, the lysosome injury and the endoplasmic reticulum injury of the cells are detected, and the toxic substance II with the effect of damaging the ultrastructure of the cells is screened out;
(3) And (3) carrying out contamination on the intestinal organoid by using the toxic substance II screened in the step (2), detecting the Golgi body injury, mitochondrial injury, lysosome injury and endoplasmic reticulum injury of cells after the contamination is finished, and evaluating the damage of the toxic substance II to the ultrastructure of the cells.
Preferably, in step (1), the concentration of the intestinal flora metabolite is 10 μm.
Preferably, in step (2), the concentration of the toxic substance I is 10nM.
Preferably, in step (3), the concentration of the toxic substance II is 10pM.
Based on the technical scheme, the invention has the following technical effects:
the invention provides an intestinal organoid toxicity evaluation system based on high content analysis, and constructs a rapid, effective and reliable screening and evaluation system for finding early intestinal injury and clarifying intestinal injury mechanism, medicine and therapy. The screening method established by the invention can realize rapid screening of the intestinal flora metabolites by using the intestinal organoids, can rapidly discover toxicity of the intestinal flora metabolites in an early stage and discover early damage indexes, and provides a basis for elucidating molecular mechanisms of the intestinal flora metabolites on intestinal tracts.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing a high content of 124 intestinal flora metabolite (10. Mu.M) cell viability screen, wherein A is a diagram showing 124 intestinal flora metabolite cell viability screen, and B is a heat diagram showing 24 intestinal flora metabolite cell viability screen;
FIG. 2 shows a high-content graph of intestinal flora metabolite (10 nM) of 79 injured cell activities, D is a lysosome screening heat map of intestinal flora metabolites of 79 damaged cell activities (10 nM), E is a mitochondrial screening heat map of intestinal flora metabolites of 79 damaged cell activities (10 nM), F is a mitochondrial screening heat map of intestinal flora metabolites of 79 damaged cell activities (10 nM), G is an endoplasmic reticulum screening heat map of intestinal flora metabolites of 79 damaged cell activities (10 nM), H is an endoplasmic reticulum screening heat map of intestinal flora metabolites of 79 damaged cell activities (10 nM);
FIG. 3 shows a 10pM screen of intestinal flora metabolites with ultrastructural damage, wherein A is golgi, B is mitochondria, C is lysosomes, and D is endoplasmic reticulum.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The technical scheme of the invention is conventional in the field, and the reagents or raw materials are purchased from commercial sources or are disclosed.
Example 1
Construction of intestinal organoids
(1) Placing 2-3C 57 mouse colorectal tissues in a 10cm culture dish, removing blood vessels and fat on the surface of the colorectal tissues, and cleaning the mouse colorectal tissues in ice PBS for about 3-5 times until the PBS is clear and transparent;
(2) 15mL of ice-free calcium-magnesium-free PBS was added to a 50mL centrifuge tube, one end of the intestine was clamped with forceps, the intestine was cut into 2mm small pieces with scissors from the bottom of the intestine, and the pieces were allowed to fall into the PBS in the tube;
(3) Pre-wetting a 10mL pipette with PBS, and blowing up and down the intestinal tract three times;
(4) When the intestinal fracture subsides to the bottom of the tube, the supernatant is gently sucked out, enough liquid is left, and the liquid level just drops out of the tissue segment;
(5) The suspended tissue fragments were blown up and down three times with 15mL of ice in PBS using a pre-moistened 10mL pipette;
(6) When the intestinal fracture subsides to the bottom of the tube, the supernatant is gently sucked out again, enough liquid is left, and the liquid level just drops out of the tissue segment;
(7) The blowing step is repeated 16 times (generally, the supernatant is clear and then washed for 4 to 5 times);
(8) The supernatant was removed and the tissue fragments were resuspended in 25mL room temperature mild cell dissociation reagent (Stemcell 100-0485) and incubated at room temperature on a shaker at 20rpm for 15min;
(9) After dissociation is completed, allowing the tissue fragments to settle by gravity, carefully sucking out and discarding the supernatant;
(10) The tissue fragments were resuspended in 10mL PBS containing 0.1% bsa ice and pipetted up and down three times and allowed to stand until the majority of the intestinal segments settled to the bottom;
(11) Carefully remove the supernatant and filter with a 70 μm filter, collect the filtrate in a clean 50mL conical tube, discard the filter and mark the filtrate as "1";
(12) Repeating the steps of blowing and filtering to obtain 2-8;
(13) Centrifuging 1-8 at 400g for 5min at 4deg.C, carefully pouring out and discarding the supernatant;
(14) The pellet was resuspended in 10mL of cold (2-8deg.C) PBS buffer containing 0.1% BSA, and the suspension of each tube was transferred to a clean 15mL conical tube and labeled with the corresponding serial number;
(15) Centrifuging at 400g at 4deg.C for 5min, and decanting the supernatant;
(16) The pellet was resuspended in 10mL of cold DMEM/F-12 medium containing 0.1% BSA;
(17) Respectively sucking 10 mu L from each group, placing on a glass slide glass or a hemocytometer for counting, and culturing organoids by taking 300-400 crypts as a hole;
(18) Re-suspending the desired crypt with 1:1 solution of culture medium and matrigel, dripping 50 μl of each well into the center of 24-well plate, standing at 37deg.C for 30minCompletely solidifying;
(19) After the droplets solidify, 500 μl of room temperature organoid growth medium is gently added to each well along the well sidewall using a pipette;
(20) Sterile PBS was added dropwise to the other wells to maintain the corresponding humidity;
(21) The plates were capped and incubated at 37℃and 5% CO 2 Culturing under the condition;
(22) The colorectal organoids generally grow to maturity completely for 7 days with one fluid change every three days, and can be passaged.
Example 2
Contamination and high content screening based on colorectal organoid intestinal flora metabolites
(1) The cultivated and matured colorectal organoids are passaged, and the colorectal organoids develop and mature about four days after passaging;
(2) Removing the culture medium from the incubator, and resuspending the colorectal organoids with ice PBS;
(3) Thoroughly wetting the 70 μm filter with PBS, and filtering the resuspended colorectal organoids with the 70 μm filter to obtain colorectal organoids below 200 μm;
(4) Centrifuging at 400g at 4deg.C for 5min, and decanting the supernatant;
(5) The pellet was resuspended in 10mL of cold DMEM/F-12 medium containing 0.1% BSA;
(6) Resuspension of the needed crypt with 1:1 solution of culture medium and matrigel, adding 10 μl of each well into the center of imaging 96-well plate, standing for 20min at 37deg.CCompletely solidifying;
(7) After the droplets solidify, 100 μl of room temperature organoid growth medium is gently added to each well along the well sidewall using a pipette;
(8) The plates were capped and incubated at 37℃and 5% CO 2 Culturing until the next day;
(9) The following day, adding intestinal flora metabolites with the concentration of 10 mu M into an intestinal organoid basic medium, wherein 100 mu L of each hole is added into colorectal organoids, and taking care that the intestinal flora metabolites can penetrate through matrigel without damaging matrigel;
(10) After all additions, 24-well plates of colorectal organoids were placed in 5% CO 2 Dyeing in an incubator at 37 ℃ for two days after contamination, and carrying out high content screening;
(11) Firstly, detecting cell activity of colorectal organoids infected with 10 mu M intestinal flora metabolites by using a Calcein/PI cell activity and cytotoxicity detection kit;
(12) Media was discarded and Calcein and PI were diluted with diluent 1:1000, adding the diluted probe into a hole containing colorectal organoids along the hole wall (to cover matrigel), taking care not to damage matrigel, incubating for 30min at 37 ℃, sucking out the probe and the diluent after incubation, cleaning three times by using normal-temperature PBS, and detecting by using high content every 5min;
(13) During detection, fluorescent intensity, the number of layers and a focusing plane are set for scanning, and when dyeing, a contrast is set in each row due to longer scanning time, so that errors caused by different dyeing time are prevented;
as shown in the figure 1, 79 intestinal flora metabolites in 124 intestinal flora metabolites reduce the cell viability, and 45 intestinal flora metabolites promote the cell viability;
(15) 79 intestinal flora metabolites with reduced cell viability were added to the intestinal organoid basal medium at 10nM, taking care not to destroy matrigel;
(16) After all additions, 24-well plates of colorectal organoids were placed in 5% CO 2 The incubator is infected for two days at 37 ℃;
(17) After the contamination, the medium was discarded and the golgi staining probe was diluted with diluent 1:200 dilution, dilution for lysosomal probe 1:20000 dilution, mitochondrial probe dilution 1:20000 dilution, dilution for endoplasmic reticulum probe 1:2000, diluting, namely adding the diluted probe into a hole containing colorectal organoids along the hole wall (to cover matrigel), taking care of not damaging the matrigel, incubating for 30min at 37 ℃, sucking out the probe and the diluent after incubation, cleaning for three times by using normal-temperature PBS, and detecting by using high content every 5min;
the high content detection results are shown in fig. 2, wherein 20 of 79 intestinal flora metabolites reducing cell viability damage mitochondria, 22 damage golgi apparatus, 11 damage lysosomes and 9 damage endoplasmic reticulum;
(19) Next, 20 damaged mitochondria, 22 damaged golgi apparatus, 11 damaged lysosomes, 9 intestinal flora metabolites that damaged endoplasmic reticulum were added to the intestinal organoid basal medium at 10pM, taking care not to damage matrigel;
(20) After all additions, 24-well plates of colorectal organoids were placed in 5% CO 2 The incubator is infected for two days at 37 ℃;
(21) After the contamination, the medium was discarded and the golgi staining probe was diluted with diluent 1:200 dilution, lysosome 1:20000 dilution, mitochondrial 1:20000 dilution, endoplasmic reticulum 1:2000, diluting, namely adding the diluted probe into a hole containing colorectal organoids along the hole wall (to cover matrigel), taking care of not damaging the matrigel, incubating for 30min at 37 ℃, sucking out the probe and the diluent after incubation, washing with PBS at normal temperature for three times, and detecting with high content every 5min;
the high content detection results are shown in FIG. 3, wherein 6 intestinal flora metabolites damage mitochondria, 5 intestinal flora metabolites damage golgi apparatus and 6 intestinal flora metabolites damage lysosomes at the concentration of 10 pm; two substances damage both ultrastructures simultaneously.
In conclusion, the invention provides an intestinal organoid toxicity evaluation system based on high content analysis, and a rapid, effective and reliable screening and evaluation system for finding early intestinal injuries and clarifying intestinal injury mechanisms, medicines and therapies is constructed. The screening method established by the invention can realize rapid screening of the intestinal flora metabolites by using the intestinal organoids, can rapidly discover toxicity of the intestinal flora metabolites in an early stage and discover early damage indexes, and provides a basis for elucidating molecular mechanisms of the intestinal flora metabolites on intestinal tracts.
It is to be understood that the above examples of the present invention are provided for clarity of illustration only and are not limiting of the embodiments of the present invention. Other variations or modifications of the above description will be apparent to persons of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (9)
1. A method for screening intestinal flora metabolites based on high content is characterized in that intestinal flora metabolites and intestinal organoids are co-cultured, physiological indexes of the intestinal organoids are detected by combining a high content technology, and the intestinal flora metabolites are screened.
2. The method of claim 1, wherein the physiological index comprises cell viability, golgi damage, mitochondrial damage, lysosomal damage, and endoplasmic reticulum damage.
3. The method for screening metabolites of intestinal flora according to claim 2, wherein said cell viability is detected by using a cell viability detection kit.
4. The method for screening intestinal flora metabolites according to claim 2 or 3, wherein the detection method for golgi damage, mitochondrial damage, lysosomal damage and endoplasmic reticulum damage comprises: detecting cell viability, golgi, mitochondria, lysosome and endoplasmic reticulum with high connotation, staining golgi, mitochondria, lysosome and endoplasmic reticulum with golgi, mitochondrial, lysosome and endoplasmic reticulum staining probes, detecting changes in golgi, mitochondria, lysosome and endoplasmic reticulum; the cell activity is detected by using a cell activity and cytotoxicity detection kit.
5. The method of claim 1-4, wherein the intestinal organoid is a colorectal organoid of 200 μm or less.
6. The method for screening metabolites of intestinal flora according to any one of claims 1-5, comprising the steps of:
(1) Co-culturing intestinal flora metabolites and intestinal organoids, detecting cell viability by using a high content technology after culturing, and screening out a toxic substance I for reducing the cell viability;
(2) The toxic substance I screened in the step (1) is utilized to infect the intestinal organoid, and after the infection is finished, the Golgi body injury, the mitochondrial injury, the lysosome injury and the endoplasmic reticulum injury of the cells are detected, and the toxic substance II with the effect of damaging the ultrastructure of the cells is screened out;
(3) And (3) carrying out contamination on the intestinal organoid by using the toxic substance II screened in the step (2), detecting the Golgi body injury, mitochondrial injury, lysosome injury and endoplasmic reticulum injury of cells after the contamination is finished, and evaluating the damage of the toxic substance II to the ultrastructure of the cells.
7. The method according to claim 6, wherein in the step (1), the concentration of the intestinal flora metabolite is 10 μm.
8. The method according to claim 7, wherein in the step (2), the concentration of the toxic substance I is 10nM.
9. The method according to claim 6, wherein in the step (3), the concentration of the toxic substance II is 10pM.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311805673.XA CN117757885A (en) | 2023-12-26 | 2023-12-26 | Intestinal flora metabolite screening method based on high content |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311805673.XA CN117757885A (en) | 2023-12-26 | 2023-12-26 | Intestinal flora metabolite screening method based on high content |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117757885A true CN117757885A (en) | 2024-03-26 |
Family
ID=90325260
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311805673.XA Pending CN117757885A (en) | 2023-12-26 | 2023-12-26 | Intestinal flora metabolite screening method based on high content |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117757885A (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112666287A (en) * | 2020-12-22 | 2021-04-16 | 上海鹿明生物科技有限公司 | GCMSMS-based high-throughput analysis method and application of animal intestinal flora metabolites |
| WO2021075528A1 (en) * | 2019-10-17 | 2021-04-22 | 公立大学法人横浜市立大学 | Method for evaluating drug toxicity |
| WO2021181388A1 (en) * | 2020-03-09 | 2021-09-16 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Methods of diagnosing and treating p53 mutant cancer |
| CN114317413A (en) * | 2022-01-17 | 2022-04-12 | 中山大学 | Culture method of intestinal organoid and application of intestinal organoid in intestinal toxicity test |
| CN114414531A (en) * | 2022-01-30 | 2022-04-29 | 福州大学 | In-situ on-line detection method and device for metabolic molecules of organoid |
| WO2023102120A1 (en) * | 2021-12-01 | 2023-06-08 | Memorial Sloan-Kettering Cancer Center | Organoid co-cultures and methods of use thereof |
| CN116875532A (en) * | 2023-07-13 | 2023-10-13 | 首都医科大学 | Environment pollutant toxicity detection method based on intestinal organoids |
| CN116904545A (en) * | 2023-07-19 | 2023-10-20 | 首都医科大学 | High-content lung injury evaluation system based on human lung organoids |
-
2023
- 2023-12-26 CN CN202311805673.XA patent/CN117757885A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021075528A1 (en) * | 2019-10-17 | 2021-04-22 | 公立大学法人横浜市立大学 | Method for evaluating drug toxicity |
| WO2021181388A1 (en) * | 2020-03-09 | 2021-09-16 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Methods of diagnosing and treating p53 mutant cancer |
| CN112666287A (en) * | 2020-12-22 | 2021-04-16 | 上海鹿明生物科技有限公司 | GCMSMS-based high-throughput analysis method and application of animal intestinal flora metabolites |
| WO2023102120A1 (en) * | 2021-12-01 | 2023-06-08 | Memorial Sloan-Kettering Cancer Center | Organoid co-cultures and methods of use thereof |
| CN114317413A (en) * | 2022-01-17 | 2022-04-12 | 中山大学 | Culture method of intestinal organoid and application of intestinal organoid in intestinal toxicity test |
| CN114414531A (en) * | 2022-01-30 | 2022-04-29 | 福州大学 | In-situ on-line detection method and device for metabolic molecules of organoid |
| CN116875532A (en) * | 2023-07-13 | 2023-10-13 | 首都医科大学 | Environment pollutant toxicity detection method based on intestinal organoids |
| CN116904545A (en) * | 2023-07-19 | 2023-10-20 | 首都医科大学 | High-content lung injury evaluation system based on human lung organoids |
Non-Patent Citations (4)
| Title |
|---|
| 孙霞;詹丽杏;: "肠道类器官的构建及应用", 结直肠肛门外科, no. 03, 28 June 2020 (2020-06-28), pages 251 - 256 * |
| 孟紫强: "《普通高等教育十四五规划教材 生态毒理学基础》", 31 January 2023, 中国环境出版集团, pages: 81 - 87 * |
| 李准多、孙辉: "肠道微生物代谢物功能筛选平台", 《生物医学转化》, vol. 3, no. 1, 20 March 2022 (2022-03-20), pages 58 - 60 * |
| 高爱社等: "《基础病理学研究进展》", 31 March 2021, 上海科学技术出版社, pages: 3 - 18 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Maridas et al. | Isolation, culture, and differentiation of bone marrow stromal cells and osteoclast progenitors from mice | |
| Durafourt et al. | Isolating, culturing, and polarizing primary human adult and fetal microglia | |
| CN114359899B (en) | Cell co-culture model, cell model construction method, computer device, and storage medium | |
| Froh et al. | Isolation of liver Kupffer cells | |
| CN109182257A (en) | A kind of mechanical environment cultural method for improving chondrocyte proliferation activity, maintaining cartilage phenotype | |
| Su et al. | Isolation, culture, and characterization of primary salivary gland cells | |
| CN104745526B (en) | A kind of zebra fish primary embryonic cells vitro differentiation is the new method of cardiac muscle cell | |
| CN102008360A (en) | Construction of artificial neural network sample catheter for repairing spinal cord injury | |
| Templeton et al. | Methods for culturing human femur tissue explants to study breast cancer cell colonization of the metastatic niche | |
| CN116486918A (en) | Stem cell quality evaluation method | |
| CN111575227B (en) | Method for establishing human-derived diabetic cardiomyopathy model | |
| CN117757885A (en) | Intestinal flora metabolite screening method based on high content | |
| CN107227296A (en) | A kind of method of myelomonocyte separation, identification and induction differentiation | |
| CN108531442A (en) | A kind of insect cell line of serum free suspension culture and its application | |
| Liebman et al. | Chondrocyte culture and assay | |
| CN102424817B (en) | Cell strain from human lung adenocarcinoma and preparation method thereof | |
| CN116904545A (en) | High-content lung injury evaluation system based on human lung organoids | |
| CN110527661A (en) | A kind of Xiphophorus helleri prelarva cell line and its construction method and application | |
| JP2010181391A (en) | Identifying method of animal cell | |
| CN116218762A (en) | Tubular organ culture medium, application thereof and tubular organ culture method | |
| CN104060329A (en) | Immortalized quality-control cell bank for chromosome karyotype analysis and construction method thereof | |
| CN116486909A (en) | Stem cell quality evaluation system | |
| CN102424816B (en) | Cell line derived from recurrent lesions after human small cell lung cancer radiotherapy and chemotherapy, and preparation method thereof | |
| Takács et al. | Isolation and micromass culturing of primary chicken chondroprogenitor cells for cartilage regeneration | |
| CN108118079B (en) | Drug hepatotoxicity evaluation method based on three-dimensional liver model of qualitative filter paper |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |