CN110004110A - Method for extracting intestinal exosomes - Google Patents

Method for extracting intestinal exosomes Download PDF

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Publication number
CN110004110A
CN110004110A CN201910278835.6A CN201910278835A CN110004110A CN 110004110 A CN110004110 A CN 110004110A CN 201910278835 A CN201910278835 A CN 201910278835A CN 110004110 A CN110004110 A CN 110004110A
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CN
China
Prior art keywords
excretion body
enteron aisle
extracting method
body extracting
intestinal
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
Application number
CN201910278835.6A
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Chinese (zh)
Inventor
狄文娟
周逸婵
夏凡
丁国宪
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Jiangsu Province Hospital First Affiliated Hospital With Nanjing Medical University
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Jiangsu Province Hospital First Affiliated Hospital With Nanjing Medical University
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Priority to CN201910278835.6A priority Critical patent/CN110004110A/en
Publication of CN110004110A publication Critical patent/CN110004110A/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Abstract

The invention provides an extraction method of an intestinal exosome, which comprises the following steps of ⑴ tubular intestinal sample treatment and cleaning, ⑵ intestinal tissue is repeatedly cleaned by using a dithiothreitol solution, ⑶ intestinal epithelial cells are separated, ⑷ intestinal epithelial cells are separated, ⑸ separating liquid is centrifuged to take supernatant for primary filtration to remove cells and cell debris, ⑹ an exosome secreted into a body is obtained.

Description

A kind of enteron aisle excretion body extracting method
Technical field
The present invention relates to a kind of extracting method, especially a kind of high efficiency, inexpensive experimental animal enteron aisle excretion body are mentioned Method is taken, the detection inspection technology field of experimental animal is belonged to.
Background technique
Enterocyte first portal extraneous as body contact, it is desirable to be able to constantly receive micro- from food, enteron aisle The stimulation of the foreign objects such as biology, and then adjust internal body and make reasonable response.Have a large amount of evidence at present and shows enteron aisle The organ and tissue at each position of body can be regulated and controled.Result of study shows that enteric epithelium can influence mind by " intestines Brain circlulation " It is closely related with neurodegenerative disease, cerebral apoplexy etc. through inflammation and mitochondrial function；Bone can also be caused with " myenteron axis " Bone flesh metabolic disorder, cell quantity and cell volume decline, and then induce the decline of muscle function；More studies have reported that, enteron aisle With ischemic is diseases related, cardiac amyloidosis, dilated cardiomyopathy and endomyocardial fibrosis incidence increase it is close It is related.Thus, enteron aisle is the important attemperator of remote organ and tissue.However, for enteron aisle regulation distal end method still not Know.
Excretion body is the extracellular nanoscale vesicles that cell is formed by " endocytosis-fusion-outlet " process, Ke Yizhuan The bioactive molecules such as fortune nucleic acid (such as Microrna, miRNA), protein and lipid are made into regulation is played in recipient cell With being the important research target spot of remote control and regulation between current organ or tissue.
Existing research person confirms that enterocyte has the excretion body for being better than other organs in enteric epithelium tumor cell line Secreting function.Enterocyte secretes excretion body with polarization mode, and on the one hand towards enteric cavity, this part excretion body may be host MicroRNAs can break through the natural mucilage barrier of enteron aisle itself, influence the main source of enteric microorganism；On the other hand it secretes Remote organ or tissue are reached to mucous membrane, and then through return-flow system under mucous membrane, realizes important remote control and regulation.
The body fluid such as autoblood, saliva, urine, cerebrospinal fluid and milk are limited primarily to the extraction of excretion body at present, although P é rez-Gonz á lez in 2017 etc. innovatively dissociates brain cell using papain, the success from brain extracellular space Excretion body is extracted, but this method is not appropriate for enteron aisle.Because intestinal mucosa is gauffer spline structure, extremely complex and and mucous membrane Lower layer is closely coupled, can not carry out mechanicalness separation.
Summary of the invention
The present invention is directed to technical problem set forth above, a kind of enteron aisle excretion body extracting method is proposed, according to gut epithelium Enteric epithelium layer and submucosa are efficiently separated using EDTA digestion method, then used from separating liquid by the tissue characteristic of amphicheirality Supercentrifugation obtains enteron aisle to the endocrine excretion body of body, obtains enteron aisle respectively to the endocrine excretion body of external and body.
The technical solution that the present invention solves the above technical problem is: providing a kind of enteron aisle excretion body extracting method, including such as Lower step:
(1) tubulose intestines sample process and cleaning；
(2) intestinal tissue is cleaned repeatedly using dithiothreitol (DTT) solution, can reduce pollution of the serosal surface cell to extraction process；
(3) the cleaning solution of collection step 2. obtains the excretion body to secreted in vitro through low temperature ultracentrifugation；
(4) separate enterocyte: by step 2. in intestinal tube move in EDTA enteric epithelium separating liquid, and be placed in and be incubated on ice 25-40min promotes epithelial layer to fall off, then using concussion to supernatant muddiness；
(5) separating liquid centrifuging and taking supernatant is subjected to primary filtration, removes cell and cell fragment；
(6) the filtered fluid 5. step obtained is obtained through low temperature ultracentrifugation to the endocrine excretion body of body.
It is of the invention to further limit technical solution, enteron aisle excretion body extracting method above-mentioned, the EDTA be it is a kind of with Mg2+、Ca2+、Mn2+、Fe2+The chelating agent that bivalent metal ion combines, passes through the Ca of the chelant ties enterocyte2+, make The Ca of enterocyte2+It is mildly separated with submucosa, discharges submucosa extracellular matrix.
Enteron aisle excretion body extracting method above-mentioned, the dithiothreitol (DTT) solution are mixed by 1 μM of dithiothreitol (DTT) with PBS solution Close preparation；Without Mg in the PBS solution2+、Ca2+、Mn2+Without Fe2+Bivalent metal ion.
5. preceding enteron aisle excretion body extracting method, the step are centrifugated 4-6min, centrifugal force sets 9000- 10000g；The primary filtration is filtered using 200nm filter.
Enteron aisle excretion body extracting method above-mentioned, the step 3. with step 6. in ultracentrifugation process be centrifugated 2- 3h, centrifugal force set 100000-120000g.The step 3. with step 6. in sediment through glutaraldehyde, sodium cacodylate and After osmium tetroxide fixing process, it can carry out verifying under Electronic Speculum.
Further, in enteron aisle excretion body extracting method above-mentioned, step tubulose intestines sample process 1. and cleaning, Tubulose enteron aisle sample is taken, is inverted it from inside to outside using ophthalmic tweezers, both ends are pricked with surgical thread and are closed.
Compared with prior art, the beneficial effects of the present invention are: the method for the present invention can be obtained without subcellular organelle or broken The pure excretion body preparation of piece, more thorough research enteron aisle remote control and regulation effect provide experiment basis and possibility.Pass through utilization EDTA digestion method efficiently separates enteric epithelium layer and submucosa, then obtains enteron aisle to body with supercentrifugation from separating liquid Endocrine excretion body.Relative to the method that conventional organization enzymic digestion obtains excretion body, the method cost is lower, the excretion that obtains Body purity is higher.
Detailed description of the invention
Fig. 1 is the exocrine excretion body figure of Electronic Speculum of embodiment of the present invention lower body.
Fig. 2 is the endocrine excretion body figure of Electronic Speculum of embodiment of the present invention lower body.
Fig. 3 be the present embodiment excretion body surface face specificity marker CD63(26KD), HSP70(70KD), TSG101(44KD) Western blotting figure.
Fig. 4 is that the present embodiment excretion body diameter measures peak figure.
Specific embodiment
The present embodiment provides a kind of enteron aisle excretion body extracting methods, comprising the following steps:
(1) intestines sample process: being taken tubulose enteron aisle sample, inverted it from inside to outside using ophthalmic tweezers, is pricked both ends with surgical thread and is closed；
(2) wash: using 1 μM of dithiothreitol (DTT) and being free of Mg2+、Ca2+、Mn2+Without Fe2+The solution of bivalent metal ion PBS is repeatedly Intestinal tissue to cleaning solution is cleaned to clarify；
(3) the excretion body to secreted in vitro: the cleaning solution of collection step 2. is obtained, is separated through low temperature ultracentrifugation and retains precipitating, from The heart separates 2.5h, and centrifugal force sets 100000g；It is washed with ice PBS solution；
(4) separate enterocyte: by step 2. in intestinal tube move in EDTA enteric epithelium separating liquid, EDTA enteric epithelium separating liquid Using 8mM EDTA and be free of Mg2+、Ca2+、Mn2+Without Fe2+The solution allocation of bivalent metal ion PBS, and the separating liquid is placed It is incubated for 30 minutes on ice；Separating liquid is abandoned, fresh ice PBS solution is added, is free of Mg in PBS solution2+、Ca2+、Mn2+Without Fe2+Two Valence metal ion；Acutely concussion makes the epithelial layer of intestinal tube fall off to supernatant muddiness；
(5) be centrifuged, filter: by step, 4. middle cleaning solution centrifuge separation takes supernatant, is centrifugated 5min, centrifugal force sets 10000g； Primary filtration is being carried out by 200nm filter,
(6) obtain to the endocrine excretion body of body: 5. filtered fluid that step is obtained is separated through low temperature ultracentrifugation, centrifuge separation 2.5h, centrifugal force set 100000g；Retain precipitating, is washed with ice PBS；
(7) verify under Electronic Speculum: the excretion body of 3., 6. intestinal epithelial tissue secretion that step obtains is precipitated through glutaraldehyde, sodium cacodylate It after osmium tetroxide fixing process, is observed under Electronic Speculum, is in concave-concave structure by the visible excretion body of Fig. 3 and Fig. 4. Western blotting detect excretion body surface face specificity marker CD63(26KD), HSP70(70KD), TSG101(44KD) Expression；Nanoparticle tracking analysis (NTA) measures the diameter of excretion body between 60-150nm, peak value In 70nm.
In addition to the implementation, the present invention can also have other embodiments.It is all to use equivalent substitution or equivalent transformation shape At technical solution, fall within the scope of protection required by the present invention.

Claims (8)

1. a kind of enteron aisle excretion body extracting method, it is characterised in that include the following steps:
(1) tubulose intestines sample process and cleaning；
(2) intestinal tissue is cleaned repeatedly using dithiothreitol (DTT) solution；
(3) the cleaning solution of collection step 2. obtains the excretion body to secreted in vitro through low temperature ultracentrifugation；
(4) separate enterocyte: by step 2. in intestinal tube move in EDTA enteric epithelium separating liquid, and be placed in and be incubated on ice 25-40min promotes epithelial layer to fall off, then using concussion to supernatant muddiness；
(5) separating liquid centrifuging and taking supernatant is subjected to primary filtration, removes cell and cell fragment；
(6) the filtered fluid 5. step obtained is obtained through low temperature ultracentrifugation to the endocrine excretion body of body.
2. enteron aisle excretion body extracting method as described in claim 1, it is characterised in that: the EDTA is a kind of and Mg2+、Ca2+、 Mn2+、Fe2+The chelating agent that bivalent metal ion combines, passes through the Ca of the chelant ties enterocyte2+, make enterocyte Ca2+It is mildly separated with submucosa, discharges submucosa extracellular matrix.
3. enteron aisle excretion body extracting method as described in claim 1, it is characterised in that: the dithiothreitol (DTT) solution is by 1 μM Dithiothreitol (DTT) is mixed with PBS solution.
4. enteron aisle excretion body extracting method as claimed in claim 3, it is characterised in that: without Mg in the PBS solution2+、Ca2+、 Mn2+Without Fe2+Bivalent metal ion.
5. enteron aisle excretion body extracting method as described in claim 1, it is characterised in that: 5. the step is centrifugated 4- 6min, centrifugal force set 9000-10000g；The primary filtration is filtered using 200nm filter.
6. enteron aisle excretion body extracting method as described in claim 1, it is characterised in that: the step 3. with step 6. in it is super Fast rotary process is centrifugated 2-3h, and centrifugal force sets 100000-120000g.
7. enteron aisle excretion body extracting method as described in claim 1, it is characterised in that: the step 3. with step 6. in it is heavy Starch can carry out verifying under Electronic Speculum after glutaraldehyde, sodium cacodylate and osmium tetroxide fixing process.
8. enteron aisle excretion body extracting method as described in claim 1, it is characterised in that: at the tubulose intestines sample of the step 1. In reason and cleaning, tubulose enteron aisle sample is taken, is inverted it from inside to outside using ophthalmic tweezers, both ends are pricked with surgical thread and are closed.
CN201910278835.6A 2019-04-09 2019-04-09 Method for extracting intestinal exosomes Pending CN110004110A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112961831A (en) * 2021-02-26 2021-06-15 仲恺农业工程学院 Preparation method of intestine-derived exosome

Citations (2)

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Publication number Priority date Publication date Assignee Title
US20140099652A1 (en) * 2012-10-09 2014-04-10 Samsung Electronics Co., Ltd. Composition for monitoring vesicle, kit and method of monitoring vesicle using the same
CN107980004A (en) * 2015-06-10 2018-05-01 得克萨斯州大学系统董事会 Purposes for the excretion body for the treatment of disease

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140099652A1 (en) * 2012-10-09 2014-04-10 Samsung Electronics Co., Ltd. Composition for monitoring vesicle, kit and method of monitoring vesicle using the same
CN107980004A (en) * 2015-06-10 2018-05-01 得克萨斯州大学系统董事会 Purposes for the excretion body for the treatment of disease

Non-Patent Citations (2)

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Title
FAN XIA等: "A High Efficient Method to Isolate Exosomes from Small Intestinal Epithelium", 《MOLECULAR BIOTECHNOLOGY》 *
GUILLAUME VAN NIEL等: "Intestinal Epithelial Cells Secrete Exosome-like Vesicles", 《GASTROENTEROLOGY》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112961831A (en) * 2021-02-26 2021-06-15 仲恺农业工程学院 Preparation method of intestine-derived exosome
CN112961831B (en) * 2021-02-26 2022-10-11 仲恺农业工程学院 Preparation method of intestine-derived exosome

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Application publication date: 20190712