CN106974937A - Application of the adipose-derived mescenchymal stem cell excretion body in treatment metabolic syndrome medicine is prepared - Google Patents
Application of the adipose-derived mescenchymal stem cell excretion body in treatment metabolic syndrome medicine is prepared Download PDFInfo
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Abstract
The invention discloses application of the adipose-derived mescenchymal stem cell excretion body in treatment metabolic syndrome medicine is prepared, the adipose-derived mescenchymal stem cell excretion body is prepared from by the following method：(1) adipose tissue of stripping is scattered in digestive juice, to produce cell suspending liquid；(2) centrifuge cell suspension, to form cell precipitation, precipitation and separation, washing obtains the vascular stroma composition containing adipose-derived mescenchymal stem cell；(3) amplification cultivation is carried out to ADSC in cultivating system in vitro, collects the supernatant after amplification cultivation and concentrated, excretion body extracts reagent is added into the supernatant after concentration, that is, extracted and obtain adipose-derived mescenchymal stem cell excretion body.The adipose-derived mescenchymal stem cell excretion body of the present invention can effectively alleviate insulin resistance and metabolic disorder, suppress metabolic syndrome to a certain extent and NASH develops.
Description
Technical field
The present invention relates to biomedicine technical field, and in particular to adipose-derived mescenchymal stem cell excretion body is controlled in preparation
Treat the application in metabolic syndrome medicine.
Background technology
Metabolic syndrome refers to that the pathology shape of metabolic disorder occurs for the materials such as the protein, fat, carbohydrate of human body
State, is a complex set of metabolic disorder disease group, is to cause diabetes, the hazards of cardiovascular and cerebrovascular disease.Insulin resistance
It is that non-alcoholic fatty liver disease, diabetes, artery are athero- with a series of metabolic syndromes such as hyperglycaemia, hypertension, dyslipidemia
" the common soil " of the disease incidences such as hardening.High admission rate, high disability rate, high fatal rate caused by metabolic syndrome have turned into
Threaten the public health problem of whole mankind's health.
Adipose-derived mescenchymal stem cell (ADSC) is present in the vascular stroma composition (SVF) of adipose tissue, with certainly
I updates and multi-lineage potential.It is different that ADSC can be induced to differentiate into fat cell, osteocyte, cartilage cell etc. in vitro
Histocyte, is the research emphasis and focus of current tissue repair and regeneration field.Adipose-derived mescenchymal stem cell excretion body
Materials such as protein and RNA with biological activity etc. can be carried, active ingredient and efficient information are transferred to recipient cell
Born of the same parents, play an important role in intercellular information interchange.
The research for adipose-derived mescenchymal stem cell relates generally to the purposes in kidney, fundus oculi disease at present
(CN102048756A), treating the purposes (CN105902568A) of erectile dysfunction, preparing treating liver fibrosis preparation
Purposes (CN103436555A), prepare treatment Osteoarthritis preparation purposes (CN105748519A), prepare liver cancer
Purposes (CN103451152A) in chemotherapy sensitizing preparation etc., but be not related to also so far adipose-derived mescenchymal stem cell and its
Report of the excretion body in terms of metabolic syndrome is treated.
The content of the invention
For above-mentioned prior art, treatment is being prepared it is an object of the invention to provide adipose-derived mescenchymal stem cell excretion body
Application in metabolic syndrome medicine.
Present invention research finds that adipose-derived mescenchymal stem cell excretion body can effectively alleviate insulin resistance and be metabolized disorderly
Disorderly, suppress metabolic syndrome to a certain extent and NASH develops, thus propose following hairs
Bright content：
The first aspect of the present invention is preparing treatment metabolic syndrome medicine there is provided adipose-derived mescenchymal stem cell excretion body
Application in thing.
In the present invention, the adipose-derived mescenchymal stem cell excretion body is prepared from by the following method：
(1) adipose tissue of stripping is scattered in digestive juice, to produce cell suspending liquid；
(2) centrifuge cell suspension, to form cell precipitation, precipitation and separation, washing is obtained containing adipose-derived mesenchyma
The vascular stroma composition (SVF) of stem cell (ADSC)；
(3) amplification cultivation is carried out to ADSC in cultivating system in vitro, collects the supernatant after amplification cultivation, concentrated, to
Excretion body extracts reagent is added in supernatant after concentration, that is, extracts and obtains adipose-derived mescenchymal stem cell excretion body.
It is preferred that, in step (1), the digestive juice is made up of KRB buffer solutions and NTx enzyme；It is furthermore preferred that KRB
The proportion of composing of buffer solution and NTx enzyme is 1ml：2mg.
It is preferred that, in step (2), centrifugal rotational speed is 2000rpm, and centrifugation time is 5 minutes.
It is preferred that, in step (3), the condition of amplification cultivation is：Trained with the DMEM of 10% hyclone without excretion body
Support base (containing bFGF) culture ADSC.
It is preferred that, in step (3), the excretion body extracts reagent is Exoquick-TCTM excretion body extracts reagents.
In the present invention, the adipose-derived mescenchymal stem cell excretion body has TSG101, CD9, CD63 and HSP90 mark
Will.
It is preferred that, in the present invention, the adipose-derived mescenchymal stem cell excretion body fatty group at mouse epididymis
Knit.
It is preferred that, in the present invention, the metabolic syndrome is the metabolic disorder of Induced by High Fat Diet.
The second aspect of the present invention is being prepared there is provided the composition comprising above-mentioned adipose-derived mescenchymal stem cell excretion body
Treat the application in metabolic syndrome medicine.
The third aspect of the present invention is preparing treatment non-alcoholic fatty there is provided adipose-derived mescenchymal stem cell excretion body
Application in the biological agent of hepatopathy.
Beneficial effects of the present invention：
Present invention firstly provides adipose-derived mescenchymal stem cell excretion body in treatment metabolic syndrome medicine is prepared
New application, and prepare treatment non-alcoholic fatty liver disease biological agent in new application.Present invention research discovery,
ADSC sources excretion body can effectively alleviate insulin resistance and metabolic disorder, suppress metabolic syndrome to a certain extent and non-
Alcoholic fatty liver develops.In addition, present invention also offers the system of above-mentioned adipose-derived mescenchymal stem cell excretion body
Preparation Method, operation is simple and feasible.
Brief description of the drawings
The Figure of description for constituting the part of the application is used for providing further understanding of the present application, and the application's shows
Meaning property embodiment and its illustrate be used for explain the application, do not constitute the improper restriction to the application.
Fig. 1：The transmission electron microscope picture of adipose-derived mescenchymal stem cell excretion body；In figure, white scale is 150nm.
Fig. 2：Excretion body mark western-blot qualification result.
Fig. 3：After the excretion soma pretreatment in ADSC sources, content of triglyceride in mice serum.
Fig. 4：After the excretion soma pretreatment in ADSC sources, the content of T-CHOL in mice serum..
Fig. 5：After the excretion soma pretreatment in ADSC sources, the change of blood glucose in the glucose tolerance test (GTT) of mouse.
Fig. 6：After the excretion soma pretreatment in ADSC sources, the change of blood glucose in the insulin tolerance tests (ITT) of mouse.
Fig. 7：After the excretion soma pretreatment in ADSC sources, the photo of mouse liver.
Fig. 8：After the excretion soma pretreatment in ADSC sources, content of triglyceride in mouse liver.
Fig. 9：After the excretion soma pretreatment in ADSC sources, mouse liver HE dyeing pictures；In figure, white scale is 100 μ
m。
Embodiment
It is noted that described further below is all exemplary, it is intended to provide further instruction to the application.Unless another
Indicate, all technologies used herein and scientific terminology are with usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe embodiment, and be not intended to restricted root
According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise singulative
It is also intended to include plural form, additionally, it should be understood that, when in this manual using term "comprising" and/or " bag
Include " when, it indicates existing characteristics, step, operation, device, component and/or combinations thereof.
Term explanation：
Metabolic syndrome：Refer to that the pathology shape of metabolic disorder occurs for the materials such as the protein, fat, carbohydrate of human body
State, is a complex set of metabolic disorder disease group, is to cause diabetes, the hazards of cardiovascular and cerebrovascular disease.
ADSC(adipose-derived mesenchymal stem cell)：Adipose-derived mescenchymal stem cell, is present
It is the adipocyte precursor in adipose tissue in adipose tissue vascular stroma composition, it is latent with self-renewing and Multidirectional Differentiation
Energy.
Excretion body (exosome)：It is the class imitated vesicle structure of the outer row of cell secretion, diameter is about 30-120nm, is included many
Kind of albumen and RNA materials, can act on corresponding tissue or cell by paracrine or endocrine mode, be between mediated cell
The important medium of information transmission.
As background technology is introduced, adipose-derived mescenchymal stem cell and its excretion are not related to also in the prior art
Report of the body in treatment metabolic syndrome.Based on this, the present invention proposes a kind of adipose-derived mescenchymal stem cell excretion body and existed
Prepare the application in treatment metabolic syndrome medicine.
In a kind of embodiment of the application, by setting up the metabolic syndrome model that high fat diet is induced, carry out
ADSC sources excretion soma is pre-, and excretion soma prognosis Triglycerides in Serum, the content of T-CHOL are detected respectively；And in excretion
Soma carries out glucose tolerance test and insulin tolerance tests before terminating in advance；Observation mouse liver after excretion soma terminates in advance
The content of triglycerides in fatty change situation and liver.
To sum up, found by the studies above：ADSC sources excretion body has the work for alleviating insulin resistance and metabolic disorder
With the treatment available for metabolic syndrome；The fatty live lesions of high fat diet induction are may also suppress, non-wine is treated available for preparing
The biological agent of essence fatty liver.
In order that the technical scheme of the application can clearly be understood by obtaining those skilled in the art, below with reference to tool
The embodiment of body describes the technical scheme of the application in detail.
Test material used is the conventional test material in this area in the embodiment of the present invention, can pass through commercial channel
It is commercially available.
Embodiment 1：ADSC extraction is with separating
(1) the separation of adipose tissue：10~12 week old C57BL/6 male mices are chosen, anaesthetizes and puts to death after mouse, are opened
Abdominal cavity, peels off fat at its epididymis, blood vessel is removed as far as possible, is weighed after fat weight, is dipped in PBS and places on ice.
(2) digestive juice is prepared：According to fat weight, 3ml KRB buffer solutions are needed according to every gram of fat, 6mg NTx enzymes
Proportions are used.
(3) adipose tissue digests：After the adipose tissue machinery peeled off is shredded, add in the digestive juice prepared, mix
After be placed in 37 DEG C of shaking tables and digest 45 minutes, 2000rpm is centrifuged 5 minutes, and the careful cell for taking out bottom is placed in not enzyme-added
In KRB, place on ice.
(4) filter：With 100 μm of aperture copper mesh filtration cell suspensions, the suspension 2000rpm after filtration is centrifuged 5 minutes, abandoned
Supernatant.
(5) wash：Sediment add double volume without enzyme KRB buffer solutions, 2000rpm after mixing is centrifuged 5 minutes, abandoned
Clearly, sediment is the vascular stroma composition (SVF) containing ADSC.
(6) plate is planted：The principle progress kind plate that cell is seeded to a hole in six orifice plates is carried according to a mouse.Addition contains
The DMEM conditioned mediums for having bFGF are resuspended.Add 2ml culture mediums per hole, 37 DEG C, cultivate under the conditions of 5%CO2,24-30 hours
After change liquid.
(7) pass on：Cell fusion degree can be passed on up to when 90%.PBS cell surface is used, appropriate pancreatin is added and disappears
Change cell, the principle reached according to every hole cell in a 10cm plate carries out Secondary Culture.The cell of forth generation carries out follow-up
Experiment.
Embodiment 2：The extraction and identification of the excretion body in ADSC sources
ADSC is cultivated with the DMEM culture mediums (containing bFGF) of 10% hyclone without excretion body.Take forth generation ADSC
Cells and supernatant, after 0.22 μm of sterilizing filter filtering, 5000g, 4 DEG C of 100K concentrations centrifuge tube is centrifuged 30 minutes, takes upper chamber
Concentrate adds Exoquick-TC in new sterile 15ml centrifuge tubesTMExcretion body extracts reagent, after being placed in 4 DEG C overnight,
5000g, 4 DEG C centrifuge 30 minutes, abandon supernatant, are resuspended with sterile PBS and precipitate and dispense, and freeze in -80 DEG C of preservations.
The excretion body extracted is detected with the method for transmission electron microscope to its direct size and form, as a result as schemed
Shown in 1；Excretion body mark TSG101, CD9, CD63, HSP90 are detected with western-blot method, further it is carried out
Identification, as a result as shown in Figure 2.
Embodiment 3：Influence of the ADSC sources excretion body to high fat diet inducible metabolism syndromes models
(1) high fat diet inducible metabolism syndromes models are set up and excretion soma is pre-
7 week old C57BL/6 male mices are used to set up metabolic syndrome model.After the pre- high fat diet of one week is adapted to,
High fat diet (HFD) (containing 60%cal) nursing is given, is fed as a control group with full diet (NCD).High-fat diet
After 12-20 weeks, (30 μ g/mouse, injection in every 3 days once, continuously injects 6-8 to excretion body prepared by intraperitoneal injection embodiment 2
Week), as HFD-exosome groups；Intraperitoneal injection of saline group is control group, i.e. HFD-NS.Full diet feeds mouse abdomen
Chamber injecting normal saline group is NCD-NS.
(2) content of triglycerides in mice serum is detected
After excretion soma terminates in advance, the content of triglycerides in mouse peripheral blood, kit detection mice serum is taken.As a result
As shown in figure 3, after the treatment of excretion body, the content of triglycerides is substantially reduced in mice serum.
(3) content of T-CHOL in mice serum is detected
After excretion soma terminates in advance, the content of T-CHOL in mouse peripheral blood, kit detection mice serum is taken.As a result
As Fig. 4 shows, after the treatment of excretion body, the content of T-CHOL is substantially reduced in mice serum.
(4) glucose tolerance test (GTT experiments)
Excretion soma terminates the last fortnight in advance, and row GTT is tested, as a result as shown in Figure 5.As seen from Figure 5, excretion body is treated
Group mouse significantly improves to the tolerance of glucose.
(5) insulin tolerance tests (ITT experiments)
Excretion soma terminates the last week in advance, row ITT experiments, as a result as shown in Figure 6.As seen from Figure 6, excretion body is treated
The insulin sensitivity of group mouse is significantly improved.
(6) the fatty change situation of mouse liver
After excretion soma terminates in advance, take mouse liver and take pictures, observe the fatty change situation of mouse liver, as a result such as Fig. 7
It is shown.As seen from Figure 7, excretion soma prognosis, mouse liver fat becomes apparent improvement.
(7) in mouse liver triglycerides content
After excretion soma terminates in advance, mouse liver is taken, extract in mouse liver triglycerides and detect glycerine with kit
The content of three esters, as a result as shown in Figure 8.As can be seen in Figure 8, excretion soma is pre- can reduce triglycerides in mouse liver
Content.
(8) hepatic tissue section is dyed
After excretion soma terminates in advance, mouse liver is taken, and carry out HE section statinings, as shown in figure 9, excretion soma prognosis,
Fat drips quantity is reduced in hepatic tissue section.
The preferred embodiment of the application is the foregoing is only, the application is not limited to, for the skill of this area
For art personnel, the application can have various modifications and variations.It is all within spirit herein and principle, made any repair
Change, equivalent substitution, improvement etc., should be included within the protection domain of the application.
Claims (10)
1. application of the adipose-derived mescenchymal stem cell excretion body in treatment metabolic syndrome medicine is prepared.
2. application as claimed in claim 1, it is characterised in that the adipose-derived mescenchymal stem cell excretion body is by such as lower section
Method is prepared from：
(1) adipose tissue of stripping is scattered in digestive juice, to produce cell suspending liquid；
(2) centrifuge cell suspension, to form cell precipitation, precipitation and separation, washing obtains dry thin containing adipose-derived mesenchyma
The adipose tissue stroma cell of born of the same parents；
(3) amplification cultivation is carried out to ADSC in cultivating system in vitro, collects the supernatant after amplification cultivation, concentration, to concentration
Excretion body extracts reagent is added in supernatant afterwards, that is, extracts and obtains adipose-derived mescenchymal stem cell excretion body.
3. application as claimed in claim 2, it is characterised in that in step (1), the digestive juice is by KRB buffer solutions and I type
Clostridiopetidase A is constituted；It is preferred that, the proportion of composing of KRB buffer solutions and NTx enzyme is 1ml：2mg.
4. application as claimed in claim 2, it is characterised in that in step (2), centrifugal rotational speed is 2000rpm, and centrifugation time is
5min。
5. application as claimed in claim 2, it is characterised in that in step (3), the condition of amplification cultivation is：With containing bFGF
10% hyclone DMEM medium cultures ADSC；Excretion body is free of in the culture medium.
6. application as claimed in claim 1, it is characterised in that the adipose-derived mescenchymal stem cell excretion body has
TSG101, CD9, CD63 and HSP90 indicate.
7. application as claimed in claim 1, it is characterised in that the adipose-derived mescenchymal stem cell excretion body is from small
Adipose tissue at mouse epididymis.
8. application as claimed in claim 1, it is characterised in that the metabolic syndrome is disorderly for the metabolism of Induced by High Fat Diet
Disorderly.
9. the composition comprising the adipose-derived mescenchymal stem cell excretion body described in claim 1 is preparing treatment Metabolic syndrome
Levy the application in medicine.
10. adipose-derived mescenchymal stem cell excretion body answering in the biological agent for preparing treatment non-alcoholic fatty liver disease
With.
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Cited By (6)
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| WO2019031729A3 (en) * | 2017-08-05 | 2019-05-16 | 주식회사 엑소코바이오 | Use of composition comprising stem cell-derived exosome as effective ingredient in alleviating non-alcoholic simple steatosis or non-alcoholic steatohepatitis |
| CN109913407A (en) * | 2019-02-22 | 2019-06-21 | 浙江大学 | The preparation method and application of adipose-derived mescenchymal stem cell class excretion body |
| CN110564682A (en) * | 2019-09-30 | 2019-12-13 | 陕西中鸿科瑞再生医学研究院有限公司 | Method for large-scale production of human adipose-derived mesenchymal stem cell exosomes |
| CN112352047A (en) * | 2020-09-08 | 2021-02-09 | 江苏大学 | Preparation method and application of exosome derived from human umbilical cord mesenchymal stem cells |
| CN112630449A (en) * | 2020-12-30 | 2021-04-09 | 广西壮族自治区水牛研究所 | Blood exosome marker for judging buffalo birth time and application thereof |
| WO2023056923A1 (en) * | 2021-10-07 | 2023-04-13 | China Medical University | Use of exosomes derived from mesenchymal stem cells for treating non-alcoholic steatohepatitis |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2019031729A3 (en) * | 2017-08-05 | 2019-05-16 | 주식회사 엑소코바이오 | Use of composition comprising stem cell-derived exosome as effective ingredient in alleviating non-alcoholic simple steatosis or non-alcoholic steatohepatitis |
| CN109913407A (en) * | 2019-02-22 | 2019-06-21 | 浙江大学 | The preparation method and application of adipose-derived mescenchymal stem cell class excretion body |
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| CN112352047A (en) * | 2020-09-08 | 2021-02-09 | 江苏大学 | Preparation method and application of exosome derived from human umbilical cord mesenchymal stem cells |
| WO2022051883A1 (en) * | 2020-09-08 | 2022-03-17 | 江苏大学 | Preparation method for and application of exosome derived from human umbilical cord mesenchymal stem cells |
| CN112630449A (en) * | 2020-12-30 | 2021-04-09 | 广西壮族自治区水牛研究所 | Blood exosome marker for judging buffalo birth time and application thereof |
| CN112630449B (en) * | 2020-12-30 | 2022-10-18 | 广西壮族自治区水牛研究所 | Blood exosome marker for judging buffalo birth time and application thereof |
| WO2023056923A1 (en) * | 2021-10-07 | 2023-04-13 | China Medical University | Use of exosomes derived from mesenchymal stem cells for treating non-alcoholic steatohepatitis |
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